Delayed synaptic transmission in Drosophila cacophonynull embryos

Ca(2+) influx through the Drosophila N-type Ca(2+) channel, encoded by cacophony (cac), triggers fast synaptic transmission. We now ask whether the cac Ca(2+) channel is the Ca(2+) channel solely dedicated for fast synaptic transmission. Because the cac(null) mutation is lethal, we used cac(null) embryos to address this question. At the neuromuscular junction in HL3 solution, no fast synchronous synaptic transmission was detected on nerve stimulation. When the wild-type cac gene was introduced in the cac(null) background, fast synaptic transmission recovered. However, even in cac(null) embryos, nerve stimulation infrequently induced delayed synaptic events in the minority of cells in 1.5 mM [Ca(2+)](e) and in the majority of cells in 5 mM [Ca(2+)](e). The number of delayed quantal events per stimulus was greater in 5 mM [Ca(2+)](e) than in 1.5 mM. Thus the delayed release is [Ca(2+)](e) dependent. Plectreurys toxin II (PLTXII) (10 nM; a spider toxin analog) depressed the frequency of delayed events, suggesting that voltage-gated Ca(2+) channels, other than cac Ca(2+) channels, are contributing to them. However, delayed events were not affected by 50 microM La(3+). The frequency of miniature synaptic currents in cac(null) embryos was approximately 1/2 of control, whereas in high K(+) solutions, it was approximately 1/135. The hypertonicity response was approximately 1/10 of control. These findings indicate that the number of release-ready vesicles is smaller in cac(null) embryos. Taken together, the cac Ca(2+) channel is indispensable for fast synaptic transmission in normal conditions, and another type of Ca(2+) channel, the non-cac, PLTXII-sensitive Ca(2+) channel, is contributing to delayed release in cac(null) embryos.     

Altered synaptic transmission in Drosophila hyperkinetic mutants

Synaptic transmission in Drosophila can be altered by mutations in specific genes. For example, mutations in the Shaker (Sh) gene, which encodes the rapidly inactivating A-type potassium channel, cause repetitive nerve firing and prolonged transmitter release at the neuromuscular junction. Here we show that mutations in the Hyperkinetic (Hk) gene also affect the properties of synaptic transmission at the neuromuscular junction. In particular, we find that whereas single or low frequency nerve stimulation evokes a wild type postsynaptic response, at higher frequencies of nerve stimulation, each stimulus results in repetitive nerve firing and increased postsynaptic response, which is similar to that observed in Sh mutants. Various experiments suggest that this increased postsynaptic response results from prolonged depolarization of the nerve terminal, leading to increased transmitter release at the neuromuscular junction. The similarity in phenotypes between Sh and Hk mutants, along with the observation that Sh is epistatic to Hk in its effects on synaptic transmission, suggest that Hk acts on synaptic transmission by an effect on A-type potassium channels.     

Optical quantal analysis of synaptic transmission in wild-type and rab3-mutant Drosophila motor axons

Synaptic transmission from a neuron to its target cells occurs via neurotransmitter release from dozens to thousands of presynaptic release sites whose strength and plasticity can vary considerably. We report an in vivo imaging method that monitors real-time synaptic transmission simultaneously at many release sites with quantal resolution. We applied this method to the model glutamatergic system of the Drosophila melanogaster larval neuromuscular junction. We find that, under basal conditions, about half of release sites have a very low release probability, but these are interspersed with sites with as much as a 50-fold higher probability. Paired-pulse stimulation depresses high-probability sites, facilitates low-probability sites, and recruits previously silent sites. Mutation of the small GTPase Rab3 substantially increases release probability but still leaves about half of the sites silent. Our findings suggest that basal synaptic strength and short-term plasticity are regulated at the level of release probability at individual sites.     

Quantal synaptic transmission in phrenic motor nucleus.

1. The quantal nature of excitatory synaptic transmission was studied in respiratory interneurons and phrenic motoneurons of intact neonatal rat brain stem-spinal cord preparations in vitro. Synaptic currents were recorded with whole-cell patch-clamp recording techniques. 2. Because the most important factor for quantal detection is the ratio of quantal size to quantal standard deviation, factors that influence this ratio were evaluated so that experimental techniques that enhance this ratio could be defined. 3. Under favorable conditions, we directly observed quantal amplitude fluctuations in spontaneous excitatory postsynaptic currents (EPSCs) in spinal cord respiratory neurons. The quantal conductance size was 55-100 pS. With fast decay of these EPSCs, the charge reaching the soma for a single quantum is only approximately 15 fC (Vh = -80 mV). 4. We also studied miniature EPSC amplitude distributions. These were skewed, as previously reported; however, distinct quantal intervals were observed. Furthermore, in three cells tested, the quantal size in the miniature EPSC amplitude distribution was similar to the quantal size in the spontaneous EPSC amplitude distribution. 5. We conclude that excitatory synaptic transmission in the mammalian spinal cord is quantal and that the apparent skewness of miniature EPSC distributions results from summation of events with multiple quantal peak amplitudes.     

Development of Drosophila larval neuromuscular junctions: maintaining synaptic strength

In spite of the available information about the development of Drosophila neuromuscular junctions, the correlation between nerve terminal morphology and maintenance of synaptic strength has still not been systematically addressed throughout larval development. We characterized the growth of the abdominal longitudinal muscle 6 (m6) and the motor terminals Ib and Is that innervate it within segment 4. In addition, we measured the evoked excitatory junction potential (EJP) amplitudes while the Ib and Is axons were selectively recruited. Regression analysis with natural log transformation of response variables indicated that the developmental curves for m6 and the motor axons Ib and Is were best fitted as second order polynomial regressions during larval development. Initially Is terminals are longer and possess more synaptic varicosities at the first instar stage. The Is terminals also grow faster in subsequent developmental stages. The growth of nerve terminals and their target m6 are not proportional although tightly correlated. This results in a larger average muscle area innervated by a single varicosity as the animal develops. The amplitudes of the EJPs of Ib and Is neurons show no developmental difference in their amplitudes from the first to the late third larval instar. The Is axon consistently produced larger EJPs than the Ib axon at each developmental stage. The time constants for both rising and decay phases of EJPs increase exponentially throughout larval development.The results presented not only help in quantifying the normal development of Drosophila neuromuscular junctions, but also provide a framework for future investigations to properly interpret developmental abnormalities that may occur in various mutants.     

Boutons originating from the same axon do not participate equally in synaptic transmission

Morphological changes in the synaptic boutons located on the ciliary neurons were observed following stimulation of the Edinger-Westphal nucleus (EWn). A reduction in the number of clear and dense core vesicles (DCVs) per unit area and an increased surface density of vacuoles were observed in boutons located proximal to the afferent axon. Only a slight reduction in the number of DCVs per unit area was observed on boutons located distally to the afferent axon. Several mechanisms are proposed to explain why all boutons do not participate equally in synaptic transmission.     

Drosophila UNC-13 is essential for synaptic transmission.

The UNC-13 protein family has been suggested to be critical for synaptic vesicle dynamics based on its interactions with Syntaxin, Munc-18 and Doc 2alpha. We cloned the Drosophila homolog (Dunc-13) and characterized its function using a combination of electrophysiology and ultrastructural analyses. Dunc-13 contained a C1 lipid-binding motif and two C2 calcium-binding domains, and its expression was restricted to neurons. Elimination of dunc-13 expression abolished synaptic transmission, an effect comparable only to removal of the core complex proteins Syntaxin and Synaptobrevin. Transmitter release remained impaired under elevated calcium influx or application of hyperosmotic saline. Ultrastructurally, mutant terminals accumulated docked vesicles at presynaptic release sites. We conclude that Dunc-13 is essential for a stage of neurotransmission following vesicle docking and before fusion.     

Electrophysiological analysis of synaptic transmission in central neurons of Drosophila larvae

We report functional neuronal and synaptic transmission properties in Drosophila CNS neurons. Whole cell current- and voltage-clamp recordings were made from dorsally positioned neurons in the larval ventral nerve cord. Comparison of neuronal Green Fluorescent Protein markers and intracellular dye labeling revealed that recorded cells consisted primarily of identified motor neurons. Neurons had resting potentials of -50 to -60 mV and fired repetitive action potentials (APs) in response to depolarizing current injection. Acetylcholine application elicited large excitatory responses and AP bursts that were reversibly blocked by the nicotinic receptor antagonist D-tubocurarine (dtC). GABA and glutamate application elicited similar inhibitory responses that reversed near normal resting potential and were reversibly blocked by the chloride channel blocker picrotoxin. Multiple types of endogenous synaptically driven activity were present in most neurons, including fast spontaneous synaptic events resembling unitary excitatory postsynaptic currents (EPSCs) and sustained excitatory currents and potentials. Sustained forms of endogenous activity ranged in amplitude from smaller subthreshold "intermediate" sustained events to large "rhythmic" events that supported bursts of APs. Electrical stimulation of peripheral nerves or focal stimulation of the neuropil evoked sustained responses and fast EPSCs similar to endogenous events. Endogenous activity and evoked responses required external Ca(2+) and were reversibly blocked by dtC application, indicating that cholinergic synaptic transmission directly underlies observed activity. Synaptic current amplitude and frequency were reduced in shibire conditional dynamin mutants and increased in dunce cAMP phosphodiesterase mutants. These results complement and advance those of recent functional studies in Drosophila embryonic neurons and demonstrate the feasibility of in-depth synaptic transmission and plasticity studies in the Drosophila CNS.     

Conditioned changes of synaptic transmission in the motor cortex of the cat

Intracellular recordings were made from 117 neurons in the motor cortex of anesthetized cats. The pyramidal tract (PT) and VL nucleus of thalamus were stimulated in order to activate the neurons from two directions. 1. PT cells were conditioned by antidromic trains (10--50 cps for 4--15 s) and by paired PT and VL stimuli with different intervals and sequences. The VL-EPSPs were examined before and after conditioning, to find differences in efficacy in giving rise to spikes. The conditioning procedures resulted in a remarkable facilitation of VL-EPSPs, manifesting itself as a significant rise of efficacy in generating spikes, a shortening of peak latency and in some cases, an enhancement of background firing. 2. In non-PT neurons the same conditioning procedures elicited heterosynaptic facilitation and a rise in firing activity. 3. Intracellularly injected square wave pulses also resulted in facilitation of VL-EPSPs. 4. Pairings of PT and VL stimuli were more effective than trains in evoking conditioned changes. 5. Plastic modifications were observed in the 13.7% of the neurons subjected to conditioning procedures. 6. The authors assume that synchronous activity of the pre- and postsynaptic neurons is a highly important condition for plastic changes in the efficacy of synaptic transmission.     

Recurrent inhibition from motor axon collaterals of transmission in the Ia inhibitory pathway to motoneurones

1. The effects of impulses in recurrent motor axon collaterals on reflex transmission from different types of primary afferents to motoneurones were investigated in the cat by conditioning of PSPs evoked in motoneurones.

2. IPSPs evoked by volleys in large muscle spindle (Ia) afferents were effectively decreased when preceded by an antidromic stimulation of ventral roots. Some IPSPs from group II muscle afferents and low threshold cutaneous afferents were also slightly depressed, while other PSPs were unaffected.

3. The depression of the IPSPs could be evoked by antidromic volleys, which produced neither conductance changes in the motoneurones nor depolarization of Ia afferent terminals.

4. The effect on the Ia IPSPs is most likely due to post-synaptic inhibition of the Ia inhibitory interneurones, evoked through α-motor axon collaterals and Renshaw cells. The depression of some IPSPs from flexor reflex afferents is explained by a convergence of excitatory effects from these afferents on the Ia inhibitory interneurones.

5. The results indicate a selective recurrent control from motor axon collaterals of the interneurones in the reciprocal Ia inhibitory pathway to motoneurones.

     

Extracellular protons reduce quantal content and prolong synaptic currents at the Drosophila larval neuromuscular junction

Fluctuations in extracellular pH occur in the nervous system in response to a number of physiological and pathological processes, such as ischemia, hypercapnea, and high-frequency activity. Using the Drosophila larval neuromuscular junction, the author has examined acute effects of low and high pH on excitability and synaptic transmission. Acidification rapidly and reversibly reduces the size of electrically evoked excitatory junctional currents (EJCs) in a concentration-dependent manner, with transmission nearly abolished at pH 5.0. Conversely, raising pH to 7.8 increases EJC amplitude significantly. Further elevation to pH 8.5 causes an initial increase in amplitude, followed by profound, long-lasting depression of the synapse. Amplitudes of spontaneous miniature EJCs (mEJCs) are modestly, but significantly reduced at pH 5.0. It is therefore the number of quanta released per action potential, rather than the size of individual quanta, that is most strongly affected. Decay times of both EJCs and mEJCs are dramatically lengthened at low pH, suggesting that glutamate remains in the synaptic cleft for much longer than normal. Presynaptic excitability is also reduced, as indicated by increased latency between nerve shock and EJC onset. The response to low pH was not altered by mutations in genes encoding Transient Receptor Potential, Mucolipin subfamily (TRPML) and Slowpoke ion channels, which had previously been implicated as possible targets of extracellular protons. The author concludes that extracellular protons have strong effects on the release of glutamate and the time course of synaptic currents. These phenotypes can be exploited to study the mechanisms of acid-mediated changes in neuronal function, and to pursue the way in which pH modulates synaptic function in normal and pathophysiological conditions.     

Development of neuromuscular transmission in a larval tunicate

1. The time sequence of the development of acetylcholinesterase (AChE), acetylcholine (ACh) receptors and functional synapses on the embryonic muscle membrane in a tunicate larva (Halocynthia roretzi) was investigated in vivo.2. The fertilized tunicate egg was incubated in natural sea water at 9 degrees C. Sixty-eight hr after fertilization the free-swimming larva was hatched, which had six striated muscle fibres in the tail. The developmental stage of the embryo was indicated by the developmental hours after fertilization.3. The transmitter at the neuromuscular junction in the hatched larva is ACh. (i) Neuromuscular transmission was completely blocked by D-tubocurarine (1-5 x 10(-5)M). (ii) Eserine (5-10 x 10(-7)M) approximately doubled the time constant of the falling phase of miniature excitatory junctional currents (e.j.c.s). (iii) The reversal potential of the membrane response to iontophoretically applied ACh was -10 mV and similar to that of e.j.c.s. (iv) AChE was present on the muscle membrane surface.4. AChE activity became visible histochemically on the embryonic cell membrane in the presumptive muscle region as early as the late gastrula stage (27 hr after fertilization, 12 hr before the ACh response appeared).5. The response to iontophoretically applied ACh was present at 39 hr after fertilization but could not be evoked at 38 hr.6. Between 39 and 41 hr after fertilization, the ACh responses increased rapidly, then remained relatively unchanged until larval hatching.7. The stage of the initial appearance of the ACh response corresponded to the stage when the Ca current abruptly increased in the muscle membrane.8. The first sign of neuromuscular transmission was appearance of a giant excitatory junctional potential (e.j.p.) with uniform amplitude (about 15-20 mV) and slow time course (time constant of the falling phase of a giant e.j.c. was 23.4 +/- 6.9 msec, mean and S.D., at -60 mV and 11 degrees C).9. Within a few hours, these giant e.j.p.s disappeared and were successively replaced by medium-sized e.j.p.s and then e.j.p.s similar to those seen in hatched larvae (time constant of the falling phase of a miniature e.j.c. was 8.5 +/- 1.8 msec at -60 mV and 11 degrees C).     

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.     

Biphasic modulation of synaptic transmission by hypertonicity at the embryonic Drosophila neuromuscular junction

Puff-application of hypertonic saline (sucrose added to external saline) causes a transient increase in the frequency of spontaneous miniature synaptic currents (mSCs) at the neuromuscular junctions of Drosophila embryos. The frequency gradually returns to pre-application levels. External Ca²⁺ is not needed for this response, but it may modify it. At 50 m m added sucrose, for example, enhanced spontaneous release was observed only in the presence of external Ca²⁺, suggesting that Ca²⁺ augments the response. In a high-K⁺ solution, in which the basal mSC frequency was elevated, higher sucrose concentrations produced an increase in mSC frequency that was followed (during and after the hypertonic exposure) by depression, with the magnitude of both effects increasing with hypertonicity between 100 and 500 m m. Evoked release by nerve stimulation showed only depression in response to hypertonicity. We do not believe that the depression of spontaneous or evoked release can be explained by the depletion of releasable quanta, however, since the frequency of quantal release did not reach levels compatible with this explanation and the enhancement and depression could be obtained independent of one another. In a mutant lacking neuronal synaptobrevin, only the depression of mSC frequency was induced by hypertonicity. Conversely, only the enhancing effect was observed in wild-type embryos when the mSC frequency was elevated with forskolin in Ca²⁺-free saline. In cultured embryonic Drosophila neurons, Ca²⁺ signals that were induced by high K⁺ and detected by Fura-2, were reduced by hypertonicity, suggesting that the depressing response is due to a direct effect of hypertonicity on Ca²⁺ influx.     

Bistable behavior originating in the axon of a crustacean motor neuron

Both vertebrate and invertebrate motor neurons can display bistable behavior in which self-sustained tonic firing results from a brief excitatory stimulus. Induction of the bistability is usually dependent on activation of intrinsic conductances located in the somatodendritic area and is commonly sensitive to action of neuromodulators. We have observed bistable behavior in a neuromuscular preparation from the foregut of the crab Cancer borealis that consists of the gastric mill 4 (gm4) muscle and the nerve that innervates it, the dorsal gastric nerve (dgn). Nerve-evoked contractions of enhanced amplitude and long duration (>30 s) were induced by extracellular stimulation when the stimulus voltage was above a certain threshold. Intracellular and extracellular recordings showed that the large contractions were accompanied by persistent firing of the dorsal gastric (DG) motor neuron that innervates gm4. The persistent firing could be induced only by stimulating a specific region of the axon and could not be triggered by depolarizing the soma, even at current amplitudes that induced high-frequency firing of the neuron. The bistable behavior was abolished in low-Ca²⁺ saline or when nicardipine or flufenamic acid, blockers of L-type Ca²⁺ and Ca²⁺-activated nonselective cation currents, respectively, was applied to the axonal stimulation region of the dgn. Negative immunostaining for synapsin and synaptotagmin argued against the presence of synaptic/modulatory neuropil in the dgn. Collectively, our results suggest that bistable behavior in a motor neuron can originate in the axon and may not require the action of a locally released neuromodulator.     

Differential modulation of synaptic strength and timing regulate synaptic efficacy in a motor network

Neuromodulators modify network output by altering neuronal firing properties and synaptic strength at multiple sites; however, the functional importance of each site is often unclear. We determined the importance of monoamine modulation of a single synapse for regulation of network cycle frequency in the oscillatory pyloric network of the lobster. The pacemaker kernel of the pyloric network receives only one chemical synaptic feedback, an inhibitory synapse from the lateral pyloric (LP) neuron to the pyloric dilator (PD) neurons, which can limit cycle frequency. We measured the effects of dopamine (DA), octopamine (Oct), and serotonin (5HT) on the strength of the LP→PD synapse and the ability of the modified synapse to regulate pyloric cycle frequency. DA and Oct strengthened, whereas 5HT weakened, LP→PD inhibition. Surprisingly, the DA-strengthened LP→PD synapse lost its ability to slow the pyloric oscillations, whereas the 5HT-weakened LP→PD synapse gained a greater influence on the oscillations. These results are explained by monoamine modulation of factors that determine the firing phase of the LP neuron in each cycle. DA acts via multiple mechanisms to phase-advance the LP neuron into the pacemaker's refractory period, where the strengthened synapse has little effect. In contrast, 5HT phase-delays LP activity into a region of greater pacemaker sensitivity to LP synaptic input. Only Oct enhanced LP regulation of cycle period simply by enhancing LP→PD synaptic strength. These results show that modulation of the strength and timing of a synaptic input can differentially affect the synapse's efficacy in the network.     

Snapin: a SNARE-associated protein implicated in synaptic transmission

Synaptic vesicle docking and fusion are mediated by the assembly of a stable SNARE core complex of proteins, which include the synaptic vesicle membrane protein VAMP/synaptobrevin and the plasmalemmal proteins syntaxin and SNAP-25. We have now identified another SNAP-25-binding protein, called Snapin. Snapin was enriched in neurons and exclusively located on synaptic vesicle membranes. It associated with the SNARE complex through direct interaction with SNAP-25. Binding of recombinant Snapin-CT to SNAP-25 blocked the association of the SNARE complex with synaptotagmin. Introduction of Snapin-CT and peptides containing the SNAP-25 binding sequence into presynaptic superior cervical ganglion neurons in culture reversibly inhibited synaptic transmission. These results suggest that Snapin is an important component of the neurotransmitter release process through its modulation of the sequential interactions between the SNAREs and synaptotagmin.