Effect of neurofibromatosis type I mutations on a novel pathway for adenylate cyclase activation requiring neurofibromin and Ras

Neurofibromatosis type I (NFI) is a common genetic disorder that causes nervous system tumors, and learning and memory defects in human and in other animal models. A novel growth factor stimulated adenylyl cyclase (AC) pathway has been identified in the Drosophila brain, which is disrupted by mutations in the epidermal growth factor receptor (EGFR), neurofibromin (NF1) and Ras, but not Galphas. This is the first demonstration in a metazoan that a receptor tyrosine kinase (RTK) pathway, acting independently of the heterotrimeric G-protein subunit Galphas, can activate AC. This study also shows that Galphas is the major Galpha isoform in fly brains, and a second AC pathway is defined stimulated by serotonin and histamine requiring NF1 and Galphas. A third, classical Galphas-dependent AC pathway, is stimulated by Phe-Met-Arg-Phe-amide (FMRFamide) and dopamine. Using mutations and deletions of the human NF1 protein (hNF1) expressed in Nf1 mutant flies, it is shown that Ras activation by hNF1 is essential for growth factor stimulation of AC activity. Further, it is demonstrated that sequences in the C-terminal region of hNF1 are sufficient for NF1/Galphas-dependent neurotransmitter stimulated AC activity, and for rescue of body size defects in Nf1 mutant flies (Hannan, 2006).

This study defines three separate pathways for AC activation: (1) a novel pathway for AC activation, downstream of growth factor stimulation of EGFR that requires both Ras and NF1, but not Galphas; (2) an NF1/Galphas-dependent AC pathway operating through the Rutabaga-AC (Rut-AC) and stimulated by serotonin and histamine, as observed in the larval brain; (3) a classical G-protein coupled receptor-stimulated AC pathway operating through Galphas alone. The Rut-AC pathway may also be stimulated by PACAP38 at the larval neuromuscular junction and in adult heads as shown in previous studies. The AC activated by NF1/Ras (AC-X), or Galphas (AC-Y), has not yet been identified (Hannan, 2006).

This study shows for the first time that Ras can stimulate AC in an NF1-dependent manner in higher organisms, via an RTK-coupled pathway that is independent of the Galphas G-protein. The functionality of human NF1 in the fly system, and the high degree of identity between human and fly NF1 (60%), suggests that similar pathways for AC activation may also operate in mammals. Previous studies failed to detect stimulation of AC by Ras in cultured vertebrate cell lines, and in Xenopus oocytes, however, these cell types may not contain sufficient NF1 to support NF1/Ras-dependent AC activation. This is consistent with the observation that levels of both Ras and NF1 are critical for stimulation of AC activity in adult head membranes. The reported EGF activation of AC in cardiac myocytes and other tissues requires both Galphas, and the juxtamembrane domain of the EGFR, which is not present in the Drosophila EGFR (Hannan, 2006).

Experiments with human NF1 mutants show that the GRD domain and the RasGAP activity of NF1 are both necessary and sufficient for growth factor-stimulated NF1/Ras-dependent AC activity. It is also concluded that C-terminal residues downstream of the GRD are critical for both body size regulation and neurotransmitter-stimulated NF1/Galphas-dependent AC activity, thus defining for the first time a region outside the GRD that contributes to this pathway. Interestingly, expression of a human NF1 GRD fragment in Nf1-/- astrocytes results in only partial restoration of NF1-mediated increases in cAMP levels in response to PACAP. Thus, regions outside the GRD also seem to be necessary for activation of AC in these mammalian cells (Hannan, 2006).

Thus, NF1, while being a negative regulator of Ras, is also actively involved in stimulation of AC activity. Moreover, it regulates AC activity through at least two different mechanisms, one of which depends on the RasGAP activity of NF1. The multifunctional nature of the NF1 protein illuminates its importance in nervous system development, tumor formation and behavioral plasticity, and may also explain the wide range of clinical manifestations in neurofibromatosis type I (Hannan, 2006).

Parallel processing of appetitive short- and long-term memories in Drosophila

It is broadly accepted that long-term memory (LTM) is formed sequentially after learning and short-term memory (STM) formation, but the nature of the relationship between early and late memory traces remains heavily debated. To shed light on this issue, this study used an olfactory appetitive conditioning in Drosophila, wherein starved flies learned to associate an odor with the presence of sugar. Advantage was taken of the fact that both STM and LTM are generated after a unique conditioning cycle to demonstrate that appetitive LTM is able to form independently of STM. More specifically, it was shown that (1) STM retrieval involves output from γ neurons of the mushroom body (MB), i.e., the olfactory memory center, whereas LTM retrieval involves output from αβ MB neurons; (2) STM information is not transferred from γ neurons to αβ neurons for LTM formation; and (3) the adenylyl cyclase RUT, which is thought to operate as a coincidence detector between the olfactory stimulus and the sugar stimulus, is required independently in γ neurons to form appetitive STM and in αβ neurons to form LTM. Taken together, these results demonstrate that appetitive short- and long-term memories are formed and processed in parallel (Trannoy, 2011).

Short-term memory (STM) forms right after learning and is based on transient molecular and cellular events lasting from a few minutes to a few hours, whereas long-term memory (LTM) forms later on and involves gene expression and de novo protein synthesis following conditioning. The nature of the links between STM and LTM has long been debated, but there is consensus that STM and LTM are sequential processes and that LTM formation is built on the short-term trace. However, other studies have led to the conclusion that the mechanisms underpinning STM and LTM in vertebrates are at least partially independent (Trannoy, 2011).

Studies in insects have highlighted that mushroom bodies (MBs) play a major role in learning and memory. In particular, in Drosophila, MBs play a key role in olfactory learning and memory. The MBs in each brain hemisphere of Drosophila consist of approximately 2,000 neurons called Kenyon cells that can be classified into three major types: αβ, whose axons branch to form a vertical (α) and a medial (β) lobe, α'β', which also form a vertical (α') and a medial (β') lobe, and γ, which form a single medial lobe in the adult (Trannoy, 2011).

Several molecular-level studies have demonstrated that the cyclic AMP (cAMP) pathway plays a pivotal role in associative learning. In particular, calcium/calmodulin-dependent adenylyl cyclase (AC) encoded by the rutabaga (rut) gene is necessary to aversive olfactory conditioning where an odorant is associated to electric shock. Rut AC was proposed to function as a coincidence detector, integrating both the olfactory information carried by projection neurons to the MB and the electric shock carried by dopaminergic neurons. Interestingly, Rut cAMP signaling is required in γ neurons to form aversive STM (Zars, 2000; Blum, 2009) and in αβ neurons to form LTM (Blum, 2009), suggesting an independence of these two memory phases. However, several results suggest that aversive STM and LTM are not processed by fully independent neuronal pathways. Thus, a more efficient rescue of rut STM or LTM defect is observed when Rut is expressed in both γ and αβ neurons, suggesting that Rut is also involved in αβ neurons for aversive STM and in γ neurons for LTM. In addition, blocking αβ neuron synaptic transmission during memory retrieval impairs both aversive STM and LTM. Moreover, the induction of aversive STM and LTM requires different conditioning protocols, because STM is induced by one cycle of conditioning, whereas LTM formation requires spaced conditioning consisting of repeated training sessions separated by 15 min rest periods. These different training protocols may induce different physiological states within the relevant neurons, making it more difficult to interpret whether LTM is or is not built upon STM (Trannoy, 2011).

In Drosophila, appetitive STM and LTM are both generated after a single session of odorant-plus-sugar association, offering a powerful situation to study the link between the short- and the long-term trace. Rut AC has been hypothesized to be the coincidence detector in olfactory appetitive memory, because rut mutants exhibit poor immediate memory. It was shown that Rut AC in MB αβ and γ neurons or in projection neurons is sufficient for appetitive learning and STM, but it remains unknown which brain structure involves Rut activity for appetitive LTM (Trannoy, 2011).

Consolidation of appetitive STM and LTM requires output from α'β' neurons for 1 hr after training but not from αβ neurons. The role of γ neurons in appetitive STM or LTM consolidation has not yet been addressed. STM retrieval involves output from αβ and/or γ neurons, but the role of α'β' neurons in STM retrieval remains unknown. LTM retrieval involves output from αβ neurons but not from α'β' neurons, and the role of γ neurons in LTM retrieval has not yet been addressed. Thus a full picture of the role of MB neurons in appetitive memory processing has yet to emerge (Trannoy, 2011).

To clarify the role of the different MB neurons in appetitive STM and LTM, the c739-GAL4 driver and the UAS-shi c739-GAL4ts (shi) transgene were used to block synaptic transmission in αβ neurons during memory retrieval. The dominant temperature-sensitive SHITS protein blocks dynamin-dependent membrane recycling and synaptic vesicle release at restrictive temperature (33°C). This effect is reversible when the temperature is shifted back to 25°C. It was previously reported that c739/+; shi/+ flies have normal olfactory acuity. This study first checked that c739/+; shi/+ flies present a normal sugar response at restrictive temperature. It was then found that blocking synaptic transmission from MB αβ neurons did not affect appetitive STM retrieval. This was a surprising observation, because it was previously shown that output from MB αβ neurons is required for appetitive LTM retrieval, which was confirmed in this study. To rule out the possibility that the LTM retrieval defect might be due to c739-driven expression of shits outside of the MB, the MB247-GAL80+ construct (MB-GAL80) was used to inhibit GAL4 activity in αβ neurons. As expected, c739/MB-GAL80; shi/+ flies showed normal LTM performance when tested at restrictive temperature. Furthermore, appetitive LTM performance of c739/+; shi/+ flies at permissive temperature was normal. Hence, these data indicate that MB αβ neuron output is required for appetitive LTM retrieval but not for STM retrieval (Trannoy, 2011).

It was previously shown that output from MB αβ and γ neurons is required for STM retrieval. Because the data established that output from αβ neurons is not required for this process, the role of γ neurons in STM retrieval was examined. shits was expressed in γ neurons using the NP21-GAL4 driver. First, it was checked whether NP21/shi flies present normal sugar response and olfactory acuity at restrictive temperature. Blocking γ neuron output during the memory test significantly impaired appetitive STM. Inhibition of NP21 activity in the MB by MB-GAL80 rescued the STM defect of NP21/shi flies. Furthermore, the STM performance of NP21/shi flies was indistinguishable from controls at permissive temperature. Interestingly, blocking γ neuron output during the test did not affect LTM retrieval. The specific role of γ neurons in appetitive STM retrieval was confirmed with another γ neuron driver, 1471-GAL4. Taken together, the results indicate that MB γ neuron output is indispensable for appetitive STM retrieval but dispensable for LTM retrieval (Trannoy, 2011).

Appetitive STM and LTM retrieval each mobilize specific subsets of MB neurons, namely γ neurons for STM and αβ neurons for LTM. This might be due to the fact that STM and LTM are actually mutually independent, being formed and stored in spatially distinct compartments. Alternatively, γ and αβ neurons might be sequentially recruited: in this scenario, STM would form in γ neurons and information would be further transferred from γ to αβ neurons during the consolidation phase to build LTM. Under this latter assumption, blocking output from γ neurons during the LTM consolidation phase should lead to an LTM defect. To discriminate between the two hypotheses, γ neuron neurotransmission was blocked during training and consolidation and then LTM performance was measured. First, it was observed that blocking γ neuron output during training and consolidation did not affect STM. Then, to test the putative role of γ neurons in LTM formation, NP21/shi flies were trained at restrictive temperature and maintained at this temperature for 14 hr during the memory consolidation phase (the flies were kept at 33oC for 14 hr and not for the full 24 hr consolidation period because they started to die after 14 hr; given that appetitive LTM is being detectable 6 hr after training, it is likely that LTM consolidation takes place during the 14 hr time-period of γ neuron neurotransmission blockade). NP21/shi flies showed a normal 24 hr memory in this condition, suggesting that γ neuron output is dispensable during appetitive LTM acquisition and consolidation (Trannoy, 2011).

To further prove that LTM could be formed independently of STM, neurotransmission was constitutively blocked from γ neurons using UAS-TNT (TNT) construct encoding the tetanus toxin [ 33]. TNT/+; NP21/+ flies are viable and present normal sugar response and olfactory acuity. Interestingly, a continuous blockade of γ neurons abolished STM but left LTM unaffected. Thus, TNT/+; NP21/+ flies trained with a single protocol showed no appetitive STM but a normal LTM at 24 hr. These results indicate that appetitive LTM formation is independent of STM and does not require synaptic communication between γ and αβ neurons (Trannoy, 2011).

Rut AC has been hypothesized to be the coincidence detector in olfactory appetitive memory. rut appetitive STM defect can be rescued by expressing Rut in αβ and γ neurons, but it had not yet been addressed whether Rut is specifically involved in γ or αβ neurons. Because circuit blocking experiments suggested that STM and LTM operate independently and involve different subsets of MB neurons, whether rut STM and LTM defects could be rescued independently by expressing Rut in γ and αβ neurons, respectively, was investigated. NP21 and c739 transactivators were used to express UAS-rut were used inrut2080 mutant flies. Expressing Rut in γ neurons fully rescued the rut STM defect, whereas expressing Rut in αβ neurons failed to rescue the rut STM defect. Conversely, expressing Rut in γ neurons failed to rescue the rut LTM defect, whereas expressing Rut in αβ neurons fully rescued the rut LTM defect. These results indicate that Rut AC is specifically required in γ neurons to form STM and in αβ neurons to form LTM, which further argues that appetitive STM and LTM are formed independently of each other (Trannoy, 2011).

The data suggest that appetitive STM and LTM are processed independently in γ and αβ neurons, respectively. Accordingly, immediate appetitive memory processing should involve γ neurons. To test this hypothesis, neurotransmission was constitutively blocked from γ neurons. As expected, TNT/+; NP21/+ flies displayed a 3 min memory defect. The involvement of γ neurons was further confirmed as 1471/+; shi/+ flies displayed a 3 min memory defect at restrictive temperature but not at permissive temperature. Strikingly, blocking neurotransmission from αβ neurons did not affect immediate memory. These results are in agreement with previous observations, suggesting that γ neurons support appetitive STM and αβ neurons support appetitive LTM. It has been shown that appetitive immediate memory is abolished by expressing SHITS in αβ and γ neurons under the MB247 driver. The partial inhibition observed with NP21 and 1471 GAL4 drivers might be due to the fact that MB247 shows a very strong expression in γ neurons, unlike 1471 or NP21. To further prove that the immediate appetitive memory forms in γ neurons, whether rut defect could be rescued was investigated by expressing Rut in γ neurons. Indeed, Rut expression under the NP21 driver restored rut immediate memory defect. On the contrary, expressing Rut in αβ neurons failed to rescue the rut immediate memory defect (Trannoy, 2011).

Appetitive conditioning offers a powerful situation for studying the link between STM and LTM, because both are formed after a single training cycle. It remained unknown whether the same MB neurons process both appetitive STM and LTM formation or whether these two memory phases are underpinned by specialized pathways. This study leads to a new understanding of the role of αβ and γ neurons in appetitive STM and LTM. Using distinct GAL4 drivers to specifically express SHITS or the tetanus toxin in either αβ or γ neurons, this study has shown that appetitive STM and LTM involve γ and αβ neurons, respectively. This study found the following: (1) immediate memory and STM processing involves Rut AC specifically in γ neurons, whereas LTM formation involves Rut in αβ neurons; (2) MB γ neuron output is required to retrieve immediate memory and STM but not LTM, and conversely, αβ neuron output is required to retrieve LTM but neither immediate memory nor STM; (3) γ neuron output is dispensable during memory consolidation, and therefore short-term information is not transferred from γ to αβ neurons to form LTM. Blocking γ neurons using tetanus toxin resulted in a striking phenotype, because flies completely deprived of appetitive STM exhibited normal LTM at 24 hr. In conclusion, this study provides strong evidence that in Drosophila, appetitive STM and LTM are two parallel and independent processes, involving different subsets of neurons within the MB (Trannoy, 2011).

The dynamics of the appetitive memory phase involve other neural circuits than just αβ and γ neurons. Blocking output from α'β' neurons for 1 hr after training affects both STM and LTM. Similarly, blocking output from dorsal paired medial (DPM) neurons, which project onto the MB lobes, for 1 hr after appetitive conditioning affects both STM and LTM. And it was recently shown that blocking GABAergic anterior paired lateral (APL) neurons, which project onto the MB lobes and dendrites, for 2 hr after appetitive conditioning affects STM but not LTM. It has been proposed that α'β'-DPM neurons form a recurrent loop that stabilizes STM and LTM and that APL activity regulates this loop for STM-related processes (Pitman, 2011). Because α'β' neurons are not required for either LTM or STM retrieval, the current results are in agreement with this scheme, where independent STM and LTM traces in γ and αβ neurons are maintained by output from α'β' neurons and MB-extrinsic neurons (Trannoy, 2011).

This model of STM and LTM independence is supported by several studies in other species. In Aplysia, synaptic connection between tail sensory neurons and motor neurons exhibits short- and long-term synaptic facilitation following learning. It has been shown that the induction of short-term synaptic plasticity is not necessary for the induction of long-term plasticity. Studies in vertebrates indicate that STM and LTM involve different biochemical pathways or distinct connected brain areas. This study goes one step further, because it identified neuronal structures that independently process STM and LTM, providing a unique opportunity to analyze biochemical and cellular processes specifically associated with STM and LTM (Trannoy, 2011).

Dopaminergic Modulation of cAMP Drives Nonlinear Plasticity across the Drosophila Mushroom Body Lobes

Activity of dopaminergic neurons is necessary and sufficient to evoke learning-related plasticity in neuronal networks that modulate learning. During olfactory classical conditioning, large subsets of dopaminergic neurons are activated, releasing dopamine across broad sets of postsynaptic neurons. It is unclear how such diffuse dopamine release generates the highly localized patterns of plasticity required for memory formation. This study has mapped spatial patterns of dopaminergic modulation of intracellular signaling and plasticity in Drosophila mushroom body (MB) neurons, combining presynaptic thermogenetic stimulation of dopaminergic neurons with postsynaptic functional imaging in vivo. Stimulation of dopaminergic neurons generated increases in cyclic AMP (cAMP) across multiple spatial regions in the MB. However, odor presentation paired with stimulation of dopaminergic neurons evoked plasticity in Ca2+ responses in discrete spatial patterns. These patterns of plasticity correlated with behavioral requirements for each set of MB neurons in aversive and appetitive conditioning. Finally, broad elevation of cAMP differentially facilitated responses in the gamma lobe, suggesting that it is more sensitive to elevations of cAMP and that it is recruited first into dopamine-dependent memory traces. These data suggest that the spatial pattern of learning-related plasticity is dependent on the postsynaptic neurons' sensitivity to cAMP signaling. This may represent a mechanism through which single-cycle conditioning allocates short-term memory to a specific subset of eligible neurons (gamma neurons) (Boto, 2014).

Dopaminergic neurons are involved in modulating diverse behaviors, including learning, motor control, motivation, arousal, addiction and obesity, and salience-based decision making. In Drosophila, dopaminergic neurons innervate multiple brain regions, including the mushroom body (MB), where they modulate aversive learning, forgetting, state-dependent modulation of appetitive memory retrieval, expression of ethanol-induced reward memory, and temperature-preference behavior (Boto, 2014).

Dopaminergic circuits play a particularly critical role in memory acquisition. During olfactory classical conditioning, where an odor (conditioned stimulus [CS]) is paired with an aversive event (e.g., electric shock; the unconditioned stimulus [US]), dopaminergic neurons respond strongly to the aversive US (Mao, 2009). Dopamine functions in concert with activity-dependent Ca2+ influx to synergistically elevate cyclic AMP (cAMP) (Tomchik, 2009) and PKA (Gervasi, 2010), suggesting that dopamine is one component of a molecular coincidence detector underlying learning. Proper dopamine signaling is necessary for aversive and appetitive memory. Moreover, driving activity of a subset of TH-GAL4+ dopaminergic neurons that differentially innervates the vertical α/α' MB lobes (with less dense innervation of the horizontal β/β'/γ lobes, peduncle, and calyx), is sufficient to induce behavioral aversion to a paired odorant in larvae and adult flies. Conversely, stimulation of a different set of Ddc-GAL4+ dopaminergic neurons, the PAM cluster that innervates mainly the horizontal β/β'/γ lobes, is sufficient to induce behavioral attraction to a paired odorant. Thus, dopaminergic neurons comprise multiple circuits with distinct roles in memory acquisition (Boto, 2014).

Multiple subsets of MB neurons receive CS and US information and express molecules associated with the coincidence detection, making them theoretically eligible to generate dopamine/cAMP-dependent plasticity. Yet only some subsets are required to support memory at any given time following conditioning, leaving open the question of how spatial patterns of plasticity are generated during conditioning. This question has been approached, by using a technique to probe the postsynaptic effects of neuronal pathway activation. Odor presentation was paired with stimulation of presynaptic dopaminergic neurons via ectopic expression of the heat-sensitive channel TRPA1, while monitoring postsynaptic effects with genetically encoded optical reporters for Ca2+, cAMP, and PKA in vivo (Boto, 2014).

The present data demonstrate four major points about how dopaminergic circuits function in neuronal plasticity underlying olfactory classical conditioning. (1) Stimulation of small subsets of dopaminergic neurons evokes consistent, compartmentalized elevations of cAMP across the MB lobes. (2) Broad stimulation of dopaminergic neurons generates broad postsynaptic elevation of cAMP, but Ca2+ response plasticity occurs in discrete spatial regions. (3) Stimulation of TH-GAL4+ neurons and Ddc/R58E02-GAL4+ neurons, which mediate opposing behavioral responses to conditioned stimuli, generates an overlapping pattern of Ca2+ response plasticity in the γ lobe, with additional regions recruited by Ddc/R58E02-GAL4+ stimulation. Finally, (4) the spatial pattern of plasticity coincides with differential sensitivity to cAMP in the γ lobe. Collectively, these data suggest that different subsets of neurons exhibit heterogeneous sensitivity to activation of second messenger signaling cascades, which might shape their responses to neuromodulatory network activity and modulate their propensity for recruitment into memory traces (Boto, 2014).

The data suggest that dopaminergic neurons mediate Ca2+ response plasticity largely in the γ lobe and suggest a potential mechanism for localization of short-term, learning-related plasticity. These data coincide with multiple previous studies that have demonstrated a critical role of γ neurons in short-term memory. Rescue of Rutabaga (Rut) in the γ lobe of rut mutants is sufficient to restore performance in short-term memory, whereas rescue in α/β lobes supports long-term memory. Rescue of the D1-like DopR receptor in the γ lobe is sufficient to rescue both short- and long-term memory in a mutant background, suggesting that the γ neurons mediate the dopaminergic input during conditioning. In addition, stimulating MP1 dopaminergic neurons innervating the heel of the γ lobe is sufficient as an aversive reinforcer. Finally, learning induces plasticity in synaptic vesicle release from MB γ lobes, which depends in part on G(o) signaling (Zhang, 2013). The data support a critical role for the γ lobe in short-term memory. Furthermore, the observation of differential sensitivity of the γ lobe to cAMP might provide an elegant explanation for why it is specifically recruited into short-term memory traces (Boto, 2014).

Direct elevation of cAMP was sufficient to generate localized, concentration-dependent Ca2+ response plasticity in the MB γ lobe in these experiments. Because applying forskolin in the bath is expected to elevate cAMP across the brain, the spatial specificity of the effect is remarkable. This was not an acute effect, because the forskolin was washed out before imaging the first postconditioning odor response. At the concentrations that were tested, only the γ lobe was facilitated. Therefore, it is concluded that the γ lobe is most sensitive to elevation of cAMP, which has the effect of differentially recruiting γ neurons into the representation of short-term memory via dopamine-mediated neuronal plasticity. It is possible that additional signaling cascades are involved in generating learning-related plasticity in α/β and α'/β' neurons, given that no Ca2+ response plasticity was observed in those neurons following forskolin application (Boto, 2014).

The dominant model for cellular mechanisms of olfactory associative learning is that integration of information about the conditioned and unconditioned stimuli are integrated by Rut, which functions as a molecular coincidence detector. This would suggest that MB neurons, which receive CS and US information, would exhibit at least somewhat uniform Ca2+ response plasticity. From this molecular and cellular perspective, the finding that the α/β and α'/β' neurons did not exhibit Ca2+ response plasticity when an odor was paired with stimulation of dopaminergic neurons is surprising. These neurons are theoretically eligible to encode memory, because they receive information about the CS and US. However, the finding that γ neurons differentially exhibit dopamine-dependent plasticity following single-cycle conditioning is consistent with the data from the behavioral experiments. In summary, the present results suggest that differential cAMP sensitivity provides a potential mechanism allowing specific subsets of eligible neurons in an array (γ neurons) to differentially encode CS-US coincidence relative to other subsets (α/β neurons) that also receive CS/US information (Boto, 2014).

Targets of Activity

DCO, the catalytic subunit of Protein kinase A, is preferentially expressed in the mushroom bodies. PKA is the target of cAMP, produced through RUT activity. PKA is a heterotetramer, consisting of two subunits of DC0 and two subunits of a regulatory subunit. Binding of cAMP to the regulatory subunits causes them to dissociate from the tetramer, activating PKA enzymatic activity. Mutants for DCO produce homozygous lethality and a 40% decrease in PKA activity in heterozygotes. This decrease has mild effects on learning but no effect on memory. However, the 80% reduction in activity obtained by constructing double mutant heteroallelic viable animals results in a dramatic learning and memory deficit. These results suggest that PKA plays a crucial role in the cAMP cascade in mushroom bodies to mediate learning and memory processes (Skoulakis, 1993).

Phototransduction in Drosophila is mediated by a G-protein-coupled phospholipase C transduction cascade in which each absorbed photon generates a discrete electrical event, the quantum bump. In whole-cell voltage-clamp recordings, cAMP, as well as its nonhydrolyzable and membrane-permeant analogs 8-bromo-cAMP (8-Br-cAMP) and dibutyryl-cAMP, slow down the macroscopic light response by increasing quantum bump latency, without changes in bump amplitude or duration. In contrast, cGMP or 8-Br-cGMP has no effect on light response amplitude or kinetics. None of the cyclic nucleotides activate any channels in the plasma membrane. The effects of cAMP are mimicked by application of the non-specific phosphodiesterase inhibitor IBMX and the adenylyl cyclase activator forskolin; zaprinast, a specific cGMP-phosphodiesterase inhibitor, is ineffective. Bump latency is also increased by targeted expression of either an activated Gsalpha subunit, which increases endogenous adenylyl cyclase activity, or an activated catalytic protein kinase A (PKA) subunit. The action of IBMX is blocked by pretreatment with the PKA inhibitor H-89. The effects of cAMP are abolished in mutants of the NinaC gene, suggesting this nonconventional myosin as a possible target for PKA-mediated phosphorylation. Dopamine (10 µM) and octopamine (100 µM) mimic the effects of cAMP. These results indicate the existence of a G-protein-coupled adenylyl cyclase pathway in Drosophila photoreceptors that modulates the phospholipase C-based phototransduction cascade (Chyb, 1999).

Protein Interactions

Studies in Aplysia and Drosophila have suggested that Ca2+/calmodulin-sensitive adenylyl cyclase may act as a site of convergence for the cellular representations of the conditioned stimulus (Ca2+ influx) and unconditioned stimulus (facilitatory transmitter) during elementary associative learning. This hypothesis predicts that the rise in intracellular free Ca2+ concentration produced by spike activity during the conditioned stimulus will cause an increase in the activity of adenylyl cyclase. However, published values for the Ca2+ sensitivity of Ca2+/calmodulin-sensitive adenylyl cyclase in mammals and in Drosophila vary widely. The difficulty in evaluating whether adenylyl cyclase would be activated by physiological elevations in intracellular Ca2+ levels is in part a consequence of the use of Ca2+/EGTA buffers, which are prone to several types of errors. Using a procedure that minimizes these errors, the Ca2+ sensitivity of adenylyl cyclase in membranes from Aplysia, Drosophila, and rat brain has been quantitified with purified species-specific calmodulins. In all three species, adenylyl cyclase is activated by an increase in free Ca2+ concentration in the range caused by spike activity. Ca2+ sensitivity is dependent on both calmodulin concentration and Mg2+ concentration. Mg2+ raises the threshold for adenylyl cyclase activation by Ca2+ but also acts synergistically with Ca2+ to activate maximally adenylyl cyclase (Yovell, 1992).

The modulatory neurotransmitters that trigger biochemical cascades underlying olfactory learning in Drosophila mushroom bodies have remained unknown. To identify molecules that may perform this role, putative biogenic amine receptors were cloned. One new receptor, DAMB, was identified as a dopamine D1 receptor by sequence analysis and pharmacological characterization. DAMB shows highly enriched expression in mushroom bodies, in a pattern coincident with the rutabaga-encoded adenylyl cyclase. The spatial coexpression of DAMB and the cyclase, along with DAMB's capacity to mediate dopamine-induced increases in cAMP make this receptor an attractive candidate for initiating biochemical cascades underlying learning (Han, 1996).

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

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