Identification of a cholinergic synapse in the giant fiber pathway of Drosophila using conditional mutations of acetylcholine synthesis

Temperature-sensitive mutations of the choline acetyltransferase (Cha) gene, which lead to reduced choline acetyltransferase (ChAT) activity and acetylcholine (ACh) levels, have been used in an attempt to identify the neurotransmitter at a chemical synapse in the giant fiber pathway (GFP) of Drosophila melanogaster. Prolonged incubation of adult mutant flies at non-permissive temperatures blocked the response of this pathway to brain stimulation, whereas shorter incubation times disturbed various parameters of the normal response. Even at permissive temperature subnormal responses were still evident. These defects in the giant fiber pathway's function suggest a specific cholinergic synapse within the pathway, the first synapse of this type implicated in Drosophila. When the function of this synapse was experimentally stressed, disruptions of GFP function paralleling decreased ChAT activity began to appear at enzyme levels estimated to be approximately 80% of wild-type.     

The Drosophila NSF protein, dNSF1, plays a similar role at neuromuscular and some central synapses.

The N-ethylmaleimide sensitive fusion protein (NSF) was originally identified as a cytosolic factor required for constitutive vesicular transport and later implicated in synaptic vesicle trafficking as well. Our previous work at neuromuscular synapses in the temperature-sensitive NSF mutant, comatose (comt), has shown that the comt gene product, dNSF1, functions after synaptic vesicle docking in the priming of vesicles for fast calcium-triggered fusion. Here we investigate whether dNSF1 performs a similar function at central synapses associated with the well-characterized giant fiber neural pathway. These include a synapse within the giant fiber pathway, made by the peripherally synapsing interneuron (PSI), as well as synapses providing input to the giant fiber pathway. The latency (delay) between stimulation and a resulting muscle action potential was used to assess the function of each class of synapses. Repetitive stimulation of the giant fiber pathway in comt produced wild-type responses at both 20 and 36 degrees C, exhibiting a characteristic and constant latency between stimulation and the muscle response. In contrast, stimulation of presynaptic inputs to the giant fiber (referred to as the "long latency pathway") revealed a striking difference between wild type and comt at 36 degrees C. Repetitive stimulation of the long latency pathway led to a progressive, activity-dependent increase in the response latency in comt, but not in wild type. Thus the giant fiber pathway, including the PSI synapse, appears to function normally in comt, whereas the presynaptic inputs to the giant fiber pathway are disrupted. Several aspects of the progressive latency increase observed in the long latency pathway can be understood in the context of the activity-dependent reduction in neurotransmitter release we observed previously at neuromuscular synapses. These results suggest that repetitive stimulation causes a progressive reduction in neurotransmitter release by presynaptic inputs to the giant fiber neuron, resulting in an increased latency preceding a giant fiber action potential. Thus synapses presynaptic to the giant fiber appear to utilize dNSF1 in a manner similar to the neuromuscular synapse, whereas the PSI chemical synapse may differ with respect to the expression or activity of dNSF1.     

Mechanisms of tonic, graded release: lessons from the vertebrate photoreceptor

The release of neurotransmitter via exocytosis is a highly conserved, fundamental feature of nervous system function. At conventional synapses, neurotransmitter release occurs as a brief burst of exocytosis triggered by an action potential. By contrast, at the first synapse of the vertebrate visual pathway, not only is the calcium-dependent release of neurotransmitter typically graded with respect to the presynaptic membrane potential, but release can be maintained throughout the duration of a sustained stimulus. The specializations that provide for graded and sustained release are not well-defined. However, recent advances in our understanding of basic synaptic vesicle dynamics and the calcium sensitivity of the release process at these and other central, glutamatergic neurons have shed some light on the photoreceptor's extraordinary abilities.     

Giant neuron pathway neurophysiological activity in per(0) mutants of Drosophila melanogaster

In Drosophila melanogaster, the clock gene period (per) has a clearly defined role in the molecular machinery involved in generating free-running circadian rhythms. per mutations also influence rhythms in the Drosophila love song and in the ultradian timescale. The relationship between these two phenomena has so far escaped satisfactory explanation. Here we analyzed the neurophysiological activity of the giant fiber neural pathway in per(0) flies. Under constant light, and at relatively low stimulation frequencies (1-2 Hz), per(01) flies habituate significantly earlier than they do under 12 h light-dark cycles. The results suggest an involvement of per in phenomena of short-term neural plasticity.     

Altered drug resistance and recovery from paralysis in Drosophila melanogaster with a deficient histamine-gated chloride channel

The recent identification and characterization of two genes, encoding histamine-gated chloride channel subunits from Drosophila melanogaster, has confirmed that histamine is a major neurotransmitter in the fruitfly. One of the cloned genes, hclA (synonyms: HisCl-alpha1; HisCl2), corresponds to ort (ora transientless), mutationsin which affect synaptic transmission in the Drosophila visual system. We identified a mutational change (a null mutation) in the genomic and RNA copies of hclA derived from mutants carrying the ort(1) allele. This correlates with new phenotypes observed in the mutant strain. We found hypersensitivity to the avermectin neurotoxins in both the ort(1) adult flies and third instar larvae compared to Oregon R wild-type animals. On the other hand, the mutation makes both male and female adult flies more resistant to treatment with diethyl ether, and the animals show substantially prolonged recovery from paralysis after diethylether anaesthesia, as well as from paralysis after mechanical shock, as revealed by the bang sensitivity test. Altogether, our data give direct evidence that in vivo a HCLA subunit-containing receptor has a distinct role in the neurotoxic action of the avermectins. They also provide new evidence for a function in the response to diethylether anaesthesia and, moreover, that HCLA function is not limited to the visual system.     

ALTERED DRUG RESISTANCE AND RECOVERY FROM PARALYSIS IN DROSOPHILA MELANOGASTER WITH A DEFICIENT HISTAMINE-GATED CHLORIDE CHANNEL

The recent identification and characterization of two genes, encoding histamine-gated chloride channel subunits from Drosophila melanogaster , has confirmed that histamine is a major neurotransmitter in the fruitfly. One of the cloned genes, hclA (synonyms: HisCl - &#102 1 ; HisCl2 ), corresponds to ort ( ora transientless ), mutationsin which affect synaptic transmission in the Drosophila visual system. We identified a mutational change (a null mutation) in the genomic and RNA copies of hclA derived from mutants carrying the ort 1 allele. This correlates with new phenotypes observed in the mutant strain. We found hypersensitivity to the avermectin neurotoxins in both the ort 1 adult flies and third instar larvae compared to Oregon R wild-type animals. On the other hand, the mutation makes both male and female adult flies more resistant to treatment with diethyl ether, and the animals show substantially prolonged recovery from paralysis after diethylether anaesthesia, as well as from paralysis after mechanical shock, as revealed by the bang sensitivity test. Altogether, our data give direct evidence that in vivo a HCLA subunit-containing receptor has a distinct role in the neurotoxic action of the avermectins. They also provide new evidence for a function in the response to diethylether anaesthesia and, moreover, that HCLA function is not limited to the visual system.     

Age affects reciprocal cellular interactions in neuromuscular synapses following peripheral nerve injury

Studies of the influence of age on regeneration and reinnervation in the peripheral nervous system (PNS) and neuromuscular junction (NMJ) are reviewed, with a particular focus on aged and denervated skeletal muscles. The morphological and functional features of incomplete regeneration and reinnervation are compared between adult and aged animals. In addition, some possible mechanisms of the age-related defects will be discussed. Increased fragmentation or damage in individual components of the NMJ (terminal Schwann cells (TSCs), axon terminals and acetylcholine receptor sites occurs during muscle reinnervation following PNS injury in the aged animals. The capacity to produce ultraterminal sprouting or multiple innervation secondary to PNS injury is maintained, but not the capacity to eliminate such anomalous axonal profiles. The frequency and accuracy of reoccupation of the synaptic sites by TSCs and axon terminals are impaired. Thus, despite the capability of extending neural processes, the rate at which regenerating nerve fibers grow, mature and precisely appose the postsynaptic muscle fiber is impaired, resulting in the failure of re-establishment of the normal single motor innervation in the NMJ. A complex set of cellular interactions in the NMJ are known to participate in the neurotrophism and neurotrophism to support growth of the regenerating and sprouting axons and their pathfinding to direct the target muscle fiber. Besides the capability of α-motoneurons, signaling originating from the TSCs and muscle may be impaired during aging.     

Retrograde regulation of motoneuron differentiation by muscle beta-catenin

Synapse formation requires proper interaction between pre- and postsynaptic cells. In anterograde signaling, neurons release factors to guide postsynaptic differentiation. However, less is known about how postsynaptic targets retrogradely regulate presynaptic differentiation or function. We found that muscle-specific conditional knockout of beta-catenin (Ctnnb1, also known as beta-cat) in mice caused both morphologic and functional defects in motoneuron terminals of neuromuscular junctions (NMJs). In the absence of muscle beta-catenin, acetylcholine receptor clusters were increased in size and distributed throughout a wider region. Primary nerve branches were mislocated, whereas secondary or intramuscular nerve branches were elongated and reduced in number. Both spontaneous and evoked neurotransmitter release was reduced at the mutant NMJs. Furthermore, short-term plasticity and calcium sensitivity of neurotransmitter release were compromised in beta-catenin-deficient muscle. In contrast, the NMJ was normal in morphology and function in motoneuron-specific beta-catenin-deficient mice. Taken together, these observations indicate a role for muscle beta-catenin in presynaptic differentiation and function, identifying a previously unknown retrograde signaling in the synapse formation and synaptic plasticity.     

The rdgB gene of Drosophila: a link between vision and olfaction.

ota1 (ota = olfactory trap abnormal), an X-linked mutation of Drosophila isolated by virtue of abnormal olfactory behavior, is shown to be an allele of rdgB (retinal degeneration B), a gene required for normal visual system physiology. rdgB function is shown to be necessary for olfactory response of both adult flies and larvae, which have distinct olfactory systems. Electrophysiological recordings from the adult antenna indicate that rdgB is required for normal response in the peripheral olfactory system: some rdgB mutants show a delayed return to the resting potential following stimulation with ethyl acetate vapor. These results indicate that rdgB is required for both visual and olfactory physiology, and they suggest that visual and olfactory transduction may share at least one common molecular step in Drosophila.     

The central release of acetylcholine during consciousness and after brain lesions

1. Acetylcholine (ACh) has been collected from the visual cortex of anaesthetized rabbits during stimulation of the lateral geniculate body and after cutting central nervous pathways. ACh has also been collected from the visual cortex of conscious, free-moving rabbits.2. After a unilateral ;vertical' lesion separating the geniculate body from more centrally situated nuclei, ACh release evoked from the contralateral cortex by geniculate body stimulation was abolished but evoked release from the ipsilateral cortex was only reduced.3. After a bilateral, ;horizontal' lesion separating the thalamic nuclei from the reticular formation, unilateral geniculate stimulation gave an increased ACh release from the ipsilateral but not from the contralateral visual cortex.4. The ;vertical' and ;horizontal' lesions had no permanent effect on the spontaneous release of ACh from the visual cortex.5. Unilateral destruction of the geniculate body reduced the spontaneous release of ACh from the ipsilateral cortex but did not affect the contralateral release.6. The spontaneous and directly evoked ACh release from chronically undercut areas of cortex was found to be considerably lower than from intact areas of cortex.7. A high output of ACh was obtained from the visual cortex of conscious, free-moving rabbits. The rate of ACh release was closely related to the activity and state of arousal of the animals.8. These results support an earlier suggestion that two major ascending cholinergic systems exist in the rabbit brain. One pathway is the non-specific reticulo-cortical tract responsible for cortical arousal and the other is the more specific thalamo-cortical pathway associated with augmenting and repetitive after-discharge responses. The functional significance of these two cholinergic pathways and their role in the conscious animal are discussed.     

Effect of neurofibromatosis type I mutations on a novel pathway for adenylyl 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 humans, and animal models. We identify a novel growth factor stimulated adenylyl cyclase (AC) pathway in the Drosophila brain, which is disrupted by mutations in the epidermal growth factor receptor (EGFR), neurofibromin (NF1) and Ras, but not Galpha(s). This is the first demonstration in a metazoan that a receptor tyrosine kinase (RTK) pathway, acting independently of the heterotrimeric G-protein subunit Galpha(s), can activate AC. We also show that Galpha(s) is the major Galpha isoform in fly brains, and define a second AC pathway stimulated by serotonin and histamine requiring NF1 and Galpha(s), as well as a third, classical Galpha(s)-dependent AC pathway, which 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, we show that Ras activation by hNF1 is essential for growth factor stimulation of AC activity. Further, we demonstrate that sequences in the C-terminal region of hNF1 are sufficient for NF1/Galpha(s)-dependent neurotransmitter stimulated AC activity, and for rescue of body size defects in Nf1 mutant flies.     

Fiber number in the mushroom bodies of adult Drosophila melanogaster depends on age, sex and experience

The mushroom bodies are two characteristically shaped structures of the insect central brain. In Drosophila melanogaster they contain more fibers in females than in males. Within the first week of adult life the total number of fibers increases by about 15% and decreases again in flies older than 3-4 weeks. The number of mushroom body fibers is significantly reduced in flies kept under social isolation or deprived of their antennal input, but not in flies subjected to visual deprivation.     

Fiber number in the mushroom bodies of adult Drosophila melanogaster depends on age, sex and experience

The mushroom bodies are two characteristically shaped structures of the insect central brain. In Drosophila melanogaster they contain more fibers in females than in males. Within the first week of adult life the total number of fibers increases by about 15% and decreases again in flies older than 3-4 weeks. The number of mushroom body fibers is significantly reduced in flies kept under social isolation or deprived of their antennal input, but not in flies subjected to visual deprivation.     

The central release of acetylcholine during stimulation of the visual pathway

1. In rabbits anaesthetized with Dial ACh has been collected from the surface of the cerebral cortex during stimulation of the visual pathways.2. The spontaneous release of ACh from the visual and non-visual areas of the cortex was found to be similar.3. Stimulation of the retinae by diffuse light produced a large increase in ACh release from the primary visual receiving areas (4.3 times the spontaneous release) and a smaller increase (1.9 times the spontaneous release) from other parts of the cortex.4. Direct unilateral electrical stimulation of the lateral geniculate body evoked a large increase in ACh release (3.4 times the spontaneous release) from the ipsilateral visual cortex and a smaller increase (1.7 times the spontaneous release) from the contralateral visual area and other regions of the cerebral cortex. The evoked increase from the contralateral cortex was not mediated by transcallosal pathways.5. The increase in ACh release evoked from the visual cortex by stimulation of the ipsilateral lateral geniculate body was dependent on the frequency of stimulation. The evoked release was smallest at low stimulus frequencies and increased to a maximum at 20 stimuli/sec.The evoked ACh release from other areas of the cortex was independent of the frequency at which the lateral geniculate body was stimulated.6. The possible central nervous pathways associated with the spontaneous release of ACh and the release evoked by stimulation of the eyes by light and by direct stimulation of the lateral geniculate body are discussed.7. It is concluded that two ascending cholinergic systems may be involved; the non-specific reticulo-cortical pathways responsible for the e.e.g arousal response, and the more specific thalamo-cortical pathways associated with augmenting and repetitive after-discharge responses. The first system is thought to be concerned with the small but widespread increase in ACh release from the cortex following stimulation of the visual pathway while the second system could give rise to the larger increases evoked from the primary receiving areas of cortex. The spontaneous release of ACh from the surface of the brain may be the result of contributions from both systems.     

Abnormalities in neuromuscular junction structure and skeletal muscle function in mice lacking the P2X2 nucleotide receptor

ATP is co-released in significant quantities with acetylcholine from motor neurons at skeletal neuromuscular junctions (NMJ). However, the role of this neurotransmitter in muscle function remains unclear. The P2X2 ion channel receptor subunit is expressed during development of the skeletal NMJ, but not in adult muscle fibers, although it is re-expressed during muscle fiber regeneration. Using mice deficient for the P2X2 receptor subunit for ATP (P2X2(-/-)), we demonstrate a role for purinergic signaling in NMJ development. Whereas control NMJs were characterized by precise apposition of pre-synaptic motor nerve terminals and post-synaptic junctional folds rich in acetylcholine receptors (AChRs), NMJs in P2X2(-/-) mice were disorganized: misapposition of nerve terminals and post-synaptic AChR expression localization was common; the density of post-synaptic junctional folds was reduced; and there was increased end-plate fragmentation. These changes in NMJ structure were associated with muscle fiber atrophy. In addition there was an increase in the proportion of fast type muscle fibers. These findings demonstrate a role for P2X2 receptor-mediated signaling in NMJ formation and suggest that purinergic signaling may play an as yet largely unrecognized part in synapse formation.     

Fiber Number in the Mushroom Bodies of Adult Drosophila melanogaster depends on Age,Sex and Experience

The mushroom bodies are two characteristically shaped structures of the insect central brain. In Drosophila melanogaster they contain more fibers in females than in males. Within the first week of adult life the total number of fibers increases by about 15% and decreases again in flies older than 3-4 weeks. The number of mushroom body fibers is significantly reduced in flies kept under social isolation or deprived of their antennal input, but not in flies subjected to visual deprivation.     

The role of integrins in the modulation of neurotransmitter release from motor nerve terminals by stretch and hypertonicity

Integrins are found at most or all synapses and play a variety of roles. At frog neuromuscular junctions, mechanical tension on integrins due to muscle stretch or hypertonicity causes a powerful modulation of release efficacy. Understanding the mechanism(s) of integrin-mediated modulation will likely further our understanding of mechanisms of neurotransmitter release. The modulation of evoked release with stretch occurs with no detectable delay, does not adapt, and bypasses the Ca(2+) triggering step in vesicle fusion. It depends primarily on integrin bonds to native ligands and requires that one or more proteins in the link between integrins and vesicle fusion be dephosphorylated. Hypertonicity, studied in both frog and Drosophila terminals, causes a larger but slower phasic-tonic change in spontaneous release, which is also Ca(2+)-independent and mostly dependent on integrins, but not dependent on the phosphorylation state of molecules in its pathway of action. In Drosophila, the integrin-dependent component involves the cAMP/PKA pathway, and is absent in mutants lacking PKA. Both stretch and hypertonicity responses in frog terminals are enhanced by agents that elevate PKA levels, suggesting that, in frogs, the cAMP/PKA cascade primarily determines the size of the pool of vesicles available for release by the integrin-mediated mechanism and is not a direct intermediary in the modulation. Evoked release is affected little or even inhibited by hypertonicity. In Drosophila, the inhibition can be explained by a decrease in Ca(2+) influx. The effect of hypertonicity on evoked release in frogs may similarly be a balance between mechanisms that enhance spontaneous release and those that suppress I (Ca).     

FIBER NUMBER IN THE MUSHROOM BODIES OF ADULT DROSOPHILA MELANOGASTER DEPENDS ON AGE,SEX AND EXPERIENCE

The mushroom bodies are two characteristically shaped structures of the insect central brain. In Drosophila melanogaster they contain more fibers in females than in males. Within the first week of adult life the total number of fibers increases by about 15% and decreses again in flies older than 3–4 weeks. The number of mushroom body fibers is significantly reduced in flies kept under social isolation or deprived of their antennal input, but not in flies subjected to visual deprivation.     

Properties of short-term synaptic depression at larval neuromuscular synapses in wild-type and temperature-sensitive paralytic mutants of Drosophila

The larval neuromuscular synapse of Drosophila serves as an important model for genetic and molecular analysis of synaptic development and function. Further functional characterization of this synapse, as well as adult neuromuscular synapses, will greatly enhance the impact of this model system on our understanding of synaptic transmission. Here we describe a form of short-term synaptic depression observed at larval, but not adult, neuromuscular synapses and explore the underlying mechanisms. Larval neuromuscular synapses exhibited a form of short-term depression that was strongly dependent on stimulation frequency over a narrow range of low frequencies (0.1-1 Hz). This form of synaptic depression, referred to here as low-frequency short-term depression (LF-STD), results from an activity-dependent reduction in neurotransmitter release. However, in contrast to the predictions of depletion models, the degree of depression was independent of the initial level of neurotransmitter release over a range of extracellular calcium concentrations. This conclusion was confirmed in two temperature-sensitive (TS) paralytic mutants, cacophony and shibire, which exhibit reduced neurotransmitter release resulting from conditional disruption of presynaptic calcium channels and dynamin, respectively. Higher stimulation frequencies (40 or 60 Hz) produced two components of depression that appeared to include LF-STD as well as a more conventional component of short-term depression. These findings reveal novel properties of short-term synaptic depression and suggest that complementary genetic analysis of larval and adult neuromuscular synapses will further define the in vivo mechanisms of neurotransmitter release and short-term synaptic plasticity.