rutabaga
RNA in situ hybridization and
immunohistochemistry demonstrate that the expression of the rutabaga gene is markedly elevated in the mushroom bodies of
normal flies (Han, 1992).
Most attempts to localize physical correlates of memory in the central nervous system (CNS) rely on ablation techniques. This approach has the limitation of defining just one of an unknown number of structures necessary for memory formation. The Drosophila rutabaga type I Ca(2+)/CaM-dependent adenylyl cyclase (AC) gene has been used to determine in which CNS region AC expression is sufficient for memory formation. Using pan-neural and restricted CNS expression with the GAL4 binary transcription activation system, the memory defect of the rutabaga mutant has been rescued in a fast robust spatial learning paradigm. The ventral ganglion, antennal lobes, and median bundle are likely the CNS structures sufficient for rutabaga AC- dependent spatial learning (Zars, 2000a).
dunce mutants, which have elevated cAMP
concentrations, show an increase in the numbers of
terminal varicosities and branches. Such increase was suppressed in dnc/rut double mutants by rut mutations, which
reduce cAMP synthesis. More profuse projections of larval motor axons have also been reported in
double-mutant combinations of ether a go-go (eag) and Shaker (Sh) alleles. These display greatly
enhanced nerve activity as a result of reduction in different K+ currents. Combinations of dnc and rut with eag and Sh mutations have been examined to explore the possible relation between
activity- and cAMP-induced morphological changes. The expanded projections in dnc
are further enhanced in double mutants of dnc with either eag or Sh, an effect that could be
suppressed by rut (Zhong, 1992).
Genetic studies using Drosophila have advanced an understanding of the molecular mechanisms upon which different
forms of learning are based, including habituation, but the relevant neural components of the learning pathways have not been as fully explored. A well defined neural circuit that underlies an escape response can be habituated, providing
excellent opportunities for studying the physiological parameters of learning in a functional circuit in the fly.
Compared with other forms of conditioning, relatively little is known of the physiological mechanisms responsible for
habituation.
The giant nerve fiber pathway mediates a jump-and-flight escape response to visual stimuli. In the tethered fly, the jump may
also be triggered electrically at multiple sites. The jump-and-flight response exhibits various parameters of habituation,
including frequency-dependent decline in responsiveness, spontaneous recovery, and dishabituation by a novel
stimulus (attributable to plasticity in the brain).
Mutations of rutabaga that diminish cAMP synthesis reduce the
rate of habituation, whereas dunce mutations that increase cAMP levels lead to a detectable but moderate
increase in habituation rates. Surprisingly, habituation is extremely rapid in dunce/rutabaga double mutants.
This corresponds to the extreme defects shown with other learning tasks in double mutants, and demonstrates that
defects of the rutabaga and dunce products interact synergistically in ways that could not have been predicted on the basis of simple counterbalancing biochemical effects.
Although habituation is localized to afferent neurons that innervate the
giant fiber, cAMP mutations also affect performance in thoracic portions of the pathway on a millisecond time
scale not otherwise accounted for by behavioral plasticity. More significantly, spontaneous recovery and dishabituation
are not as clearly affected as is habituation in mutants; this indicates that these processes may not overlap entirely in
terms of cAMP-regulating mechanisms. The analysis of the habituation of the giant fiber response in available
learning and memory mutants could be a crucial step toward realizing the promise of memory mutations to
elucidate mechanisms in neural circuits that underlie behavioral plasticity (Engel, 1996).
In both dunce and rutabaga mutant larvae, voltage-clamp analysis
of neuromuscular transmission reveals impaired synaptic facilitation and post-tetanic potentiation as
well as abnormal responses to direct application of dibutyryl cAMP. In addition, the calcium
dependence of transmitter release is shifted in dunce (Zhong, 1991).
Four
distinct K+ currents have been identified in Drosophila larval muscle fibers, i.e. the voltage-activated
transient IA and delayed IK and the Ca(2+)-activated fast ICF and slow ICS. Both IA and IK are increased in dnc alleles. Normal muscle
fibers treated with cyclic AMP show a similar increase of IA, but no significant effect on IK.
In contrast to the dnc alleles, the rut mutations appear to enhance ICS greatly while leaving the
amplitude of other currents largely unchanged. In addition, the cAMP-induced increase in
IA is not observed in rut mutant fibers. The fact that not all dnc and rut mutant defects can be mimicked or reversed by acute
application of cAMP suggests that long-term modulation of K+ currents by cAMP may involve
mechanisms distinct from the short-term effect of cAMP (Zhong, 1993)
Selection of mutations that suppress dunce
sterility has led to the isolation of two rutabaga alleles. The alleles (rut2 and rut3) decrease basal
adenylate cyclase activity but, unlike the original rutabaga mutation, leave the
calcium/calmodulin-stimulated activity intact. Behaviorally, the two alleles also differ from rut1. One
of the mutations partially rescues the dunce learning defect, and flies bearing both alleles learn.
Calcium responsiveness may thus be the crucial component of adenylate cyclase activity required for
associative learning (Feany, 1990).
Females homozygous for dunce null mutations that abolish PDE activity do not deposit
eggs. The suppressors exhibit differential effects on egg deposition and production of progeny;
double-mutant females deposit many eggs that fail to hatch, but some develop to adults. These adult
progeny exhibit morphological defects that are confined mostly to the second and third thoracic
segments or to the first five abdominal segments. Mutant alleles of rutabaga act in the germ line cells to partially suppress the
developmental defects caused by dunce mutations. Thus the rutabaga gene, as well as the dunce
gene, functions in both somatic and germ line cells (Bellen, 1987).
Mutants of the Drosophila dunce and rutabaga genes, which encode a cAMP-specific
phosphodiesterase and a calcium/calmodulin-responsive adenylyl cyclase, respectively, are deficient in
short-term memory. Altered synaptic plasticity has been demonstrated at neuromuscular junctions in
these mutants, but little is known about how their central neurons are affected. This
problem was examined by using the "giant" neuron culture, which offers a unique opportunity to analyze mutational
effects on neuronal activity and the underlying ionic currents in Drosophila. On the basis of
instantaneous frequency and first latency of spikes evoked by current steps, four categories of firing
patterns (tonic, adaptive, delayed, and interrupted) were identified in wild-type neurons, revealing
interesting parallels to those commonly observed in vertebrate CNS neurons. The distinct firing
patterns are correlated with expression of different ratios of 4-aminopyridine- and
tetraethylammonium-sensitive K+ currents. Subsets of dnc and rut neurons display abnormal
spontaneous spikes and altered firing patterns. Altered frequency coding in mutant neurons was
demonstrated further by using stimulation protocols involving conditioning with previous activity.
Abnormal spike activity and reduced K+ current remain in double-mutant neurons, suggesting that
the opposite effects on cAMP metabolism by dnc and rut do not counterbalance the mutual functional
defects. The aberrant spontaneous activity and altered frequency coding in different stimulus
paradigms may present problems in the stability and reliability of neural circuits for information
processing during certain behavioral tasks, raising the possibility of modulation in neuronal excitability
as a cellular mechanism underlying learning and memory (Zhao, 1997).
In response to suprathreshold step current injections, wild-type
neurons of different categories follow a defined temporal pattern in
firing frequency, and each operates within a restricted frequency range. In contrast, erratic firing patterns in subsets
of dnc and rut mutant neurons deviate from a
clear scheme of frequency coding for each cell category.
Some details of the abnormalities are noteworthy. (1) The periodic
bursting activity of single mutant neurons reaches an instantaneous
spike frequency as high as 120 Hz, whereas the maximum
instantaneous frequency seldom approaches 30 Hz in wild-type controls.
Such bursting activities apparently occur more frequently in tonic
and delayed neurons than in adaptive neurons. (2) Unlike wild-type
neurons that return to quiescence at the termination of stimulation, some mutant neurons frequently generate prolonged firing
activities outlasting current steps for seconds. These long-lasting potentials seem to be more
frequent in neurons of rut than those of dnc.
(3) Extreme cases of abnormal patterns of regenerative potentials
were found in subpopulations of mutant neurons that do not fall into
the four categories in response to step current injections. Additional subtleties of mutational effects on neuronal excitability have been revealed with stimulation paradigms involving preconditioning, such as a progressive increment of stimulation strength in the ramp or long-duration depolarization in the twin-pulse protocol. In general, mutant neurons displayed in these two paradigms show considerably greater variability than wild-type controls. Moreover, the overall trend found in each category of wild-type
controls with a twin-pulse paradigm becomes blurred in dnc
and rut mutant neurons. So far, these paradigms have examined only short-term plasticity in neuronal excitability. The
long-term effects of conditioning by prolonged previous activity on
firing patterns in Drosophila neurons must await further
investigation (Zhao, 1997 and references).
Synchronous activities and oscillations at characteristic firing
frequencies in neuronal populations are thought to be important for the
proper functioning of isolated neuronal networks of the rat hippocampus
and neocortex. Recently, theoretical analysis and computational modeling have proposed that
multiple short-term memory events could be represented by oscillatory
activities in a network, with each memory event stored at a different
high-frequency subcycle imbedded in a low-frequency oscillation. Progress made in insects reveals that the frequency
of field potential oscillations in the mushroom bodies of the locust is
odor-dependent, with the processing of
different features of olfactory information distributed among neural
subassemblies. The observed aberrant
spontaneous activity, disrupted frequency coding, and abnormal
modulation by previous conditioning in dnc and
rut neurons of Drosophila might present problems
in the stability of neural circuits and the reliability of information
processing, causing
poor performance in certain learning tasks in mutants. These results thus
lend strong support for the notion that in addition to the well
established synaptic mechanisms, modulation of neuronal excitability
represents a potentially important cellular mechanism for learning and
memory processes (Zhao, 1997 and references).
Upon exposure to ethanol, adult Drosophila display behaviors that are similar to acute ethanol intoxication in rodents and humans. Within minutes of exposure to ethanol vapor, flies first become hyperactive and disoriented and then uncoordinated and sedated. After approximately 20 min of exposure they become immobile, but nevertheless recover 5-10 min after ethanol is withdrawn. cheapdate, a mutant with enhanced sensitivity to ethanol, has been identified as a contributory factor, using an inebriometer to measure ethanol-induced loss of postural control. An inebriometer is a device that allows a quantitative assessment of ethanol-induced loss of postural control. The inebriometer is an approximately 4 ft long glass column containing multiple oblique mesh baffles through which ethanol vapor is circulated. To begin a "run," about 100 flies are introduced into the top of the inebriometer. With time, flies lose their ability to stand on the baffles and gradually tumble downward. As they fall out of the bottom of the inebriometer, a fraction collector is used to gather them at 3 min intervals, at which point they are counted. The elution profile of wild-type control flies follows a normal distribution; the mean elution time (MET), approximately 20 min at a standard ethanol vapor concentration, is inversely proportional to their sensitivity to ethanol.
A genetic screen was carried out to isolate P element-induced mutants with altered sensitivity to ethanol intoxication using the inebriometer as the behavioral assay. One X-linked mutation isolated in this screen was named cheapdate (chpd) to reflect the increased ethanol sensitivity displayed by hemizygous mutant male flies. chpd males elute from the inebriometer with a MET of 15 min compared with 20 min for the wild-type controls. This increased ethanol sensitivity of chpd males was observed at all ethanol vapor concentrations tested. Genetic and molecular analyses reveals that cheapdate is an allele of the memory mutant amnesiac. amnesiac has been postulated to encode a neuropeptide that activates the cAMP pathway. Consistent with this, it is found that the enhanced ethanol sensitivity of cheapdate can be reversed by treatment with agents that increase cAMP levels or PKA activity. Conversely, genetic or pharmacological reduction in PKA activity results in increased sensitivity to ethanol (Moore, 1998).
Flies carrying mutations in three molecules involved in cAMP signaling were tested for response to ethanol: (1) rutabaga (rut), encoding the Ca2+-calmodulin-sensitive AC; (2) dunce (dnc), encoding the major cAMP-phosphodiesterase (PDE), and (3) DCO, encoding the major catalytic subunit of cAMP-dependent protein kinase (PKA-C1). Males hemizygous for rut mutations display an ethanol-sensitive phenotype similar to that of amn mutants. Flies heterozygous for the loss-of-function DCO alleles, which show reduced cAMP-stimulated PKA activity, also display increased ethanol sensitivity (homozygotes cannot be tested because they die as embryos). Ethanol sensitivity of males hemizygous for dnc mutations, however, are nearly normal. These data show that flies unable to increase cAMP levels normally (such as rut and possibly amn) or to respond properly to increased cAMP levels (such as DCO/+) are more sensitive to ethanol-induced loss of postural control. The converse, however, is not observed; dnc flies, whose cAMP levels are several times higher than wild type, display nearly normal ethanol sensitivity, a phenotype that is also observed in males doubly mutant for dnc and amn. Unexpectedly, whereas both rut and amn are ethanol sensitive, males doubly mutant for rut and amn are not significantly different from control (Moore, 1998).
In mammalian cells and tissues, ethanol potentiates receptor-mediated cAMP signal transduction; the mechanisms underlying this effect, however, remain poorly understood. While a direct link between cAMP signaling and ethanol-induced behaviors has not been established in mammals, the responses to acute ethanol are thought to be mediated by alterations in the function of various ligand-gated ion channels. Certain subtypes of GABAA and NMDA receptors are potentiated and inhibited by ethanol, respectively, and both these channels can be phosphorylated by PKA in cells, tissues, or heterologous expression systems. It is tempting to speculate that PKA phosphorylation of neurotransmitter receptors is altered by ethanol and that this contributes to the behavior of the inebriated animal (Moore, 1998 and references).
A hybrid system was used to explore the relationship between Neurofibromin 1
and the PKA pathway. PACAP38 is mammalian protein that belongs to the vasoactive intestinal polypeptide-secretin-glucagon peptide family. Mutations in Drosophila genes rutabaga, Ras1 and Raf1 eliminate the response of flies to PACAP38. PACAP38 functions as a ligand for G protein-coupled receptors in vertebrates and in flies is known to stimulate cAMP synthesis inducing a 100-fold enhancement in K+ currents by coactivating both Rutabaga-adenylyl cyclase-cAMP and Ras-Raf kinase pathways (Zhong, 1995a). Mutations in rutabaga, Ras1 and Raf1 eliminate the response to PACAP38. Activation of both cAMP and Ras-Raf pathways together, but not alone, mimics the PACAP38 response (Zhong, 1995a). The involvement of Ras in the PACAP38 response has led to an investigation into the effect of Nf1 mutations. The purpose of this study was to further test whether Nf1 acts in the Ras or PKA pathways (Guo, 1997).
The PACAP38 enhancement of potassium currents is eliminated in Nf1 mutants. Perfusion of PACAP38 to the neuromuscular junction induces an inward current followed by a 100-fold enhancement of K+ currents in wild-type larvae. In Nf1 mutants, the inward current remains mostly intact, but the enhancement of K+ currents is abolished. Because the inward current is not affected in Nf1 mutants, it appears that PACAP38 receptors are normally activated by the peptide in these mutants. To control for the involvement of potential developmental effects of Nf1 mutants, wild type heat shock inducable Nf1 was induced in Nf1 mutants and the response to PACAP38 was studied. The PACAP mediated enhancement requires heat shock induction of Nf1. Because PACAP38 is a vertebrate peptide, the response induced by endogenous PACAP38-like neuropeptide (Zhong, 1995b) was tested.
High-frequency stimulation (40Hz) applied to motor axons through a suction pipette increases K+ currents, presumably by causing the release of PACAP38-like peptides (Zhong, 1995b). The evoked PACAP38-like response is also eliminated in NF1 mutants. Induced expression of constitutively active Ras or active Raf neither blocks nor mimicks the PACAP38 response, suggesting that failure to negatively regulate Ras-Raf signaling does not explain the defective PACAP38 response in Nf1 mutants (Guo, 1997).
What is the role of the cyclic AMP pathway in PACAP38 responses? Application of cAMP analogs to Nf1 mutants restores the normal response to PACAP38. The cAMP analogs are effective if applied any time before or within 2 min after application of PACAP38. Application of cAMP analogs also restores the response to PACAP38 in rutabaga mutants, but not in Ras mutants. Thus, Nf1 appears to regulate the rutabaga-encoded adenylyl
cyclase rather than the Ras-Raf pathway. Moreover, the Nf1 defect is rescued by the exposure of
cells to pharmacological treatment that increased concentrations of cAMP, such as forskolin, which stimulates G-protein coupled adenylyl cyclase activity. Exploration of the mechanism by which NF1 influences G protein-mediated activation of adenylyl cyclase may lead to new insights into the mechanisms of G protein-mediated signal transduction and the pathogenesis, and possibly the treatment, of human type 1 neurofibromatosis (Guo, 1997).
The tumor-suppressor gene Neurofibromatosis 1 (Nf1) encodes a Ras-specific GTPase activating protein (Ras-GAP). In addition to being involved
in tumor formation, NF1 has been reported to cause learning defects in humans and Nf1 knockout mice. However, it remains to be
determined whether the observed learning defect is secondary to abnormal development. The Drosophila NF1 protein is highly conserved, showing
60% identity of its 2,803 amino acids with human NF1. Previous studies have suggested that Drosophila NF1 acts not only as a Ras-GAP but
also as a possible regulator of the cAMP pathway that involves the rutabaga (rut)-encoded adenylyl cyclase. Because rut was isolated as a learning
and short-term memory mutant, the hypothesis has been pursued that NF1 may affect learning through its control of the Rut-adenylyl
cyclase/cAMP pathway. NF1 has been shown to affect learning and short-term memory independent of its developmental effects. G-protein-activated adenylyl cyclase activity consists of NF1-independent and NF1-dependent components, and the mechanism of the
NF1-dependent activation of the Rut-adenylyl cyclase pathway is essential for mediating Drosophila learning and memory (Guo, 2000).
To test whether the NF1-dependent learning defect involves the cAMP pathway,
learning scores of NF1 P2 and
rut 1 single-mutant, and rut 1; NF1 P2 double-mutant flies were compared. The learning scores of all three mutant genotypes are very similar. The learning score of another double mutant, dunce (dnc) rut 1, is reduced when compared
with either single mutants, which indicates that the two mutations
exert additive effects on learning even though both gene products are involved
in the cAMP cascade (Rut-adenylyl cyclase (AC) for synthesizing cAMP, and Dnc-phosphodiesterase for degrading cAMP.
Therefore, the absence of any further reduction of learning in the double
mutant rut 1;NF1 P2
suggests that both gene products function closely in the cAMP pathway (Guo, 2000).
This idea is supported by studies of NF1 mutant flies carrying a
transgene encoding a mutant catalytic subunit of cAMP-dependent protein kinase
(PKA*), which is constitutively active. Sustained expression
of this PKA subunit rescues the small body size phenotype of NF1 mutants. Heat-shock induction of the constitutively active PKA should,
in principle, bypass the requirement for the Rut-AC and all other molecules
upstream of normal PKA activation. The hsp70-PKA* transgene completely
rescues the learning defect of NF1P1 when the
flies are raised at room temperature.
NF1P2 mutants are partially rescued by the transgene
at room temperature, but show complete rescue with heat shock (37°C,
30 min), or with a shift to 25°C overnight before being
tested. In addition, NF1 mutations
also cause a short-term memory defect (3- and 8-h retention) that is also
fully rescued by heat-shock induction of PKA*. To determine whether expression
of hsp70-PKA* induces a nonspecific enhancement of learning, leaky or induced expression of hsp-PKA* in the wild-type background
does not increase the learning score even if flies are undertrained. For undertraining, flies were subjected to 3 repeats
of electric shock in a single training trial instead of 12 trials. It is concluded that the PKA* effect is not nonspecific
and that the learning defect observed in NF1 mutants can be rescued
by induction of PKA activity. Therefore, the biochemical deficiency in the
NF1 mutants must reside upstream of PKA induction in the cAMP pathway (Guo, 2000).
These behavioral analyses corroborate electrophysiological
data that indicate that NF1 might exert its effect through
regulation of the activation of Rut-AC. Biochemical assays provide direct
evidence to support the idea. Previous experiments have shown that Rut-AC
expressed in a cell line can be stimulated not only by Ca2+/calmodulin,
but also by reagents that stimulate G-proteins, including GTPgammaS and
AIF-4. AC activity has been examined in membrane fractions of adult brain tissues.
The basal level of AC activity is very similar in the control (K33) and
NF1 mutant membranes, but the GTPgammaS-stimulated AC activity is markedly
reduced in NF1P1 and NF1P2
mutant membranes. However, significant GTPgammaS-stimulated
activity occurs above the basal level in the mutants. Overexpression of
NF1 in control flies does not increase AC activity, whereas
the reduction in stimulated AC activity seen in NF1 mutants is mostly
rescued by acutely induced expression of the NF1 transgene, indicating that NF1 is indeed able to regulate cAMP synthesis.
Thus, GTPgammaS-stimulated AC activity consists of two components: one that
is NF1 dependent and one that is NF1 independent. To determine whether the
NF1-dependent AC activity is due to Rutabaga, rut
1 and rut 1;NF1
P2 mutant flies were examined. The basal and GTPgammaS-stimulated levels of
AC activity are very similar in the single mutant, rut 1
, and in the double mutant, rut 1;NF1 P2. Thus, the NF1 mutation has no impact on AC activity in the absence of Rut-AC. In other words, the NF1-dependent cAMP activity is mediated through Rut-AC (Guo, 2000).
The Ca2+ dependence of the NF1 effect was examined to
determine how Rut-AC is involved. Membrane fractions extracted from abdominal
tissues were used because the Ca2+-dependent Rut-AC activity
is easier to detect. The data are consistent with a report that the Ca
2+-dependent peak of AC activity is missing in rut mutants.
Again, the NF1 mutation has no effects on AC activity across Ca
2+ concentrations without Rut-AC.
Moreover, the Ca2+-dependent peak of Rut-AC activity is dependent
on G-protein stimulation but not on the presence of NF1 (Guo, 2000).
Together, these results reveal a new mechanism that describes how G-proteins activate
the cAMP pathway for normal learning and memory. The G-protein-activated AC activity is both NF1 dependent and NF1 independent. The NF1-dependent component involves Rut-AC. One possibility for how NF1 regulates AC activity is that NF1 acts as a GAP not only for the small G-protein Ras but also for heterotrimeric
G-proteins. Thus, NF1 is required for a functional interaction between AC and heterotrimeric G-proteins, similar to the involvement of IRA, a Ras-GAP that is distantly related to NF1, in the Ras-activated cAMP pathway in yeast (Guo, 2000).
Another possibility is that NF1 regulates AC activity independent of its role as a Ras-GAP. Therefore, NF1 may be important for coordinating activities of multiple signal-transduction pathways. Nevertheless, the expression of the tumor-suppressor gene NF1 and its regulation of the Rut-AC signal-transduction pathway are critical to the biochemical processes underlying olfactory learning in Drosophila. Similar mechanisms are to be expected in vertebrates (Guo, 2000).
Relatively little is known about the regulation of ion channels, particularly that of Ca2+ channels, in Drosophila. Physiological and pharmacological differences between invertebrate and mammalian L-type Ca2+ channels raise questions on the extent of conservation of Ca2+ channel modulatory pathways. An examination was made of the role of the cyclic adenosine monophosphate (cAMP) cascade in modulating the dihydropyridine (DHP)-sensitive Ca2+ channels in the larval muscles of Drosophila, using mutations and drugs that disrupt specific steps in this pathway. The L-type (DHP-sensitive) Ca2+ channel current is increased in dunce mutants, which have high cAMP concentration owing to cAMP-specific phosphodiesterase (PDE) disruption. The current is decreased in the rutabaga mutants, where adenylyl cyclase (AC) activity is altered, thereby decreasing the cAMP concentration. The dunce effect is mimicked by 8-Br-cAMP, a cAMP analog, and IBMX, a PDE inhibitor. The rutabaga effect is rescued by forskolin, an AC activator. H-89, an inhibitor of protein kinase-A (PKA), reduces the current and inhibits the effect of 8-Br-cAMP. The data suggest modulation of L-type Ca2+ channels of Drosophila via a cAMP-PKA mediated pathway. While there are differences in L-type channels, as well as in components of cAMP cascade, between Drosophila and vertebrates, main features of the modulatory pathway have been conserved. The data also raise questions on the likely role of DHP-sensitive Ca2+ channel modulation in synaptic plasticity, and learning and memory, processes disrupted by the dnc and the rut mutations (Bhattacharya, 1999).
The effects of chronically lowered cyclic adenosine monophosphate (cAMP) on the morphology and physiology of the Drosophila larval neuromuscular junction has been investigated using two fly lines in which cAMP is significantly lower than normal in the nervous system: (1) transgenic flies in which Dunce is overexpressed in the nervous system, and (2) flies mutant for the
rutabaga gene (rut1) that have reduced adenylyl cyclase activity. In comparison with controls, larvae with reduced cAMP exhibit a
smaller number of synaptic varicosities. This effect is more pronounced in transgenic larvae, in which the reduction of neural cAMP
is more pronounced. Synaptic transmission is also reduced in both cases, as evidenced by smaller excitatory junctional potentials
(EJPs). Synaptic currents recorded from individual synaptic varicosities of the neuromuscular junction indicate almost normal
transmitter release properties in transgenic larvae and a modest impairment in rut1 larvae. Thus, reduction in EJP amplitude in
transgenic larvae is primarily due to reduced innervation, while in rut1 larvae it is attributable to the combined effects of reduced
innervation and a mild impairment of transmitter release. It is concluded that the major effect of chronically lowered cAMP is reduction
of innervation rather than impairment of transmitter release properties (Cheung, 1999).
At Drosophila neuromuscular junctions, there are two synaptic vesicle pools, namely the exo/endo cycling pool (ECP) and the reserve pool (RP). An extracellularly applied fluorescent dye, FM1-43, is incorporated into synaptic vesicles in nerve terminals during endocytosis and subsequently released by exocytosis. Using this dye, the two synaptic vesicle pools, ECP and RP have been identified in the larval NMJ. Vesicles in ECP can be loaded with FM1-43 by high K+ stimulation and are located at the periphery of individual boutons. Loaded dye is completely released by a second challenge of high K+ saline. Both pools are loaded with FM1-43 by enhancing endocytosis with cyclosporin A or by incubating at room temperature after complete depletion of vesicles in shibire at a nonpermissive temperature. The dye in ECP can be unloaded by a second challenge of high K+ saline, while vesicles in RP still maintain the dye. The RP appears to be more broadly distributed toward the center of individual boutons. In this report, this distribution is referred to as being in the center of the bouton because of its appearance in fluorescence microscopy without intending any implication as to its composition or exact boundary. In the animals whose RP is disconnected from the cycling pathway by treatment with cytochalasin D, high-frequency stimulation causes an accelerated decline of synaptic potentials, while low-frequency stimulation does not, suggesting that RP is required for sustaining high rate release of transmitter (Kuromi, 2000 and references therein).
During high-frequency nerve stimulation, vesicles in RP are recruited for release, and endocytosed vesicles are incorporated into both pools, whereas with low-frequency stimulation, vesicles are incorporated into and released from ECP. Release of vesicles from RP can be detected electrophysiologically after emptying vesicles in the ECP of transmitter by a H+ pump inhibitor. Recruitment from RP is depressed by
inhibitors of steps in the cAMP/PKA cascade and enhanced by their activators. In rutabaga (rut) mutants, which have low cAMP levels, mobilization of vesicles from RP during tetanic stimulation is depressed, while it is enhanced in dunce (dnc) mutants, which have high cAMP levels (Kuromi, 2000).
The present electrophysiological studies have shown that, in wild type larvae, recruitment of synaptic vesicles from RP induced by 10 Hz stimulation
for 10 s continues for some period, even after tetanic stimulation, but such recruitment is not observed in rut and does not continue after tetanic stimulation in dnc. Reduced recruitment of vesicles from RP in rut mutants may be caused by a failure in Ca2+/calmodulin-dependent cAMP production. However, even in rut, repeated tetanic stimulation does recruit vesicles from RP, and enhanced synaptic potentials continue after tetanic stimulation. A
similar phenomenon is observed in rut after treatment with db-cAMP. Thus, it is likely even in rut that cAMP production can occur through another pathway
during tetanic stimulation. FM1-43 loading experiments show that during high-frequency stimulation, some recycling vesicles are incorporated into RP in wild
type, whereas recycling vesicles are minimally stored in RP in dnc mutants. Although RP may be refilled slowly with vesicles recycled or transported by axonal flow, the high level of cAMP produced by tetanic stimulation causes a marked translocation of vesicles from RP to ECP, resulting in no net accumulation of recycling vesicles into RP in dnc. Together, these findings suggest that recruitment of vesicles from RP to ECP is one of the mechanisms that control synaptic efficacy and plasticity (Kuromi, 2000).
Larval molting in Drosophila, as in other insects, is initiated by the coordinated release of the steroid hormone ecdysone, in response
to neural signals, at precise stages during development. Using genetic and molecular methods, the
roles played by two major signaling pathways in the regulation of larval molting have been examined in Drosophila. Previous studies have shown that
mutants for the Inositol 1,4,5-trisphosphate receptor gene (Itpr) are larval lethals. In addition, they exhibit delays in molting that can be
rescued by exogenous feeding of 20-hydroxyecdysone. Mutants for adenylate cyclase (rut) synergize, during
larval molting, with Itpr mutant alleles, indicating that both cAMP and InsP3 signaling pathways function in this process. The two pathways act in parallel to affect
molting, as judged by phenotypes obtained through expression of dominant negative and dominant active forms of protein kinase A (PKA) in tissues that normally express
the InsP3 receptor. Furthermore, these studies predict the existence of feedback inhibition through protein kinase A on the InsP3 receptor by increased levels of
20-hydroxyecdysone (Venkatesh, 2001).
Interestingly, steroid secretion by the adrenal fasciculata-reticularis cells of mammalian adrenal glands in response to adrenocorticotrophic hormone occurs through the cAMP pathway, while InsP3-mediated Ca2+ release is required for the steroidogenic action of Angiotensin II on adrenal glomerulosa cells. An increase in cytosolic Ca2+ levels is thought to affect multiple steps in mammalian steroid biosynthesis, including one crucial step that requires the transfer of endogenous cholesterol from the outer to the inner mitochondrial membrane. The data presented in this study support a similar model in which 20-hydroxyecdysone levels are regulated through activation of both InsP3 and cAMP signals. The presence of multiple genes encoding adenylate cyclases allows rut mutant alleles to proceed through molting normally. Presumably, however, activity of the alternate adenylate cyclase(s) is dependent on InsP3 receptor function since removal of the Itpr gene in rut mutant backgrounds leads to phenotypes that are synergistic. Activation of the two second messenger pathways probably occurs in the ring gland via PTTH and other as yet unidentified neural factors. Alternate explanations whereby InsP3 and/or cAMP signaling are required for PTTH release from neurons or during conversion of 20-hydroxyecdysone precursors to 20-hydroxyecdysone cannot be ruled out at this stage. In either event the two pathways act in parallel to maintain 20-hydroxyecdysone levels perhaps via nonoverlapping downstream targets (Venkatesh, 2001).
The dunce and rutabaga mutations of Drosophila affect a cAMP-dependent phosphodiesterase and a Ca2+/CaM-regulated adenylyl cyclase, respectively. These mutations cause deficiencies in several learning paradigms and alter synaptic transmission, growth cone motility, and action potential generation. The cellular phenotypes either are Ca2+ dependent (neurotransmission and motility) or mediate a Ca2+ rise (action potential generation). However, interrelations among these defects have not been addressed. Conditions have been established for fura-2 imaging of Ca2+ dynamics in the 'giant' neuron culture system of Drosophila. Using high K+ depolarization of isolated neurons, a larger, faster, and more dynamic response was observed from the growth cone than the cell body. This Ca2+ increase depends on an influx through Ca2+ channels and is suppressed by the Na+ channel blocker TTX. Altered cAMP metabolism by the dnc and rut mutations reduces response amplitude in the growth cone while prolonging the response within the soma. The enhanced spatial resolution of these larger cells allow an analysis of Ca2+ regulation within distinct domains of mutant growth cones. Modulation by a previous conditioning stimulus was altered in terms of response amplitude and waveform complexity. Furthermore, rut disrupts the distinction in Ca2+ responses observed between the periphery and central domain of growth cones with motile filopodia (Berke, 2002).
This study describes the first use of fura-2 imaging for
intracellular Ca2+ in a dissociated culture system of Drosophila. Unlike in vivo preparations such as the neuromuscular junction, optical
imaging in culture can relate subcellular Ca2+ dynamics throughout different regions of single neurons. The spatial resolution offered by optical
imaging complements electrophysiological studies of
Drosophila giant neurons in culture and may indicate the
functional significance of membrane excitability differences in
subneuronal regions. The two approaches in combination will greatly
enhance the neurogenetic study of Ca2+-dependent processes involved in
neuronal development and physiology (Berke, 2002).
The initial characterization of regional differences in high
K+-induced Ca2+ regulation in the soma and growth
cone indicates that both Ca2+
and Na+ channels are involved.
Drosophila central neurons in culture contain two types of
Ca2+ currents (L and T type) distinguished
by their activation voltage, decay kinetics, voltage dependence, and
underlying single-channel activities. In other species, electrical
recordings from the growth cone indicate that similar L- and T-type Ca2+
channels are expressed throughout the neuron, but that channel density
may be higher and the channels more clustered in the growth cone. Such
variation may relate to findings that the growth cone and soma
differ in sensitivity and response kinetics during depolarization. In future investigations, it will be interesting to use identified mutations in Na+ and Ca2+ channel genes to dissect their
involvement in Ca2+ regulation throughout the neuron (Berke, 2002).
Local Ca2+ levels regulate filopodial
formation and play an important role in directing growth cone turning in
cultured neurons from other species. The data from Drosophila show that the magnitude
of the high K+ response is larger in the
periphery of motile, as opposed to nonmotile, growth cones. Distinct
regions within the growth cone exhibit differences in the localization
of cytoskeletal elements and cytoplasmic organelles. It may be
questioned whether the cytokinesis inhibition technique used to
generate giant neurons affects the distribution of
Ca2+ channels in the soma and growth cone
because of actin cytoskeletal disruption. However, previous
electrophysiological studies did not detect differences in action
potential and ion current properties with and without the removal of
cytochalasin B. This result is consistent
with a preliminary study indicating that differences in response
characteristics between the soma and growth cone are still evident in
embryonic cultures made in the absence of CCB. In
future studies, untreated larval neurons in culture can be used to
examine Ca2+ signaling in the same
dnc and rut growth cones that display retarded motility (Berke, 2002).
The analysis of these well studied mutants by fura-2 imaging has
revealed previously unknown mutant phenotypes and suggests the usefulness of Ca2+ imaging for a wide range of other mutations. dnc and
rut decrease sensitivity to high K+ stimulation for a cytosolic
Ca2+ increase while affecting both the activity dependence and spatial distribution of [Ca2+] within motile and nonmotile
growth cones. Chronic changes in cAMP metabolism imposed by
the dnc and rut mutations decrease sensitivity
most strongly in the growth cone while prolonging the
Ca2+ increase only in the soma (Berke, 2002).
It is known that dnc and rut alter the modulation
of K+ currents gated by the Sh and eag channel subunits. Moreover, enhanced spike activity has been detected with patch-clamp recordings from the soma of dnc and rut giant neurons. Such altered excitability may explain the prolonged soma response observed and should be further examined during patch-clamp experiments with high K+ depolarization. Electrophysiological studies of other currents in dnc and rut neurons have been lacking, but a Ba2+ current flowing through wild-type Ca2+ channels increases during the application of cAMP analogs. Further support for Ca2+ channel defects stems from findings that dnc and rut increase and
decrease an L-type (dihydropyridine-sensitive) Ca2+ current in larval muscle, phenotypes that are mimicked by short-term pharmacological manipulations on
wild-type muscles. However, some dnc and rut physiological phenotypes at the larval neuromuscular junction cannot be mimicked by acute pharmacological treatments and are attributed to long-term, developmental effects of the mutations. Different aspects of the Ca2+ signaling phenotypes may be caused by either acute or chronic effects. Therefore, the combination of genetic and pharmacological analyses will offer a more comprehensive picture of how the cAMP pathway regulates neuronal Ca2+ (Berke, 2002).
In Drosophila, rest shares features with mammalian sleep, including prolonged immobility, decreased sensory responsiveness and a homeostatic rebound after deprivation. To understand the molecular regulation of sleep-like rest, the involvement of a candidate gene, cAMP response-element binding protein (CREB), was investigated. The duration of rest is inversely related to cAMP signaling and CREB activity. Acutely blocking CREB activity in transgenic flies does not affect the clock, but increases rest rebound. CREB mutants also have a prolonged and increased homeostatic rebound. In wild types, in vivo CREB activity increases after rest deprivation and remains elevated for a 72-hour recovery period. These data indicate that cAMP signaling has a non-circadian role in waking and rest homeostasis in Drosophila (Hendricks, 2001).
The daily rest of flies carrying mutations and/or transgenes that alter cAMP signaling was examined at several points in the pathway. dunce flies have a mutation in the phosphodiesterase enzyme and therefore have increased cAMP. The null mutant (dncML) rests significantly less than the background yw strain. Similarly, increasing PKA activity in flies with a heat-shock-inducible transgene of the catalytic subunit of PKA significantly decreases daily rest durations compared to pre-heat-shock rest levels. Decreased adenylyl cyclase enzyme activity and thus decreased cAMP characterize rutabaga (rut) mutants, which rest more than the Canton S background strain. Similarly, S162 flies that carry a mutation that abolishes dCREB2 activity rest more than their comparison group (siblings without the mutation). The mutation is a stop codon just upstream of the basic leucine-zipper motif of the dCREB2 gene (Hendricks, 2001).
Habituation is a fundamental form of behavioral plasticity that permits organisms to ignore inconsequential stimuli. This study describes the habituation of a locomotor response to ethanol and other odorants in Drosophila, measured by an automated high-throughput locomotor tracking system. Flies exhibit an immediate and transient startle response upon exposure to a novel odor. Surgical removal of the antennae, the fly's major olfactory organs, abolishes this startle response. With repeated discrete exposures to ethanol vapor, the startle response habituates. Habituation is reversible by a mechanical stimulus and is not due to the accumulation of ethanol in the organism, nor to non-specific mechanisms. Ablation or inactivation of the mushroom bodies, central brain structures involved in olfactory and courtship conditioning, results in decreased olfactory habituation. In addition, olfactory habituation to ethanol generalizes to odorants that activate separate olfactory receptors. Finally, habituation is impaired in rutabaga, an adenylyl cyclase mutant isolated based on a defect in olfactory associative learning. These data demonstrate that olfactory habituation operates, at least in part, through central mechanisms. This novel model of olfactory habituation in freely moving Drosophila provides a scalable method for studying the molecular and neural bases of this simple and ubiquitous form of learning (Cho, 2004).
The development of the nervous system is influenced by environmental factors.
Among all environmental factors, temperature belongs to a unique category.
Besides activating some specific sensory pathways, it exerts nonspecific,
pervasive effects directly on the entire nervous system, especially in
exothermic species. This study uses mutants to genetically discover how
temperature affects nerve terminal arborization at larval neuromuscular
junctions of Drosophila. It is known that hyperexcitability in K+ channel
mutants leads to enhanced ramification of larval nerve terminals. Elevated cAMP
levels in dunce mutants with reduced phosphodiesterase activity also cause
enhanced arborization. These genetic alterations are thought to perturb
mechanisms relevant to activity-dependent neural plasticity, in which neuronal
activity activates the cAMP pathway, and consequently affect nerve terminal
arborization by regulating expression of adhesion molecules. This study demonstrates
the robust influence of rearing temperature on motor nerve terminal
arborization. Analysis of ion channel and cAMP pathway mutants indicates that
this temperature-dependent plasticity is mediated via neuronal activity changes
linked to mechanisms controlled by the rutabaga-encoded adenylyl cyclase (Zhong, 2004 ).
From these observations, four conclusions can be drawn. (1) Developmental
temperature is a robust environmental factor that influences neuronal outgrowth
in larval neuromuscular junctions of Drosophila. (2) Critical
temperature-dependent neuronal growth is mediated by neural activity, although
temperature may exert nonspecific pervasive effects on cellular or molecular
activities. (3) Nerve terminal arborization increases with activity level but
becomes suppressed beyond an optimal activity level. (4) Rut-regulated cAMP
pathways play an essential role in mediating activity-dependent nerve terminal
arborization. The result suggests that presynaptic Rut activity is critical (Zhong, 2004).
It is conceivable that neuronal activity may be generally increased at higher
rearing temperatures in flies. For instance, transient K+ currents
inactivate faster at increased temperatures, which should allow higher-frequency
firing of action potentials. It is also noted that a wings-down phenotype
presumably resulting from extreme hyperexcitability is observed only in eag
Sh double mutants but not in the corresponding single mutants at room
temperature. However, this phenotype could be found among a fraction of
eag or Sh single mutants reared at 30°C.
Thus, it is logical to speculate that increased neural activity at
higher rearing temperatures leads to modification of nerve terminal
arborization. The present study provides several lines of evidence in support of
this idea, as summarized below. It appears that temperature increase and
hyperexcitability mutations exert similar influences on nerve terminal
arborization. The effect of a small increase in temperature (from RT to
25°C) is equivalent to that of single eag or Sh mutations,
whereas a large increase (from RT to 30°C) affects arborization similar to
that of the double mutants. More conclusive
evidence comes from the observation that temperature-induced enhancement of
arborization can be suppressed by the no action potential (nap) mutation,
in which neuronal activity is lowered because of a reduced number of
Na+ channels. Moreover, both activity- and temperature-dependent
arborization are linked to the cAMP pathway. Both Sh (at 25°C)- and
temperature (at 30°C)-induced enhancement in arborization are suppressed by
the rut mutation (Zhong, 2004).
The cAMP pathway has been suggested to be a necessary component in visual
experience-dependent cortical plasticity of ocular dominance and has been shown
to be a critical signal transduction pathway in mediating synaptic
reorganization during long-term memory formation in Aplysia.
Previous studies have indicated that elevated cAMP
levels in dnc mutants lead to enhancement of arborization at the larval
neuromuscular junction, and this enhanced ramification in dnc mutants can
be suppressed by the rut mutation, as shown in dnc rut double
mutants. This establishes that cAMP is
able to influence arborization, but its role in mediating this
activity-dependent arborization has not been resolved previously. In this study,
it is clearly demonstrated in rut and rut Sh double mutants that
arborization is not enhanced (even at high temperatures or in hyperexcitability
mutants) if rut-encoded adenylyl cyclase activity is removed.
In contrast, dnc-encoded cAMP-specific
phosphodiesterase is not a component directly mediating activity-dependent
plasticity. Arborization in dnc mutants still varies with temperature in
a striking manner, whereas hyperexcitability and
temperature are unable to alter arborization in rut mutants (Zhong, 2004).
It is interesting to note that motor nerve terminal arborization is reduced in
dnc, Sh, and eag Sh mutants reared at 30°C.
This observation has prompted the proposal
that there is an optimal level of activity, hence of cAMP, for promoting axon
outgrowth and arborization.
In other words, there is a bell-shaped relationship curve between neuronal
activity and ramification of arbors: motor nerve terminal arborization is
enhanced with an increase in activity and will become suppressed with additional
increases in activity. In fact, a similar relationship has been suggested
between intracellular calcium concentrations and growth cone formation and
neurite outgrowth in cultured neurons. In
summary, the results presented demonstrate that developmental temperature is a
robust environmental factor that influences neuronal outgrowth, and that
temperature-dependent neuronal growth is mediated by neural activity. The effect
of rut and dnc at different developmental temperatures and their
interaction with channel mutations demonstrate an essential role of the
Rut-regulated cAMP pathway in developmental neural plasticity in response to
environmental changes (Zhong, 2004).
The GAL4-based Gene-Switch system has been engineered to regulate transgene expression in Drosophila in both time and space. A Gene-Switch transgene was constructed in which Gene-Switch expression is restricted spatially by a defined mushroom body enhancer. This system allows Gene-Switch to be active only in the mushroom bodies and only on administration of the pharmacological Gene-Switch ligand RU486. This line was used to drive the expression of a rutabaga cDNA in otherwise rutabaga mutant flies. Induction of the rutabaga cDNA in the mushroom bodies only during adulthood, or during adulthood along with the larval and pupal developmental stages, corrects the olfactory memory impairment found in rutabaga mutants. Induction of the cDNA only during the larval and pupal stages was inconsequential to performance in olfactory memory tasks. These data indicate that normal rutabaga function must be expressed in adulthood for normal memory and conclusively delimit the time and space expression requirements for correcting the rutabaga memory impairment. Such combined pharmacogenetic regulation of transgene expression now allows this time and space dissection to be made for other behavioral mutants (Mao, 2004; full text of article).
Rutabaga is the most thoroughly characterized of all Drosophila memory mutants. Previous studies have shown that the gene encodes for a calcium:calmodulin-dependent adenylyl cyclase and that this cyclase is preferentially expressed in the axons of mushroom body neurons. These observations, combined with the knowledge that the products of other learning genes like dunce show preferential expression in the mushroom bodies, prompted two major and important questions. First, are mushroom bodies the site of action of the adenylyl cyclase in terms of promoting memory formation? This possibility was strongly predicted to be the case, given the preferential expression in these neurons and the fact that other gene products important for learning exhibit a similar expression pattern. This prediction was confirmed through standard GAL4-mediated rescue experiments. The second question, whose answer, surprisingly, was unknown, was whether the adenylyl cyclase is important for the development of brain structures such as mushroom bodies that are required for memory, or whether the requirement of the cyclase is restricted to adult stages. The possibility that developmental defects underlie the impairment in memory was made stronger by studies that demonstrated that the mutants do have an altered nervous system and the discovery that mutation of the rut homolog in the mouse produces an alteration in barrel fields of the somatosensory cortex and in the refinement of axonal projections from retinal ganglion cells (Mao, 2004).
This study has provided through the Gene-Switch pharmacogenetic approach described here, an answer to this question. Expression of rut only in the adult mushroom bodies is sufficient for the rescue of the memory impairment of rut mutants, and expression of rut in the mushroom bodies during development is inconsequential to adult odor memory formation. This result was achieved in part through the construction of an RU486-inducible, mushroom body Gene-Switch line P{MB-Switch}12-1. This line should be very valuable for similar pharmacogenetic studies in which the experimenter requires control over the timing of gene expression in the mushroom bodies. Moreover, nearly identical results were obtained with a second system for controlling transgene expression in time and space. This system, named TARGET, utilizes a temperature-sensitive repressor of GAL4 (GAL80ts) to modulate GAL4 activity with temperature shifts (Mao, 2004).
Even though the requirement for adenylyl cyclase activity for odor learning has been delimited to the adult brain, its specific role in learning processes is still uncertain. The most attractive possibility is that the adenylyl cyclase is coupled to neurotransmitter receptors that register the unconditioned stimulus during learning, or provide a neuromodulatory role for the actual association of the conditioned stimulus and the unconditioned stimulus. An alternative idea is that the adenylyl cyclase is required for the maturation of some other physiological process or cellular components and, in its absence, the neurons remain incompetent to support memory formation (Mao, 2004).
The Gene-Switch system, like TARGET, requires several hours to days for complete induction. This time course is to be expected for any transcriptionally based system, which requires an applied inducer to alter the activity of a transcription factor to initiate the subsequent processes of transcription, RNA processing, translation, posttranslational modification, and cellular targeting. The system should be extremely effective for answering questions regarding broad temporal expression requirements such as the one posed here. Questions that require more acute induction and deinduction, such as whether any given gene product is required for the formation of memories, their stability, or their retrieval, will likely require the development of gene expression systems that operate at the posttranslational level, to turn on or turn off the activity of any given protein (Mao, 2004).
Olfactory learning assays in Drosophila have revealed that distinct brain structures known as mushroom bodies (MBs) are critical for the associative learning and memory of olfactory stimuli. However, the precise roles of the different neurons comprising the MBs are still under debate. The confusion surrounding the roles of the different neurons may be due, in part, to the use of different odors as conditioned stimuli in previous studies. This study investigated the requirements for the different MB neurons, specifically the α/ß versus the γ neurons, and whether olfactory learning is supported by different subsets of MB neurons irrespective of the odors used as conditioned stimuli. The rutabaga (rut)-encoded adenylyl cyclase was expressed in either the γ or α/ß neurons and the effects were examined on restoring olfactory associative learning and memory of rut mutant flies. A temperature-sensitive shibire (shi) transgene was expressed in these neuron sets and the effects of disrupting synaptic vesicle recycling on Drosophila olfactory learning was examined. These results indicate that although odor-pair-specific learning was not detected using GAL4 drivers that primarily express in γ neurons, expression of the transgenes in a subset of α/ß neurons resulted in both odor-pair-specific rescue of the rut defect as well as odor-pair-specific disruption of learning using shits1 (Akalal, 2006).
Drosophila olfactory learning is typically assayed using olfactory classical conditioning. Using this assay, several memory mutants and the genes involved in olfactory memory formation have been identified. This assay has also been used to investigate the roles of the different MB lobes. In each case, GAL4 drivers that express in distinct lobes of the MBs were used. When G-protein signaling was disrupted using pan-MB GAL4 drivers (238y, c747, and c309) to express a constitutively activated stimulatory heterotrimeric GTP-binding protein α-subunit (Gαs*), associative olfactory learning was reported to be completely abolished. Using 201y-GAL4, a line that expresses extensively in the γ lobes but only in the narrow core elements of the α/ß lobes, learning was only reduced by ~50%. In a study that investigated the effects of expressing shits1 using c739-GAL4, an α/ß driver, and 201y-GAL4, a significant impairment of performance was observed when neurotransmission was transiently inactivated through the α/ß lobes with only a slight, but nonsignificant, decrease in memory performance observed using 201y-GAL4, suggesting a greater role for MB α/ß lobes in olfactory memory. Other experiments involving rescue of the rut mutant defect by expressing a wild-type rut cDNA showed that memory was restored to wild-type levels using broad-MB GAL4 drivers (247, c772, and 30y) and the γ lobe driver H24-GAL4. However, memory was only partially rescued using 201y-GAL4, and no rescue was observed using the GAL4 drivers 189y and 17d, which both express primarily in the MB α/ß lobes. This, therefore, suggested a greater role of MB γ lobes in olfactory learning. The apparent contradictions among each of these studies could be due to the fact that in each of these experiments different combinations of odors were used for the olfactory learning assay: MCH-OCT, MCH-BEN, and OCT-BEN. To resolve the apparent discrepancies and inconsistencies among the different studies, this study used three commonly used odor combinations (MCH-OCT, MCH-BEN, and OCT-BEN) and two different assays, shits1 inactivation of neurotransmission as well as rescue of the rut memory defect, to examine the roles of the different neurons comprising the MB lobes (Akalal, 2006).
Expressing transgenes using the two γ lobe GAL4 drivers NP1131 and H24 did not produce any odor-pair-specific learning effects. Using these γ drivers to express a rut cDNA in a rut mutant background results in a partial rescue of the learning defect for each of the odor combinations tested. The γ driver of choice has typically been H24-GAL4, but the expression pattern of this driver is not limited to the γ neurons. In fact, it expresses very robustly in the ALs, and one concern is that this might affect performance scores since the learning assay is based on olfactory cues. The use of NP1131-GAL4 that expresses primarily in the γ neurons and a small subset of α'/ß' neurons is important to validate the results for H24-GAL4. A prior study using H24-GAL4 to rescue the rut learning defect resulted in performance scores that were statistically indistinguishable from wild-type flies using MCH and BEN. However, among the different lines that were observed to rescue the rut defect, H24-GAL4 yielded the lowest scores, and another γ driver, 201y-GAL4, used in the same study failed to rescue the defect. Yet another study that used H24-GAL4 to restore a rut cDNA in a rut mutant background failed to produce rescue of the defect using MCH and OCT as odorants. The current data suggest that driving a rut cDNA in γ neurons results in rut levels that partially rescue the defect. Although no odor-pair-specific learning was detected using the two γ drivers tested, this does not preclude the possibility that when γ drivers that express in other subsets of γ neurons are discovered, this would remain the case. It is likewise important to note that the three odorants used in in the current study do not represent the whole repertoire of odors that a fly responds and learns to. Thus, it remains possible that the γ neurons labeled by NP1131-GAL4 and H24-GAL4 may still be involved in the odor-pair-specific associative learning of other smells (Akalal, 2006).
Rut rescue using GAL4 drivers that express in the α/ß neurons reveals some odor-pair-specific effects. Performance scores for flies carrying c739-GAL4 together with the UAS-rut transgene in a rut mutant background show no rescue of the rut defect for all odor combinations tested. In contrast, driving a rut cDNA using 17d-GAL4 shows partial rescue of the rut defect when MCH-BEN and OCT-BEN are used for the assay but not for MCH-OCT. To address the observed contrast in the rescue using the different odor combinations for these two lines, the expression patterns for the two α/ß drivers was compared. The expression level for c739-GAL4 is greater than 17d-GAL4, and it appears that only a subset of neurons, a thin core, is labeled for 17d-GAL4. Using antibodies raised against glutamate, a further subdivision of the lobes has been described as a slender core of glutamatergic neurons, the α and ß core neurons (αc and ßc), that lie posteriorly and are partly enclosed by the α and ß neurons. Whether the core neurons that are marked by 17d-GAL4 correspond to the glutamatergic subdivision of the α/ß lobes remains to be determined. One explanation for the observed odor-pair-specific effects is that 17d-GAL4 and c739-GAL4 express in nonoverlapping regions of the MB α/ß neurons. Behavioral experiments were performed on flies that were 2–5 d old, and although the neuron counts make clear that the expression of c739-GAL4 is broader than 17d-GAL4, it was not possible to confirm whether there is nonoverlapping expression at the α/ß core. Preliminary data suggest, however, that during this time period, the spatial expression of 17d-GAL4 is in the core region, while c739-GAL4 expression is more peripheral with a slight overlap of expression as a ring around this core. Additional experiments are needed to extend these observations (Akalal, 2006).
What is the significance of organizing the Drosophila MBs into different lobes, and is there differential representation of odors in the different lobes? These findings indicate that to answer this question one must take into account the choice of odor pairs used in the olfactory assay. The differential effects seen when using different odor combinations for 17d-GAL4 suggest that even though this driver expresses in a subset of α/ß neurons, some of these neurons must be important in MCH-BEN and OCT-BEN learning since driving rut using c739-GAL4, a line that expresses in a greater number of α/ß neurons, is insufficient to see a partial rescue of the rut learning defect. Clearly, this partial rescue is more than a mass effect of rut-expressing MB neurons. This raises an important point: It may not be accurate to describe GAL4 driver patterns based solely on lobe-specificity, since different GAL4 drivers may highlight subsets of the neurons comprising individual lobes. The significance of achieving complete rescue when the double GAL4 c739;H24 was used to express UAS-rut in a rut mutant background is still unclear. Although it is tempting to speculate that this complete rescue occurs by completing a spatial circuit that involves odorant representation in both α/ß and γ neurons, more experiments need to be performed to investigate whether it may just represent the massed effect of expression in more MB neurons (Akalal, 2006).
Examination of the odor-evoked activity in Drosophila MB neurons by expressing a green fluorescent protein-based Ca2+ indicator, G-CaMP, have revealed remarkable spatial stereotypy. In fact, several studies have shown that stereotypical anatomical and functional organization can be found at the different levels of the insect olfactory pathway. Each olfactory receptor neuron (ORN) likely expresses a single olfactory receptor (OR) gene, and ORNs that express the same OR genes converge on a common glomerulus in the AL, resulting in a stereotyped projection pattern. A near complete map of ORN connectivity constructed through a systematic survey of Drosophila OR expression has validated the principles of 'one neuron-one receptor' and 'one glomerulus-one receptor.' Different odors activate different combinations of ORs, and individual receptors can mediate both excitatory and inhibitory responses to different odors in the same cell. In addition, a topographic organization of the AL has been described wherein ORNs in distinct sensilla types project into distinct regions of the AL. At the level of the MB neuron cell bodies and the calyx, different odors evoked distribution patterns of fluorescence that were odor-specific and conserved across flies, resulting in stereotyped responses for BEN-, MCH-, and OCT-evoked fluorescence activity at both the wide-field and single-neuronal level. The current results demonstrate an odor-pair-specific effect with 17d-GAL4 using two odor combinations that have BEN as one of the odors. Several studies have indicated that Drosophila processes BEN differently from other odors. In fact, although surgical removal of the antennae and palps of wild-type flies results in the abolishment of MCH and OCT avoidance, BEN avoidance is only partially affected in both T-maze and arena paradigms, suggesting that BEN is sensed through other nonolfactory pathways. To eliminate naive odor bias, experiments are usually performed in a counterbalanced design, with half of the flies used in the calculation of the performance index being trained to the first odor and the other half to the second odor. An examination of the half P.I. scores for these experiments does not reveal obvious asymmetries between BEN and the two counter-odors. Moreover, the fact that the odor-pair-specific effects are seen in both rescue as well as disruption of memory experiments suggests that it is the expression of transgenes in the subset of neurons defined by 17d-GAL4 that confers the odor-pair-specific behavioral phenotypes observed in this study (Akalal, 2006).
rutabaga:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| References
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