The Drosophila NSF Protein, dNSF1, Plays a Similar Role at Neuromuscular and Some Central Synapses

Fumiko Kawasaki and Richard W. Ordway

Department of Biology, The Pennsylvania State University, University Park, Pennsylvania 16802


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Kawasaki, Fumiko and Richard W. Ordway. The Drosophila NSF Protein, dNSF1, Plays a Similar Role at Neuromuscular and Some Central Synapses. J. Neurophysiol. 82: 123-130, 1999. 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°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°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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The N-ethylmaleimide sensitive fusion protein (NSF) acts within a protein complex that functions in the docking and fusion of intracellular vesicles with the appropriate target membrane. This complex is thought to form by assembly of membrane proteins of the vesicle and target membranes called SNAREs (soluble NSF attachment protein receptors), followed by recruitment of NSF and the soluble NSF attachment proteins (SNAPs). The ATPase activity of NSF is thought to play a critical role in the structure and function of this complex on the basis of both in vitro and in vivo evidence indicating that ATP hydrolysis by NSF leads to complex disassembly (reviewed in Hanson et al. 1997; Hay and Scheller 1997; Rothman 1994; Rothman and Wieland 1996; Südhof 1995).

Despite substantial in vitro biochemical evidence supporting a role for NSF in synaptic vesicle trafficking, only recently has NSF been shown to play an in vivo role in this process [see Kawasaki et al. (1998) for a recent review of literature relevant to this study]. In part, this progress developed from analysis of a Drosophila NSF mutant, comatose (comt). This mutant was originally identified in a screen for temperature-sensitive paralytic mutants and was shown to exhibit a conditional neurophysiological defect (Siddiqi and Benzer 1976). Our previous work demonstrated that mutations in the dNSF1 gene are responsible for temperature-sensitive paralysis in comt (Ordway et al. 1994; Pallanck et al. 1995a). More recently, our electrophysiological and ultrastructural analysis at neuromuscular synapses in comt has revealed that dNSF1 functions in the priming of docked vesicles for fast calcium-triggered fusion (Kawasaki et al. 1998). Work at the squid giant synapse using injection of NSF peptides has led to some similar conclusions, with some important differences (Schweizer et al. 1998). Although recent experiments suggest that NSF proteins may also function postsynaptically (Lin and Sheng 1998; Nishimune et al. 1998; Osten et al. 1998; Song et al. 1998), our experiments in comt indicate a strictly presynaptic role for dNSF1 (Kawasaki et al. 1998; Kawasaki and Ordway 1999).

Biochemical analysis in comt has also been informative. Recent work by the Pallanck laboratory (Tolar and Pallanck 1998) has characterized neural SNARE complexes in Drosophila and carefully analyzed the status of this complex in comt. These studies show that exposure of comt to restrictive temperature results in accumulation of SNARE complex on the plasma membrane. A different conclusion has been reported elsewhere on the basis of a less exhaustive biochemical analysis in comt (Littleton et al. 1998).

Taken together, the electrophysiological, ultrastructural, and biochemical analyses in comt lead to the conclusion that the priming of docked vesicles relies on dNSF1-mediated disassembly of plasma membrane SNARE complexes. Models of NSF function incorporating these observations are presented elsewhere (Kawasaki et al. 1998; Tolar and Pallanck 1998).

Despite this progress, a number of unresolved issues remain. For example, early models of NSF function have proposed that it is a general purpose protein, perhaps a single protein performing the same function in membrane trafficking events throughout the organism. However, previous work has demonstrated the existence of two closely related NSF genes in Drosophila, dNSF1 and dNSF2, as well as multiple transcripts derived from the dNSF2 gene (Pallanck et al. 1995b). This diversity raises the question of whether specific NSF gene products may perform specialized functions within the organism. Thus our recent progress defining the synaptic function of a specific NSF gene product, dNSF1, provides an opportunity to address whether this single NSF protein performs an analogous function at all synapses. Here we utilize comt mutations and the well-characterized giant fiber neural pathway in Drosophila to compare the function of dNSF1 at different chemical synapses. Our results show that dNSF1 appears to function in a similar manner at neuromuscular and some central synapses, and that one synapse may differ with respect to the function or expression of dNSF1.

Some of this work has been communicated in preliminary form (Kawasaki and Ordway 1997, 1998).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Dissection

Male or female flies 1-4 days of age were anesthetized using CO2 and then mounted over a hole in an air tube and secured with wax as described (Koenig et al. 1989). Air was delivered to the tracheal system using an aquarium pump. The fly was mounted laterally and dissected in recording solution (see Microelectrode recordings) to expose the lateral surface of one set of the dorsal longitudinal flight muscles (DLMs) as well as the thoracic ganglion of the CNS. For simultaneous recordings from the DLM and tergotrochanter muscle (TTM), TTMs were exposed and the first dorsoventral muscles were removed to expose the anterior part of the DLM. Temperature was maintained at 20°C during dissection.

Brain stimulation

The head of the fly was twisted and mounted ventral side up. The membrane between the prementum and the anterior tentorial pit was cut along the midline to make an approach for a stimulation electrode to the head capsule. The glass stimulation electrode filled with recording solution was inserted through the incision and advanced between the frontal tracheal sacs to the brain. The tip diameter of the stimulation electrode was ~80 µm. The duration of the stimulus was 0.2 ms. Stimulation voltages used to elicit the long latency and giant fiber pathway responses were typically 10-15 and 90-100 V, respectively.

DLM motor axon stimulation

The posterior dorsal mesothoracic nerve (PDMN), through which the DLM motor axons project from the thoracic ganglion to the muscle, was cut and pulled into a suction electrode for stimulation.

Temperature control

Temperature control was achieved using a Medical Systems TC-202 temperature controller and PDMI micro-incubator (Medical Systems, Greenvale, NY). Temperature shifts from 20 to 36°C typically required ~4.5 min.

Microelectrode recordings

Microelectrode recordings were carried out by conventional methods with the use of a Dagan LX2-700 amplifier (Dagan, Minneapolis, MN) and glass microelectrodes filled with 3 M KCI (~30 MOmega ). The recording solution consisted of (in mM) 128 NaCl, 2 KCI, 4 MgCl2, 1.8 CaC12, 5 HEPES, and 36 sucrose. The pH was adjusted to 7.0 using NaOH. During recording, the 3-ml preparation chamber was perfused at 0.5 ml/min.

Electrophysiological data acquisition and analysis

Data were acquired on-line using a Power Macintosh 7500 computer, Pulse software (HEKA elektronik, Lambrecht, Germany), and an Instrutech ITC-16 laboratory interface (Instrutech, Great Neck, NY). Data were low-pass filtered at 3 kHz and acquired at 10 or 20 kHz. Stimulation was achieved using the Pulse program to trigger a Dagan S-900/S-910 stimulator (Dagan, Minneapolis, MN). Latency measurements were carried out using cursor measurements in the Pulse program. Microsoft Excel was utilized for data tabulation and statistical analysis. With the use of an unpaired Student's t-test, statistical significance was assigned to comparisons with P <=  0.05.

Drosophila lines and transformation rescue

All Drosophila lines used were cultured at 20°C. The three temperature-sensitive comt alleles used in this study contain point mutations leading to single amino acid changes. Missense mutations in comtST17 and comtST53 have been reported previously (Pallanck et al. 1995a); comtTP7 contains a missense mutation converting proline 398 to serine. Rescue experiments utilized a transgene construct containing a wild-type dNSF1 cDNA under the control of an hsp70 heat shock promoter (Pallanck et al. 1995a). For rescue experiments, comt flies bearing a transgene were subjected to three heat shocks separated by 24-h intervals (all heat shocks were at 38°C). A 15-min heat shock on the first day was followed by 30-min heat shocks on the second and third days. Recordings were obtained ~24 h after the last heat shock. In the case of the comtST53 rescue experiments, the third heat shock was omitted. As is the case for temperature-sensitive paralytic behavior (Pallanck et al. 1995a), controls in which comt flies lacking the transgene were heat shocked demonstrated that phenotypic rescue was entirely dependent on the presence of the transgene.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Giant fiber and long latency pathways

The morphology and physiology of the giant fiber pathway was originally defined in the pioneering work of the Wyman (King and Wyman 1980; Sun and Wyman 1997; Tanouye and Wyman 1980) and Ikeda (Ikeda and Koenig 1988; Ikeda et al. 1980) laboratories and has been subjected to further detailed characterization in a number of other studies (e.g., see Bacon and Strausfeld 1986; Engel and Wu 1996; Gorczyca and Hall 1984; Phelan et al. 1996; Trimarchi and Schneiderman 1995). As shown schematically in Fig. 1C (see legend), the giant fiber axon projects from the brain to the thoracic ganglion, where it forms an electrical synapse with a peripherally synapsing interneuron (PSI). The PSI forms a chemical synapse on each motor neuron innervating the DLMs, and this represents the only chemical synapse within the giant fiber pathway to the DLM motor neuron. The giant fiber neuron also makes an electrical synapse on the TTM motor neuron. Elements of the giant fiber pathway to be discussed here include the giant fiber neuron, the PSI, and the DLM and TTM motor neurons.



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Fig. 1. Giant fiber and long latency pathways. A: recordings of dorsal longitudinal flight muscle (DLM) postsynaptic potentials elicited by brain stimulation of the giant fiber and long latency pathways (indicated in gray and black, respectively) in a wild-type fly. Fifteen traces were superimposed for each type of response. B: long latency response results from stimulation of presynaptic inputs to the giant fiber pathway. Simultaneous recordings from the DLM and tergotrochanter muscle (TTM) showing the response to stimulation of the long latency pathway. The long latency response in the DLM occurred 0.4 ms after the TTM potential. C: a highly simplified schematic representation of important connections within the giant fiber pathway (after Tanouye and Wyman 1980). The actual morphology of the entire pathway has been clearly presented in detail (Ikeda and Koenig 1988; King and Wyman 1980; Sun and Wyman 1997). Chemical synapses are represented as triangles, electrical synapses are represented as small rectangles. PSI Interneuron, the peripherally synapsing interneuron.

Excitation of the giant fiber pathway can be elicited by electrical stimulation of the brain and monitored by intracellular recording of postsynaptic action potentials in the DLMs. By varying the stimulus intensity, the giant fiber neuron may be stimulated directly or, alternatively, indirectly through stimulation of its presynaptic inputs. Higher stimulus intensities activate the giant fiber neuron directly and thus produce a DLM potential through subsequent activation of the PSI and the DLM motor neuron. This giant fiber response indicated in gray in Fig. 1A exhibits a characteristic latency between brain stimulation and the DLM potential (~1.2 ms) as described previously (Tanouye and Wyman 1980). As discussed below, lower stimulus intensities activate the giant fiber neuron indirectly through its presynaptic inputs; this long latency response exhibits a latency of ~2.0 ms and is indicated in black in Fig. 1A.

A previously described long latency response (Tanouye and Wyman 1980) exhibiting a latency of ~2.0 ms was attributed to stimulation of presynaptic inputs to the giant fiber pathway on the following basis. After activation of the giant fiber neuron, action potentials appear in both the DLM and TTM. Because the pathway to the DLM includes an additional chemical synapse, the DLM potential occurs ~0.4 ms after the TTM potential (Tanouye and Wyman 1980). Thus this difference in TTM and DLM response latencies serves as a signature of the giant fiber pathway. To confirm that we observe the same long latency response previously described, we examined whether this response results from activation of inputs to the giant fiber pathway. Simultaneous DLM and TTM recordings were performed using lower intensity stimulation to produce long latency responses. Both DLM and TTM potentials were elicited with the 0.4-ms difference in latencies characteristic of the giant fiber pathway (Fig. 1B). Thus we define long latency responses as those elicited by stimulation of presynaptic inputs to the giant fiber pathway.

comt exhibits a conditional neurophysiological defect presynaptic to the giant fiber pathway

Our previous work at neuromuscular synapses in comt has defined a role for dNSF1 in the priming of docked synaptic vesicles for fast calcium-triggered fusion (Kawasaki et al. 1998; Kawasaki and Ordway 1999). These experiments utilized direct stimulation of the motor axon, together with microelectrode and voltage-clamp recordings, to demonstrate a progressive, activity-dependent reduction in neurotransmitter release at restrictive temperature. In microelectrode recordings from the DLM, the reduction in neurotransmitter release is seen as a graded decrease and then loss of the DLM action potential, revealing the underlying postsynaptic potential, which continues to decrease in amplitude. This reduction was dependent on the stimulation frequency. comtST17 was most sensitive, exhibiting a marked reduction in response to 1-Hz stimulation. In comtST53 and comtTP7, 10-Hz, but not 1-Hz, stimulation markedly reduced neurotransmitter release. Thus, as shown in Fig. 2 (see caption), when giant fiber or long latency responses are elicited at 1 Hz in comtTP7 or comtST53, the DLM action potential is not markedly reduced.

Giant fiber and long latency responses were examined in wild-type and comt flies at both permissive temperature (20°C) and a restrictive temperature (36°C) at which comt is paralyzed (Fig. 2). In wild-type and comt flies at 20°C, the latencies of the giant fiber and long latency responses were constant for a 1-Hz train of stimuli (Fig. 2, A and D). Also in comt flies at 36°C, the giant fiber pathway exhibited wild-type latencies (Fig. 2, B and E). In contrast, stimulation of the long latency pathway under the same conditions produced responses with increased and variable latencies, ranging from wild-type to very long latency responses (Fig. 2, B and E). These responses were attributed to the long latency pathway on the basis of the following three observations. 1) They were elicited with the same characteristic stimulation threshold observed for long latency responses in wild type and in comt at 20°C (the giant fiber pathway also exhibits the same stimulation threshold in comt and wild type). 2) The shortest latencies observed were very similar to wild type. 3) Longer latency responses always appeared in place of, rather than in addition to, responses exhibiting wild-type latencies. The increase and variation in latency were reversible, recovering over ~20 min after the preparation was returned to 20°C (Fig. 2F).



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Fig. 2. Conditional neurophysiological defect in the long latency pathway of comt. Recordings of the giant fiber and long latency responses (indicated in gray and black, respectively). Each panel shows superimposed traces during a 1-Hz stimulation train. The stimulation trains at restrictive temperature were initiated after 5 min at 36°C in both wild type (WT) and comtTP7. Recovery of comtTP7 is shown 21 min after return to 20°C. For both WT and comtTP7, the data shown were obtained from the same preparation. As discussed below, spontaneous neural activity is present in comt at 36°C and may contribute to the slight reduction in the amplitude of the DLM action potential observed in E.

The latency increase and variation seen in the long latency response of comt contrasts the constant latency responses observed in the giant fiber pathway under the same conditions. As described earlier, both response pathways include activation of the PSI chemical synapse with the DLM motor neuron, whereas only the long latency response includes activation of chemical synapses presynaptic to the giant fiber pathway. Thus under these conditions, the chemical synapse between the PSI and the DLM motor neuron appears to be functioning normally, indicating that the observed latency changes occur in the presynaptic inputs to the giant fiber pathway. As described below, the persistence of apparently wild-type neurotransmission at PSI chemical synapses in comt was also observed at higher stimulation frequencies.

Loss of dNSF1 function is responsible for increased latencies in the long latency pathway

The same conditional neurophysiological defect observed in the long latency pathway of comtTP7 was also observed in two additional comt alleles, comtST17 and comtST53. A comparison of latency values in comtTP7, comtST53, and wild type are shown in Table 1. The similarity of results obtained in multiple comt alleles indicates that this phenotype results from a general loss of dNSF1 function rather than from any specific amino acid substitution in the dNSF1 protein. To further confirm that the comt mutations in dNSF1 are responsible for the latency changes, long latency responses were recorded from flies in which a transgene expressing the wild-type dNSF1 protein was introduced into a comt mutant background. Expression of wild type dNSF1 in a comtTP7 genetic background dramatically rescued both the temperature-sensitive paralytic behavior and the latency increases (Fig. 3 and Table 1). Although not shown, the behavioral and neurophysiological phenotypes in comtST17 and comtST53 were also completely rescued. These results indicate that the synaptic defect presynaptic to the giant fiber pathway results entirely from mutations in the dNSF1 gene.


                              
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Table 1. Response latencies during 1-Hz stimulation of the long latency pathway



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Fig. 3. Transformation rescue of latency increases and temperature-sensitive paralysis. A: long latency responses recorded from a comtTP7 fly and a transgenic fly expressing wild-type dNSF1 protein in a comtTP7 mutant background (comtTP7 HS dNSF1). Stimulation trains (1 Hz) were initiated after 5 and 6 min at 36°C in comtTP7 and comtTP7 HS dNSF1, respectively. B: time required for paralysis at 38°C. Each bar represents the mean time (±SE) for 50% of the flies to become paralyzed. In each case 6 trials were performed using 6 flies for each trial. The behavioral tests for comtTP7 HS dNSF1 and WT flies were terminated at 50 min.

Temperature-induced spontaneous neural activity in comt

Virtually no spontaneous DLM action potentials were observed at 20°C in either comt or wild type. At 36°C, comt flies exhibited a markedly elevated frequency of spontaneous DLM action potentials, whereas spontaneous activity in wild type was only slightly elevated (Fig. 4). Because this spontaneous activity was eliminated by cutting the DLM motor axon, it clearly represents an increase in neural activity. The spontaneous activity in comt often occurred as periodic high-frequency bursts of action potentials separated by intervals of lower frequency activity (Fig. 4). During this spontaneous activity, the amplitude of the DLM action potential was reduced during a burst and gradually recovered between bursts. This observation is consistent with two previous results obtained using direct stimulation of the DLM motor axon (Kawasaki et al. 1998). First, an activity-dependent reduction in neurotransmitter release at DLM neuromuscular synapses was observed during repetitive stimulation at restrictive temperature. Second, this reduction in neurotransmitter release was shown to recover at restrictive temperature in the absence of repetitive stimulation.



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Fig. 4. Increased spontaneous neural activity at restrictive temperatures in comt. Continuous recordings from the DLM in WT and comtST53 were obtained after 5 and 2 min at 36°C, respectively.

In the experiment in Fig. 2E at restrictive temperature in comtTP7, a typical level of spontaneous neural activity was present and may have contributed to the observed reduction in action potential amplitude (compare Fig. 2, B and E). The frequency of spontaneous activity observed in this experiment was ~2.6 Hz, at which we estimate that <3% of traces contain a spontaneous event. Thus >97% (perhaps all) of the DLM potentials shown in Fig. 2E are evoked. Consistent with this, the responses are not randomly distributed. Each trace contains a single event, no events occur before stimulation, and the responses are clustered near the latency typically observed in the long latency pathway of wild type.

Importantly, the temperature-induced spontaneous activity in comt (as well as the small increase observed in wild type) recovered immediately on returning the preparation to 20°C, and thus latency values taken during recovery (as shown in Table 1) were recorded in the absence of spontaneous activity. As discussed below, this rapid recovery was further utilized to examine latency changes in the absence of spontaneous activity. Finally, the temperature-induced spontaneous activity in comt was rescued by transgenic expression of the wild-type dNSF1 protein.

Reduction in postsynaptic potential amplitude in comt delays action potential firing at central and peripheral synapses

Although no obvious pattern was observed in the latency increases occurring in the long latency pathway of comt, any such pattern may have been obscured by the effects of temperature-induced increases in spontaneous activity. To address this issue, response latencies were recorded in comt under conditions in which spontaneous DLM potentials are greatly reduced in frequency or absent. As mentioned earlier, the increased spontaneous activity observed at restrictive temperature in comt recovers immediately on return to permissive temperature. In contrast, the latency changes observed in the long latency pathway require >20 min for recovery. Thus long latency responses were recorded in the absence of spontaneous activity at 20°C during recovery from exposure to restrictive temperature. These experiments revealed a striking pattern in the latency changes: a progressive, activity-dependent increase in the response latency during the stimulus train (Fig. 5A and Table 1).



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Fig. 5. Reduction in postsynaptic potential amplitude in comt delays action potential firing at central and peripheral synapses. A: central synapse phenotype. Long latency responses recorded from comtST53 after 15 min at 20°C during recovery from a 10 min exposure to 36°C. During this recording no spontaneous activity was observed. Every 3rd trace is shown from 1st 20 responses in a 1-Hz stimulus train. B: peripheral synapse phenotype. Postsynaptic potentials elicited by direct stimulation of the DLM motor axon in comtST53. A 10-Hz stimulation train was initiated after 15 min at 20°C during recovery from a 6-min exposure to 38°C. The delay in the DLM action potential is more striking than reported previously at restrictive temperature (Kawasaki et al. 1998) because of the lower recording temperature in this experiment. Representative traces from the 1st 30 responses are superimposed. The final trace shows failure to trigger the DLM action potential.

To compare central and neuromuscular synapses under similar conditions, we also directly stimulated the DLM motor axon at 20°C during recovery from exposure to restrictive temperature. As expected from our previous results in comtST53 (Kawasaki et al. 1998), stimulation of the motor axon at 10 Hz, but not 1 Hz, caused a progressive reduction of the DLM action potential and the underlying postsynaptic potential. As seen in Fig. 5B, this progressive decrease in the postsynaptic potential delayed firing of the muscle action potential, resulting in a progressive increase in the latency between motor axon stimulation and the DLM action potential. Thus the progressive increase in latency observed in the long latency pathway of comt (Fig. 5A) is precisely what would be predicted for an activity-dependent decrease in neurotransmitter release at presynaptic inputs to the giant fiber pathway. These observations indicate that both the DLM neuromuscular synapse and the presynaptic inputs to the giant fiber neuron exhibit a progressive, activity-dependent decrease in the postsynaptic potential, resulting in a progressive increase in the delay preceding the postsynaptic action potential. A further similarity between the behavior of these synapses is that the latency increase observed in the long latency pathway, like the activity-dependent reduction in neurotransmitter release at the DLM neuromuscular synapse (Kawasaki et al. 1998), recovers at restrictive temperature in the absence of stimulation. Thus synapses presynaptic to the giant fiber pathway appear to utilize dNSF1 as defined at the DLM neuromuscular synapse, in the priming of docked vesicles for fast calcium-triggered fusion.

PSI chemical synapse appears to function normally in comt

As discussed above, 1-Hz stimulation of the long latency pathway at restrictive temperature in either comtTP7 or comtST53 appears to produce an activity-dependent reduction in neurotransmitter release at chemical synapses presynaptic to the giant fiber neuron. In contrast, 1-Hz stimulation of the DLM motor axon in these alleles does not markedly reduce neurotransmitter release at the neuromuscular synapse under the same conditions (Kawasaki et al. 1998). Similarly, the PSI chemical synapse appears to function normally during 1-Hz stimulation at restrictive temperature in comt, as indicated by the constant latency observed when the giant fiber pathway is stimulated directly (Fig. 2E). However, it is not clear from these observations whether the PSI chemical synapse, like the DLM neuromuscular synapse, might exhibit an activity-dependent phenotype at higher stimulation frequencies. Thus we examined whether conditions that strongly reduce neurotransmitter release at the DLM neuromuscular synapse might also perturb the function of the PSI chemical synapse.

In the most severe temperature-sensitive comt allele, comtST17, 1-Hz stimulation of the DLM motor axon at restrictive temperature elicits a progressive and marked reduction in neurotransmitter release (Kawasaki et al. 1998). Recovery from exposure to restrictive temperature is also slower in comtST17, requiring up to 1 h for full recovery at 20°C. To examine the function of the PSI chemical synapse under conditions that severely affect neurotransmitter release at the DLM neuromuscular synapse, 10-Hz stimulation of the giant fiber pathway was performed in comtST17 during recovery from exposure to restrictive temperature. These conditions produce a striking reduction in neurotransmitter release at the DLM neuromuscular synapse, and also eliminate spontaneous neural activity. Stimulation of the giant fiber pathway in comtST17 under these conditions (Fig. 6) caused a progressive reduction in the DLM postsynaptic potential, resulting in an increase in the latency between initiation of the postsynaptic potential and the muscle action potential (as also shown for motor axon stimulation in Fig. 5B). However, despite the strong phenotype observed at the neuromuscular synapse, a constant latency was observed between giant fiber stimulation and initiation of the DLM postsynaptic potential in the same experiment, suggesting that the PSI chemical synapse functions normally under these conditions.



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Fig. 6. PSI synapses on the DLM motor neurons appear to function normally in comt. Giant fiber responses recorded from comtST17 after 35 min at 20°C during recovery from a 10-min exposure to 36°C. Representative traces from the 1st 100 responses are shown during a 10-Hz stimulation train. Note the constant latency between giant fiber stimulation and initiation of the DLM postsynaptic potential. Similar results were obtained in 4 experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The work presented here extends our earlier investigation of dNSF1 function at DLM neuromuscular synapses by analyzing the role of dNSF1 at chemical synapses associated with the giant fiber neural pathway. These experiments indicate that chemical synapses presynaptic to the giant fiber neuron utilize dNSF1 as described for the DLM neuromuscular synapse, in the priming of docked synaptic vesicles for fast calcium-triggered exocytosis (Fig. 7).



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Fig. 7. Model of dNSF1 function in the giant fiber pathway to the DLM.

One important observation is that the long latency pathway in comt exhibits a progressive, activity-dependent increase in response latency during repetitive stimulation at restrictive temperature. This increase in latency occurs in the presynaptic inputs to the giant fiber neuron and can be understood in the context of our previous observations at the DLM neuromuscular synapse. We suggest that a progressive, activity-dependent reduction in neurotransmitter release at presynaptic inputs to the giant fiber neuron results in a reduction in the postsynaptic potential and a progressive delay in the latency preceding the giant fiber neuron action potential.

The schematic diagrams of the giant fiber pathway shown here are highly simplistic representations of a much more complex pathway. In fact, the electrophysiological characteristics of the long latency pathway and the latency increases observed in comt raise the question of whether the long latency pathway includes only a single chemical synapse presynaptic to the giant fiber neuron. Although this issue remains unresolved, the electrophysiological observations allow some speculation. The difference between the response latency observed for the giant fiber and long latency pathways defines the latency between brain stimulation and activation of the giant fiber neuron. These values typically fall within the range of 0.6-0.9 ms. By comparison with the 0.4-ms time delay introduced by the PSI chemical synapse, these observations suggest that the long latency pathway may include two chemical synapses presynaptic to the giant fiber neuron. Such a structure would be consistent with the most extreme latencies seen in the long latency pathway of comt, which typically exceed those observed at the DLM neuromuscular synapse between initiation of the postsynaptic potential and the action potential.

A second interesting observation is that comt exhibits a marked temperature-sensitive increase in spontaneous neural activity. While the mechanisms responsible for this phenotype remain unclear, one possibility is that it represents an enhancement of the small temperature-induced increase observed in wild type. In this case, inhibitory synapses might limit the level of activity observed at elevated temperatures in wild type, and impairment of the these synapses in comt might produce a large increase in spontaneous activity. In this case the rapid recovery of this phenotype at 20°C in comt might reflect the same process responsible for rapid recovery in wild type, rather than the time course for recovery of dNSF1 activity at inhibitory synapses. Together with the observed activity-dependent decrease in neurotransmitter release, this increased spontaneous activity may be an important factor contributing to paralysis. As suggested by Fig. 4, spontaneous bursts of action potentials may lead to a reduction in the readily releasable pool of vesicles and compromise synaptic transmission. Thus the kinetics of paralysis in comt probably do not reflect the mechanism of NSF function alone, but rather result from complex interactions within the nervous system.

A final important observation is that direct stimulation of the giant fiber pathway elicits a DLM postsynaptic potential with wild-type latency, even under conditions that produce a marked reduction in neurotransmitter release at both the neuromuscular synapse and presynaptic inputs to the giant fiber pathway. We cannot conclude from this experiment that the PSI synapse does not use dNSF1 in synaptic vesicle trafficking; however, the extreme resistance of the PSI to activity-dependent latency changes suggests this as one possibility. Alternatively, the activity of dNSF1 may differ at this synapse, such that it plays a less critical role in priming.

Diversity in NSF function is suggested by the identification of a second Drosophila NSF gene, dNSF2, which exhibits 84% amino acid identity to dNSF1 (Boulianne and Trimble 1995; Pallanck et al. 1995b). At present, it is unclear whether these NSFs operate completely independently or whether both dNSF1 and dNSF2 may function at the same synapse. We have observed that the activity-dependent reduction in neurotransmitter release at both the presynaptic inputs to the giant fiber and the DLM neuromuscular synapse recovers at restrictive temperature in the absence of stimulation. Thus it appears that a residual vesicle priming process occurs at restrictive temperature. Whether this priming represents residual dNSF1 activity, dNSF2 activity, or priming by another mechanism remains unresolved; however, at present we favor the idea that a dNSF1-independent priming process is responsible (Kawasaki et al. 1998). Localization of the dNSF1 and dNSF2 proteins will begin to clarify whether synapses may differ in their complement of NSF proteins, and whether these differences may lead to diversity in synaptic mechanisms.


    ACKNOWLEDGMENTS

This work was supported by National Science Foundation Grant IBN-9514485.


    FOOTNOTES

Address for reprint requests: R. W. Ordway, Dept. of Biology, The Pennsylvania State University, University Park, PA 16802.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 21 January 1999; accepted in final form 10 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society