Calcium channels involved in synaptic transmission from reticulospinal axons in lamprey

The pharmacology of calcium channels involved in glutamatergic synaptic transmission from reticulospinal axons in the lamprey spinal cord was analyzed with specific agonists and antagonists of different high-voltage activated calcium channels. The N-type calcium channel blocker omega-conotoxin GVIA (omega-CgTx) induced a large decrease of the amplitude of reticulospinal-evoked excitatory postsynaptic potentials (EPSPs). The P/Q-type calcium channel blocker omega-agatoxin IVA (omega-Aga) also reduced the amplitude of the reticulospinal EPSPs, but to a lesser extent than omega-CgTx. The dihydropyridine agonist Bay K and antagonist nimodipine had no effect on the amplitude of the reticulospinal EPSP. Combined application of omega-CgTx and omega-Aga strongly decreased the amplitude the EPSPs but was never able to completely block them, indicating that calcium channels insensitive to these toxins (R-type) are also involved in synaptic transmission from reticulospinal axons. We have previously shown that the group III metabotropic glutamate receptor agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) mediates presynaptic inhibition at the reticulospinal synapse. To test if this presynaptic effect is mediated through inhibition of calcium influx, the effect of L-AP4 on reticulospinal transmission was tested before and after blockade of N-type channels, which contribute predominantly to transmitter release at this synapse. Blocking the N-type channels with omega-CgTx did not prevent inhibition of reticulospinal synaptic transmission by L-AP4. In addition, L-AP4 had no affect on the calcium current recorded in the somata of reticulospinal neurons or on the calcium component of action potentials in reticulospinal axons. These results show that synaptic transmission from reticulospinal axons in the lamprey is mediated by calcium influx through N-, P/Q- and R-type channels, with N-type channels playing the major role. Furthermore, presynaptic inhibition of reticulospinal transmission by L-AP4 appears not to be mediated through inhibition of presynaptic calcium channels.     

The lack of effect of baclofen on action potentials of spinal motor axon collateral terminations in vivo

 In the lumbar ventral horn of pentobarbitone-anaesthetised cats, (-)-baclofen reduces both the synaptic release of excitatory transmitter from muscle group Ia afferent terminations and the duration of the presynaptic action potentials of these terminations, presumably by interfering with the influx of calcium ions through voltage-activated channels. Baclofen, however, has little or no effect on cholinergic excitation at motor axon collateral synapses on spinal Renshaw cells and, in the present study, was found not to reduce the duration of the action potential of axon collateral terminations located in the vicinity of Renshaw cells in pentobarbitone-anaesthetised cats. Furthermore, in contrast to group Ia terminations, a 4-aminopyridine-sensitive potassium conductance could not be detected as contributing to axon collateral termination action potentials. These results suggest that there may be differences in presynaptic ion fluxes associated with transmitter release at the intraspinal terminations of group Ia afferent fibres and motor axon collaterals in the cat spinal cord. Received: 16 October 1996 / Accepted: 5 December 1996     

Control by Preynaptic Correlation: a mechanism affecting information transmission from Ia fibers to motoneurons.

1. In the unanesthetized spinal cord of the cat, simultaneous intracellular recordings were made from two motoneurons belonging to the gastronemius motor nucleus. 2. Supramaximal iterative stimulation of small branches of the gastrocnemius nerve produced monosynaptic EPSPs (Ia EPSPs) of varying amplitude superimposed on a fluctuating base line. 3. In most cases the variance of the motoneuron membrane potential was increased above base-line levels with a time course approximately matching the Ia EPSP. This suggests that Ia EPSP fluctuations are greater than can be accounted for by the base-line fluctuations alone. 4. For a given series of Ia EPSPs, the smaller responses in the series had about the same decay phase as the larger EPSPs, suggesting that most of the Ia EPSP fluctuations were not due to systematic changes in postsynaptic conductances produced by ongoing activity, but rather to a presynaptic mechanism. 5. Simultaneous recording from two motoneurons showed that base-line fluctuations were positively correlated. In most cases, however, there was an additional increased correlation above base-line levels resembling the time course of the Ia EPSPs, indicating positive correlation between EPSP fluctuations which is attributed to a presynaptic mechanism. 6. Conditioning volleys to group I muscle afferents or to low-threshold cutaneous afferents reduced the variance of the Ia EPSPs and also their correlation in motoneuron pairs, often without changing the mean Ia EPSPs. 7. It is concluded that, in the unanesthetized spinal cord, in addition to the random process which governs transmitter release intrinsic to a given synaptic terminal, there is another stochastic process affecting, in a correlated manner, transmitter release in large sets of Ia synaptic terminals. Most likely, the correlation in transmitter release is achieved by membrane potential fluctuations imposed on the Ia terminal arborizations by ongoing activity of the segmental mechanism mediating primary afferent depolarization. 8. The effects of such a correlating influence on cell firing behavior have been analyzed. The results suggest that this mechanism, referred to as control by presynaptic correlation, is able to modulate the information transmitted from Ia fibers to motoneurons.     

Baclofen: reduction of presynaptic calcium influx in the cat spinal cord in vivo

 In the ventral horn of the lumbar spinal cord of cats anaesthetised with pentobarbitone sodium, microelectrophoretically administered (–)-baclofen, but not (+)-baclofen, reversibly reduced the duration of the orthodromic action potential of muscle group Ia afferent terminations, but not those of muscle group I afferent myelinated fibres. The presumably submicromolar concentrations are already known to reversibly reduce excitatory transmitter release from muscle group Ia afferent terminations. Action potential durations were estimated from threshold recovery curves after an orthodromic impulse using an extracellular microstimulation technique. Both of these presynaptic effects of (–)-baclofen were blocked by baclofen antagonists, and neither appeared to be reduced by the potassium channel blocking agents tetraethylammonium and 4-aminopyridine. Tetraethylammonium and 4-aminopyridine also did not significantly modify the reduction by (–)-baclofen of monosynaptic field potentials in the lumbar cord of rats anaesthetised with pentobarbitone sodium. In the cat the maximum reduction by (–)-baclofen of termination action potentials was considerably less than that produced by cadmium ions, which, unlike (–)-baclofen, also reduced the action potential duration of group I myelinated fibres. These findings are consistent with a reduction by (–)-baclofen of the influx of calcium through voltage-activated channels in the membrane of group Ia terminations, a proposal which also accounts for the reduction by (–)-baclofen of the release of GABA at axo-axonic depolarizing synapses on these terminations. The results are discussed in relation to the mode of action of (–)-baclofen and the different sensitivities of transmitter release at various central synapses. Received: 30 May 1996 / Accepted: 11 September 1996     

Facilitation at single synapses probed with optical quantal analysis

Many synapses can change their strength rapidly in a use-dependent manner, but the mechanisms of such short-term plasticity remain unknown. To understand these mechanisms, measurements of neurotransmitter release at single synapses are required. We probed transmitter release by imaging transient increases in [Ca(2+)] mediated by synaptic N-methyl-D-aspartate receptors (NMDARs) in individual dendritic spines of CA1 pyramidal neurons in rat brain slices, enabling quantal analysis at single synapses. We found that changes in release probability, produced by paired-pulse facilitation (PPF) or by manipulation of presynaptic adenosine receptors, were associated with changes in glutamate concentration in the synaptic cleft, indicating that single synapses can release a variable amount of glutamate per action potential. The relationship between release probability and response size is consistent with a binomial model of vesicle release with several (>5) independent release sites per active zone, suggesting that multivesicular release contributes to facilitation at these synapses.     

Presynaptic Ca(2+) influx at the inhibitor of the crayfish neuromuscular junction: a photometric study at a high time resolution

Presynaptic calcium influx at the inhibitor of the crayfish neuromuscular junction was investigated by measuring fluorescence transients generated by calcium-sensitive dyes. This approach allowed us to correlate presynaptic calcium influx with transmitter release at a high time resolution. Systematic testing of the calcium indicators showed that only low-affinity dyes, with affinities in the range of micromolar, should be used to avoid saturation of dye binding and interference with transmitter release. Presynaptic calcium influx was regulated by slowly increasing the duration of the action potential through progressive block of potassium channels. The amplitude of the calcium transient, measured from a cluster of varicosities, was linearly related to the duration of the action potential with a slope of 1.2. Gradual changes in potassium channel block allowed us to estimate the calcium cooperativity of transmitter release over a 10-fold range in presynaptic calcium influx. Calcium cooperativity measured here exhibited one component with an average value of 3.1. Inspection of simultaneously recorded presynaptic calcium transients and inhibitory postsynaptic currents (IPSCs) showed that prolonged action potentials were associated with a slow rising phase of presynaptic calcium transients, which were matched by a slow rate of rise of IPSCs. The close correlation suggests that fluorescence transients provide information on the rate of calcium influx. Because there is an anatomic mismatch between the presynaptic calcium transient, measured from a cluster of varicosities, and IPSC, measured with two-electrode voltage clamp, macropatch recording was used to monitor inhibitory postsynaptic responses from the same cluster of varicosities from which the calcium transient was measured. Inhibitory postsynaptic responses recorded with the macropatch method exhibited a faster rising phase than that recorded with two-electrode voltage clamp. This difference could be attributed to slight asynchrony of transmitter release due to action potential conduction along fine branches. In conclusion, this report shows that fluorescence transients generated by calcium-sensitive dyes can provide insights to the properties of presynaptic calcium influx, and its correlation with transmitter release, at a high time resolution.     

John Eccles' studies of spinal cord presynaptic inhibition

Presynaptic inhibition is one of many areas of neurophysiology in which Sir John Eccles did pioneering work. Frank and Fuortes first described presynaptic inhibition in 1957. Subsequently, Eccles and his colleagues characterized the process more fully and showed its relationship to primary afferent depolarization. Eccles' studies emphasized presynaptic inhibition of the group Ia monosynaptic reflex pathway but also included group Ib, II and cutaneous afferent pathways, and the dorsal column nuclei. Presynaptic inhibition of the group Ia afferent pathway was demonstrated by depression of monosynaptic excitatory postsynaptic potentials and inhibition of monosynaptic reflex discharges. Primary afferent depolarization was investigated by recordings of dorsal root potentials, dorsal root reflexes, cord dorsum and spinal cord field potentials, and tests of the excitability of primary afferent terminals. Primary afferent depolarization was proposed to result in presynaptic inhibition by reducing the amplitude of the action potential as it invades presynaptic terminals. This resulted in less calcium influx and, therefore, less transmitter release. Presynaptic inhibition and primary afferent depolarization could be blocked by antagonists of GABA(A) receptors, implying a role of interneurons that release gamma aminobutyric acid in the inhibitory circuit. The reason why afferent terminals were depolarized was later explained by a high intracellular concentration of Cl(-) ions in primary sensory neurons. Activation of GABA(A) receptors opens Cl(-) channels, and Cl(-) efflux results in depolarization. Another proposed mechanism of depolarization was an increase in extracellular concentration of K(+) following neural activity. Eccles' work on presynaptic inhibition has since been extended in a variety of ways.     

Inhibition [corrected] of olfactory receptor neuron input to olfactory bulb glomeruli mediated by suppression of presynaptic calcium influx

We investigated the cellular mechanism underlying presynaptic regulation of olfactory receptor neuron (ORN) input to the mouse olfactory bulb using optical-imaging techniques that selectively report activity in the ORN presynaptic terminal. First, we loaded ORNs with calcium-sensitive dye and imaged stimulus-evoked calcium influx in a slice preparation. Single olfactory nerve shocks evoked rapid fluorescence increases that were largely blocked by the N-type calcium channel blocker omega-conotoxin GVIA. Paired shocks revealed a long-lasting suppression of calcium influx with approximately 40% suppression at 400-ms interstimulus intervals and a recovery time constant of approximately 450 ms. Blocking activation of postsynaptic olfactory bulb neurons with APV/CNQX reduced this suppression. The GABA(B) receptor agonist baclofen inhibited calcium influx, whereas GABA(B) antagonists reduced paired-pulse suppression without affecting the response to the conditioning pulse. We also imaged transmitter release directly using a mouse line that expresses synaptopHluorin selectively in ORNs. We found that the relationship between calcium influx and transmitter release was superlinear and that paired-pulse suppression of transmitter release was reduced, but not eliminated, by APV/CNQX and GABA(B) antagonists. These results demonstrate that primary olfactory input to the CNS can be presynaptically regulated by GABAergic interneurons and show that one major intracellular pathway for this regulation is via the suppression of calcium influx through N-type calcium channels in the presynaptic terminal. This mechanism is unique among primary sensory afferents.     

Posttetanic potentiation of group Ia EPSPs: possible mechanisms for differential distribution among medial gastrocnemius motoneurons

We have reinvestigated the phenomenon of posttetanic potentiation (PTP) of group Ia monosynaptic excitatory postsynaptic potentials (EPSPs) in medial gastrocnemius (MG) alpha-motoneurons of pentobarbital-anesthetized cats. The results generally confirm earlier reports by Lüscher and colleagues (43, 44) of a negative correlation between the maximum percentage potentiation of Ia EPSP amplitude (Pmax) and 1) the mean amplitude of the pretetanic control EPSP in the same cell and 2) the input resistance of the postsynaptic motoneuron. These negative correlations, which we will refer to as "differential distribution of PTP" within the MG motor pool, were less strong in the present work than reported by Lüscher et al. (43, 44). We also found a relatively strong negative correlation between posttetanic EPSP depression, assessed by the amplitude of the first posttetanic EPSP, and the level of Pmax subsequently attained. We found no evidence that posttetanic depression is caused by failure of presynaptic action potentials. We investigated a second type of depression, referred to as "specific" synaptic depression, in which the second EPSP of paired responses (interval 250 ms) is, on average, smaller in peak amplitude than the first EPSP. This phenomenon appears to reflect decreases in the probability of transmitter release from previously activated synapses. Specific synaptic depression was consistently increased when paired responses were conditioned by a high-frequency tetanus. This is most easily explained by postulating that PTP results, at least in part, from an increase in the statistical probability of transmitter liberation from group Ia synapses that are activated (i.e., presumably invaded by action potentials) both before and after afferent tetanization. On the basis of the present results and other available evidence, we conclude that the differential distribution of PTP can be explained by two main factors: 1) the nonlinear relation between conductance and voltage changes inherent in all chemical synapses and 2) systematic variations in the properties of group Ia synapses that innervated different motoneurons, which remain to be clarified.     

Contribution of presynaptic calcium-activated potassium currents to transmitter release regulation in cultured Xenopus nerve-muscle synapses

Using Xenopus nerve-muscle co-cultures, we have examined the contribution of calcium-activated potassium (K(Ca)) channels to the regulation of transmitter release evoked by single action potentials. The presynaptic varicosities that form on muscle cells in these cultures were studied directly using patch-clamp recording techniques. In these developing synapses, blockade of K(Ca) channels with iberiotoxin or charybdotoxin decreased transmitter release by an average of 35%. This effect would be expected to be caused by changes in the late phases of action potential repolarization. We hypothesize that these changes are due to a reduction in the driving force for calcium that is normally enhanced by the local hyperpolarization at the active zone caused by potassium current through the K(Ca) channels that co-localize with calcium channels. In support of this hypothesis, we have shown that when action potential waveforms were used as voltage-clamp commands to elicit calcium current in varicosities, peak calcium current was reduced only when these waveforms were broadened beginning when action potential repolarization was 20% complete. In contrast to peak calcium current, total calcium influx was consistently increased following action potential broadening. A model, based on previously reported properties of ion channels, faithfully reproduced predicted effects on action potential repolarization and calcium currents. From these data, we suggest that the large-conductance K(Ca) channels expressed at presynaptic varicosities regulate transmitter release magnitude during single action potentials by altering the rate of action potential repolarization, and thus the magnitude of peak calcium current.     

Expression of the P/Q (Cav2.1) calcium channel in nodose sensory neurons and arterial baroreceptors

The predominant calcium current in nodose sensory neurons, including the subpopulation of baroreceptor neurons, is the N-type channel, Cav2.2. It is also the primary calcium channel responsible for transmitter release at their presynaptic terminals in the nucleus of the solitary tract in the brainstem. The P/Q channel, Cav2.1, the other major calcium channel responsible for transmitter release at mammalian synapses, represents only 15–20% of total calcium current in the general population of sensory neurons and makes a minor contribution to transmitter release at the presynaptic terminal. In the present study we identified a subpopulation of the largest nodose neurons (capacitance > 50 pF) in which, surprisingly, Cav2.1 represents over 50% of the total calcium current, differing from the remainder of the population. Consistent with these electrophysiological data, anti-Cav2.1 antibody labeling was more membrane delimited in a subgroup of the large neurons in slices of nodose ganglia. Data reported in other synapses in the central nervous system assign different roles in synaptic information transfer to the P/Q-type versus N-type calcium channels. The study raises the possibility that the P/Q channel which has been associated with high fidelity transmission at other central synapses serves a similar function in this group of large myelinated sensory afferents, including arterial baroreceptors where a high frequency regular discharge pattern signals the pressure pulse. This contrasts to the irregular lower frequency discharge of the unmyelinated fibers that make up the majority of the sensory population and that utilize the N-type channel in synaptic transmission.     

Developmental changes in release properties of the CA3-CA1 glutamate synapse in rat hippocampus

Developmental changes in release probability (Pr) and paired-pulse plasticity at CA3-CA1 glutamate synapses in hippocampal slices of neonatal rats were examined using field excitatory postsynaptic potential (EPSP) recordings. Paired-pulse facilitation (PPF) at these synapses was, on average, absent in the first postnatal week but emerged and became successively larger during the second postnatal week. This developmental increase in PPF was associated with a reduction in Pr, as indicated by the slower progressive block of the N-methyl-D-aspartate (NMDA) EPSP by the noncompetitive NMDA receptor antagonist MK-801. This developmental reduction in Pr was not homogenous among the synapses. As shown by the MK-801 analysis, the Pr heterogeneity observed among adult CA3-CA1 synapses is present already during the first postnatal week, and the developmental Pr reduction was found to be largely selective for synapses with higher Pr values, leaving Pr of the vast majority of the synapses essentially unaffected. A reduction in Pves, the release probability of the individual vesicle, possibly caused by reduction in Ca2+ influx, seems to explain the reduction in Pr. In vivo injection of tetanus toxin at the end of the first postnatal week did not prevent the increase in PPF, indicating that this developmental change in release is not critically dependent on normal neural activity during the second postnatal week.     

Postsynaptic induction and presynaptic expression of group 1 mGluR-dependent LTD in the hippocampal CA1 region

Activation of metabotropic glutamate receptors (mGluRs) with the group I mGluR selective agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) induces a long-term depression (LTD) of excitatory synaptic transmission in the CA1 region of the hippocampus. Here we investigated the potential roles of pre- and postsynaptic processes in the DHPG-induced LTD at excitatory synapses onto hippocampal pyramidal cells in the mouse hippocampus. Activation of mGluRs with DHPG, but not ACPD, induced LTD at both Schaffer collateral/commissural fiber synapses onto CA1 pyramidal cells and at associational/commissural fiber synapses onto CA3 pyramidal cells. DHPG-induced LTD was blocked when the G-protein inhibitor guanosine-5'-O-(2-thiodiphosphate) was selectively delivered into postsynaptic CA1 pyramidal cells via an intracellular recording electrode, suggesting that DHPG depresses synaptic transmission through a postsynaptic, GTP-dependent signaling pathway. The effects of DHPG were also strongly modulated, however, by experimental manipulations that altered presynaptic calcium influx. In these experiments, we found that elevating extracellular Ca(2+) concentrations ([Ca(2+)](o)) to 6 mM almost completely blocked the effects of DHPG, whereas lowering [Ca(2+)](o) to 1 mM significantly enhanced the ability of DHPG to depress synaptic transmission. Enhancing Ca(2+) influx by prolonging action potential duration with bath applications of the K(+) channel blocker 4-aminopyridine (4-AP) also strongly reduced the effects of DHPG in the presence of normal [Ca(2+)](o) (2 mM). Although these findings indicate that alterations in Ca(2+)-dependent signaling processes strongly regulate the effects of DHPG on synaptic transmission, they do not distinguish between potential pre- versus postsynaptic sites of action. We found, however, that while inhibiting both pre- and postsynaptic K(+) channels with bath-applied 4-AP blocked the effects of DHPG; inhibition of postsynaptic K(+) channels alone with intracellular Cs(+) and TEA had no effect on the ability of DHPG to inhibit synaptic transmission. This suggests that presynaptic changes in transmitter release contribute to the depression of synaptic transmission by DHPG. Consistent with this, DHPG induced a persistent depression of both AMPA and N-methyl-D-aspartate receptor-mediated components of excitatory postsynaptic currents in voltage-clamped pyramidal cells. Together our results suggest that activation of postsynaptic mGluRs suppresses transmission at excitatory synapses onto CA1 pyramidal cells through presynaptic effects on transmitter release.     

Distinct transmitter release properties determine differences in short-term plasticity at functional and silent synapses

Recent evidence suggests that functional and silent synapses are not only postsynaptically different but also presynaptically distinct. The presynaptic differences may be of functional importance in memory formation because a proposed mechanism for long-term potentiation is the conversion of silent synapses into functional ones. However, there is little direct experimentally evidence of these differences. We have investigated the transmitter release properties of functional and silent Schaffer collateral synapses and show that on the average functional synapses displayed a lower percentage of failures and higher excitatory postsynaptic current (EPSC) amplitudes than silent synapses at +60 mV. Moreover, functional but not silent synapses show paired-pulse facilitation (PPF) at +60 mV and thus presynaptic short-term plasticity will be distinct in the two types of synapse. We examined whether intraterminal endoplasmic reticulum Ca2+ stores influenced the release properties of these synapses. Ryanodine (100 microM) and thapsigargin (1 microM) increased the percentage of failures and decreased both the EPSC amplitude and PPF in functional synapses. Caffeine (10 mM) had the opposite effects. In contrast, silent synapses were insensitive to both ryanodine and caffeine. Hence we have identified differences in the release properties of functional and silent synapses, suggesting that synaptic terminals of functional synapses express regulatory molecular mechanisms that are absent in silent synapses.     

Mechanisms underlying cannabinoid inhibition of presynaptic Ca2+ influx at parallel fibre synapses of the rat cerebellum

Activation of CB1 cannabinoid receptors in the cerebellum acutely depresses excitatory synaptic transmission at parallel fibre–Purkinje cell synapses by decreasing the probability of glutamate release. This depression involves the activation of presynaptic 4-aminopyridine-sensitive K⁺ channels by CB1 receptors, which in turn inhibits presynaptic Ca²⁺ influx controlling glutamate release at these synapses. Using rat cerebellar frontal slices and fluorometric measures of presynaptic Ca²⁺ influx evoked by stimulation of parallel fibres with the fluorescent dye fluo-4FF, we tested whether the CB1 receptor-mediated inhibition of this influx also involves a direct inhibition of presynaptic voltage-gated calcium channels. Since various physiological effects of CB1 receptors appear to be mediated through the activation of PTX-sensitive proteins, including inhibition of adenylate cyclases, activation of mitogen-activated protein kinases (MAPK) and activation of G protein-gated inwardly rectifying K⁺ channels, we also studied the potential involvement of these intracellular signal transduction pathways in the cannabinoid-mediated depression of presynaptic Ca²⁺ influx. The present study demonstrates that the molecular mechanisms underlying the CB1 inhibitory effect involve the activation of the PTX-sensitive G_i/G_o subclass of G proteins, independently of any direct effect on presynaptic Ca²⁺ channels (N, P/Q and R (SNX-482-sensitive) types) or on adenylate cyclase or MAPK activity, but do require the activation of G protein-gated inwardly rectifying (Ba²⁺- and tertiapin Q-sensitive) K⁺ channels, in addition to 4-aminopyridine-sensitive K⁺ channels.     

The Hodgkin–Huxley–Katz Prize Lecture

During the last decade, advances in experimental techniques and quantitative modelling have resulted in the development of the calyx of Held as one of the best preparations in which to study synaptic transmission. Here we review some of these advances, including simultaneous recording of pre- and postsynaptic currents, measuring the Ca²⁺ sensitivity of transmitter release, reconstructing the 3-D anatomy at the electron microscope (EM) level, and modelling the buffered diffusion of Ca²⁺ in the nerve terminal. An important outcome of these studies is an improved understanding of the Ca²⁺ signal that controls phasic transmitter release. This article illustrates the spatial and temporal aspects of the three main steps in the presynaptic signalling cascade: Ca²⁺ influx through voltage-gated calcium channels, buffered Ca²⁺ diffusion from the channels to releasable vesicles, and activation of the Ca²⁺ sensor for release. Particular emphasis is placed on how presynaptic Ca²⁺ buffers affect the Ca²⁺ signal and thus the amplitude and time course of the release probability. Since many aspects of the signalling cascade were first conceived with reference to the squid giant presynaptic terminal, we include comparisons with the squid model and revisit some of its implications. Whilst the characteristics of buffered Ca²⁺ diffusion presented here are based on the calyx of Held, we demonstrate the circumstances under which they may be valid for other nerve terminals at mammalian CNS synapses.     

Differential expression of calcium channels in sympathetic and parasympathetic preganglionic inputs to neurons in paracervical ganglia of guinea-pigs

Neurons in pelvic ganglia receive nicotinic excitatory post-synaptic potentials (EPSPs) from sacral preganglionic neurons via the pelvic nerve, lumbar preganglionic neurons via the hypogastric nerve or both. We tested the effect of a range of calcium channel antagonists on EPSPs evoked in paracervical ganglia of female guinea-pigs after pelvic or hypogastric nerve stimulation. omega-Conotoxin GVIA (CTX GVIA, 100 nM) or the novel N-type calcium channel antagonist, CTX CVID (100 nM) reduced the amplitude of EPSPs evoked after pelvic nerve stimulation by 50-75% but had no effect on EPSPs evoked by hypogastric nerve stimulation. Combined addition of CTX GVIA and CTX CVID was no more effective than either antagonist alone. EPSPs evoked by stimulating either nerve trunk were not inhibited by the P/Q calcium channel antagonist, omega-agatoxin IVA (100 nM), nor the L-type calcium channel antagonist, nifedipine (30 microM). SNX 482 (300 nM), an antagonist at some R-type calcium channels, inhibited EPSPs after hypogastric nerve stimulation by 20% but had little effect on EPSPs after pelvic nerve stimulation. Amiloride (100 microM) inhibited EPSPs after stimulation of either trunk by 40%, while nickel (100 microM) was ineffective. CTX GVIA or CTX CVID (100 nM) also slowed the rate of action potential repolarization and reduced afterhyperpolarization amplitude in paracervical neurons. Thus, release of transmitter from the terminals of sacral preganglionic neurons is largely dependent on calcium influx through N-type calcium channels, although an unknown calcium channel which is resistant to selective antagonists also contributes to release. Release of transmitter from lumbar preganglionic neurons does not require calcium entry through either conventional N-type calcium channels or the variant CTX CVID-sensitive N-type calcium channel and seems to be mediated largely by a novel calcium channel.     

Sustained granule cell activity disinhibits juvenile mouse cerebellar stellate cells through presynaptic mechanisms

GABA release from cerebellar molecular layer interneurons can be modulated by presynaptic glutamate and/or GABA B receptors upon perfusing the respective agonists. However, it is unclear how release and potential spillover of endogenous transmitter lead to activation of presynaptic receptors. High frequency firing of granule cells, as observed in vivo upon sensory stimulation, could lead to glutamate and/or GABA spillover. Here, we established sustained glutamatergic activity in the granule cell layer of acute mouse cerebellar slices and performed 190 paired recordings from connected stellate cells. Train stimulation at 50 Hz reduced by about 30% the peak amplitude of IPSCs evoked by brief depolarization of the presynaptic cell in 2-week-old mice. A presynaptic mechanism was indicated by changes in failure rate, paired-pulse ratio and coefficient of variation of evoked IPSCs. Furthermore, two-photon Ca2+ imaging in identified Ca2+ hot spots of stellate cell axons confirmed reduced presynaptic Ca2+ influx after train stimulation within the granular layer. Pharmacological experiments indicated that glutamate released from parallel fibres activated AMPARs in stellate cells, evoking GABA release from surrounding cells. Consequential GABA spillover activated presynaptic GABA B Rs, which reduced the amplitude of eIPSCs. Two-thirds of the total disinhibitory effect were mediated by GABA B Rs, one-third being attributable to presynaptic AMPARs. This estimation was confirmed by the observation that bath applied baclofen induced a more pronounced reduction of evoked IPSCs than kainate. Granule cell-mediated disinhibition persisted at near-physiological temperature but was strongly diminished in 3-week-old mice. At this age, GABA release probability was not reduced and presynaptic GABA B Rs were still detectable, but GABA uptake appeared to be advanced, attenuating GABA spillover. Thus, sustained granule cell activity modulates stellate cell-to-stellate cell synapses, involving transmitter spillover during a developmentally restricted period.     

Synapse-to-synapse variation of calcium channel subtype contributions in large mossy fiber terminals of mouse hippocampus

Both N- and P/Q-type voltage-dependent calcium channels are involved in fast transmitter release in the hippocampus, but are differentially regulated. Although variable contributions of voltage-dependent calcium channel subtypes to presynaptic Ca2+ influx have been suggested to give a neural network of great diversity, their presence has only been demonstrated in a culture system and has remained unclear in the brain. Here, the individual large mossy fiber presynaptic terminal was labeled with Ca2+/Sr2+-sensitive fluorescent dextrans in the hippocampal slice of the mouse. The fractional contribution of voltage-dependent calcium channel subtypes to presynaptic Ca2+/Sr2+ influx was directly measured by the sensitivity of Ca2+/Sr2+-dependent fluorescent increment to subtype-selective neurotoxins, omega-conotoxin GVIA (an N-type selective blocker), omega-agatoxin IVA (a P/Q-type selective blocker) and SNX-482 (an R-type selective blocker). Synapse-to-synapse comparison of large mossy fiber terminals revealed that the contributions of N- and R-type voltage-dependent calcium channels varied more widely than that of P/Q-type. Even two large mossy fiber presynaptic terminals neighboring on the same axon differed in the fractional contributions of N- and R-type voltage-dependent calcium channels. On the other hand, these terminals were similar in the fractional contributions of P/Q-type voltage-dependent calcium channels. These results provide direct evidence that individual large mossy fiber synapses are differential in the contribution of N- and R-type voltage-dependent calcium channel subtypes to presynaptic Ca2+/Sr2+ influx. We suggest that the synapse-to-synapse variation of presynaptic voltage-dependent calcium channel subtype contributions may be one of the mechanisms amplifying diversity of the hippocampal network.     

Rab3a deletion reduces vesicle docking and transmitter release at the mouse diaphragm synapse

Rab3a is a small GTP binding protein associated with presynaptic vesicles that is thought to regulate vesicle targeting to active zones. Although this rab3a function implies that vesicle docking and action potential-evoked release might be inhibited in rab3a gene-deleted synapses, such inhibition has never been demonstrated. To investigate vesicle docking at the neuromuscular junction of rab3a gene-deleted (rab3a(-)) mice, we performed electron microscopy analysis of the diaphragm slow-fatigue (type I) synapses. We found a significant (26%) reduction in the number of vesicles docked to the presynaptic membrane in rab3a(-) terminals, although intraterminal vesicles were not affected. Aiming to detect possible changes in quantal release due to rab3a gene deletion, we minimized the variability between preparations employing focal recordings of synaptic responses from visualized type I endplates. We found a significant decrease in both evoked (27% reduction in quantal content) and spontaneous (28% reduction in mini frequency) quantal release. The decrease in the evoked release produced by rab3a deletion was most pronounced at reduced extracellular Ca(2+) concentrations (over 50% decrease at 0.5 and 0.2 mM Ca(2+)). By manipulating extracellular calcium, we demonstrated that calcium cooperativity is not altered in rab3a(-) synapses, however calcium sensitivity of quantal release is affected. Thus, we demonstrated that rab3a positively regulates docking and basal quantal release at the mouse neuromuscular junction. This result is consistent with the proposed role of rab3a in trafficking and targeting vesicles to the active zones.