InteractiveFly: GeneBrief

foraging: Biological Overview | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - foraging

Synonyms - dg2

Cytological map position - 24A2--4

Function - signaling

Keywords - feeding behavior, locomotory behavior, synapse

Symbol - for

FlyBase ID: FBgn0000721

Genetic map position - 2-10

Classification - cGMP-dependent protein kinase activity

Cellular location - cytoplasm



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Philippe, A.S., Jeanson, R., Pasquaretta, C., Rebaudo, F., Sueur, C. and Mery, F. (2016). Genetic variation in aggregation behaviour and interacting phenotypes in Drosophila. Proc Biol Sci 283 (1827). PubMed ID: 27009219
Summary:
This study used a combination of approaches to study how genetic variation and social environment interact to influence aggregation dynamics in Drosophila. The study used two different natural lines of Drosophila that arise from a polymorphism in the foraging gene (rovers and sitters). Groups of flies were placed in a heated arena. Flies could freely move towards one of two small, cooler refuge areas. In groups of the same strain, sitters were found to have a greater tendency to aggregate. The observed behavioural variation was based on only two parameters: the probability of entering a refuge and the likelihood of choosing a refuge based on the number of individuals present. Aggregation behaviour of each line was strongly affected by the presence of the other strain, without changing the decision rules used by each. Individuals obeying local rules shaped complex group dynamics via a constant feedback loop between the individual and the group. This study could help to identify the circumstances under which particular group compositions may improve individual fitness through underlying aggregation mechanisms under specific environmental conditions. 

Peng, Q., Wang, Y., Li, M., Yuan, D., Xu, M., Li, C., Gong, Z., Jiao, R. and Liu, L. (2016). cGMP-dependent protein kinase encoded by foraging regulates motor axon guidance in Drosophila by suppressing Lola function. J Neurosci 36: 4635-4646. PubMed ID: 27098704
Summary:
Correct pathfinding and target recognition of a developing axon are exquisitely regulated processes that require multiple guidance factors. Among these factors, the second messengers, cAMP and cGMP, are known to be involved in establishing the guidance cues for axon growth through different intracellular signaling pathways. However, whether and how cGMP-dependent protein kinase (PKG) regulates axon guidance remains poorly understood. This study shows that the motor axons of intersegmental nerve b (ISNb) in the Drosophila embryo display targeting defects during axon development in the absence of foraging (for), a gene encoding PKG. In vivo tag expression reveals PKG to be present in the ventral nerve code at late embryonic stages, supporting its function in embryonic axon guidance. Mechanistic studies show that the transcription factor longitudinal lacking (lola) genetically interacts with for. PKG physically associates with the LolaT isoform via the C-terminal zinc-finger-containing domain. Overexpression of PKG leads to the cytoplasmic retention of LolaT in S2 cells, suggesting a role for PKG in mediating the nucleocytoplasmic trafficking of Lola. Together, these findings reveal a novel function of PKG in regulating the establishment of neuronal connectivity by sequestering Lola in the cytoplasm.

Krill, J. L. and Dawson-Scully, K. (2016). cGMP-dependent protein kinase inhibition extends the upper temperature limit of stimulus-evoked calcium responses in motoneuronal boutons of Drosophila melanogaster larvae. PLoS One 11: e0164114. PubMed ID: 27711243
Summary:
While the mammalian brain functions within a very narrow range of oxygen concentrations and temperatures, the fruit fly, Drosophila melanogaster, has employed strategies to deal with a much wider range of acute environmental stressors. The foraging (for) gene encodes the cGMP-dependent protein kinase (PKG), has been shown to regulate thermotolerance in many stress-adapted species, including Drosophila, and could be a potential therapeutic target in the treatment of hyperthermia in mammals. Whereas previous thermotolerance studies have looked at the effects of PKG variation on Drosophila behavior or excitatory postsynaptic potentials at the neuromuscular junction (NMJ), little is known about PKG effects on presynaptic mechanisms. This study characterized presynaptic calcium ([Ca2+]i) dynamics at the Drosophila larval NMJ to determine the effects of high temperature stress on synaptic transmission. The neuroprotective role of PKG modulation was investigated both genetically using RNA interference (RNAi), and pharmacologically, to determine if and how PKG affects presynaptic [Ca2+]i dynamics during hyperthermia. PKG activity was found to modulate presynaptic neuronal Ca2+ responses during acute hyperthermia, where PKG activation makes neurons more sensitive to temperature-induced failure of Ca2+ flux and PKG inhibition confers thermotolerance and maintains normal Ca2+ dynamics under the same conditions. Targeted motoneuronal knockdown of PKG using RNAi demonstrated that decreased PKG expression was sufficient to confer thermoprotection. These results demonstrate that the PKG pathway regulates presynaptic motoneuronal Ca2+ signaling to influence thermotolerance of presynaptic function during acute hyperthermia.
Allen, A. M., Anreiter, I., Neville, M. C. and Sokolowski, M. B. (2016). Feeding-related traits are affected by dosage of the foraging gene in Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 28007892
Summary:
Nutrient acquisition and energy storage are critical parts of achieving metabolic homeostasis. The foraging gene in Drosophila melanogaster has previously been implicated in multiple feeding-related and metabolic traits. Before foraging's functions can be further dissected, a precise genetic null mutant is needed to definitively map its amorphic phenotypes. This study used homologous recombination to precisely delete foraging, generating the for0 null allele, and used recombineering to re-integrate a full copy of the gene, generating the {forBAC} rescue allele. Total loss of foraging expression in larvae results in reduced larval path length and food intake behavior, while conversely showing an increase in triglyceride levels. Furthermore, varying foraging gene dosage demonstrates a linear dose-response on these phenotypes in relation to foraging gene expression levels. These experiments have unequivocally proven a causal, dose-dependent relationship between the foraging gene and its pleiotropic influence on these feeding-related traits. In that regard, this analysis of foraging's transcription start sites, termination sites, and splicing patterns using RACE and full length cDNA sequencing, revealed 4 independent promoters, pr1-4, that produce 21 transcripts with 9 distinct ORFs. The use of alternative promoters and alternative splicing at the foraging locus creates diversity and flexibility in the regulation of gene expression, and ultimately function. Future studies will exploit these genetic tools to precisely dissect the isoform- and tissue-specific requirements of foraging's functions and shed light on the genetic control of feeding-related traits involved in energy homeostasis.
Wang, S. and Sokolowski, M. B. (2017). Aggressive behaviours, food deprivation and the foraging gene. R Soc Open Sci 4(4): 170042. PubMed ID: 28484630
Summary:
A pleiotropic gene governs multiple traits, which might constrain the evolution of complexity due to conflicting selection on these traits. However, if the pleiotropic effect is modular, then this can facilitate synergistic responses to selection on functionally related traits, thereby leveraging the evolution of complexity. To understand the evolutionary consequence of pleiotropy, the relation among functionally different traits governed by the same gene is key. This study examined a pleiotropic function of the foraging (for) gene with its rover and sitter allelic variants in fruit fly, Drosophila melanogaster. for's effect on adult male aggressive behaviours was measured and whether this effect was shaped by for's known role in food-related traits. Rover exhibited higher levels of offensive behaviour than sitters and by s2, a sitter-like mutant on rover genetic background. With a Markov chain model, w the rate of aggression escalation was measure, and the rover pattern of aggressive escalation was found to more rapidly intensify fights. Subsequent analysis revealed that this was not caused by for's effect on food-related traits, suggesting that for might directly regulate aggressive behaviours. Food deprivation did not elevate aggression, but reduced intermediate-level aggressive behaviours. Aggression and other foraging-related behaviour might comprise a synergistic trait module underlaid by this pleiotropic gene.
McConnell, M. W. and Fitzpatrick, M. J. (2017). 'Foraging' for a place to lay eggs: A genetic link between foraging behaviour and oviposition preferences. PLoS One 12(6): e0179362. PubMed ID: 28622389
Summary:
Gravid female arthropods in search of egg-laying substrates embark on foraging-like forays: they survey the environment assessing multiple patches, tasting each with their tarsi and proboscis, and then, if interested, they deposit an egg (or eggs). In fruit flies, Drosophila melanogaster, allelic variation in the foraging gene (for) underlies the rover/sitter foraging behaviour polymorphism. Rover flies (forR) are more active foragers (both within and between food patches) compared to sitters (fors). In nematodes, Caenorhabditis elegans, a mutation in egl-4, the ortholog of for, leads to aberrations in egg laying. Given this and the notion that females may 'forage' for a place to oviposit, it was hypothesized that for may underlie egg-laying decisions in the fruit fly. Indeed, when given a choice between patches of low- and high-nutrient availability, rovers lay significantly more eggs on the low-nutrient patches than sitters and also a sitter mutant (fors2). This study confirmed the role of for by inducing rover-like oviposition preferences in a sitter fly using the transgenic overexpression of for-mRNA in the nervous system.
Allen, A. M., Anreiter, I., Vesterberg, A., Douglas, S. J. and Sokolowski, M. B. (2018). Pleiotropy of the Drosophila melanogaster foraging gene on larval feeding-related traits. J Neurogenet: 1-11. PubMed ID: 30303018
Summary:
Little is known about the molecular underpinning of behavioral pleiotropy. The Drosophila melanogaster foraging gene is highly pleiotropic, affecting many independent larval and adult phenotypes. Included in foraging's multiple phenotypes are larval foraging path length, triglyceride levels, and food intake. foraging has a complex structure with four promoters and 21 transcripts that encode nine protein isoforms of a cGMP dependent protein kinase (PKG). This study examined if foraging's complex molecular structure underlies the behavioral pleiotropy associated with this gene. Using a promotor analysis strategy, DNA fragments upstream of each of foraging's transcription start sites was cloned and four separate forpr-Gal4s were generated. Supporting the hypothesis of modular function, they had discrete, restricted expression patterns throughout the larva. Promoter specific expression was found in the larval fat body, salivary glands, and body muscle. The modularity of foraging's molecular structure was also apparent in the phenotypic rescues. Path length, triglyceride levels (bordered on significance), and food intake were rescued of forpr0 null larvae using different forpr-Gal4s to drive UAS-for(cDNA). The results refine the spatial expression responsible for foraging's associated phenotypes, as well as the sub-regions of the locus responsible for their expression. foraging's pleiotropy arises at least in part from the individual contributions of its four promoters.
BIOLOGICAL OVERVIEW

Naturally occuring polymorphisms in behavior are difficult to map genetically and thus are refractory to molecular characterization. An exception is the Drosophila melanogaster foraging (for) gene, which has two naturally occurring variants relating to food-search behavior: 'rover' and 'sitter'. Molecular mapping placed foraging mutations in the dg2 gene, which encodes a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG). Rovers have higher PKG activity than sitters, and transgenic sitters expressing a dg2 complementary DNA from rover show transformation of behavior to rover type. Thus, PKG levels affect food-search behavior, and natural variation in PKG activity accounts for a behavioral polymorphism (Osborne, 1997).

Interest in the cGMP-dependent serine/threonine kinase, or PKG, has grown with the awareness of the diversity of biochemical pathways that involve cGMP. PKG has been shown to influence characteristics involved in both functional and developmental plasticity of neural circuits. In Drosophila, one form of PKG (known as dg2; Kalderon, 1989) is encoded by the foraging gene (Osborne, 1997), which takes its name from a behavioral phenotype, the degree of locomotion while feeding, indicated by larval and adult foraging trail lengths (Sokolowski 1980; de Belle, 1987; de Belle, 1989; Pereira, 1993. Two naturally occurring variants, forR (rovers, with long foraging trails) and fors (sitters, with short foraging trails), have high and low PKG levels, respectively. Rovers and sitters do not differ in general activity in the absence of food. Both rovers and sitters are wild-type forms that exist at appreciable frequencies. Several mutations of the locus map with the naturally occurring alleles in the 24A3-5 region of the D. melanogaster polytene chromosomes. This region contains dg2, one of two cGMP-dependent protein kinase (PKG) genes in Drosophila. The dg2 gene has three major transcripts, T1, T2, and T3, and the for mutations are localized to this region. The P[GAL4] transposable element in 189Y was inserted in the 5' end of the dg2 T2 transcript. This homozygous viable insertion identified a new for allele, because P-element excision reverts larval foraging behavior from the sitter to the rover phenotype. As is the case with other sitter alleles, locomotion of the 189Y larvae is not reduced in the absence of food, indicating that the change in behavior is foraging-specific (Osborne, 1997).

To determine whether PKG is directly responsible for the foraging polymorphism in Drosophila, dg2 was overexpressed in sitter larvae. This results in a change of behavior to the rover phenotype. The transgenic strain contains four copies of a heat shock-driven dg2-cDNA. The basal level of PKG expression in this transgenic strain is sufficient to rescue rover larval behavior, thus eliminating the lethal and sublethal effects of heat on the dg2-transgenic larvae. As expected, the PKG enzyme activities of the dissected larval central nervous systems (CNSs) show that without heat shock, the dg2-cDNA transgenic strain have levels of PKG similar to those of forR and significantly higher than those of the sitter control strain (Osborne, 1997).

The basis for the dg2 activity difference between forR and fors was further addressed by measurement of RNA levels and PKG protein. Northern (RNA) analysis revealed that fors and fors2 show a small but consistent reduction in the abundance of T1 RNA relative to that in forR. T2 and T3 RNA are also reduced in these strains, but to a lesser extent. To assess protein levels, extracts of adult heads were subjected to protein immunoblot analysis by probing with an antibody to bovine PKG, or the extracts were affinity-purified by chromatography on cGMP-sepharose, labeled, and electrophoresed. In both experiments, a prominent band at a molecular mass of 80,000 Daltons was found. This is the only band strongly induced by heat shock in the dg2-cDNA transgenic strain, and it is less intense in fors than forR. (This band is also somewhat less intense in fors2 and nearly absent in 189Y homozygotes). Taken together, these results argue that the difference between the naturally occurring alleles forR and fors is in the level of expression of the enzyme (Osborne, 1997).

The assignment of mutations in the for gene to the dg2 locus not only establishes the identification of PKG mutations but also implicates the cGMP signal transduction pathway in the regulation of food-search behavior in D. melanogaster. Small but significant differences in the levels of this kinase affect the naturally occurring behavioral polymorphism. These small differences in PKG are even detectable in homogenates, indicating that the differences in PKG level in rovers and sitters might be larger in cells relevant to the expression of the foraging behavior. These results suggest that the amount of kinase activity affects larval food-search behavior. Indeed, even modest quantitative changes in kinase activity affect behavior. Induced mutations that affect behavioral phenotypes often lie in signal transduction pathways. For example, the cyclic adenosine monophosphate (cAMP) system influences associative learning in flies, and genetic variants in two other serine/threonine kinases: the calcium/calmodulin-dependent protein kinase II and protein kinase C affect learning and behavioral plasticity in flies and mice. The finding that for encodes a PKG shows that a naturally occurring genetic polymorphism in behavior involves these pathways. PKG has a variety of pleiotropic cellular regulatory functions that are also typical of signal transduction components. Electrophysiological studies have shown that injected kinase affects neuronal membrane conductance in snails and mammals; that inhibitors of PKG block long-term potentiation in mammalian hippocampus and that PKG is involved in presynaptic long-term potentiation in cultured hippocampal neurons. Outside the nervous system, PKG has also been implicated in controlling proliferation of smooth muscle cells and neutrophil degranulation. These findings assign behavioral functions to this relatively scarce member of the serine/threonine kinases and show that subtle differences in PKG can lead to naturally occurring variation in behavior (Osborne, 1997 and references).

A cGMP-dependent protein kinase gene, foraging, modifies habituation-like response decrement of the giant fiber escape circuit in Drosophila

The Drosophila giant fiber jump-and-flight escape response is a model for genetic analysis of both the physiology and the plasticity of a sensorimotor behavioral pathway. The electrically induced giant fiber response in intact tethered flies has been established as a model for habituation, a form of nonassociative learning. The rate of stimulus-dependent response decrement of this neural pathway in a habituation protocol is correlated with PKG (cGMP-Dependent Protein Kinase) activity and foraging behavior. Response decrement was assayed for natural and mutant rover and sitter alleles of the foraging (for) gene that encodes a Drosophila PKG. Rover larvae and adults, which have higher PKG activities, travel significantly farther while foraging than sitters with lower PKG activities. Response decrement is most rapid in genotypes previously shown to have low PKG activities and sitter-like foraging behavior. Differences were found in spontaneous recovery (the reversal of response decrement during a rest from stimulation) and a dishabituation-like phenomenon (the reversal of response decrement evoked by a novel stimulus). This electrophysiological study in an intact animal preparation provides one of the first direct demonstrations that PKG can affect plasticity in a simple learning paradigm. It increases understanding of the complex interplay of factors that can modulate the sensitivity of the giant fiber escape response, and it defines a new adult-stage phenotype of the foraging locus. Finally, these results show that behaviorally relevant neural plasticity in an identified circuit can be influenced by a single-locus genetic polymorphism existing in a natural population of Drosophila (Engel, 2000).

Habituation is a form of nonassociative learning in which a behavioral response is reduced or disappears with repeated stimulation. Nonassociative conditioning is of interest as a simple manifestation of physiological mechanisms that also may underlie more complex associative learning paradigms. Habituation may be mediated by a variety of mechanisms, including homosynaptic depression and extrinsic inhibition. Habituation is phylogenetically widespread and has functional significance in modulating both the gain and sensitivity of behavioral responses (Engel, 2000 and references therein).

The Drosophila giant fiber pathway that mediates the visually induced startle reflex, a jump-and-flight escape response, has been studied extensively at the levels of neural physiology and development. The response can be evoked by electrical stimulation to the brain in an intact animal, and this has allowed the bypassing of visual input and facilitated the focusing on central and motor stages of neural processing in an intact, behaviorally relevant circuit. The response likelihood diminishes with repeated electrical stimulation. This response decrement shows most of the typical characteristics of behavioral habituation including frequency dependence, strength dependence, habituation beyond zero response, spontaneous recovery, faster rehabituation, dishabituation, and habituation of dishabituation (Engel, 1996, 1998). Because electrical stimulation recruits the escape response circuit after initial stages of sensory processing, this report refers to modification patterns resembling 'habituation' and 'dishabituation' as 'response decrement' and 'evoked recovery,' respectively. Nevertheless, conformity to the characteristics of a widely studied learning paradigm makes the giant fiber response a useful model for genetic analyses of behavioral plasticity and its physiological correlates at the circuit level (Engel, 1996, 1998). This approach has provided evidence that Drosophila mutants defective in associative learning paradigms (in genes affecting cAMP metabolism) also display abnormal response decrement of the giant fiber response in a habituation protocol (Engel, 1996, 1998, 2000).

Several gene loci have been identified that influence habituation-like decrement of the giant fiber response, with products that include adenylyl cyclase (rutabaga) and cAMP phosphodiesterase (dunce; Engel, 1996), K+ channel subunits with distinct physiological properties including voltage activation (Shaker, ether à go-go), calcium activation (slowpoke), and channel modulation (Hyperkinetic; Engel, 1998), and now PKG (foraging). Like the cAMP pathway genes that affect learning, foraging has pleiotropic effects with potential fitness consequences (Hughes, 1996; Sokolowski, 1997; Wingrove, 1999). This pleiotropy is paralleled at the cellular level in which these gene products have diverse molecular targets and actions. PKG serine/threonine kinases have numerous targets that could affect neuronal function and growth, such as ion channels (Stockand, 1996; Carrier, 1997; Taguchi, 1997; Alioua, 1998; Han, 1998; Vaandrager, 1998; Wexler, 1998), ATPases (e.g., Uneyama, 1998), and regulators of gene expression (Gudi, 1997; Idriss, 1999). PKG may interact with other second messenger systems such as PKA, either by regulating such other systems (Moon, 1998) or by phosphorylating common targets (Lengyel, 1999). It is interesting that mutations of dunce that increase cAMP abundance lead to more rapid stimulus-dependent response decrement (Engel, 1996), opposite that of the effect of increased PKG activity in foraging rover genotypes (Engel, 2000).

Thus, a picture has emerged in which the molecular mechanisms that underlie response decrement in a habituation paradigm, like other neural plasticity such as LTP, are influenced by multiple biochemical and genetic factors. The redundancy of pathways influencing response modification therefore could allow habituation of the escape behavior to be modified and fine-tuned over the course of generations for more adaptive matching to ecologically relevant stimuli. An important point is that the foraging locus is known to be polymorphic in wild populations. This suggests that habituation of escape could vary among flies in a natural population. The foraging locus may be part of the genetic architecture through which plasticity and sensitivity of the escape response have been fine-tuned over evolutionary time (Engel, 2000).

The rate of response decrement has been found to be correlated with PKG activity and foraging behavior: decrement of the electrically induced response was most rapid in genotypes previously shown to have low PKG activity and sitter-like foraging behavior. Differences in spontaneous recovery from response decrement during a rest from stimulation and in dishabituation-like recovery were evoked by a novel stimulus (a puff of air). The data suggest that these differences in spontaneous recovery and evoked recovery may be secondary consequences of differing rates of response decrement. This indicates the interdependence of multiple processes of plasticity in stimulus-dependent response decrement of the giant fiber response. The data further raise the possibility that two processes with different time courses contribute to the response decrement (Engel, 2000).

Overall, the results show that PKG affects habituation-like response decrement in an identified neural circuit of intact tethered flies. From this it can be hypothesized that PKG also may be involved in other forms of learning. It has been shown that cAMP signaling pathways, which play an essential role in associative learning in flies, also affect stimulus-dependent decrement of the giant fiber response (Engel, 1996). The present results suggest that modulation of the escape response could involve the counterbalancing of multiple second messenger systems. A new adult-stage phenotype of the foraging locus has been defined. Finally, it has been shown that behaviorally relevant neural plasticity in an identified circuit can be influenced by a single-locus genetic polymorphism (Engel, 2000).

The response decrement of the long-latency giant fiber response induced has been examined by electrical stimulation; this treatment bypasses the initial stages of visual processing to recruit afferents to the descending giant fibers (Engel, 1996). Rates of response decrement are strongly affected by allelic variation in the foraging gene. Sitter stocks show more rapid response decrement than rovers in comparisons between the two artificially induced alleles or the two natural alleles. The most dramatic difference was between alleles generated artificially by P-element insertion and excision. for189Y showed more rapid response decrement than any other line in this study. The abundance of foraging PKG is quite low in for189Y (Osborne, 1997). In contrast, forE1 showed scarcely any response decrement at the standard stimulation frequency of 5 Hz. In fact, some forE1 flies could be driven at stimulus rates of 30 Hz or higher without showing failures. forE1 arose by excision of the P-element from the foraging locus in for189Y; rover behavior and high abundance of PKG are restored in forE1 relative to for189Y (Engel, 2000).

More subtle differences were observed between the naturally occurring alleles. fors flies showed more rapid response decrement than forR. Flies homozygous for each of the two foraging alleles forR and fors differ in their degrees of PKG activity (Osborne, 1997). forR is genetically dominant to fors for the larval foraging phenotype (de Belle, 1987) but intermediate for adult foraging (Pereira, 1993). As was the case for adult foraging behavior, heterozygous F1 progeny (forR/fors) showed a rate of response decrement intermediate between the parental stocks, suggesting semidominance for this response modification phenotype (Engel, 2000).

The experiments described in this study were conducted within a single year (1999). The forR and fors stocks also were tested in this habituation-like protocol in 1996. In these earlier tests, the absolute resistance to response decrement was greater for both genotypes than in 1999, but fors again showed more rapid response decrement than forR. Similarly, repeated measurements of larval and adult foraging behavior have shown that it is the relative differences between rovers and sitters, not the absolute mean behavioral scores, that are maintained across tests performed at different times or in different laboratories (Engel, 2000).

Differences were observed in spontaneous recovery from decrement of the long-latency electrically induced response. Flies first were stimulated to a response decrement criterion of five consecutive failures (indicating a low response likelihood). One measure of spontaneous recovery is the response likelihood for the first stimulus given after 5 sec of rest. A 5-sec rest period is ordinarily sufficient for the response likelihood to return to nearly 100%, even in genotypes with very rapid response decrement (Engel, 1996, 1998). Full recovery of the response was observed for forR and fors flies as well as forR/fors heterozygotes. However, for189Y flies did not recover fully in 5 sec (Engel, 2000).

A second measure of recovery is the resistance to response decrement within a subsequent stimulus episode. This was quantified as the number of responses evoked before reaching the five-failure decrement criterion during stimulus bouts delivered after different recovery intervals. The resistance to response decrement integrates performance over the entire stimulus bout rather than just the first stimulus. The for genotypes showed clear differences in their degree of recovery to initial rates of response decrement. Absolute postrecovery response numbers were highest for forR, the most slowly decrementing stock, and were progressively lower for more rapidly decrementing genotypes. However, when mean response numbers were divided by first-bout response numbers to give normalized recovery indices, this ranking was reversed: the highest recovery indices were shown by rapidly decrementing sitter genotypes, particularly after 30- and 120-sec recovery intervals. When postrecovery response scores were log-transformed, effectively normalizing the results within genotypes while retaining scale differences between genotypes, the kinetic profiles of recovery showed a similar ranking pattern, with the greatest degrees of recovery after 30- and 120-sec intervals being shown by rapidly decrementing genotypes (Engel, 2000).

The slight degree of spontaneous recovery between 30 and 120 sec suggests that, in addition to a short-term component of response decrement that recovers in less than 30 sec, there is also a long-term component of response decrement with slower onset and recovery kinetics that becomes stronger over multiple stimulus bouts and recovers with a time course exceeding 120 sec. In previous work, 30- or 120-sec recovery intervals were tested after a single prior stimulus bout (in different groups of flies), and with that protocol the recovery to first-bout response decrement rates was nearly complete (Engel, 1996, 1998). In the present experiments, each fly received four stimulus bouts separated by intervals of 5, 30, and 120 sec, so that 30- and 120-sec recovery intervals were tested after two or three prior stimulus bouts (instead of one prior bout as in the earlier studies). It appears that additional prior stimulus bouts affected the state of the response pathway even though every bout ended with a consistent response decrement criterion of five failures (Engel, 2000).

A slowly developing component of response decrement could be most apparent in slowly decrementing flies, because they are exposed to a greater number of stimuli in the two or three bouts preceding the recovery interval. Consistent with this, the lowest 30- and 120-sec recovery indices were shown by the most slowly decrementing genotypes. To examine this relationship more directly, normalized recovery indices for individual flies of all genotypes were plotted against the total number of stimuli given in bouts before the recovery interval. After 30- or 120-sec intervals, recovery indices were inversely related to the number of prior stimuli. This relationship was most evident for the range of 50 to 300 prior stimuli, suggesting that this slow component of response decrement became saturated after 300 stimuli and that other factors contributed more to response variation with fewer than 50 prior stimuli. After the shortest recovery interval of 5 sec, the relationship was weak. This suggests that recovery from a short-term process of response decrement is the predominant factor during the first 5 sec after the end of a bout (Engel, 2000).

The potential to distinguish multiple components of habituation-like response decrement in this system will require further study. Here, it is most important to note that for genotypes showed differences in recovery when tested under a consistent protocol (Engel, 2000).

Recovery of the long-latency giant fiber response can be evoked by a novel stimulus such as an airpuff in a dishabituation protocol (Engel, 1996, 1998). Clear evoked recovery could be shown in each strain except for189Y. The number of responses for the 20 stimuli after an airpuff or 'sham puff' (each averaged from five repetitions) was divided by the number of responses at the beginnings of bouts, giving test and control scores, respectively. The operational criterion for evoked recovery was a test score greater than double the control score. Evoked recovery was observed most often in slowly decrementing genotypes (forR and forR/fors). Among flies that did show evoked recovery by this definition, the magnitude of recovery (the test score) was also greatest in slowly decrementing genotypes (Engel, 2000).

Few forE1 flies showed response decrement to five-failure criterion at the standard stimulation frequency of 5 Hz. However, with higher stimulus frequencies forE1 flies did display habituation-like response decrement, characterized by synchronous loss of responses in DLM (Dorsal Longitudinal Muscle) and TTM (Tergotrochanteral Muscle), spontaneous recovery, and recovery evoked by an airpuff (Engel, 2000).

Latency and refractory period are indicators of the integrity of neural connectivity and signal transmission in the giant fiber pathway. Two response classes, evoked by different stimulus voltages, give information about different parts of the circuit. Weak stimuli evoke a long-latency response by recruiting afferent neurons upstream of the giant fibers, whereas stronger stimuli trigger a short-latency response by directly activating the giant fibers (Engel, 1996). The long-latency response can reveal properties of connections in the brain that do not contribute to the short-latency response. The thoracic portion of the circuit (activated in both long- and short-latency responses) can give information about how mutations affect neural functioning within a network of identified neurons. The TTM branch has a single electrochemical neuronal synapse onto the TTM motoneuron, whereas the DLM branch includes two synapses, an apparent electrochemical synapse of the cervical giant fiber onto the peripherally synapsing interneuron (PSI) neuron and cholinergic synapses of the PSI onto the DLM motoneurons (Engel, 2000 and references therein).

Response latencies differed between forE1 and for189Y for the long-latency response but not the short-latency one. Latency (but not refractory period or response decrement in a habituation protocol) is significantly influenced by ambient temperature (Engel, 1996). The response latencies was tested for forE1 and for189Y under similar temperature conditions and during the same period of days. Response latencies did not differ when other genotypes were compared (Engel, 2000).

The twin-pulse refractory period of the short-latency response, mediated in the thoracic portion of the giant fiber pathway, has proven to be a sensitive indicator of deficits in basic physiological properties such as transmitter processing and ion channel function. Short-latency response refractory periods were not significantly affected by allelic variation at the foraging locus. The refractory period of the long-latency response, mediated in the afferent portion of the pathway, is an indicator of properties of the brain portion of the circuit (Engel, 1996, 1998). The long-latency refractory period tended to be shorter in genotypes with slower stimulus-dependent response decrement. This is most clear when forE1 and for189Y are compared (Engel, 2000).

It is interesting that forE1 and for189Y showed differences in response properties that were restricted to the afferent portion of the neural pathway, because these stocks showed an extreme difference in response decrement in the habituation protocol, which also is mediated in the afferent portion of the pathway. Despite these differences, it is clear that the giant fiber pathway is fundamentally sound in all the foraging genotypes tested. The extreme effects on response latency or short-latency refractory period that have been reported using mutations affecting ion channels or synaptic integrity were not found in genotypes differing in PKG activity (Engel, 2000).

These results strongly indicate that the foraging PKG affects habituation-like response decrement in the electrically induced giant fiber response. Artificially induced alleles (forE1 and for189Y) defined the influence of PKG in response decrement of the giant fiber response, and more modest naturally occurring genetic variants (forR and fors) showed similar but more subtle effects. In comparisons between different genotypes at the PKG foraging locus, response decrement was slower in genotypes with more abundant PKG (forE1 and forR) than in genotypes with less abundant PKG (for189Y and fors). It is interesting that rate of response decrement, response latency, and refractory period were all more extreme in forE1 than the wild rover genotype forR. It is possible that imprecise excision of the P-element from for189Y resulted in a more highly expressing allele in forE1 than the original parental for allele from which for189Y arose. Sequencing of forE1, currently in progress, should help to resolve this possibility. Differences in rate of response decrement followed a semidominant mode of inheritance as shown by forR/fors heterozygotes. Semidominant inheritance also has been reported (Pereira, 1993) for the adult rover and sitter foraging phenotypes (Engel, 2000).

Recovery results indicate that foraging affects spontaneous recovery from stimulus-dependent response decrement. The results also imply the existence of distinct components of this habituation-like response decrement with different kinetics of onset and recovery that could partly account for genetic differences in recovery phenotypes. A long-term component of response decrement is suggested by the similarity of recovery indices after either 30- or 120-sec recovery intervals. For those intervals, recovery of the resistance to subsequent response decrement is correlated with the number of stimuli that were given before the recovery rest interval (Engel, 2000).

Sitter genotypes with low PKG expression showed the greatest recovery of resistance to response decrement after 30- and 120-sec intervals. However, these flies also showed more rapid response decrement in initial stimulus bouts and experienced fewer stimuli in all bouts before recovery testing, and in consequence may have had less exposure to a long-term component of response decrement. Therefore, differences in rates of response decrement may have contributed indirectly to the observed genetic differences in recovery indices for 30 and 120 sec. This would not preclude the possibility that PKG also could play a role in physiological processes that underlie spontaneous recovery per se (Engel, 2000).

Early recovery after stimulus-dependent response decrement appears to be dominated by a short-term component of response decrement. Recovery indices increased substantially between 5 and 30 sec after ending the preceding stimulus bout, and response likelihood did not recover to 100% after 5 sec in some genotypes. Response likelihood for the first stimulus following a 5-sec recovery interval showed complete recovery in fors and forR but did not recover completely in for189Y flies, which showed the most rapid response decrement in this study and have low PKG expression (Osborne, 1997). In contrast to the recovery of resistance to subsequent response decrement, this genetic effect could not be a consequence of differences in exposure to a long-term response decrement process, because for189Y flies actually experienced the smallest numbers of stimuli before the 5-sec recovery interval. This result suggests that PKG may facilitate recovery of the likelihood of responding to a single stimulus after prior response decrement (Engel, 2000).

Evoked recovery in a dishabituation protocol was weakest in the most rapidly decrementing foraging genotypes. These results may point to a direct involvement of PKG pathways in evoked recovery. Alternatively, a more rapid rate of response decrement in sitter genotypes could have reduced evoked recovery in an indirect manner as follows. Assuming an equivalent activation of recovery processes by an airpuff in all genotypes, more rapid response decrement after the puff could diminish the amount of recovery observed. Furthermore, because a standard decrement criterion of five consecutive failures preceded the puff in all genotypes, a rapid rate of 'latent' response decrement during the five criterion stimuli could induce a deeper level of response decrement for the circuit to recover from at the time of the airpuff (Engel, 2000).

These results suggest that the foraging PKG could affect the observed levels of spontaneous recovery and evoked recovery in part through altering the rate of stimulus-dependent response decrement. Similar correspondences between response decrement rates, spontaneous recovery, and evoked recovery may be seen for cAMP metabolic mutants. This highlights the interrelatedness of these three processes in the giant fiber system. One goal for the future is to determine the extent to which these phenomena can be altered independently by mutations and thus may involve independent molecular mechanisms (Engel, 2000).

Differences were seen in the response latencies and refractory periods of the forE1 and for189Y genotypes. These effects were seen in the long-latency response but not the short-latency response, indicating that they are mediated in the afferent or brain segment of the giant fiber pathway in which habituation-like response decrement also is mediated (Engel, 1996, 1998). However, response decrement rate and long-latency refractory period may not be functionally related. Earlier studies with mutations affecting cAMP cascades (Engel, 1996) and K+ channels (Engel, 1998) have not shown a strong correlation between response decrement in a habituation protocol and refractory period. Moreover, flies bearing Shaker and ether à go-go K+ channel mutations have refractory periods comparable to forE1 but show much more rapid response decrement (Engel, 1998). Previous studies (Engel, 1992, 1996, 1998) have indicated that the thoracic portion of the giant fiber pathway may have qualitatively normal characteristics even in genotypes with very rapid response decrement. Consistent with this, short-latency refractory periods and latencies did not differ between foraging genotypes, even the very rapidly decrementing genotype for189Y (Engel, 2000).

The results associate high PKG expression with a slow rate of response decrement in a habituation protocol but do not indicate the mechanism underlying this association. PKG may play a direct role in plasticity, either by down-regulating a physiological process that underlies response decrement or by enhancing a concomitant process of response sensitization as in a dual process model. Alternatively, high PKG expression could influence response decrement in a less direct manner by modifying the physiological or developmental context in which it occurs. For instance, if PKG enhanced basic properties of neural conduction or synaptic transmission so that the neural signal is stronger to begin with, then it could take longer for normally functioning mechanisms underlying stimulus-dependent response decrement to lead to failed responses. Enhancement of neural response properties would be consistent with the forE1 phenotype of shortened latency and refractory period of the long-latency response, parameters that are mediated in the afferent part of the giant fiber pathway, just as is the habituation-like response decrement is (Engel, 2000).

PKG appears to affect such basic functional properties differently in different parts of the fly nervous system. Variation in foraging genotype did not affect latency or refractory period of the short-latency response, parameters that are mediated in the thoracic portion of the pathway. Moreover, reduced PKG activity in sitter genotypes is associated with hyperexcitability and enhanced nerve terminal sprouting at larval neuromuscular junctions and with reduced K+ currents and increased membrane excitability in a significant population of neurons in dissociated embryonic cultures (Renger, 1999). These observations suggest a widely distributed role for PKG in the nervous system of flies (Engel, 2000).

Social environment influences performance in a cognitive task in natural variants of the foraging gene

In Drosophila melanogaster, natural genetic variation in the foraging gene (for) affects the foraging behaviour of larval and adult flies, larval reward learning, adult visual learning, and adult aversive training tasks. Sitters (fors) are more sedentary and aggregate within food patches whereas rovers (forR) have greater movement within and between food patches, suggesting that these natural variants are likely to experience different social environments. It is hypothesized that social context would differentially influence rover and sitter behaviour in a cognitive task. Adult rover and sitter performance was measured in a classical olfactory training test in groups and alone. All flies were reared in groups, but fly training and testing were done alone and in groups. Sitters trained and tested in a group had significantly higher learning performances compared to sitters trained and tested alone. Rovers performed similarly when trained and tested alone and in a group. In other words, rovers learning ability is independent of group training and testing. This suggests that sitters may be more sensitive to the social context than rovers. These differences in learning performance can be altered by pharmacological manipulations of PKG activity levels, the foraging gene's gene product. Learning and memory is also affected by the type of social interaction (being in a group of the same strain or in a group of a different strain) in rovers, but not in sitters. These results suggest that for mediates social learning and memory in D. melanogaster (Kohn, 2013).

This study found that associative learning in D. melanogaster was influenced by social context and that this effect could be modulated by natural variation in the foraging gene which encodes PKG. The sitters, with lower PKG, when trained and tested alone, had significantly lower PI than sitters who were trained and tested in groups. Group versus individual training and testing did not affect the PI of rovers. Also, a change in social context between training and testing affected sitter but not rover performance. That is, sitter performance was significantly lower when trained in a group and then tested alone (or vice-versa). This research not only extends previous work showing that social experience affects memory retrieval in D. melanogaster, but also demonstrates within-species variation in these effects (Kohn, 2013).

To better understand how PKG impacts the effects of social context, PKG levels weew pharmacologically manipulated in individual flies to 'transform' rovers into sitters, and sitters into rovers (Dawson-Scully, 2010). PKG was found to modulate associative learning in a social context. High PKG levels appear to enhance learning in a solitary context, as sitter performance was improved when PKG activity was increased. In contrast, pharmacologically lowering PKG activity decreased rover performance. This indicates that PKG activity levels can buffer the lack of learning performance found when sitters are alone (Kohn, 2013).

The results suggest that sitter variants are more sensitive to social context than rovers. Given what is known about differences in sitter and rover movement patterns during foraging, this finding may not be surprising. Rovers visit more patches, travel greater distances, and tend not to revisit previous patches compared to sitters. Therefore, sitters, which tend to be more gregarious, may experience less variation in social interactions. Social isolation -- particularly in a learning context -- could thus constitute distractions or stresses to sitters (Kohn, 2013).

The results also suggest that, after training, rovers use personal information over public information. Their performance index (PI) was not diminished by the potentially distracting presence of naive flies during testing. Surprisingly, the PI of individual rovers was also greater when tested in a group of trained sitters compared to a group of trained rovers. Learning and memory are known to be facilitated or impaired by the general level of stress. In Drosophila, mechanical shocks induce the release of 'Drosophila stress odorant' (mainly CO2). Variation between rover and sitter strains in the release of or sensitivity to stress odorants could potentially affect their performance in the t-maze. This potential form of context-dependent social facilitation requires future investigation (Kohn, 2013).

Interestingly, when only public information was available rover flies tended to use this information whereas sitter flies did not. Naïve rovers introduced into a group of trained sitters had a positive PI, suggesting that they followed trained sitters. Naïve rovers introduced into a group of trained rovers did not have a positive PI. Sitters, however, did not follow trained rovers or trained sitters. This rover behaviour could indicate a type of sharing of 'public information' from sitters. Information sharing has been observed in D. melanogaster courtship behaviour. It is important to note that trained rovers did not follow trained or naive sitters, so the effect depends on the state of the individual rover. It may be that rovers use a strategy of 'copying when uncertain' (Kohn, 2013).

In nature, D. melanogaster adults aggregate at food sources where a number of social behaviours take place including feeding, courtship, mating, and oviposition. Previous research on flies suggests that the complexity of food search behaviour is not an individual process but requires social interactions between individuals (Tinette, 2004, Tinette 2007). For example, social interactions between flies impacts what specific food patches are chosen. First, 'primer flies' randomly search the environment and land on different food patches. Groups of follower flies then aggregate on the most favourable patches of food based on social interactions. Several learning mutants (e.g., dunce and rutabaga) have been shown to have considerable effects on these complex search-aggregation social interactions involved in food search behaviour. This suggests a possible overlap in the signalling pathways of the decision making processes involved in food search behaviour and those involved in learning (Kohn, 2013).

More recent investigations suggest that a nitric oxide (NO) signalling pathway may also be involved in social interactions related to search-aggregation behaviour (Tinette, 2007). Mutants of soluble-guanylatecyclase (sGC) showed marked defects in a number of the search-aggregation behaviours including a decreased performance in exploratory aggregation, a disconnect of exploration from odour and taste cues, and a lack of preference for aggregated food patches. Similarly, the current results found that PKG signalling is important for performance in a decision making task where learning and memory are important. Pharmacological treatments showed that individual flies deficient in PKG were unable to perform in a learning task. Whether these two behavioural paradigms implicating NO and PKG signalling in social decision-making tasks are indicative of a similar underlying mechanism of signalling and sensory integration remains to be determined (Kohn, 2013).

In the ant, Phidole pallidudal, social experience is known to effect brain PKG enzyme activity (Lucas, 2009). In honey bees, foraging gene RNA expression and PKG activity differs depending on a bees role in the hive which is socially determined. PKG is known to both phosphorylate proteins and act transcriptionally. The present study provides further evidence for a conserved role for PKG in social functioning. The molecular and cellular mechanisms by which social context acts to affect PKG remains to be determined (Kohn, 2013).

Social context affects learning, and this in part can be modulated by natural variation in a single gene. Variation in the foraging gene can affect not only how flies learn, but also how social context influences that learning. Along with previous evidence on how for affects social roles in honeybees and ants, this study provides new evidence for a social role in Drosophila suggesting for is a candidate gene for the genetic conservation of social behaviour. Taken together, these results contribute to the burgeoning field of socially-influenced behaviour in D. melanogaster, and demonstrate the effectiveness of the fruit fly as a model for studies on social behaviour. Understanding how genes function in social contexts is fundamental to understanding how social behaviour operates, as well as how it evolved (Kohn, 2013).

Epigenetic mechanisms modulate differences in Drosophila foraging behavior

Little is known about how genetic variation and epigenetic marks interact to shape differences in behavior. The foraging (for) gene regulates behavioral differences between the rover and sitter Drosophila melanogaster strains, but the molecular mechanisms through which it does so have remained elusive. This study shows that the epigenetic regulator G9a interacts with for to regulate strain-specific adult foraging behavior through allele-specific histone methylation of a for promoter (pr4). Rovers have higher pr4 H3K9me dimethylation, lower pr4 RNA expression, and higher foraging scores than sitters. The rover-sitter differences disappear in the presence of G9a null mutant alleles, showing that G9a is necessary for these differences. Furthermore, rover foraging scores can be phenocopied by transgenically reducing pr4 expression in sitters. This compelling evidence shows that genetic variation can interact with an epigenetic modifier to produce differences in gene expression, establishing a behavioral polymorphism in Drosophila (Anreiter, 2017).

Rovers and sitters have a natural difference in adult foraging behavior that is caused by differences in G9a-dependent expression of the for pr4 transcripts. pr4 is differentially methylated by G9a in rovers and sitters, and this study demonstrates that pr4 is solely responsible for the rover-sitter behavioral polymorphism in adult foraging behavior. Nevertheless, G9a is not the sole transcriptional regulator, or the sole H3K9 methyltransferase, regulating pr4 expression. The results show that the loss of G9a can result in more or less H3K9me2 at pr4, depending on the for allele present. This dual function of G9a has been previously shown in mice, where G9a is able to both repress and activate gene expression through interactions with other proteins in its regulatory complex. While pr4 is responsible for regulating the rover-sitter difference in adult foraging behavior, other for promoters likely regulate other for-related phenotypes. In fact, expression data show that other for promoters are differentially expressed in rovers and sitters. For example, pr2 is highly expressed in sitters and not expressed at all in rovers. The pr2 expression difference also correlates with H3K9me2, but cannot be explained solely by G9a. pr2 and pr4 transcribe different isoforms of for (P1 and P4, respectively) that might differ in function. These results suggest that the expression of for's four promoters might be regulated by distinct regulatory complexes, and that each promoter might influence distinct behavioral phenotypes (Anreiter, 2017).

The difference in pr4 expression is tissue-specific, being driven by the brain and ovaries. The central nervous system and ovaries might be linked in regulating feeding behavior, since reproduction constitutes the major energy expenditure of female flies, and sex peptide signaling in the reproductive organs affects the feeding behavior of female flies. This work highlights the complex epigenetic architecture that underlies behavioral regulation (Anreiter, 2017).

The lack of a DNA-binding domain suggests that G9a is targeted to specific DNA regions through interactions with DNA-binding proteins, such as transcription factors. SNPs in the promoter region could lead to differential binding of DNA-binding proteins that recruit G9a. For instance, the SNP in pr4 lies within a conserved site of a putative Mad binding motif, and potentially could affect Mad binding. If Mad is one of the elements in the G9a complex, then less binding of Mad in the rover strain (which would be predicted from the SNP) potentially could explain the lower pr4 H3K9me2 levels in rovers. Like G9a, Mad has been shown to act as both a repressor and an activator of gene transcription, depending on context. Although Mad is best known for its role in development, some studies suggest that it might have regulatory functions in the mature nervous system (Anreiter, 2017).

In conclusion, the mechanisms by which epigenetic regulation influences behavioral differences are poorly understood. Epigenetic regulation has been shown to be a mechanism through which animals adjust their behavior and physiology to the environment in which they live. Not all individuals respond similarly to the same environmental cue, however. In this case, epigenetic-by-genetic interaction would be an important but neglected component of gene-by-environment interactions. The deposition of epigenetic marks can depend on underlying genetic differences, and genetic variation likely plays an important role in moderating epigenetic differences between individuals. Importantly, epigenetic-by-genetic interactions present an avenue through which genetic variation outside of gene coding regions can modulate phenotypic variability. Two other noteworthy studies in humans and prairie voles have reported associations among genetic variation, DNA methylation, and behavior. This study used the fruit fly to establish molecular causality, and provide definitive evidence for how the complex interactions among genetics, epigenetics, and isoform-specific gene regulation causes variation in naturally occurring behavioral polymorphisms (Anreiter, 2017).


DEVELOPMENTAL BIOLOGY

The nitric oxide-cyclic GMP pathway regulates FoxO and alters dopaminergic neuron survival in Drosophila

Activation of the forkhead box transcription factor FoxO is suggested to be involved in dopaminergic (DA) neurodegeneration in a Drosophila model of Parkinson's disease (PD), in which a PD gene product LRRK2 activates FoxO through phosphorylation. In the current study that combines Drosophila genetics and biochemical analysis, it was shown that cyclic guanosine monophosphate (cGMP)-dependent kinase II (cGKII) also phosphorylates FoxO at the same residue as LRRK2, and Drosophila orthologues of cGKII and LRRK2, DG2/For and dLRRK, respectively, enhance the neurotoxic activity of FoxO in an additive manner. Biochemical assays using mammalian cGKII and FoxO1 reveal that cGKII enhances the transcriptional activity of FoxO1 through phosphorylation of the FoxO1 S319 site in the same manner as LRRK2. A Drosophila FoxO mutant resistant to phosphorylation by DG2 and dLRRK (dFoxO S259A corresponding to human FoxO1 S319A) suppressed the neurotoxicity and improved motor dysfunction caused by co-expression of FoxO and DG2. Nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) also increased FoxO's activity, whereas the administration of a NOS inhibitor L-NAME suppressed the loss of DA neurons in aged flies co-expressing FoxO and DG2. These results strongly suggest that the NO-FoxO axis contributes to DA neurodegeneration in LRRK2-linked PD (Kanao, 2012).

The Gyc76C receptor Guanylyl cyclase and the Foraging cGMP-dependent kinase regulate extracellular matrix organization and BMP signaling in the developing wing of Drosophila melanogaster

The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, this study has identified Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. This study identified a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases and shows that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. Parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues are discussed (Schleede, 2015).

The vein cells that develop from the ectodermal epithelia of the Drosophila melanogaster wing are positioned, elaborated and maintained by a series of well-characterized intercellular signaling pathways. The wing is easily visualized, and specific mutant vein phenotypes have been linked to changes in specific signals, making the wing an ideal tissue for examining signaling mechanisms, for identifying intracellular and extracellular crosstalk between different pathways, and for isolating new pathway components (Schleede, 2015).

The venation defect, the loss of the posterior crossvein (PCV), is used to identify and characterize participants in Bone Morphogenetic Protein (BMP) signaling. The PCV is formed during the end of the first day of pupal wing development, well after the formation of the longitudinal veins (LVs, numbered L1-L6), and requires localized BMP signaling in the PCV region between L4 and L5. Many of the homozygous viable crossveinless mutants identified in early genetic screens have now been shown to disrupt direct regulators of BMP signaling, especially those that bind BMPs and regulate BMP movement and activity in the extracellular space. The PCV is especially sensitive to loss of these regulators because of the long range over which signaling must take place, and the role many of these BMP regulators play in the assembly or disassembly of a BMP-carrying 'shuttle' (Schleede, 2015).

The BMP Decapentaplegic (Dpp) is secreted by the pupal LVs, possibly as a heterodimer with the BMP Glass bottom boat (Gbb). This stimulates autocrine and short-range BMP signaling in the LVs that is relatively insensitive to extracellular BMP regulators. However, Dpp and Gbb also signal over a long range by moving into the intervein tissues where the PCV forms. In order for this to occur, the secreted BMPs must bind the D. melanogaster Chordin homolog Short gastrulation (Sog) and the Twisted gastrulation family member Crossveinless (Cv, termed here Cv-Tsg2 to avoid confusion with other 'Cv' gene names). The Sog/Cv-Tsg2 complex facilitates the movement of BMPs from the LVs through the extracellular space, likely by protecting BMPs from binding to cell bound molecules such as their receptors. In order to stimulate signaling in the PCV, BMPs must also be freed from the complex. The Tolloid-related protease (Tlr, also known as Tolkin) cleaves Sog, lowering its affinity for BMPs, and Tsg family proteins help stimulate this cleavage. Signaling is further aided in the PCV region by a positive feedback loop, as BMP signaling increases localized expression of the BMP-binding protein Crossveinless 2 (Cv-2, recently renamed BMPER in vertebrates). Cv-2 also binds Sog, cell surface glypicans and the BMP receptor complex, and likely acts as a co-receptor and a transfer protein that frees BMPs from Sog. The lipoprotein Crossveinless-d (Cv-d) also binds BMPs and glypicans and helps signaling by an unknown mechanism (Schleede, 2015).

PCV development takes place in a complex and changing extracellular environment, but while there is some evidence that PCV-specific BMP signaling can be influenced by changes in tissue morphology or loss of the cell-bound glypican heparan sulfate proteoglycans, other aspects of the environment have not been greatly investigated. During the initial stages of BMP signaling in the PCV, at 15-18 hours after pupariation (AP), the dorsal and ventral wing epithelia form a sack that retains only a few dorsal to ventral connections from earlier stages; the inner, basal side of the sack is filled with extracellular matrix (ECM) proteins, both diffusely and in laminar aggregates. As BMP signaling in the PCV is maintained and refined, from 18-30 hours AP, increasing numbers of dorsal and ventral epithelial cells adhere, basal to basal, flattening the sack. The veins form as ECM-filled channels between the two epithelia, while in intervein regions scattered pockets of ECM are retained basolaterally between the cells within each epithelium; a small amount of ECM is also retained at the sites of basal-to-basal contact. This changing ECM environment could potentially alter BMP movement, assembly of BMP-containing complexes, and signal reception, as has been demonstrated in other developmental contexts in Drosophila (Schleede, 2015).

This study demonstrates the strong influence of the pupal ECM on PCV-specific long-range BMP signaling, through the identification of a previously unknown ECM-regulating pathway in the wing. In a screen conducted for novel crossveinless mutations on the third chromosome, a mutation was found in the guanylyl cyclase at 76C (gyc76C) locus, which encodes one of five transmembrane, receptor class guanylyl cyclases in D. melanogaster. Gyc76C has been previously characterized for its role in Semaphorin-mediated axon guidance; Malpighian tubule physiology, and the development of embryonic muscles and salivary glands. Like the similar mammalian natriuretic peptide receptors NPR1 and NPR2, the guanylyl cyclase activity of Gyc76C is likely regulated by secreted peptides, and can act via a variety of downstream cGMP sensors (Schleede, 2015).

The evidence suggests that Gyc76C influences BMP signaling in the pupal wing by changing the activity of the cGMP-dependent kinase Foraging (For; also known as Dg2 or Pkg24A), also a novel role for this kinase. But rather than controlling BMP signal transduction in a cell-autonomous manner, evidence is provided that Gyc76C and Foraging regulate BMP signaling non-autonomously by dramatically altering the wing ECM during the period of BMP signaling in the PCV. This effect is largely mediated by changing the levels and activity of matrix metalloproteinases (Mmps), especially Drosophila Mmp2. Genetic interactions suggest that the ECM alterations affect the extracellular mobility and activity of the BMP-binding protein Sog (Schleede, 2015).

This provides the first demonstration of Gyc76C and For activity in the developing wing, and the first evidence these proteins can act by affecting Mmp activity. Moreover, the demonstration of in vivo link from a guanylyl cyclase to Mmps and the ECM, and from there to long-range BMP signaling, may have parallels with findings in mammalian cells and tissues. NPR and NO-mediated changes in cGMP activity can on the one hand change matrix metalloproteinase expression secretion and activity, and on the other change in BMP and TGFβ signaling (Schleede, 2015).

Mutation 3L043, uncovered by a genetic screen to identify homozygous lethal mutations required for PCV development, is a novel allele of gyc76C, a transmembrane peptide receptor that, like vertebrate NPRs, acts as a guanylyl cyclase. gyc76C is likely linked by cGMP production to the activity of the cGMP-dependent kinase For, and that Gyc76C and For define a new pathway for the regulation of wing ECM. This pathway appears to act largely through changes in the activity of ECM-remodeling Mmp enzymes. Loss of gyc76C or For alter both the organization of the wing ECM and the levels of the two D. melanogaster Mmps, and the gyc76C knockdown phenotype can be largely reversed by knockdown of Mmp2. This is the first indication of a role for cGMP, Gyc76C and For function in the developing wing, and their effects on the ECM provides a novel molecular output for each (Schleede, 2015).

Gyc76C and For are necessary for the normal refinement and maintenance of long-range BMP signaling in the posterior crossvein region of the pupal wing; in fact, crossvein loss is the most prominent aspect of the adult gyc76C knockdown phenotype. The evidence suggests that this effect is also mediated by changes in Mmp activity, and most likely the Mmp-dependent reorganization of the ECM. In fact, analysis using genetic mosaics finds no evidence for a reliable, cell autonomous effect of cGMP activity on BMP signal transduction in the wing. Thus, this apparent crosstalk between receptor guanylyl cyclase activity and BMP signaling in the wing is mediated by extracellular effects (Schleede, 2015).

It is noteworthy that the cGMP activity mediated by NPR or nitric oxide signaling can change also Mmp gene expression, secretion or activation in many different mammalian cells and tissues. Both positive and negative effects have been noted, depending on the cells, the context, and the specific Mmp. Given the strong role of the ECM in cell-cell signaling, the contribution of cGMP-mediated changes in Mmp activity to extracellular signaling may be significant. There is also precedent for cGMP activity specifically affecting BMP and TGFβ signaling in mammals. cGMP-dependent kinase activity increases BMP signaling in C2C12 cells, and this effect has been suggested to underlie some of the effects of nitric oxide-induced cGMP on BMP-dependent pulmonary arterial hypertension. Conversely, atrial natriuretic peptide stimulates the guanylyl cyclase activities of NPR1 and NPR2 and can inhibit TGFβ activity in myofibroblasts; this inhibition has been suggested to underlie the opposing roles of atrial natriuretic peptide and TGFβ during hypoxia-induced remodeling of the pulmonary vasculature. However, unlike the pathway observed in the fly wing, these mammalian effects are thought to be mediated by the intracellular modulation of signal transduction, with cGMP-dependent kinases altering BMP receptor activity or the phosphorylation and nuclear accumulation of receptor-activated Smads. Nonetheless, it remains possible that there are additional layers of regulation mediated through extracellular effects, underscoring the importance of testing cell autonomy (Schleede, 2015).

Aside from its role in adult Malpighian tubule physiology, Gyc76C was previously shown to have three developmental effects: in the embryo it regulates the repulsive axon guidance mediated by Semaphorin 1A and Plexin A, the proper formation and arrangement of somatic muscles, and lumen formation in the salivary gland. All these may have links to the ECM. Loss of gyc76C from embryonic muscles affects the distribution and vesicular accumulation of the βintegrin Mys, and reduces laminins and the integrin regulator Talin in the salivary gland. The axon defects likely involve a physical interaction between Gyc76C and semaphorin receptors that affects cGMP levels; nonetheless, gyc76C mutant axon defects are very similar to those caused by loss of the perlecan Trol (Schleede, 2015).

The parallels between the different contexts of Gyc76C action are not exact, however. First, only the wing phenotype has been linked to a change in Mmp activity. Second, unlike the muscle phenotype, the wing phenotype is not accompanied by any obvious changes in integrin levels or distribution, beyond those caused by altered venation. Finally, most gyc76C mutant phenotypes are reproduced by loss of the Pkg21D (Dg1) cytoplasmic cGMP-dependent kinase, instead of For (Dg2, Pkg24A) as found in the wing, and thus may be mediated by different kinase targets (Schleede, 2015).

For has been largely analyzed for behavioral mutant phenotypes, and the overlap between Pkg21D and For targets is unknown. While many targets have been identified for the two mammalian cGMP-dependent kinases, PRKG1 (which exists in alpha and beta isoforms) and PRKG2, it is not clear if either of these is functionally equivalent to For. One of the protein isoforms generated by the for locus has a putative protein interaction/dimerization motif with slight similarity to the N-terminal binding/dimerization domains of alpha and beta PRKG1, but all three For isoforms have long N-terminal regions that are lacking from PRKG1 and PRKG2. In fact, a recent study suggested that For is instead functionally equivalent to PRKG2: Like PRKG2, For can stimulate phosphorylation of FOXO, and is localized to cell membranes in vitro. But For apparently lacks the canonical myristoylation site that is thought to account for the membrane localization and thus much of the target specificity of PRKG2. FOXO remains the only identified For target, and foxo null mutants are viable with normal wings (Schleede, 2015).

The loss of long range BMP signaling in the PCV region caused by knockdown of gyc76C can, like the ECM, be largely rescued by knockdown of Mmp2. Two results suggest that it is the alteration to the ECM that affects long-range BMP signaling, rather than some independent effect of Mmp2. First, the BMP signaling defects caused by gyc76C knockdown were rescued by directly manipulating the ECM through the overexpression of the perlecan Trol. Second, when Mmp activity is inhibited by overexpression of the diffusible Mmp inhibitor TIMP, this not only rescued the PCV BMP signaling defects caused by gyc76C knockdown, but also led to ectopic BMP signaling, not throughout the region of TIMP expression, but only in those regions with abnormal accumulation of ECM (Schleede, 2015).

The Mmp2-mediated changes in the ECM likely affect long-range BMP signaling by altering the activity of extracellular BMP-binding proteins, particularly Sog. The BMPs Dpp and Gbb produced in the LVs bind Sog and Cv-Tsg2, shuttle into the PCV region, and are released there by Tlr-mediated cleavage of Sog and transfer to Cv-2 and the receptors. Genetic interaction experiments suggest that knockdown of gyc76C both increases Sog's affinity for BMPs and reduces the movement of the Sog/Cv-Tsg2/BMP complex into the crossvein region (Schleede, 2015).

Collagen IV provides the best-studied example for how the ECM might affect Sog activity. The two D. melanogaster collagen IV chains regulate BMP signaling in other contexts, and they bind both Sog and the BMP Dpp. Results suggest that collagen IV helps assemble and release a Dpp/Sog/Tsg shuttling complex, and also recruits the Tld protease that cleaves Sog cleavage and releases Dpp for signaling. D. melanogaster Mmp1 can cleave vertebrate Collagen IV. Since reduced Gyc76C and For activity increases abnormal Collagen IV aggregates throughout the wing and diffuse Collagen IV in the veins, it ishypothesized that these Collagen IV changes both foster the assembly or stability of Sog/Cv-Tsg2/BMP complexes and tether them to the ECM, favoring the sequestration of BMPs in the complex and reducing thelong-range movement of the complex into the region of the PCV (Schleede, 2015).

While few other D. melanogaster Mmp targets have been identified, it is likely that Mmp1 and Mmp2 share the broad specificity of their mammalian counterparts, so other ECM components, known or unknown, might be involved. For instance, vertebrate Perlecan and can be cleaved by Mmps. Trol regulates BMP signaling in other D. melanogaster contexts, and Trol overexpression rescue gyc76C knockdown's effects on BMP signaling. But while null trol alleles are lethal before pupal stages, normal PCVs were formed in viable and even adult lethal alleles like trolG0023, and actin-Gal 4-driven expression of trol-RNAi using any of four different trol-RNAi lines did not alter adult wing venation. Loss of the D. melanogaster laminin B chain shared by all laminin trimers strongly disrupts wing venation, and a zebrafish laminin mutation can reduce BMP signaling (Schleede, 2015).

Finally, it was recently shown that Dlp, one of the two D. melanogaster glypicans, can be removed from the cell surface by Mmp2. While gyc76C knockdown did not detectably alter anti-Dlp staining in the pupal wing, it is noteworthy that Dlp and the second glypican Dally are required non-autonomously for BMP signaling in the PCV and that they bind BMPs and other BMP-binding proteins.


EFFECTS OF MUTATION

Two larval foraging strategies in Drosophila have been identified, 'rover' and 'sitter'. 'Rovers' traverse a large area while feeding whereas 'sitters' cover a small area. The difference between 'rovers' and 'sitters' was analyzed genetically by chromosomal substitutions between isogenic stocks. Differences in larval locomotor behavior ('crawling behavior') can be attributed to the second chromosome, the 'rover' strategy being dominant over the 'sitter' strategy. Differences in feeding rate ('shoveling behavior') are affected additively by both the second and third chromosomes. Natural populations of Drosophila larvae were sampled three times over a 2-month period' 'rovers' and 'sitters' were at constant frequencies in these populations. The two foraging strategies are discussed in the light of resource utilization in environments where food is distributed continuously or discontinuously (Sokolowski, 1980).

Localizing genes for quantitative traits by conventional recombination mapping is a formidable challenge because environmental variation, minor genes, and genetic markers have modifying effects on continuously varying phenotypes. The method of 'lethal tagging' is described; it was used in conjunction with deficiency mapping for localizing major genes associated with quantitative traits. Rover/sitter is a naturally occurring larval foraging polymorphism in Drosophila that has a polygenic pattern of inheritance comprised of a single major gene (foraging) and minor modifier genes. The lethal tagged foraging (for) gene has been successfully localized by deficiency mapping to 24A3-C5 on the polytene chromosome map (de Belle, 1989).

There is a correlation between the locomotory component of larval and adult foraging behavior in the fruit fly. This relationship is far more than mere correlation. It can be attributable to different alleles at the same genetic locus of the behavioral gene foraging (for). The for gene offers an unique opportunity to study the genetic basis and evolutionary significance of a naturally occurring behavioral polymorphism. Until now, only the effect of for on Drosophila larval behavior was studied. Larvae with the rover allele (forR) move significantly more while eating during a set time period than those homozygous for the sitter alleles (fors). Rover and sitter larval strains derived from nature differ in the distance adults walk after feeding per unit time and this variation results from different alleles at the foraging locus, the very gene originally defined on the basis of larval behavior. It is hypothesized that for may be involved in the way flies evaluate a food resource (Pereira, 1993).

The foraging gene affects adult but not larval olfactory-related behavior in Drosophila melanogaster

The ability of larvae and adult rover and sitter flies to detect and migrate towards the source of a fly medium attractant was investigated using larval plate assays and an adult olfactory trap assay. Allelic variation at the foraging locus does not affect larval olfactory response in the larval plate assays. In contrast, adult males of the sitter mutant fors2 exhibit an olfactory trap response (OTR) which is significantly greater than that of males of the wild type forR strain from which fors2 was derived and further genetic analysis shows that this is attributable to the fors2 allele. The olfactory responses of fbrR and fors2 flies to three odors (propionic acid, ethyl acetate and acetone) in a T-maze assay is normal, indicating that they do not have general olfactory deficits. The finding that adult flies who differ in their PKG enzyme activities differ in foraging behaviors and olfactory trap responses to yeast odors, suggests that PKG signalling pathways are involved in olfactory related responses to food (Shaver, 1998).

Neuronal polymorphism among natural alleles of a cGMP-dependent kinase gene, foraging, in Drosophila

Natural variation in neuronal excitability and connectivity has not been extensively studied. In Drosophila, a naturally maintained genetic polymorphism at a cGMP-dependent protein kinase (PKG) gene, foraging, is associated with alternative food search strategies among the allelic variants Rover (forR; higher PKG activity) and sitter (fors; lower PKG activity). Physiological and morphological variations were examined in nervous systems of these allelic variants isolated from natural populations. Whole-cell current clamping revealed distinct excitability patterns, with spontaneous activities and excessive evoked firing in cultured sitter, but not Rover, neurons. Voltage-clamp examination demonstrated reduced voltage-dependent K(+) currents in sitter neurons. Focal recordings from synapses at the larval neuromuscular junction demonstrate spontaneous activity and supernumerary discharges with increased transmitter release after nerve stimulation. Immunolabeling show more diffuse motor axon terminal projections with increased ectopic nerve entry points in sitter larval muscles. The differences between the two natural alleles were enhanced in laboratory-induced mutant alleles of the for gene. The pervasive effects of the for-PKG on neuronal excitability, synaptic transmission, and nerve connectivity illustrate the magnitude of neuronal variability in Drosophila that can be attributed to a single gene. These findings establish the consequences in cellular function for natural variation in an isoform of PKG and suggest a role for natural selection in maintaining variation in neuronal properties (Renger, 1999).

Activity of cGMP-dependent protein kinase (PKG) affects sucrose responsiveness and habituation

The cGMP-dependent protein kinase (PKG) has many cellular functions in vertebrates and insects that affect complex behaviors such as locomotion and foraging. The foraging (for) gene encodes a PKG in Drosophila melanogaster. A function for the for gene in sensory responsiveness and nonassociative learning has been demonstrated. Larvae of the natural variant sitter (fors) show less locomotor activity during feeding and have a lower PKG activity than rover (forR) larvae. rover and sitter adult flies were used to test whether PKG activity affects (1) responsiveness to sucrose stimuli applied to the front tarsi, and (2) habituation of proboscis extension after repeated sucrose stimulation. To determine whether the differences observed resulted from variation in the for gene, fors2, a sitter mutant produced on a rover genetic background was tested. Rovers (forR) are more responsive to sucrose than sitters (fors and fors2) at 1-, 2-, and 3-wk old. This is true for both sexes. Differences in sucrose responsiveness between rovers and sitters are greater after 2 h of food deprivation than after 24 h. Of flies with similar sucrose responsiveness, forR rovers showed less habituation and generalization of habituation than fors and fors2 sitters. These results show that PKG independently affects sensory responsiveness and habituation in Drosophila melanogaster (Scheiner, 2004).

Evolution of foraging behavior in Drosophila by density dependent selection

One of the rare examples of a single major gene underlying a naturally occurring behavioral polymorphism is the foraging locus of Drosophila. Larvae with the rover allele, forR, have significantly longer foraging path lengths on a yeast paste than do those homozygous for the sitter allele, fors. These variants do not differ in general activity in the absence of food. The evolutionary significance of this polymorphism is not as yet understood. The effect of high and low animal rearing densities on the larval foraging path-length phenotype has been examined; density-dependent natural selection produces changes in this trait. In three unrelated base populations the long path (rover) phenotype was selected for under high-density rearing conditions, whereas the short path (sitter) phenotype was selected for under low-density conditions. Genetic crosses suggested that these changes resulted from alterations in the frequency of the fors allele in the low-density-selected lines. Further experiments showed that density-dependent selection during the larval stage rather than the adult stage of development is sufficient to explain these results. Density-dependent mechanisms may be sufficient to maintain variation in rover and sitter behavior in laboratory populations (Sokolowski, 1997).

Morphological polymorphisms have been shown to be affected by density-dependent selection in, for example, aphids, damsel flies, and ungulates. However, the importance of genetically based behavioral polymorphisms for population regulation has rarely been investigated. Populations of wild house mice, Mus musculus domesticus, exhibit two heritable alternative strategies called 'aggressive or active copers' and 'nonaggressive or passive copers'. 'Active copers' have a fitness advantage in established (called K type strategist) populations, whereas 'passive copers' are better at establishing new populations (called r type strategist) (Benus, 1991). In the current study, the rover phenotype was selected under high-density (K type) conditions, whereas the sitter was selected under low-density (r type) conditions. Both studies show a clear relationship between individual behavior and population dynamics (density- dependent selection). This study is the first to show a genetic basis to this relationship. One theoretical framework that these studies contribute to is the Chitty hypothesis (also called the self-regulation or the genetic control hypothesis) for population regulation that is based on the idea that populations can be regulated by factors intrinsic to the organism. In this scenario, directional selection is thought to act on behavioral morphs in a density-dependent manner such that the gene pool changes with population size. Thus, as in the present study, behavioral morphs are differentially selected under high (K-selected) as compared with low (r-selected) population densities. Although foraging behavior has been studied in detail by behavioral ecologists, little is known about the heritable basis of this trait and under what conditions individual differences in foraging behavior contribute to fitness. Two exceptions to this are the rover/sitter polymorphism investigated in this study and the foraging behavior of zebra finches (Taeniopygia guttato), which show heritable differences in the ability to discriminate between patches of food that differ in quality (Sokolowski, 1997 and references therein).

In the current study, density-dependent selection in the laboratory resulted in differences in larval foraging behavior in three independent experiments using three unrelated base populations. Low-density conditions selected for significantly shorter paths (sitter phenotype) in the m, mr, and UU lines and longer paths (rover phenotype) in the K, mK, and CU lines. The differences in behavior between the UU and CU lines suggested that density-dependent selection is important during the larval stages of development. Results of genetic crosses between the mr and mK lines and a strain carrying a deficiency of for demonstrated that some of the differences in behavior are attributable to variation at the for locus. The present study is of particular interest because it involves natural selection in the laboratory and not artificial selection. No artificial selection pressure on larval path length was placed on the high- and low-density treatment lines. The differences in path length resulted from high- compared with low-density rearing conditions, and these conditions constituted the selection pressure (Sokolowski, 1997).

It is difficult to make a case for the direct action of selection on a trait. Indeed, a number of factors could be responsible for density-dependent selection on larval path length. High-density compared with low-density cultures would differ in, for example, the distribution and concentration of food, waste products, and abiotic factors such as the moisture content of the medium. Larval density in the medium is an important biotic factor that varies in time and space. It is low during the early stages of medium infestation but higher during later stages. Variation in larval density also occurs within and between fruits (Sokolowski, 1997).

Drosophila larvae spend most of their lives foraging for food. They move through the food by extending their anterior end and retracting their posterior ends. They feed by shovelling the food (yeast) with their mouth hooks. In the absence of food, both rover and sitter larvae have long paths that do not differ from each other. Within a patch of food rover larvae exhibit significantly longer foraging trails than sitters. When food has a patchy distribution, rover larvae forage for food by moving between patches, whereas sitters forage within a patch. At high densities, larvae are required to crawl around other larvae, drowned pupae, and adults to reach a nearby food patch. In contrast, under low-density conditions, increased locomotory behavior (rover behavior) is unnecessary, since food is continuously distributed and of relatively higher quantity and quality. Thus patch size, patch quality, and interpatch distance would differ in high-density compared with low-density cultures. Indeed, the energetic cost of locomotion in Drosophila larvae is extremely high. In addition, larvae may be forced to look elsewhere for food under high-density conditions when food is limited. These factors should influence the success of rover compared with sitter larval behavior in high-density compared with low-density conditions (Sokolowski, 1997).

Density-dependent selection has been shown to influence a number of traits in Drosophila populations. Larval crowding increases pupation height (the distance larvae pupate from the food in vials) and larval feeding rate. The for gene does not have pleiotropic effects on these traits, although phenotypic correlations between larval path length and pupation behavior have been found in natural populations. Pupation height and larval feeding rate are polygenic characters influenced by many genes with additive effects on the major autosomes in Drosophila. From the perspective of larval fitness, feeding and moving are two of the most important behaviors performed in the larval period. Both larval behaviors (feeding rate and locomotion while foraging) show a significant response to density-dependent selection from larval crowding. Indeed, K lines evolved increased larval viability at high densities relative to the r lines (Sokolowski, 1997).

Density-dependent selection on the frequency of the rover and sitter phenotypes is a mechanism that could act both in the field and in the laboratory. In the laboratory, density-dependent selection likely arises from fly rearing methods. In the laboratory, 50-100 flies are placed in a bottle with culture medium, where they mate and lay eggs. Eggs are laid on the surface of the medium for several weeks. The larval period lasts 4-5 days at 25°C. The first larvae to hatch will experience low larval density conditions in contrast to larvae that hatch later. Thus larval density is low in the initial stages but high in the later stages of culture growth. Surveys of laboratory and natural populations have shown that some are polymorphic for rover/sitter behavior. Population numbers and larval density of D. melanogaster in the field also fluctuate both temporally (e.g., over the season) and spatially (between fruits). Indeed, population numbers may fluctuate dramatically as a consequence of density-dependent regulating mechanisms (Sokolowski, 1997 and references therein).

It should be possible to determine whether density-dependent selection affects the rover/sitter polymorphism in field populations because this polymorphism is found as a single gene (for). However, performing behavioral assays for rover/sitter phenotypic frequencies in the field is a difficult task. This is because the character being measured is a behavioral one and phenotypic frequencies will not always be representative of the true underlying genotypic frequencies due to, for example, incomplete penetrance. Proper aging of the larva and strict control of environmental conditions (i.e., food availability) are important for minimizing the probability of misclassifying a rover as a sitter or vice versa. These types of controls are impossible to implement in the field. Thus DNA probes are being developed for rover and sitter alleles to assess their frequencies in the field. These probes can then be used to address the density-dependent selection hypothesis in a variety of natural populations whose density varies temporally and/or spatially. Experimental manipulations of population densities in the field along with careful monitoring of the polymorphism should enable a furthering of understanding of how density-dependent selection contributes to the maintenance of the rover/sitter polymorphism (Sokolowski, 1997).

The for gene confers a renal phenotype in Drosophila by modulation of cGMP-specific phosphodiesterase

Fluid transport in Drosophila tubules is regulated by guanosine 3',5'-cyclic monophosphate (cGMP) signalling. The functional effects on tubules of different alleles of the foraging gene were compared; the fors allele confers an epithelial phenotype. This manifests itself as hypersensitivity of epithelial fluid transport to the nitridergic neuropeptide, capa-1, which acts through nitric oxide and cGMP. However, there was no significant difference in tubule cGK activity between fors and forR adults. Nonetheless, fors tubules contained higher levels of cGMP-specific phosphodiesterase (cG-PDE) activity compared to forR. This increase in cGMP-PDE activity suffices to decrease cGMP content in fors tubules compared to forR. Challenge of tubules with capa-1 increases cGMP content in both fors and forR tubules, although the increase from resting cGMP levels is greater in fors tubules. Capa-1 stimulation of tubules reveals a potent inhibition of cGMP-specific phosphodiesterase in both lines, although this is greater in fors; and is sufficient to explain the hypersensitive transport phenotype observed. Thus, polymorphisms at the for locus do indeed confer a cGMP-dependent transport phenotype, but this can best be ascribed to an indirect modulation of cGMP-specific phosphodiesterase activity, and thence cGMP homeostasis, rather than a direct effect on cGK levels (MacPherson, 2004).

Natural variation in Drosophila larval reward learning and memory due to a cGMP-dependent protein kinase

Animals must be able to find and evaluate food to ensure survival. The ability to associate a cue with the presence of food is advantageous because it allows an animal to quickly identify a situation associated with a good, bad, or even harmful food. Identifying genes underlying these natural learned responses is essential to understanding this ability. This study investigated whether natural variation in the foraging (for) gene in Drosophila larvae is important in mediating associations between either an odor or a light stimulus and food reward. It was found that for influences olfactory conditioning and that the mushroom bodies play a role in this for-mediated olfactory learning. Genotypes associated with high activity of the product of for, cGMP-dependent protein kinase (PKG), showed greater memory acquisition and retention compared with genotypes associated with low activity of PKG when trained with three conditioning trials. Interestingly, increasing the number of training trials resulted in decreased memory retention only in genotypes associated with high PKG activity. The difference in the dynamics of memory acquisition and retention between variants of for suggests that the ability to learn and retain an association may be linked to the foraging strategies of the two variants (Kaun, 2007).

While many studies have suggested the importance of cAMP signaling in associative learning, the current results implicate the cGMP signaling pathway in mediating reward learning and memory. Specifically, the results suggest a novel role for the foraging gene in larval learning and memory. for plays a more robust role in larval olfactory reward conditioning than larval visual reward conditioning. It was also found that the role of for in larval olfactory reward conditioning involves the mushroom bodies. These results suggest that the role of for in olfactory memory is dependent on training experience, suggesting that for may be involved in multiple mechanisms that influence formation of olfactory associations. The evidence suggests that these mechanisms affect the training experience necessary for memory acquisition and retention. For example, fors (the sitter variants) larvae respond to an increase in the number of training trials compared with forR (the rover variants) larvae. Interestingly, for also plays a role in short- and long-term memory in Drosophila adults, suggesting that these mechanisms may be conserved throughout development (Kaun, 2007).

for may also play a role in the mechanisms that affect the time-course of training necessary to produce memory acquisition and retention. The results suggest that increased PKG activity is associated with faster learning, and decreased PKG activity is associated with slower learning. As mentioned above, these differences may be related to the alternative foraging strategies of forR and fors larvae. forR larvae move more when foraging, and thus, potentially come across many food patches, whereas sitters move less and potentially stay close to a smaller number of food patches. Thus, it may be advantageous for forR larvae to quickly form an association between a new food source and odor and then use this new information to assess the quality of future food sources. Decreased memory retention may be advantageous for rovers, as previous studies suggest that formation and retention of formed associations can be energetically costly. Conversely, staying longer in a single food patch may result in a delay in the ability to form an association between a food source and odor in fors larvae. However, it may aid in the ability of fors larvae to remember which nearby food patches had been depleted (Kaun, 2007).

Intriguingly, rover adult flies show a slower rate of habituation compared with sitter adult flies upon repetitive presentation of stimuli. forR flies show less habituation and generalization of habituation to repeated sucrose stimuli compared with fors and fors2 flies. This is consistent with a more rapid response decrement of the giant-fiber escape circuit in fors and fors2 flies compared with forR flies as measured electrophysiologically. Since repetitive stimuli in the form of odor-reward pairings are used in the current study, the decrease in LI seen after increasing the amount of odor-reward pairings may, in part, represent a response decrement to the stimuli (Kaun, 2007).

The differences in olfactory learning and memory due to for may be inextricably linked to the different foraging strategies of the two natural variants. This leads to the question of whether for may play a role in other behaviors associated with foraging and food such as path-integration, territoriality, aggression, and courtship. If so, for may play a role in higher-order reward pathways that mediate these individual behaviors. Interestingly, the mushroom bodies play a role in such complex behaviors, including courtship, courtship conditioning, spatial learning, aggression, and sleep. Accordingly, the role of the mushroom bodies in for-dependent larval reward learning hints at a role of for in more complex behaviors involving higher-order reward pathways. Future research will provide insight as to whether a role of for in a variety of complex behaviors exists (Kaun, 2007).

Natural polymorphism affecting learning and memory in Drosophila

Knowing which genes contribute to natural variation in learning and memory would help in the understanding of how differences in these cognitive traits evolve among populations and species. A natural polymorphism at the foraging (for) locus, which encodes a cGMP-dependent protein kinase (PKG), affects associative olfactory learning in Drosophila melanogaster. In an assay that tests the ability to associate an odor with mechanical shock, flies homozygous for one natural allelic variant of this gene (forR) showed better short-term but poorer long-term memory than flies homozygous for another natural allele (fors). The fors allele is characterized by reduced PKG activity. forR-like levels of both short-term learning and long-term memory can be induced in fors flies by selectively increasing the level of PKG in the mushroom bodies, which are centers of olfactory learning in the fly brain. Thus, the natural polymorphism at for may mediate an evolutionary tradeoff between short- and long-term memory. The respective strengths of learning performance of the two genotypes seem coadapted with their effects on foraging behavior: forR flies move more between food patches and so could particularly benefit from fast learning, whereas fors flies are more sedentary, which should favor good long-term memory (Mery, 2007b).

The cellular functions of PKG are poorly elucidated, and its downstream effectors, for the most part, are unknown. Mammalian PKG (also called cGKI) plays a role in synaptic plasticity (long-term potentiation and depression) and seems to act both pre- and postsynaptically as a downstream component of nitric oxide signaling. Mice deficient in cGKI are defective in a cerebellum-dependent motor-learning task (Feil, 2003), but their performance in hippocampus-dependent learning is apparently not affected (Kleppisch, 2003). However, pharmacological potentiation of NO-cGMP signaling was reported to improve the performance of mice in a hippocampus-dependent learning task, the water maze (Chien, 2005), whereas pharmacological inhibition of PKG impaired memory retrieval in chickens (Edwards, 2002). Finally, NO-cGMP signaling was recently shown to interact with a cAMP-dependent mechanism in long-term memory formation in crickets (Matsumoto, 2006). Although pharmacological inhibition of PKG had no effect on memory in that study, it indicated that memory processes dependent on cGMP may run in parallel to processes mediated by other signaling pathways, like the rut adenylate cyclase-dependent memory trace (Mery, 2007b).

Interactions between such parallel pathways might be responsible for the antagonistic effects of for-PKG on short- and long-term memory, which are reported here. Yet, long-term memory is thought to form on the basis of short-term memory (with middle-term memory being an intermediate step), so one would rather expect them to be positively correlated. A positive correlation between short-term learning performance and long-term memory was observed in fly populations subject to selection for improved performance in an ecologically relevant oviposition learning task (Mery, 2007a). The antagonistic effects of FOR on short-term learning performance and long-term memory reported here are thus unexpected and call for more research on the role of cGMP-dependent processed in memory formation (Mery, 2007b).

Although the mechanism by which for-PKG acts to modulate learning and memory are unknown, these findings clearly point to mushroom bodies as the spatial focus of its action. FOR expressed in all three subsets of mushroom body neurons (α/β, α'/β', and γ). However, transgenic expression of forT2 transcript restricted to the α/β neurons was sufficient to induce forR-like pattern of short- and long-term memory in fors flies. The α/β neurons play a central role in olfactory memory: memory retrieval relies on synaptic output from these neurons, and the α lobes contain a long-term memory trace. However, a role of FOR in α'/β' and γ neurons, which have also been implicated in olfactory learning, cannot be excluded. In particular, the GAL4 driver 201Y shows only weak expression in the α/β neurons and apparently only in those that project to the cores of the α/β lobes; this line seems to express more strongly in the γ neurons. Yet, using it to drive the expression of forT2 had the same effect on learning performance as that using the other two driver lines. This implies either that a low level of forT2 expression in the α/β neurons is sufficient for the full effect on short- and long-term memory, or that forT2 expression in the γ neurons also contributes to this effect. Identification of GAL4 lines specific to α'/β' and γ neurons will help to resolve the effect of for expression in those neurons on memory formation and consolidation (Mery, 2007b).

There is a wealth of evidence for the ecological significance of learning in a variety of insects. In Drosophila, experimental data suggest that larvae use learning to find food and avoid predators; oviposition substrate choice of females is modified by experience; and males learn to discriminate against heterospecific females and to recognize unreceptive females of their own species, as well as refine their courtship behavior. Thus, even though the understanding of ecological aspects of learning in fruit flies is still rudimentary, there is evidence that it contributes to their fitness under natural conditions. This would explain why Drosophila are capable of learning, despite learning ability being a costly adaptation. But is the effect of for polymorphism on learning and memory ecologically relevant? The classical conditioning paradigm used in this paper allows control of the amount of shock and odors received by the flies and dissection of the memory dynamics, but its relevance to situations in which Drosophila learn in nature is unclear. Nonetheless, different forms of olfactory learning, involving different contexts and stimuli, rely at least in part on the same genes and neural circuits and are affected by the same naturally occurring genetic variation. In accord with that notion, the for alleles also affect larval appetitive learning. Thus, it is reasonable to expect that the learning and memory differences among for genotypes will affect their learning performance in nature and thus may contribute to natural selection on this polymorphism (Mery, 2007b).

The extent to which learning ability is favored by natural selection and which aspects are favored should depend on the environment. In particular, fast learning would be highly advantageous if the environment changed frequently within the lifetime of an individual, whereas good long-term memory would be particularly useful in more stable environments. Arguably, rover (forR) flies are more prone to encounter different environments within their lifetime than sitter (fors) flies; they spend less time feeding at one location, both as larvae and as adults, and are more likely to leave a patch of food in search of another one. It is thus tempting to speculate that the superior short-term learning performance of forR flies and the good long-term memory of fors flies form elements of complex rover and sitter evolutionary strategies, respectively adapted to variable and constant environments. However, one might also argue that rover flies would benefit from good long-term memory if they revisit places visited previously; resolving this argument would require a better understanding of Drosophila field ecology than currently is currently available. In the absence of evidence, it is more parsimonious to regard the antagonistic effects of the for alleles on short-term learning and long-term memory as a mechanistic consequence of the role of PKG in neuronal processes. As discussed above, too little is known about this role to understand the mechanism of this antagonism. It is also not clear whether this antagonism is typical for natural allelic variants, leading to a strong tradeoff between short- and long-term memory. The pattern of genetic correlations among different memory phases in natural gene pools has not been investigated, except for one study (Mery, 2007a) where both short-term learning rate and long-term memory improved in response to selection on learning performance in an ecologically relevant task (Mery, 2007b).

Whether they form part of coadapted alternative strategies or are mechanistic consequences of differences in PKG activity, the learning and long-term memory differences among for genotypes are likely to contribute to natural selection on the allelic variants of for polymorphism. However, in addition to its effect on learning, the for polymorphism influences a number of other behavioral and physiological traits of ecological relevance. It also affects larval competitive ability in a density-dependent manner, whereby high population density favors the forR allele and low density favors the fors allele. Furthermore, under some circumstances, negative frequency-dependent selection seems to favor whichever of the two alleles is currently rare, likely contributing to the maintenance of this polymorphism in nature. Thus, the overall force of selection acting on the for alleles will reflect the aggregate impact of their manifold pleiotropic effects on survival and reproduction. If such a high degree of pleiotropy were typical of natural alleles affecting learning, there would be two important consequences for evolution of cognitive traits. First, evolutionary changes in learning ability would be associated with changes in other ecologically relevant traits. Second, improved learning or memory might evolve as a byproduct of natural selection on other traits rather than because of fitness advantages of learning itself (Mery, 2007b).

Foraging alters resilience/vulnerability to sleep disruption and starvation in Drosophila

Recent human studies suggest that genetic polymorphisms allow an individual to maintain optimal cognitive functioning during sleep deprivation (SD). If such polymorphisms were not associated with additional costs, selective pressures would allow these alleles to spread through the population such that an evolutionary alternative to sleep would emerge. To determine whether there are indeed costs associated with resiliency to sleep loss, natural allelic variants of the foraging gene (for) were challenged with either sleep deprivation or starvation. Flies with high levels of Protein Kinase G (PKG) (forR; R is for 'rover') do not display deficits in short-term memory following 12 h of sleep deprivation. However, short-term memory is significantly disrupted when forR flies are starved overnight. In contrast, flies with low levels of PKG (fors, fors2; s is for 'sitter') show substantial deficits in short-term memory following sleep deprivation but retain their ability to learn after 12 h of starvation. It was found that forR phenotypes could be largely recapitulated in fors flies by selectively increasing the level of PKG in the α/β lobes of the mushroom bodies, a structure known to regulate both sleep and memory. Together, these data indicate that whereas the expression of for may appear to provide resilience in one environmental context, it may confer an unexpected vulnerability in other situations. Understanding how these tradeoffs confer resilience or vulnerability to specific environmental challenges may provide additional clues as to why an evolutionary alternative to sleep has not emerged (Donlea, 2012).

The results not only show that the naturally occurring foraging polymorphism modulates sleep homeostasis but also demonstrate that the resistance to sleep loss conferred by higher levels of foraging has a tradeoff that is revealed as an increased vulnerability to starvation. In contrast, lower levels of foraging are associated with resistance to starvation and a corresponding tradeoff, as indicated by an increased vulnerability to SD. Importantly, the phenotypes seen in foraging alleles can be largely recapitulated by overexpressing or reducing foraging in the α/β lobes of the MBs (Donlea, 2012).

Does the variability in resilience to sleep loss that is conferred by the for polymorphism have ecological relevance? Currently, it is not clear whether the ability to withstand sleep loss can confer an advantage in reproductive fitness and, thus, influence natural selection at the for locus. Furthermore, the for locus is notably pleiotropic and has been implicated in modulation of learning and memory as well as metabolic plasticity, making it difficult to specify which phenotype might respond to a given selection pressure aimed at changing the allelic variation at the for locus. It has been established, however, that flies carrying a given for allele have a relative fitness advantage when that allele is more rare (Donlea, 2012).

This finding is consistent with the idea that flies might exploit the resiliencies conferred by their for genotype to increase their chances of reproduction. For example, if a rover fly lives in a population where the fors allele is most frequent, it might increase its reproductive fitness by forgoing sleep to mate at night while its sitter neighbors must rest. This strategy may allow the rover to reduce the competition for a mate yet still maintain optimal functioning the following day. Conversely, a sitter fly might outcompete rover rivals by forgoing a feeding to mate. Under this hypothesis, both natural for alleles (along with their associated resiliencies) could be maintained within a given population of flies (Donlea, 2012).

Ecological pressures have been shown to affect cavefish, which have moved from living near the surface of lakes to deeper inside caves. Shifting ecological pressures have independently led each of these populations to sleep less than their surface-dwelling ancestors and, importantly, all three have converged upon similar genetic adaptations to adapt to a decrease in sleep time. It is possible that the polymorphism in for evolved in response to such ecological conditions, such as a prolonged food shortage that might select for animals able to withstand starvation or to seasonal changes in the length of nights that might place constraints on sleep time (Donlea, 2012).

Ultimately, the extent to which resiliency to sleep loss contributes to the frequency of for alleles in clinically varying natural populations of flies remains to be determined. It is important to note, however, that roles for PKG in sleep regulation have been identified in Caenorhabditis elegans and in mice, indicating that the influence of PKG on sleep regulation is likely to be evolutionarily conserved (Donlea, 2012).

Human studies indicate that individuals vary greatly in their vulnerability to sleep loss. With that in mind, several laboratories have begun to examine naturally occurring polymorphisms in humans to determine their role in this differential sensitivity. For example, a polymorphism in PERIOD3 (PER3) is associated with larger cognitive deficits following SD. Similarly, a functional polymorphism in adenosine deaminase results in increased sleep pressure and increased sensitivity to SD. Moreover, a functional polymorphism in brain-derived neurotrophic factor alters EEG slow-wave activity during both baseline and recovery following SD. Pharmaceuticals are commonly used to offset the negative results of SD. Not surprisingly, polymorphisms also influence the efficacy of drugs to improve performance during SD. In this context, the present study suggests that cGMP signaling and PKG are a candidate pathway for sleep resilience (Donlea, 2012).

Single-nucleotide polymorphism in the human for ortholog PRKG1 could be investigated for association with sleep loss. Thus, human studies have begun to identify molecular pathways that alter not only sleep time but resilience to sleep loss. Unfortunately, determining whether a polymorphism in humans is also associated with unexpected tradeoffs is time-consuming and costly. However, such experiments are tractable in the fly. Indeed, a previous report has found natural genetic variants that contribute to baseline sleep time during the fly's primary waking period (Donlea, 2012).

The data extend these findings to show that naturally occurring polymorphisms alter sleep homeostasis and, importantly, can confer resilience to sleep loss. In addition, the data suggest that the power of Drosophila genetics can be applied to these questions to determine the mechanismand extent to which a polymorphismhas unexpected tradeoffs. Understanding how these tradeoffs confer resilience or vulnerability to specific environmental challenges is highly relevant for understanding both the importance of sleep during evolution and translational sleep research (Donlea, 2012).

A genetic screen for olfactory habituation mutations in Drosophila: analysis of novel foraging alleles and an underlying neural circuit

Habituation is a form of non-associative learning that enables animals to reduce their reaction to repeated harmless stimuli. When exposed to ethanol vapor, Drosophila show an olfactory-mediated startle response characterized by a transient increase in locomotor activity. Upon repeated exposures, this olfactory startle attenuates with the characteristics of habituation. This study describes the results of a genetic screen to identify olfactory startle habituation (OSH) mutants. One mutation is a transcript specific allele of foraging (for) encoding a cGMP-dependent kinase. This allele of for reduces expression of a for-T1 isoform expressed in the head and functions normally to inhibit OSH. for-T1 function was localized to a limited set of neurons that include olfactory receptor neurons (ORNs) and the mushroom body (MB). Overexpression of for-T1 in ORNs inhibits OSH, an effect also seen upon synaptic silencing of the ORNs; for-T1 may therefore function in ORNs to decrease synaptic release upon repeated exposure to ethanol vapor. Overall, this work contributes to understanding of the genes and neurons underlying olfactory habituation in Drosophila (Eddison, 2012).

This study describes the isolation of Drosophila mutants that disrupt olfactory startle habituation (OSH); of these 26 mutants, the majority showed enhanced OSH. Additional targeted analysis also identified several strains carrying mutations in genes that play a role in septate junctions thus implicating this structure in regulating OSH. Two mutations were characterized in for that enhanced OSH due to reduced expression of a specific for product, FOR-T1. for-T1 limits OSH by functioning in a subset of neurons that include ORNs and the MB. The data further map for-T1 function primarily to ORNs, implying that OSH can occur in the sensory neurons of the olfactory circuit (Eddison, 2012).

for encodes several isoforms of protein kinase G (PKG), a cGMP-dependent serine/threonine kinase that regulates neuronal excitability. With respect to habituation, the natural variant (fors) with reduced PKG activity also has reduced habituation of the giant-fiber system, which mediates escape responses to visual stimuli and the gustatory-based PER , implying that for limits these behaviors. This study shows that for also limits OSH; thus for appears to be a central suppressor of habituation, regardless of sensory modality. A question remains as to whether for isoforms and their function is similar in these separate neuronal populations. Interestingly, the mammalian PKG with highest homology to for, PRKG1, has been associated with Attention Deficit/Hyperactivity Disorder, a condition characterized by a persistent lack of attention possibly due to a failure to habituate to large amounts of information received from the environment (Eddison, 2012).

Ethanol activates several olfactory receptors (ORs): OR7a, OR22a, OR35a, OR85b. Although, curiously, activity of the ORNs expressing these ORs does not appear to be needed for flies to initially sense the smell of ethanol, as the magnitude of the initial startle response was unaffected by synaptic silencing using Orco-GAL4. Interestingly, one glomerulus that appeared labeled in for11.247-GAL4 heterozygotes is VC31, which expresses OR35a, the OR most strongly activated by acute ethanol. Therefore, VC31 maybe a glomerulus mediating ethanol-induced OSH. It is also worth noting that, in addition to activating particular ORs, ethanol is also a known GABAA receptor agonist and may also act on GABAA receptors expressed in LNs and PNs that promote OSH (Eddison, 2012).

How might for-T1 function in ORNs to limit OSH? Since for-T1 overexpression in ORNs, or their synaptic silencing, reduced OSH, for-T1 may limit OSH by decreasing synaptic release. Indeed, cultured neurons of fors flies with reduced PKG activity exhibit increased excitability, resulting in increased spontaneous and evoked activity. for-T1 may achieve decreased synaptic release by modulating cAMP levels, as PKG does in mammalian ORNs. Alternatively, as in the mammalian neurons, it may phosphorylate a number of possible substrates including: TRPC channels, which regulate Ca2+ influx, SEPTIN3, a regulator of vesicle targeting or tethering, or transporters of serotonin , a neurotransmitter implicated in presynaptic inhibition in the AL(Eddison, 2012).

Finally, these data suggest that olfactory habituation can occur in the 1st order neurons of the olfactory circuit (the ORNs), while several recent papers demonstrate that the 2nd order neurons of the olfactory circuit (the LNs and PNs) are key players in olfactory habituation. MB silencing and ablation experiments also suggest that these 3rd order neurons are also involved. Indeed, studies in the rat show that olfactory cortex and not peripheral circuits, regulate olfactory habituation. Therefore, the capacity to habituate to olfactory cues appears to be distributed throughout the olfactory circuit. Indeed, synaptic silencing of either the ORNs or the MB did not completely block OSH, as one might expect if habituation occurred at a singular point in the circuit. This distributed mechanism of habituation may allow the fruit fly a greater flexibility in the interplay between its innate responses and learnt experience (Eddison, 2012).

Controlling anoxic tolerance in adult Drosophila via the cGMP-PKG pathway

A cGMP-dependent protein kinase (PKG) cascade is a biochemical pathway critical for controlling low-oxygen tolerance in the adult fruit fly, Drosophila melanogaster. Even though adult Drosophila can survive in 0% oxygen (anoxia) environments for hours, air with less than 2% oxygen rapidly induces locomotory failure resulting in an anoxic coma. Natural genetic variation and an induced mutation in the foraging (for) gene, which encodes a Drosophila PKG, were used to demonstrate that the onset of anoxic coma is correlated with PKG activity. Flies that have lower PKG activity demonstrate a significant increase in time to the onset of anoxic coma. Further, in vivo pharmacological manipulations reveal that reducing either PKG or protein phosphatase 2A (PP2A) activity increases tolerance of behavior to acute hypoxic conditions. Alternatively, PKG activation and phosphodiesterase (PDE5/6) inhibition significantly reduce the time to the onset of anoxic coma. By manipulating these targets in paired combinations, a specific PKG cascade was characterized, with upstream and downstream components. Further, using genetic variants of PKG expression/activity subjected to chronic anoxia over 6 h, approximately 50% of animals with higher PKG activity survive, while only approximately 25% of those with lower PKG activity survive after a 24 h recovery. Therefore, this report describes the PKG pathway and the differential protection of function vs survival in a critically low oxygen environment (Dawson-Scully, 2010).

A cGMP-dependent protein kinase (PKG) controls synaptic transmission tolerance to acute oxidative stress at the Drosophila larval neuromuscular junction

Increasing evidence demonstrates that modulating the cGMP-dependent protein kinase G (PKG) pathway produces an array of behavioral phenotypes in the fruit fly, Drosophila melanogaster. Altering PKG activity, either genetically via the foraging (for) gene or using pharmacology modifies tolerance to acute abiotic stresses such as hyperthermia and hypoxia. PKG signaling has been shown to modulate neuroprotection in many experimental paradigms of acute brain trauma and chronic neurodegenerative diseases. However, relatively little is known about how this stress-induced neuroprotective mechanism affects neural communication. This study investigated the role PKG activity has on synaptic transmission at the Drosophila larval neuromuscular junction (NMJ) during acute oxidative stress; the application of 2.25 mM hydrogen peroxide (H2O2) disrupts synaptic function by rapidly increasing the rate of neuronal failure. This study reports that reducing PKG activity through either natural genetic variation or an induced mutation of the for gene increases synaptic tolerance during acute oxidative conditions. Furthermore, pharmacological manipulations revealed that neurotransmission is significantly extended during acute H2O2 exposure upon inhibition of the PKG pathway. Conversely, activation of this signaling cascade using either genetics or pharmacology significantly reduced the time until synaptic failure. Therefore, these findings suggest a potential role for PKG activity to regulate the tolerance of synaptic transmission during acute oxidative stress, where inhibition promotes functional protection while activation increases susceptibility to neurotransmission breakdown (Caplan, 2013).

The visual orientation memory of Drosophila requires Foraging (PKG) upstream of Ignorant (RSK2) in ring neurons of the central complex

Orientation and navigation in a complex environment requires path planning and recall to exert goal-driven behavior. Walking Drosophila flies possess a visual orientation memory for attractive targets which is localized in the central complex of the adult brain. This study shows that this type of working memory requires the cGMP-dependent protein kinase encoded by the foraging gene in just one type of ellipsoid-body ring neurons. Moreover, genetic and epistatic interaction studies provide evidence that Foraging functions upstream of the Ignorant Ribosomal-S6 Kinase 2, thus revealing a novel neuronal signaling pathway necessary for this type of memory in Drosophila (Kuntz, 2012).

For signaling has previously been implicated in different types of memories; however, in contrast to the working memory in the detour paradigm, these memories require a longer time frame to be established. In mammals, nitric oxide, the initiating molecule of the cGMP/PKG-pathway, is thought to act as a retrograde messenger during the induction of long-term potentiation (LTP). A LTP enhancement has been reported after adding PKG activators and a long-term depression after the addition of PKG inhibitors. Mice carrying a knock-out for the Pkg gene show reduced ability of motor learning due to a loss of synaptic plasticity in the cerebellum. Furthermore, mice lacking Pkg in the amygdala exhibit an impairment in fear conditioning and cGMP/PKG signaling in the hippocampus is required for novel object recognition. In insects, For is involved in different types of food searching behavior and associative memories in which establishing the learning traces takes at least seconds. In contrast, the orientation memory observed in the detour paradigm presented in this study represents a form of working memory which has to be updated continuously in fractions of seconds. Whereas the phosphorylation and activation of For and Ignorant might be the mechanism by which these kinases affect longer-lasting memories, it is thought unlikely that this mechanism is involved in the constantly and rapidly changing orientation memory. Both kinases would have to be activated or inactivated in an online fashion during every turn of the fly. On the other hand, RSK2 has been implicated in multiple cellular processes and transcriptional control. It is therefore speculated that the biochemical pathway both kinases work in is necessary to endow the ring neurons with the capacity to efficiently change signaling rapidly to encode orientation. For instance, ring neurons might need a higher density of synaptic release sites and/or dendritic neurotransmitter receptors to exert their specific function (Kuntz, 2012).


EVOLUTIONARY HOMOLOGS

PKG functions in invertebrates

Noxious stimulation can trigger persistent sensitization of somatosensory systems that involves memory-like mechanisms. Noxious stimulation of the mollusc Aplysia produces transcription-dependent, long-term hyperexcitability (LTH) of nociceptive sensory neurons that requires a nitric oxide (NO)-cyclic GMP-protein kinase G (PKG) pathway. Injection of cGMP induces LTH, whereas antagonists of the NO-cGMP-PKG pathway prevent pinch-induced LTH. Co-injection of calcium/cAMP-responsive-element (CRE) blocks both pinch-induced LTH and cAMP-induced LTH, but antagonists of protein kinase A (PKA) fail to block pinch-induced LTH. Thus the NO-cGMP-PKG pathway and at least one other pathway, but not the cAMP-PKA pathway, are critical for inducing LTH after brief, noxious stimulation (Lewin, 1999).

Genes can affect natural behavioral variation in different ways. Allelic variation causes alternative behavioral phenotypes, whereas changes in gene expression can influence the initiation of behavior at different ages. The age-related transition by honey bees from hive work to foraging is associated with an increase in the expression of the foraging (for) gene, which encodes a guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (PKG). cGMP treatment elevates PKG activity and causes foraging behavior. Previous research has shown that allelic differences in PKG expression result in two Drosophila foraging variants. The same gene can thus exert different types of influence on a behavior (Ban-Shahar, 2002).

Division of labor in honey bee colonies is influenced by the foraging gene (Amfor), which encodes a cGMP-dependent protein kinase (PKG). Amfor upregulation in the bee brain is associated with the age-related transition from working in the hive to foraging for food outside, and cGMP treatment (which increases PKG activity) causes precocious foraging. Two lines of evidence are presented in support of the hypothesis that Amfor affects division of labor by modulating phototaxis. (1) A subset of worker bees involved in the removal of corpses from the hive had forager-like brain levels of Amfor brain expression despite being middle aged; age-matched food-handlers, who do not leave the hive to perform their job, had low levels of Amfor expression. This finding suggests that occupations that involve working outside the hive are associated with high levels of Amfor in brain. (2) Foragers were much more positively phototactic than hive bees in a laboratory assay, and cGMP treatment (bees were treated orally with a 50% sucrose solution containing 8-Br-cGMP) caused a precocious onset of positive phototaxis. The cGMP effect was not due to a general increase in behavioral activity; cGMP treatment had no effect on locomotor activity under either constant darkness or a light:dark regime. The cGMP effect also was not due to changes in circadian rhythmicity; neither age at onset of locomotor circadian rhythmicity nor the period of rhythmicity was affected by cGMP treatment. The effects of Amfor on phototaxis are not related to peripheral processing; electroretinogram analysis revealed no effect of cGMP treatment on photoreceptor activity and no differences between untreated hive bees and foragers. The cAMP/PKA pathway does not appear to be playing a similar role to cGMP/PKG; cAMP treatment (oral administration) did not affect phototaxis and gene expression analysis revealed task-related differences only for the gene encoding the regulatory subunit, but not the catalytic subunit, of PKA. These findings implicate one neural process associated with honey bee division of labor that can be affected by naturally occurring changes in the expression of Amfor (Ben-Shahar, 2003).

The dynamic regulation of nitric oxide synthase (NOS) activity and cGMP levels suggests a functional role in the development of nervous systems. NO/cGMP signalling cascade plays a key role in regulating migration of postmitotic neurons in the enteric nervous system of the embryonic grasshopper. During embryonic development, a population of enteric neurons migrates several hundred micrometers on the surface of the midgut. These midgut neurons (MG neurons) exhibit nitric oxide-induced cGMP-immunoreactivity coinciding with the migratory phase. Using a histochemical marker for NOS, potential sources were identified of NO in subsets of the midgut cells below the migrating MG neurons. Pharmacological inhibition of endogenous NOS, soluble guanylyl cyclase (sGC) and protein kinase G (PKG) activity in whole embryo culture significantly blocks MG neuron migration. This pharmacological inhibition can be rescued by supplementing with protoporphyrin IX free acid, an activator of sGC, and membrane-permeant cGMP, indicating that NO/cGMP signalling is essential for MG neuron migration. Conversely, the stimulation of the cAMP/protein kinase A signalling cascade results in an inhibition of cell migration. Activation of either the cGMP or the cAMP cascade influences the cellular distribution of F-actin in neuronal somata in a complementary fashion. The cytochemical stainings and experimental manipulations of cyclic nucleotide levels provide clear evidence that NO/cGMP/PKG signalling is permissive for MG neuron migration, whereas the cAMP/PKA cascade may be a negative regulator. These findings reveal an accessible invertebrate model in which the role of the NO and cyclic nucleotide signalling in neuronal migration can be analyzed in a natural setting (Haase, 2003).

The induction of a long-term hyperexcitability (LTH) in vertebrate nociceptive sensory neurons (SNs) after nerve injury is an important contributor to neuropathic pain in humans, but the signaling cascades that induce this LTH have not been identified. In particular, it is not known how injuring an axon far from the cell soma elicits changes in gene expression in the nucleus that underlie LTH. The nociceptive SNs of Aplysia (ap) develop an LTH with electrophysiological properties after axotomy similar to those of mammalian neurons and are an experimentally useful model to examine these issues. An Aplysia PKG (cGMP-dependent protein kinase; protein kinase G) has been cloned that is homologous to vertebrate type-I PKGs; apPKG is activated at the site of injury in the axon after peripheral nerve crush. The active apPKG is subsequently retrogradely transported to the somata of the SNs, but apPKG activity does not appear in other neurons whose axons are injured. In the soma, apPKG phosphorylates apMAPK (Aplysia mitogen-activated protein kinase), resulting in its entry into the nucleus. Surprisingly, studies using recombinant proteins in vivo and in vitro indicate that apPKG directly phosphorylates the threonine moiety in the T-E-Y activation site of apMAPK when the -Y- site contains a phosphate. Inhibitors of nitric oxide synthase, soluble guanyl cyclase, or PKG were used after nerve injury; each prevents the appearance of the LTH. Moreover, blocking apPKG activation prevents the nuclear import of apMAPK. Consequently, the nitric oxide-PKG-MAPK pathway is a potential target for treatment of neuropathic pain (Sung, 2004).

Molecular basis for changes in behavioral state in ant social behaviors

A hallmark of behavior is that animals respond to environmental change by switching from one behavioral state to another. However, information on the molecular underpinnings of these behavioral shifts and how they are mediated by the environment is lacking. The ant Pheidole pallidula with its morphologically and behaviorally distinct major and minor workers is an ideal system to investigate behavioral shifts. The physically larger majors are predisposed to defend the ant nest, whereas the smaller minors are the foragers. Despite this predisposition, majors are able to shift to foraging according to the needs of the colony. The ant foraging (ppfor) gene, which encodes a cGMP-dependent protein kinase (PKG), mediates this shift. Majors have higher brain PKG activities than minors, and the spatial distribution of the PPFOR protein differs in these workers. Specifically, majors express the PPFOR protein in 5 cells in the anterior face of the ant brain, whereas minors do not. Environmental manipulations show that PKG is lower in the presence of a foraging stimulus and higher when defense is required. Finally, pharmacological activation of PKG increases defense and reduces foraging behavior. Thus, PKG signaling plays a critical role in P. pallidula behavioral shifts (Lucas, 2009).

Developmental expression of PKG

cDNA clones (PKG Ia and PKG Ib) for medaka fish cGMP-dependent protein kinase (PKG) Ia and Ib were isolated and characterized, and both were demonstrated to be expressed in the embryos after late gastrula stage. The transcripts of the PKG Ia gene are present in the spinal cord and gill arch, while those of the PKG Ib gene are only weakly expressed in these organs, but highly expressed in the otic vesicles. Injection of PKG Ia-specific morpholino antisense oligonucleotides (Ia-MO) into two-cell stage medaka fish embryos caused severe abnormalities in the developing embryos, such as the development of a hammer-like head, fusion of the developing eyes, and degeneration of cells around the eyes, while injection of PKG Ib-specific morpholino antisense oligonucleotides (Ib-MO) caused fewer abnormalities in the embryos, even when injected at higher concentrations than Ib-MO. The PKG I-overexpressing embryos exhibited smaller eyes and enlargement of the forebrain, a phenotype similar to that observed in the cAMP-dependent protein kinase (PKA)-depressed embryos. In the PKG-deficient embryos, a shh-target gene, HNF-3b was expressed weakly; this phenotype is similar to that observed in the PKA-overexpressing embryos suggesting that the cGMP/PKG signaling pathway is involved in some steps of shh signaling. Gli proteins, shh-downstream molecules, are phosphorylated by the NO/cGMP signaling pathway, probably by PKG in NG108-15 neuroblastoma cells. These results imply that PKG and PKA share common substrates and work in an opposite manner during the early embryogenesis of medaka fish (Yamamoto, 2005).

Dimerization of PKG

All mammalian cGMP-dependent protein kinases (PKGs) are dimeric. Dimerization of PKGs involves sequences located near the amino termini, which contain a conserved, extended leucine zipper motif. In PKG Ibeta this includes eight Leu/Ile heptad repeats, and in the present study, deletion and site-directed mutagenesis have been used to systematically delete these repeats or substitute individual Leu/Ile. The enzymatic properties and quaternary structures of these purified PKG mutants have been determined. All had specific enzyme activities comparable to wild type PKG. Simultaneous substitution of alanine at four or more of the Leu/Ile heptad repeats of the motif produces a monomeric PKG Ibeta. Mutation of two Leu/Ile heptad repeats can produce either a dimeric or monomeric PKG. Point mutation of Leu-17 or Ile-24 does not disrupt dimerization. These results suggest that all eight Leu/Ile heptad repeats are involved in dimerization of PKG Ibeta. Six of the eight repeats are sufficient to mediate dimerization, but substitutions at some positions appear to have greater impact than others on dimerization. The Ka of cGMP for activation of monomeric mutants is 2- to 3-fold greater than that for wild type dimeric PKG Ibeta, and there is a corresponding 2- to 3-fold increase in cGMP-dissociation rate of the high affinity cGMP-binding site of these monomers. These results indicate that dimerization increases sensitivity for cGMP activation of the enzyme (Richie-Jannetta, 2003).

PKG mutation

The cGMP-dependent protein kinase (PKG) is the main mediator of nitric oxide-induced relaxation of smooth muscle. Although this pathway is well established, the cellular action of PKG, nitric oxide, and cGMP is complex and not fully understood. A cross-talk between the cGMP-PKG and other pathways (e.g. cAMP-protein kinase A) seems to exist. cGMP- and cAMP-dependent relaxation of smooth muscle has been examined using PKG-deficient mice (cGKI-/-). In intact ileum strips of wild type mice (cGKI+/+), 8-Br-cGMP inhibited the sustained phase of carbachol contractions by approximately 80%. The initial peak was less inhibited (approximately 30%). This relaxation was associated with a reduction in intracellular [Ca2+] and decreased Ca2+ sensitivity. Contractions of cGKI-/- ileum were not influenced by 8-Br-cGMP. EC50 for 8-Br-cGMP for PKG was estimated to be 10 nm. PKG-independent relaxation by 8-Br-cGMP had an EC50 of 10 microm. Relaxation by cAMP (approximately 50% at 100 microm), Ca2+ sensitivity of force, and force potentiation by GTPgammaS were similar in cGKI+/+ and cGKI-/- tissues. The results show that PKG is the main target for cGMP-induced relaxation in intestinal smooth muscle. cGMP desensitize the contractile system to Ca2+ via PKG. PKG-independent pathways are activated at 1000-fold higher cGMP concentrations. Relaxation by cAMP can occur independently of PKG. Long term deficiency of PKG does not lead to an apparent up-regulation of the cAMP-dependent pathways or changes in Ca2+ sensitivity (Bonnevier, 2004).

cGMP-dependent protein kinase I (PKG-I) has been suggested to contribute to the facilitation of nociceptive transmission in the spinal cord presumably by acting as a downstream target of nitric oxide. However, PKG-I activators cause conflicting effects on nociceptive behavior. In the present study PKG-I-/- mice were used to further assess the role of PKG-I in nociception. PKG-I deficiency is associated with reduced nociceptive behavior in the formalin assay and zymosan-induced paw inflammation. However, acute thermal nociception in the hot-plate test was unaltered. After spinal delivery of the PKG inhibitor, Rp-8-Br-cGMPS, nociceptive behavior of PKG-I(+/+) mice was indistinguishable from that of PKG-I-/- mice. In contrast, the PKG activator, 8-Br-cGMP (250 nmol intrathecally) caused mechanical allodynia (I.F. editor's note: condition in which pain results from a non-injurious stimulus to the skin) only in PKG-I(+/+) mice, indicating that the presence of PKG-I is essential for this effect. Immunofluorescence studies of the spinal cord reveal additional morphological differences. In the dorsal horn of 3- to 4-week-old PKG-I-/- mice, laminae I-III are smaller and contain fewer neurons than controls. Furthermore, the density of substance P-positive neurons and fibers is significantly reduced. The paucity of substance P in laminae I-III may contribute to the reduction of nociception in PKG-I-/- mice and suggests a role of PKG-I in substance P synthesis (Tegeder, 2004).

Regulation of cGMP-dependent protein kinase expression by soluble guanylyl cyclase

Vascular smooth muscle cells (VSMC) undergo many phenotypic changes when placed in culture. Several studies have shown that the levels of expression of soluble guanylyl cyclase (sGC) or cGMP-dependent protein kinase (PKG) are altered in cultured VSMC. In this study, the mechanisms involved in the coordinated expression of sGC and PKG were examined. Pro-inflammatory cytokines that increase the expression of type II NO synthase (inducible NO synthase or iNOS) decreased PKG expression in freshly isolated, non-passaged bovine aortic SMC. However, in several passaged VSMC lines (i.e., bovine aortic SMC, human aortic SMC, and A7r5 cells), PKG protein expression was not suppressed by cytokines or NO. sGC was highly expressed in non-passaged bovine aortic SMC but not in passaged cell lines. Restoration of expression of sGC to passaged bovine SMC using adenovirus encoding the a1 and b1 subunits of sGC restored the capacity of the cells to increase cGMP in response to NO. Furthermore, treatment of these sGC-transduced cells with NO donors for 48 hours results in decreased PKG protein expression. In contrast, passaged rat aortic SMC expressed high levels of NO-responsive sGC, but demonstrated reduced expression of PKG. Adenovirus-mediated expression of the PKG catalytically active domain in rat aortic SMC caused a reduction in the expression of sGC in these cells. These results suggest that there is a mechanism for the coordinated expression of sGC and PKG in VSMC, and that prolonged activation of sGC down-regulates PKG expression. Likewise, the loss of PKG expression appears to increase sGC expression. These effects may be an adaptive mechanism allowing growth and survival of VSMC in vitro (Browner, 2004).

Circadian clock-controlled regulation of cGMP-protein kinase G

The suprachiasmatic nucleus (SCN) circadian clock exhibits a recurrent series of dynamic cellular states, characterized by the ability of exogenous signals to activate defined kinases that alter clock time. To explore potential relationships between kinase activation by exogenous signals and endogenous control mechanisms, clock-controlled protein kinase G (PKG) regulation in the mammalian SCN were examined. Signaling via the cGMP-PKG pathway is required for light- or glutamate (GLU)-induced phase advance in late night. Spontaneous cGMP-PKG activation occura at the end of subjective night in free-running SCN in vitro. Phasing of the SCN rhythm in vitro is delayed by approximately 3 hr after treatment with guanylyl cyclase (GC) inhibitors, PKG inhibition, or antisense oligodeoxynucleotide (alphaODN) specific for PKG, but not PKA inhibitor or mismatched ODN. This sensitivity to GC-PKG inhibition was limited to the same 2 hr time window demarcated by clock-controlled activation of cGMP-PKG. Inhibition of the cGMP-PKG pathway at this time caused delays in the phasing of four endogenous rhythms: wheel-running activity, neuronal activity, cGMP, and Per1. Timing of the cGMP-PKG-necessary window in both rat and mouse depends on clock phase, established by the antecedent light/dark cycle rather than solar time. Because behavioral, neurophysiological, biochemical, and molecular rhythms show the same temporal sensitivities and qualitative responses, it is predicted that clock-regulated GC-cGMP-PKG activation may provide a necessary cue as to clock state at the end of the nocturnal domain. Because sensitivity to phase advance by light-GLU-activated GC-cGMP-PKG occurs in juxtaposition, these signals may induce a premature shift to this PKG-necessary clock state (Tischkau, 2003).

Circadian clocks comprise a cyclic series of dynamic cellular states, characterized by the changing availability of substrates that alter clock time when activated. To determine whether circadian clocks, like the cell cycle, exhibit regulation by key phosphorylation events, endogenous kinase regulation of timekeeping was examined in the mammalian suprachiasmatic nucleus (SCN). Short-term inhibition of PKG-II but not PKG-Ibeta using antisense oligodeoxynucleotides delayed rhythms of electrical activity and Bmal1 mRNA. Phase resetting was rapid and dynamic; inhibition of PKG-II forced repetition of the last 3.5 hr of the cycle. Chronic inhibition of PKG-II disrupted electrical activity rhythms and tonically increased Bmal1 mRNA. PKG-II-like immunoreactivity was detected after coimmunoprecipitation with CLOCK, and CLOCK is phosphorylated in the presence of active PKG-II. PKG-II activation may define a critical control point for temporal progression into the daytime domain by acting on the positive arm of the transcriptional/translational feedback loop (Tischkau, 2004).

PKG targets channels and receptors

Native large conductance, voltage-dependent, and Ca2+-sensitive K+ channels are activated by cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: (1) direct phosphorylation of the channel and (2) indirect via PKG-dependent activation of a phosphatase. To scrutinize the first possibility, at the molecular level, the human pore-forming alpha-subunit of the Ca2+-sensitive K+ channel, Hslo, and the alpha-isoform of cGMP-dependent protein kinase I were used. In cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP significantly increased the macroscopic currents. This increase in current was due to an increase in the channel voltage sensitivity by approximately 20 mV and was reversed by alkaline phosphatase treatment after patch excision. In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase applied to the cytoplasmic side of reconstituted channels increased their open probability. This stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show that the purified kinase incorporates gamma-33P into the immunopurified Hslo band of approximately 125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation experiments. These results demonstrate that the alpha-subunit of large conductance Ca2+-sensitive K+ channels is substrate for G-Ialpha kinase in vivo and support direct phosphorylation as a mechanism for PKG-Ialpha-induced activation of maxi-K channels (Alioua, 1998).

Nitric oxide (NO) is thought to play an essential role in neuronal processing, but the downstream mechanisms of its action remain unclear. NO analogs reduce GABA-gated currents in cultured retinal amacrine cells via two distinct, but convergent, cGMP-dependent pathways. Either extracellular application of the NO-mimetic S-nitroso-N-acetyl-penicillamine (SNAP) or intracellular perfusion with cGMP depresses GABA currents. This depression is partially blocked by a pseudosubstrate peptide inhibitor of cGMP-dependent protein kinase (PKG), suggesting both PKG-dependent and independent actions of cGMP. cAMP-dependent protein kinase (PKA) is known to enhance retinal GABA responses. The membrane permiable 8-Bromoinosine 3',5'-cyclic monophosphate (8Br-cIMP), which activates a type of cGMP-stimulated phosphodiesterase that hydrolyzes cAMP, also significantly reduces GABA currents. 1-Methyl-3-isobutylxanthine (IBMX), a nonspecific phosphodiesterase (PDE) inhibitor, blocks both the action of 8Br-cIMP and the portion of SNAP-induced depression that is not blocked by PKG inhibition. These results suggest that NO depresses retinal GABAA receptor function by simultaneously upregulating PKG and downregulating PKA (Wexler, 1998).

A recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, has been implicated as the mediator of cGMP-provoked intestinal Cl- secretion, based on cGK localization in the apical membrane of enterocytes and on the cGK capacity to activate cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In contrast, the soluble type I cGK is unable to activate CFTR in intact cells, although both cGK I and cGK II can phosphorylate CFTR in vitro. To investigate the molecular basis for the cGK II isotype specificity of CFTR channel gating, cGK II or cGK I mutant protein variants, possessing different membrane binding properties, were expressed using adenoviral vectors in a CFTR-transfected intestinal cell line, and the ability of cGMP to phosphorylate and activate the Cl- channel was examined. Mutation of the cGK II N-terminal myristoylation site (Gly2 --> Ala) reduced cGK II membrane binding and severely impaired cGK II activation of CFTR. Conversely, a chimeric protein, in which the N-terminal membrane-anchoring domain of cGK II was fused to the N terminus of cGK Ibeta, acquired the ability to associate with the membrane and activate the CFTR Cl- channel. The potency order of cGK constructs for activation of CFTR (cGK II > membrane-bound cGK I chimer >> nonmyristoylated cGK II > cGK Ibeta) correlated with the extent of 32P incorporation into CFTR observed in parallel measurements. These results strongly support the concept that membrane targeting of cGK is a major determinant of CFTR Cl- channel activation in intact cells (Vaandrager, 1998).

Canonical transient receptor potential (TRPC) channels are Ca2+-permeable nonselective cation channels that are widely expressed in numerous cell types. Seven different members of TRPC channels have been isolated. The activity of these channels is regulated by the filling state of intracellular Ca2+ stores and/or diacylglycerol and/or Ca2+/calmodulin. However, no evidence is available as to whether TRPC channels are regulated by direct phosphorylation on the channels. In the present study, TRPC isoform 3 (TRPC3) gene was overexpressed in HEK293 cells that were stably transfected with protein kinase G (PKG). The overexpressed TRPC3 mediates store-operated Ca2+ influx and this type of Ca2+ influx is inhibited by cGMP. The inhibitory effect of cGMP was abolished by KT5823 or H8. Point mutations at two consensus PKG phosphorylation sites (T11A and S263Q) of TRPC3 channel markedly reduced the inhibitory effect of cGMP. In addition, TRPC3 proteins were purified from HEK293 cells that were transfected with either wild-type or mutant TRPC3 constructs, and an in vitro PKG phosphorylation assay was carried out. It was found that wild-type TRPC3 could be directly phosphorylated by PKG in vitro and that the phosphorylation was abolished in the presence of KT5823. The phosphorylation signal was greatly reduced in mutant protein T11A or S263Q. Taken together, TRPC3 channels can be directly phosphorylated by PKG at position T11 and S263, and this phosphorylation abolishes the store-operated Ca2+ influx mediated by TRPC3 channels in HEK293 cells (Kwan, 2004).

Nitric oxide mediates local activity-dependent excitatory synapse developmen

Learning related paradigms play an important role in shaping the development and specificity of synaptic networks, notably by regulating mechanisms of spine growth and pruning. The molecular events underlying these synaptic rearrangements remain poorly understood. In this study, carried out in mice, NO signaling was identified as a key mediator of activity-dependent excitatory synapse development. Chronic blockade of NO production in vitro and in vivo interferes with the development of hippocampal and cortical excitatory spine synapses. The effect results from a selective loss of activity-mediated spine growth mechanisms and is associated with morphological and functional alterations of remaining synapses. These effects of NO are mediated by a cGMP cascade and can be reproduced or prevented by postsynaptic expression of vasodilator-stimulated phosphoprotein phospho-mimetic or phospho-resistant mutants. In vivo analyses show that absence of NO prevents the increase in excitatory synapse density induced by environmental enrichment and interferes with the formation of local clusters of excitatory synapses. It is concluded that NO plays an important role in regulating the development of excitatory synapses by promoting local activity-dependent spine-growth mechanisms (Nikonenko, 2013).

Additional cytoplasmic activities of PKG

Cyclic-GMP-dependent protein kinase (PKG) is widely appreciated as having diverse roles in a variety of cell types. Many reports have indicated that PKG might regulate cell function by activating members of the mitogen-activated protein kinase (MAPK) family of signaling proteins. Stimulation of HEK-293 cells with nitric oxide (NO) was found to induce a rapid accumulation of phosphorylated p38 MAPK. The involvement of PKG in this process was confirmed by cotransfection of a dominant negative PKG construct (G1alphaR-GFP), which was able to block cGMP-induced p38 MAPK activation. Transfection of cells to express dominant negative Rac1(T17N) is also able to dose-dependently block cGMP-stimulated activation of p38 MAPK, thus indicating the importance of this pathway downstream of PKG. GST-PDB affinity-precipitation experiments have revealed that stimulation of HEK293 cells with either nitric oxide or 8-Br-cGMP results in a rapid and transient activation of Rac1 with kinetics similar to p38 MAPK phosphorylation. Moreover, using in vitro kinase assays it was found that cGMP also stimulates the activity of the Rac1 effector Pak1. The activation of both Rac1 and Pak1 by 8-Br-cGMP is completely abolished by transfection of the cells with G1alphaR-GFP. Expression of the Rac1(T17N) mutant inhibits PKG-dependent activation of PAK1 indicating that Rac1 functions upstream of PAK1 in this pathway. Immunofluorescence experiments demonstrate clear colocalization of PKG and Rac1 in membrane ruffles and dynamic membrane regions, supporting a functional interaction. However, in vitro kinase assays demonstrate that Rac1 is not a substrate for PKG, suggesting an indirect activation mechanism. Taken together these data demonstrate a novel PKG-dependent pathway by which the Rac1/Pak1 pathway is activated. Furthermore, this pathway is central to the activation of p38 MAPK by PKG in these cells (Hou, 2004).

Agrin acting through nitric oxide induces the formation of AChR aggregates on myotubes in culture. Soluble guanylyl cyclase (sGC), which is present at the neuromuscular junction, is a common target of NO. Therefore, it was hypothesized that sGC and cGMP are involved in the agrin signaling cascade. Inhibition of sGC hinders AChR aggregation in both agrin- and NO donor-treated cultured myotubes; whereas, a cGMP analog is able to induce the formation of AChR aggregates on naive muscle cells. Due to the presence of cyclic GMP-dependent protein kinase (PKG) at the neuromuscular junction, the ability of a PKG inhibitor to alter the agrin signaling cascade was tested. PKG inhibition does not prevent nascent AChR aggregate formation; however, these aggregates were diffuse and composed of numerous microaggregates consistent with incomplete maturation. Thus, it is concluded that cGMP is important for the initiation of AChR aggregation, while PKG is involved in the maturation of AChR aggregates (Jones, 2004).

Platelet secretion (exocytosis) is critical in amplifying platelet activation, stabilizing thrombus, and in arteriosclerosis and vascular remodeling. The signaling mechanisms leading to secretion have not been well defined. PKG plays a stimulatory role in platelet activation via the glycoprotein Ib-IX pathway. PKG also plays an important stimulatory role in mediating aggregation-dependent platelet secretion and secretion-dependent second wave platelet aggregation, particularly those induced via Gq-coupled agonist receptors, the thromboxane A2 (TXA2) receptor and protease-activated receptors (PAR). PKG I knockout mouse platelets and PKG inhibitor-treated human platelets show diminished aggregation-dependent secretion, and also show diminished secondary wave of platelet aggregation induced by a TXA2 analog and thrombin-receptor activating peptides that is rescued by the granule content ADP. Low dose collagen-induced platelet secretion and aggregation are also reduced by PKG inhibitors. Furthermore PKG I knockout and PKG inhibitors significantly attenuate activation of the Gi pathway that is mediated by secreted ADP. These data unveil a novel PKG-dependent platelet secretion pathway and a mechanism by which PKG promotes platelet activation (Li, 2004).

Cyclic GMP-dependent protein kinase I (PKGI) mediates vascular relaxation by nitric oxide and related nitrovasodilators and inhibits vascular smooth muscle cell (VSMC) migration. To identify VSMC proteins that interact with PKGI, the N-terminal protein interaction domain of PKGIalpha was used to screen a yeast two-hybrid human aortic cDNA library. The formin homology (FH) domain-containing protein, FHOD1, was found to interact with PKGIalpha in this screen. FH domain-containing proteins bind Rho-family GTPases and regulate actin cytoskeletal dynamics, cell migration, and gene expression. Antisera to FHOD1 were raised and used to characterize FHOD1 expression and distribution in vascular cells. FHOD1 is highly expressed in human coronary artery, aortic smooth muscle cells, and in human arterial and venous endothelial cells. In glutathione S-transferase pull-down experiments, the FHOD1 C terminus (amino acids 964-1165) binds full-length PKGI. Both in vitro and intact cell studies demonstrate that the interaction between FHOD1 and PKGI is decreased 3- to 5-fold in the presence of the membrane permiable PKG activator, 8Br-cGMP. Immunofluorescence studies of human VSMC show that FHOD1 is cytoplasmic and is concentrated in the perinuclear region. PKGI also directly phosphorylates FHOD1, and studies with wild-type and mutant FHOD1-derived peptides identify Ser-1131 in the FHOD1 C terminus as the unique PKGI phosphorylation site in FHOD1. These studies demonstrate that FHOD1 is a PKGI-interacting protein and substrate in VSMCs and show that cyclic GMP negatively regulates the FHOD1-PKGI interaction. Based on the known functions of FHOD1, the data are consistent with a role for FHOD1 in cyclic GMP-dependent inhibition of VSMC stress fiber formation and/or migration (Y. Wang, 2004).

Transcriptional regulation induced downstream of PKG

Nitric oxide (NO) regulates the expression of multiple genes but in most cases its precise mechanism of action is unclear. Baby hamster kidney (BHK) cells, which have very low soluble guanylate cyclase and cGMP-dependent protein kinase (G-kinase) activity, and CS-54 arterial smooth muscle cells, which express these two enzymes, were used to study NO regulation of the human fos promoter. The NO-releasing agent Deta-NONOate [ethanamine-2,2'-(hydroxynitrosohydrazone)bis-] has no effect on a chloramphenicol acetyltransferase (CAT) reporter gene under control of the fos promoter in BHK cells transfected with an empty vector or in cells transfected with a G-kinase Ibeta expression vector. In BHK cells transfected with expression vectors for guanylate cyclase, Deta-NONOate markedly increases the intracellular cGMP concentration and causes a small (2-fold) increase in CAT activity; the increased CAT activity appears to be from cGMP activation of cAMP-dependent protein kinase. In BHK cells co-transfected with guanylate cyclase and G-kinase expression vectors, CAT activity was increased 5-fold in the absence of Deta-NONOate and 7-fold in the presence of Deta-NONOate. Stimulation of CAT activity in the absence of Deta-NONOate appears to be largely from endogenous NO since it was found that: (1) BHK cells produced high amounts of NO; (2) CAT activity was partially inhibited by a NO synthase inhibitor; and (3) the inhibition by the NO synthase inhibitor is reversed by exogenous NO. In CS-54 cells, NO was found to increase fos promoter activity and the increase was prevented by a guanylate cyclase inhibitor. In summary, NO was found to activate the fos promoter by a guanylate cyclase- and G-kinase-dependent mechanism (Idriss, 1999).

Transcriptional regulation of the fos promoter by nitric oxide and cGMP can occur by nuclear translocation of cGMP-dependent protein kinase I. To identify nuclear targets of G-kinase I, a yeast two-hybrid screen was performed with G-kinase I beta as bait. G-kinase I beta was found to interact specifically with TFII-I, an unusual transcriptional regulator that associates with multiple proteins to modulate both basal and signal-induced transcription. By using purified recombinant proteins, the interaction was mapped to the N-terminal 93 amino acids of G-kinase I beta and one of six 95-amino acid repeats found in TFII-I. In baby hamster kidney cells, cGMP analogs enhanced co-immunoprecipitation of G-kinase I beta and TFII-I by inducing co-localization of both proteins in the nucleus, but in other cell types containing cytoplasmic TFII-I, the G-kinase-TFII-I interaction was largely cGMP-independent. G-kinase phosphorylates TFII-I in vitro and in vivo on Ser(371) and Ser(743) outside of the interaction domain. G-kinase strongly enhances TFII-I transactivation of a serum-response element-containing promoter in COS7 cells, and this effect is lost when Ser(371) and Ser(743) of TFII-I are mutated. TFII-I by itself has little effect on a full-length fos promoter in baby hamster kidney cells, but it synergistically enhances transcriptional activation by G-kinase I beta. Binding of G-kinase to TFII-I may position the kinase to phosphorylate and regulate TFII-I and/or factors that interact with TFII-I at the serum-response element (Casteel, 2002).

Activation of the arterial baroreceptors induces expression of the proto-oncogene c-fos in the nucleus tractus solitarii (NTS), the terminal site of baroreceptor afferents in the medulla oblongata. This induced expression is an intracellular event that is crucial to long-term maintenance of stable blood pressure. Using Sprague-Dawley rats maintained under propofol anesthesia, the role of nitric oxide (NO) in this process was evaluated. Baroreceptor activation induced by 30 min of sustained hypertension significantly and sequentially increased the level of cyclic GMP-dependent protein kinase I (PKG-I), phosphorylated cyclic AMP response element-binding protein (pCREB), c-fos mRNA, and Fos protein in the NTS. All of these up-regulated expressions were significantly attenuated in animals that were pretreated immediately before baroreceptor activation with bilateral microinjection into the NTS of a selective neuronal nitric-oxide synthase (nNOS) inhibitor or a soluble guanylyl cyclase (sGC) inhibitor. Bilateral NTS microinjection of a cell-permeable cGMP analog significantly elevated the level of pCREB or c-fos mRNA in the NTS. In contrast, the up-regulated CREB phosphorylation or c-fos induction evoked in the dorsomedial medulla by baroreceptor activation is significantly antagonized by NTS application of a cell-permeable cGMP antagonist or a PKG inhibitor. It is concluded that NO derived from nNOS in the NTS on baroreceptor activation may participate in c-fos expression via phosphorylation of CREB in a process that engages the sGC/cGMP/PKG-I signaling cascade (Chan, 2004).

The transcription factor NFAT (nuclear factor of activated T-cells) is implicated in cardiac hypertrophy and vasculogenesis. NFAT activation, reflecting dephosphorylation by the calcium-dependent phosphatase, calcineurin, and subsequent nuclear localization, is generally thought to require a sustained increase in intracellular calcium. However, in smooth muscle it was found that elevation of calcium by membrane depolarization fails to induce an increase in nuclear localization of the NFATc3 isoform. Physiological intravascular pressure (100 mm Hg) induces an increase in NFATc3 nuclear localization in mouse cerebral arteries. Pressure-induced NFATc3 nuclear accumulation is abrogated by endothelial denudation and by nitric-oxide synthase, cGMP-dependent kinase (PKG), and voltage-dependent calcium channels inhibition. It is shown that exogenous nitric oxide, in combination with an elevation in calcium, is an effective stimulus for NFATc3 nuclear accumulation. c-Jun terminal kinase 2 (JNK) activity, which has been shown to regulate NFATc3 nuclear export, is also reduced by pressure, an effect that is prevented by pretreatment with a PKG inhibitor. Consistent with this, pressure-induced NFATc3 nuclear accumulation is independent of PKG in arteries from JNK2-/- mice. Collectively, these results indicate that both activation of the NO/PKG pathway and elevation of smooth muscle calcium are required for NFATc3 nuclear accumulation and that PKG inhibits JNK2 to decrease NFAT nuclear export. These findings suggest that at physiological intravascular pressures NFATc3 is localized to the nucleus in smooth muscle cells of intact arteries and indicate a novel and unexpected role for nitric oxide/PKG in NFAT activation (Gonzalez Bosc, 2004).

Thrombospondin 1 (TSP1) transcription is stimulated by glucose, resulting in increased TGF-beta activation and matrix protein synthesis. Inducible expression of the catalytic domain of cGMP-dependent protein kinase (PKG) inhibits glucose-regulated TSP1 transcription and transforming growth factor (TGF)-beta activity in stably transfected rat mesangial cells. However, the molecular mechanisms by which PKG represses glucose-regulated TSP1 transcription are unknown. Using a luciferase-promoter deletion assay, a single region of the human TSP1 promoter (-1172 to -878, relative to the transcription start site) was identified that is responsive to glucose. Further characterization of this region identified an 18-bp sequence that specifically binds nuclear proteins from mesangial cells. Moreover, binding is significantly enhanced by high glucose treatment and is reduced by increased PKG activity. Gel mobility shift and supershift assays show that the nuclear proteins binding to the 18-bp sequence are USF1 and -2. USF1 and USF2 binds to the endogenous TSP1 promoter using a chromatin immunoprecipitation assay. Glucose stimulates nuclear USF2 protein accumulation through protein kinase C, p38 MAPK, and extracellular signal-regulated kinase pathways. Increased PKG activity down-regulates USF2 protein levels and its DNA binding activity under high glucose conditions, resulting in inhibition of glucose-induced TSP1 transcription and TGF-beta activity. Overexpression of USF2 reversed the inhibitory effect of PKG on glucose-induced TSP1 gene transcription and TGF-beta activity. Taken together these data present the first evidence that USF2 mediates glucose-induced TSP1 expression and TSP1-dependent TGF-beta bioactivity in mesangial cells, suggesting that USF2 is an important transcriptional regulator of diabetic complications (S. Wang, 2004).

PKG function in neurons

The second messengers cAMP and inositol-1,4,5-triphosphate have been implicated in olfaction in various species. The odorant-induced cGMP response was investigated using cilia preparations and olfactory primary cultures. Odorants cause a delayed and sustained elevation of cGMP. A component of this cGMP response is attributable to the activation of one of two kinetically distinct cilial receptor guanylyl cyclases by calcium and a guanylyl cyclase-activating protein (GCAP). cGMP thus formed serves to augment the cAMP signal in a cGMP-dependent protein kinase (PKG) manner by direct activation of adenylate cyclase. cAMP, in turn, activates cAMP-dependent protein kinase (PKA) to negatively regulate guanylyl cyclase, limiting the cGMP signal. These data demonstrate the existence of a regulatory loop in which cGMP can augment a cAMP signal, and in turn cAMP negatively regulates cGMP production via PKA. Thus, a small, localized, odorant-induced cAMP response may be amplified to modulate downstream transduction enzymes or transcriptional events (Moon, 1998).

Norepinephrine (NE) induces a sustained potentiation of transmitter release in the chick ciliary ganglion through a mechanism pharmacologically distinct from any known adrenergic receptors. The adrenergic potentiation of transmitter release is enhanced by a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and by zaprinast, an inhibitor of cGMP-selective phosphodiesterase. Exogenous application of the membrane-permeable cGMP, 8-bromo-cGMP (8Br-cGMP), potentiates the quantal transmitter release, and after potentiation, the addition of NE is no longer effective. In contrast, 8Br-cAMP neither potentiates the transmitter release nor occludes the NE-induced potentiation. The NE-induced potentiation is blocked by neither nitric oxide (NO) synthase inhibitor nor NO scavenger. The quantal transmitter release is not potentiated by NO donors, e.g., sodium nitroprusside. The NE-induced potentiation and its enhancement by IBMX is antagonized by two inhibitors of protein kinase G (PKG). As with NE-induced potentiation, the effects of 8Br-cGMP on both the resting intraterminal [Ca2+] ([Ca2+]i) and the action potential-dependent increment of [Ca2+]i (DeltaCa) in the presynaptic terminal are negligible. The reduction of the paired pulse ratio of EPSC is consistent with the notion that the NE- and cGMP-dependent potentiation of transmitter release is attributable mainly to an increase of the exocytotic fusion probability. These results indicate that NE binds to a novel adrenergic receptor that activates guanylyl cyclase and that accumulation of cGMP activates PKG, which may phosphorylate a target protein involved in the exocytosis of synaptic vesicles (Yawo, 1999).

The septins are a family of GTPase enzymes required for cytokinesis and play a role in exocytosis. Among the ten vertebrate septins, Sept5 (CDCrel-1) and Sept3 (G-septin) are primarily concentrated in the brain, wherein Sept3 is a substrate for PKG-I (cGMP-dependent protein kinase-I) in nerve terminals. There are two motifs for potential PKG-I phosphorylation in Sept3, Thr-55 and Ser-91, but phosphoamino acid analysis revealed that the primary site is a serine. Derivatization of phosphoserine to S-propylcysteine followed by N-terminal sequence analysis revealed Ser-91 as a major phosphorylation site. Tandem MS revealed a single phosphorylation site at Ser-91. Substitution of Ser-91 with Ala in a synthetic peptide abolishes phosphorylation. Mutation of Ser-91 to Ala in recombinant Sept3 also abolishes PKG phosphorylation, confirming that Ser-91 is the major site in vitro. Antibodies raised against a peptide containing phospho-Ser-91 detect phospho-Sept3 only in the cytosol of nerve terminals, whereas Sept3 is located in a peripheral membrane extract. Therefore Sept3 is phosphorylated on Ser-91 in nerve terminals and its phosphorylation may contribute to the regulation of its subcellular localization in neurons (Xue, 2004).

PKG in learning and LTP

Several lines of evidence suggest that cyclic GMP might be involved in long-term potentiation (LTP) in the hippocampus. Arachidonic acid, nitric oxide and carbon monoxide, three molecules that have been proposed to act as retrograde messengers in LTP, all activate soluble guanylyl cyclase. An inhibitor of guanylyl cyclase blocks the induction of LTP in the CA1 region of hippocampal slices. Conversely, cGMP analogues produce long-lasting enhancement of the excitatory postsynaptic potential if they are applied at the same time as weak tetanic stimulation of the presynaptic fibres. The enhancement is spatially restricted, is not blocked by valeric acid (APV), nifedipine, or picrotoxin, and partially occludes LTP. This synaptic enhancement may be mediated by the cGMP-dependent protein kinase (PKG). Inhibitors of PKG block the induction of LTP, and activators of PKG produce activity-dependent long-lasting enhancement. These results suggest that guanylyl cyclase and PKG contribute to LTP, possibly as activity-dependent presynaptic effectors of retrograde messengers (Zhuo, 1994).

Hippocampal cyclic GMP (cGMP) has been recently postulated to participate in an early phase of memory consolidation of an inhibitory avoidance learning in rats. This study reports the effects of the intrahippocampal infusion of a soluble guanylyl cyclase inhibitor (LY 83583) in the consolidation of one-trial step-down inhibitory avoidance and on the effect of this task on hippocampal cGMP levels and cGMP-dependent protein kinase (PKG) activity. Bilateral intrahippocampal administration of LY 83583 (2.5 micrograms per side) caused full amnesia for inhibitory avoidance when given immediately (0 min) after training, but not 30 min post-training. Rats submitted to the inhibitory avoidance task showed a significant increase in both cGMP levels and in PKG activity in the hippocampus at 0 min after training. No changes were observed 30 min after training. These findings provide further evidence that the hippocampal cGMP/PKG cascade is involved in the early stages of memory formation of an inhibitory avoidance task in rats (Bernabeu, 1997).

The involvement of the cGMP-protein kinase G (PKG) signaling pathway in the induction of long-term depression (LTD) and long-term potentiation (LTP) was investigated in the medial perforant path of the dentate gyrus in vitro. Low-frequency stimulation (LFS)-induced LTD of field EPSPs is inhibited by bath perfusion of a selective soluble guanylyl cyclase inhibitor ODQ. LFS-induced LTD of EPSPs and whole-cell patch-clamped EPSCs is also blocked by bath perfusion and postsynaptic intracellular injection, respectively, of the selective PKG inhibitor KT5823. Elevation of intracellular cGMP by perfusion of the cGMP phosphodiesterase inhibitor zaprinast results in induction of LTD of field EPSPs and EPSCs. Occlusion experiments show mutual inhibition between LFS-induced LTD and zaprinast-induced LTD. The zaprinast-induced LTD of field EPSPs is inhibited by perfusion of ODQ and KT5823. In addition, zaprinast-induced LTD of EPSCs is inhibited by postsynaptic application of KT5823. Glutamate receptor stimulation, especially that of metabotropic glutamate receptors (mGluRs), is required for zaprinast-induced LTD, because cessation of test stimulation or perfusion with the mGluR antagonist MCPG inhibits zaprinast-induced LTD. No inhibitory effect of ODQ or KT5823 on the induction of LTP of EPSPs or EPSCs was found. These data indicate that the cGMP-guanyly cyclase-PKG signaling pathway in the dentate gyrus is essential for induction of LTD, although not of LTP, in the dentate gyrus (Wu, 1998).

PKG and addiction

Nitric oxide (NO) and the C-type natriuretic peptide (CNP) exert their action on brain via the cGMP signaling pathway. NO, by activating soluble guanylyl cyclase, and CNP, by stimulating membrane-bound guanylyl cyclase, causes intracellular increases of cGMP, activating cGMP-dependent protein kinases (PKGs). Injection of CNP into the rat ventral tegmental area strongly reduces cocaine-induced egr-1 expression in the nucleus accumbens in a dose-dependent manner. The effect of CNP is reversed by the previous injection of a selective PKG inhibitor, KT5823. Activation of PKG by 8-bromo-cGMP reduces, like CNP, cocaine-induced gene transcription in dopaminergic structures. To confirm the involvement of PKG, this was overexpressed in either the mesencephalon or the caudate-putamen. Using the polyethyleneimine delivery system, an active protein was expressed by injecting a plasmid vector containing the human PKG-Ialpha cDNA. PKG was overexpressed in dopaminergic and GABAergic neurons when the plasmid was injected in the ventral tegmental area, whereas overexpression was observed in medium spiny GABAergic neurons and in both cholinergic and GABAergic interneurons when the PKG vector was injected into the caudate-putamen. Activation of the overexpressed PKG reduces cocaine-induced egr-1 expression in dopaminergic structures and affects behavior (i.e., locomotor activity). These effects were again reversed by previous injection of the selective PKG inhibitor. The current data suggest that NO and the neuropeptide CNP are potential regulators of cocaine-related effects on behavior (Jouvert, 2004).

A GluR1-cGKII interaction regulates AMPA receptor trafficking

Trafficking of AMPA receptors (AMPARs) is regulated by specific interactions of the subunit intracellular C-terminal domains (CTDs) with other proteins, but the mechanisms involved in this process are still unclear. This study found that the GluR1 CTD binds to cGMP-dependent protein kinase II (cGKII) adjacent to the kinase catalytic site. Binding of GluR1 is increased when cGKII is activated by cGMP. cGKII and GluR1 form a complex in the brain, and cGKII in this complex phosphorylates GluR1 at S845, a site also phosphorylated by PKA. Activation of cGKII by cGMP increases the surface expression of AMPARs at extrasynaptic sites. Inhibition of cGKII activity blocks the surface increase of GluR1 during chemLTP and reduces LTP in the hippocampal slice. This work identifies a pathway, downstream from the NMDA receptor (NMDAR) and nitric oxide (NO), which stimulates GluR1 accumulation in the plasma membrane and plays an important role in synaptic plasticity (Serulle, 2007).

NMDAR stimulation activates nNOS and production of NO, which results in cGMP production and cGKII activation. A major mechanism for expression of NMDAR-dependent LTP involves the synaptic insertion of GluR1. This study reports that, following activation by the NMDAR, cGKII binds to GluR1 and phosphorylates S845, leading to an increase of GluR1 in the plasma membrane. Notably, a cGKII dominant-negative inhibitor peptide blocked the cGMP-dependent increase of GluR1 surface expression, prevented the increase in amplitude and frequency of mEPSCs after chemLTP, and strongly reduced LTP in hippocampal slices. These results demonstrate a mechanism in which the NMDAR regulates AMPAR trafficking during LTP via NO and cGKII (Serulle, 2007).

Because NO is produced at postsynaptic sites and can diffuse through lipid membranes, initial studies of NO-dependent plasticity focused on presynaptic NO function through retrograde mechanisms. Some results were controversial, possibly because different methodologies were employed. Indeed, cGMP derivatives only facilitate LTP maximally if briefly applied when the NMDA receptor is active, and deviating protocols would lead to conflicting results. More recently, the use of new NO donors and NOS antagonists (Bon, 2003; Puzzo, 2005), both in vitro and in vivo (Feil, 2005), has demonstrated a role of the NO cascade in synaptic plasticity. Interestingly, as reported here, both the sGC inhibitor ODQ and the cGK inhibitor KT5823 were found to block LTP. Nonetheless, specific molecular mechanisms underlying the effects of NO, in particular in NO control of AMPAR trafficking in LTP, have been wanting. S-nitrosylation of NSF enhances NSF binding to GluR2 and regulates GluR2 surface expression (Huang, 2005). Also, activation of the NO-cGMP-cGKI pathway increases both GluR1 and synaptophysin puncta and the phosphorylation of VASP in hippocampal neurons (Wang, 2005). However, as yet, a specific pathway for NO control of activity-dependent GluR1 trafficking to synapses, an essential component of LTP, has not been reported. The interaction of cGKII with GluR1 reported here, and its consequent effect on GluR1 surface levels, directly link the actions of NO to LTP via GluR1 trafficking (Serulle, 2007).

A physical association of cGKII with GluR1 enables the kinase to phosphorylate GluR1 at S845. This phosphorylation is required for cGMP-dependent GluR1 surface accumulation, since block of the phosphorylation by the S845A GluR1 mutation blocked the surface increase. Phosphorylation of S845 accompanies increases in GluR1 surface levels and is necessary for GluR1 synaptic insertion during LTP. S845 is dephosphorylated during hippocampal LTD, and S845 phosphorylation on its own is sufficient for increase of GluR1 in the extrasynaptic plasma membrane. Thus far only PKA phosphorylation of S845 has been considered, perhaps because it was the initial kinase shown to phosphorylate this site. The present study demonstrates that cGKII activity also phosphorylates S845 (Serulle, 2007).

Increases of surface GluR1 following both PKA and cGKII phosphorylation are restricted to extrasynaptic sites , and AMPAR synaptic incorporation requires at least one additional step, possibly mediated by S818 phosphorylation. Interestingly, although 8-Br-cGMP on its own did not enhance hippocampal synaptic responses, when paired with a weak tetanus that by itself does not enhance responses, 8-Br-cGMP produced an immediate potentiation. This suggests that cGMP can prime the system for potentiation by a weak tetanic stimulation, possibly by increasing the extrasynaptic surface AMPAR population (Serulle, 2007).

The NMDAR and nNOS mutually interact with PSD-95, and Ca2+ fluxes through the NMDAR activate nNOS in this complex to produce NO, which induces sGC to produce cGMP, which activates cGKII. Ca2+ fluxes also stimulate Ca2+-regulated adenylate cyclases, which produce cAMP, which activates PKA, which also phosphorylates S845. PKA binds the A kinase anchoring protein, AKAP79, which in turn binds the PDZ domain scaffolding protein, SAP97, which binds the GluR1 CTD, thus targeting PKA to the GluR1 CTD and facilitating phosphorylation of S845 (Serulle, 2007).

Unlike the SAP97-AKAP-PKA pathway, the NO-cGMP-cGKII pathway does not rely on a scaffold since the kinase binds the receptor directly. Interestingly, a knockin mouse expressing GluR1 that lacks the last 7 aa of its CTD and does not bind SAP97 exhibited normal hippocampal LTP and GluR1 trafficking. This is explained if the NO-cGMP-cGKII pathway phosphorylates S845 in this mutant (Serulle, 2007).

GluR1 interacts with cGKII via auxiliary and core contact CTD sequences that flank S845. Interestingly, a CTD contact sequence resembles an AI domain sequence of cGKII, suggesting that to bind the catalytic domain, GluR1 mimics the AI domain. Also, this receptor-kinase interaction resembles the well-studied CaMKII binding to the NR2B (Serulle, 2007).

In the absence of cGMP, cGKII is inactive. Following NMDAR stimulation, binding of cGMP to cGKII induces a cGKII conformational change that causes AI domain autophosphorylation, AI domain release from the catalytic domain, and elongation of the kinase. The GluR1 CTD binds the newly exposed cGKII catalytic domain, facilitating GluR1 phosphorylation and the increase of surface GluR1. In one model for this increase, S845 phosphorylation promotes GluR1 trafficking to the plasma membrane, perhaps by releasing of GluR1 from a cytosolic retention factor. Alternatively, GluR1 may cycle into and out of the plasma membrane constitutively, and S845 phosphorylation may stabilize the receptor at the neuron surface. With either model, S845 phosphorylation would regulate the size of an extrasynaptic pool from which receptors may be inserted into the synapse during LTP. Such transport may depend on additional GluR1 phosphorylation. Because a highly selective peptide block of cGKII strongly reduces LTP, such an increase in an extrasynaptic receptor pool is likely to be a requirement for the synaptic potentiation associated with LTP. The present work demonstrates that the NMDAR can control the size of such a receptor pool, acting through nNOS, NO, and cGMP production and the activation of cGKII (Serulle, 2007).


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