Presynaptic N-type and P/Q-type Ca2+ channels mediating synaptic transmission at the calyx of Held of mice

At the nerve terminal, both N- and P/Q-type Ca²⁺ channels mediate synaptic transmission, with their relative contribution varying between synapses and with postnatal age. To clarify functional significance of different presynaptic Ca²⁺ channel subtypes, we recorded N-type and P/Q-type Ca²⁺ currents directly from calyces of Held nerve terminals in α_1A-subunit-deficient mice and wild-type (WT) mice, respectively. The most prominent feature of P/Q-type Ca²⁺ currents was activity-dependent facilitation, which was absent for N-type Ca²⁺ currents. EPSCs mediated by P/Q-type Ca²⁺ currents showed less depression during high-frequency stimulation compared with those mediated by N-type Ca²⁺ currents. In addition, the maximal inhibition by the GABA_B receptor agonist baclofen was greater for EPSCs mediated by N-type channels than for those mediated by P/Q-type channels. These results suggest that the developmental switch of presynaptic Ca²⁺ channels from N- to P/Q-type may serve to increase synaptic efficacy at high frequencies of activity, securing high-fidelity synaptic transmission.     

Force generation examined by laser temperature-jumps in shortening and lengthening mammalian (rabbit psoas) muscle fibres

The modulation of synaptic transmission by presynaptic ionotropic and metabotropic receptors is an important means to control and dynamically adjust synaptic strength. Even though synaptic transmission and plasticity at the hippocampal mossy fibre synapse are tightly controlled by presynaptic receptors, little is known about the downstream signalling mechanisms and targets of the different receptor systems. In the present study, we identified the cellular signalling cascade by which adenosine modulates mossy fibre synaptic transmission. By means of electrophysiological and optical recording techniques, we found that adenosine activates presynaptic A₁ receptors and reduces Ca²⁺ influx into mossy fibre terminals. Ca²⁺ currents are directly modulated via a membrane-delimited pathway and the reduction of presynaptic Ca²⁺ influx can explain the inhibition of synaptic transmission. Specifically, we found that adenosine modulates both P/Q- and N-type presynaptic voltage-dependent Ca²⁺ channels and thereby controls transmitter release at the mossy fibre synapse.     

Glycinergic synaptic transmission to bullfrog retinal bipolar cells is input-specific

Glycinergic inhibitory postsynaptic currents (IPSCs) focally elicited at the dendrites and axon terminals were recorded from bipolar cells in the bullfrog retinal slice, using the whole-cell clamp technique. IPSCs driven by input from interplexiform cells at bipolar cell dendrites (ipc-IPSCs) had a much slower decay time constant (25.2 +/- 7.8 ms) than IPSCs driven by input from amacrine cells at bipolar cell axon terminals (ac-IPSCs) (14.7 +/- 5.5 ms). Furthermore, peak-scaled non-stationary noise analysis revealed that the weighted mean single-channel conductance of the glycine receptors underlying bipolar cell dendritic ipc-IPSCs (20.8 +/- 6.6 pS) was significantly larger than that of those underlying bipolar cell axon terminal ac-IPSCs (12.9 +/- 2.9 pS). These results demonstrate that glycinergic synaptic transmission with different properties at bipolar cell dendrites and axon terminals differentially mediates intraretinal centrofugal signal transfer from the inner retina to the outer retina provided by interplexiform cells and lateral inhibition offered by amacrine cells in the inner retina.     

Dual Ca2+ modulation of glycinergic synaptic currents in rodent hypoglossal motoneurones

Glycinergic synapses are implicated in the coordination of reflex responses, sensory signal processing and pain sensation. Their activity is pre- and postsynaptically regulated, although mechanisms are poorly understood. Using patch-clamp recording and Ca²⁺ imaging in hypoglossal motoneurones from rat and mouse brainstem slices, we address here the role of cytoplasmic Ca²⁺ (Ca_i) in glycinergic synapse modulation. Ca²⁺ influx through voltage-gated or NMDA receptor channels caused powerful transient inhibition of glycinergic IPSCs. This effect was accompanied by an increase in both the failure rate and paired-pulse ratio, as well as a decrease in the frequency of mIPSCs, suggesting a presynaptic mechanism of depression. Inhibition was reduced by the cannabinoid receptor antagonist SR141716A and occluded by the agonist WIN55,212-2, indicating involvement of endocannabinoid retrograde signalling. Conversely, in the presence of SR141716A, glycinergic IPSCs were potentiated postsynaptically by glutamate or NMDA, displaying a Ca²⁺-dependent increase in amplitude and decay prolongation. Both presynaptic inhibition and postsynaptic potentiation were completely prevented by strong Ca_i buffering (20 m m BAPTA). Our findings demonstrate two independent mechanisms by which Ca²⁺ modulates glycinergic synaptic transmission: (i) presynaptic inhibition of glycine release and (ii) postsynaptic potentiation of GlyR-mediated responses. This dual Ca²⁺-induced regulation might be important for feedback control of neurotransmission in a variety of glycinergic networks in mammalian nervous systems.     

Properties of glycine receptors underlying synaptic currents in presynaptic axon terminals of rod bipolar cells in the rat retina

The excitability of presynaptic terminals can be controlled by synaptic input that directly targets the terminals. Retinal rod bipolar axon terminals receive presynaptic input from different types of amacrine cells, some of which are glycinergic. Here, we have performed patch-clamp recordings from rod bipolar axon terminals in rat retinal slices. We used whole-cell recordings to study glycinergic inhibitory postsynaptic currents (IPSCs) under conditions of adequate local voltage clamp and outside-out patch recordings to study biophysical and pharmacological properties of the glycine receptors with ultrafast application. Glycinergic IPSCs, recorded in both intact cells and isolated terminals, were strychnine sensitive and displayed fast kinetics with a double-exponential decay. Ultrafast application of brief (∼1 ms) pulses of glycine (3 m m ) to patches evoked responses with fast, double-exponential deactivation kinetics, no evidence of desensitization in double-pulse experiments, relatively low apparent affinity (EC₅₀∼100 μ m ), and high maximum open probability (∼0.9). Longer pulses evoked slow, double-exponential desensitization and double-pulse experiments indicated slow, double-exponential recovery from desensitization. Non-stationary noise analysis of IPSCs and patch responses yielded single-channel conductances of ∼41 pS and ∼64 pS, respectively. Directly observed single-channel gating occurred at ∼40–50 pS and ∼80–90 pS in both types of responses, suggesting a mixture of heteromeric and homomeric receptors. Synaptic release of glycine leads to transient receptor activation, with about eight receptors available to bind transmitter after release of a single vesicle. With a low intracellular chloride concentration, this leads to either hyperpolarizing or shunting inhibition that will counteract passive and regenerative depolarization and depolarization-evoked transmitter release.     

Adenosine A1 receptor-mediated presynaptic inhibition at the calyx of Held of immature rats

At the calyx of Held synapse in brainstem slices of 5- to 7-day-old (P5–7) rats, adenosine, or the type 1 adenosine (A₁) receptor agonist N ⁶-cyclopentyladenosine (CPA), inhibited excitatory postsynaptic currents (EPSCs) without affecting the amplitude of miniature EPSCs. The A₁ receptor antagonist 8-cyclopentyltheophylline (CPT) had no effect on the amplitude of EPSCs evoked at a low frequency, but significantly reduced the magnitude of synaptic depression caused by repetitive stimulation at 10 Hz, suggesting that endogenous adenosine is involved in the regulation of transmitter release. Adenosine inhibited presynaptic Ca²⁺ currents ( I _pCa) recorded directly from calyceal terminals, but had no effect on presynaptic K⁺ currents. When EPSCs were evoked by I _pCa during simultaneous pre- and postsynaptic recordings, the magnitude of the adenosine-induced inhibition of I _pCa fully explained that of EPSCs, suggesting that the presynaptic Ca²⁺ channel is the main target of A₁ receptors. Whereas the N-type Ca²⁺ channel blocker ω-conotoxin attenuated EPSCs, it had no effect on the magnitude of adenosine-induced inhibition of EPSCs. During postnatal development, in parallel with a decrease in the A₁ receptor immunoreactivity at the calyceal terminal, the inhibitory effect of adenosine became weaker. We conclude that presynaptic A₁ receptors at the immature calyx of Held synapse play a regulatory role in transmitter release during high frequency transmission, by inhibiting multiple types of presynaptic Ca²⁺ channels.     

Functional roles of presynaptic GABAA receptors on glycinergic nerve terminals in the rat spinal cord

GABA_A receptor-mediated presynaptic depolarization is believed to induce presynaptic inhibition of excitatory synaptic transmission. We report here the functional roles of presynaptic GABA_A receptors in glycinergic transmission of the rat spinal cord. In mechanically dissociated rat sacral dorsal commissural nucleus (SDCN) neurons attached with native glycinergic and GABAergic nerve terminals, glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were isolated from a mixture of both glycinergic and GABAergic sIPSCs by perfusing the SDCN nerve cell body with ATP-free internal solution. Under such experimental conditions, exogenously applied muscimol (0.5 μM) depolarized glycinergic presynaptic nerve terminals and significantly increased glycinergic sIPSC frequency to 542.7 ± 47.3 % of the control without affecting the mean current amplitude. The facilitatory effect of muscimol on sIPSC frequency was completely blocked by bicuculline (10 μM) or SR95531 (10 μM), selective GABA_A receptor antagonists. This muscimol-induced presynaptic depolarization was due to a higher intraterminal Cl⁻ concentration, which is maintained by a bumetanide-sensitive Na-K-Cl cotransporter. On the contrary, when electrically evoked, this muscimol-induced presynaptic depolarization was found to decrease the action potential-dependent glycine release evoked by focal stimulation of a single terminal. The results suggest that GABA_A receptor-mediated presynaptic depolarization has two functional roles: (1) presynaptic inhibition of action potential-driven glycinergic transmission, and (2) presynaptic facilitation of spontaneous glycinergic transmission.     

Glycinergic input of widefield, displaced amacrine cells of the mouse retina

Glycine receptors (GlyRs) of displaced amacrine cells of the mouse retina were analysed using whole cell recordings and immunocytochemical staining with subunit-specific antibodies. During the recordings the cells were filled with a fluorescent tracer and 11 different morphological types could be identified. The studies were performed in wild-type mice and in mutant mice deficient in the GlyRα1 ( Glra1^spd-ot , 'oscillator' mouse), the GlyRα2 ( Glra2 ^−/−) and the GlyRα3 subunit ( Glra3 ^−/−). Based on their responses to the application of exogenous glycine in the retinas of wild-type and mutant mice, the cells were grouped into three major classes: group I cells (comprising the morphological types MA-S5, MA-S1, MA-S1/S5, A17, PA-S1, PA-S5 and WA-S1), group II cells (comprising the morphological types PA-S4, WA-S3 and WA-multi) and ON-starburst cells. For further analysis, spontaneous inhibitory postsynaptic currents (sIPSCs) were measured both in wild-type and mutant mouse retinas. Glycinergic sIPSCs and glycine induced currents of group I cells remained unaltered across wild-type and the three mutant mice (mean decay time constant of sIPSCs, τ∼25 ms). Group II cells showed glycinergic sIPSCs and glycine induced currents in wild-type, Glra1^spd-ot and Glra3 ^−/− mice (τ∼25 ms); however, glycinergic currents were absent in group II cells of Glra2 ^−/− mice. Glycine induced currents and sIPSCs recorded from ON-starburst amacrine cells did not differ significantly between wild-type and the mutant mouse retinas (τ∼50–70 ms). We propose that GlyRs of group II cells are dominated by the α2 subunit; GlyRs of ON-starburst amacrine cells appear to be dominated by the α4 subunit.     

Modulation of Ca2+-activated K+ currents and Ca2+-dependent action potentials by exocytosis in goldfish bipolar cell terminals

Retinal bipolar cells convey light-evoked potentials from photoreceptors to ganglion cells and mediate the initial stages of visual signal processing. They do not fire Na⁺-dependent action potentials (APs) but the Mb1 class of goldfish bipolar cell exhibits Ca²⁺-dependent APs and regenerative potentials that originate in the axon terminal. I have examined the properties of Ca²⁺-dependent APs in isolated bipolar-cell terminals in goldfish retinal slices. All recorded terminals fired spontaneous or evoked APs at frequencies of up to 15 Hz. When an AP waveform was used as a voltage stimulus, exocytosis was evoked by single APs, maintained throughout AP trains and modulated by AP frequency. Furthermore, feedback inhibition of the Ca²⁺ current ( I _Ca) by released vesicular protons reduced depression of exocytosis during AP trains. In the absence of K⁺ current inhibition, step depolarizations and AP waveforms evoked a rapidly activated outward current that was dependent on Ca²⁺ influx ( I _K(Ca)). I therefore investigated whether proton-mediated feedback inhibition of I _Ca affected the activation of I _K(Ca). A transient inhibition of I _K(Ca) was observed that was dependent on exocytosis, blocked by high-pH extracellular buffer, of similar magnitude to inhibition of I _Ca but occurred with a delay of 2.7 ms. In addition, the amplitude of APs evoked under current clamp was inhibited by the action of vesicular protons released by the APs. Protons released via exocytosis may therefore be a significant modulator of Ca²⁺-dependent currents and regenerative potentials in bipolar-cell terminals.     

Heterogeneity of Postsynaptic Receptor Occupancy Fluctuations among Glycinergic Inhibitory Synapses in the Zebrafish Hindbrain

The amplitude of glycinergic miniature inhibitory postsynaptic currents (mIPSCs) varies considerably in neurons recorded in the isolated hindbrain of 50-h-old zebrafish larvae. At this age, glycinergic synapses are functionally mature. In order to measure the occupancy level of postsynaptic glycine receptors (GlyRs) and to determine the pre- and/or postsynaptic origin of its variability, we analysed mIPSCs within bursts evoked by α-latrotoxin (0.1–1 n m ). Two types of burst were observed according to their mIPSC frequencies: 'slow' bursts with clearly spaced mIPSCs and 'fast' bursts characterised by superimposed events. Non-stationary noise analysis of mIPSCs in some 'slow' bursts recorded in the presence or in the absence of Ca²⁺ denoted that mIPSC amplitude variance did not depend on the quantity of neurotransmitters released (presynaptic origin), but rather on intrinsic stochastic behaviour of the same group of GlyRs (postsynaptic origin). In these bursts, the open probability measured at the peak of the mIPSCs was close to 0.5 while the maximum open probability is close to 0.9 for the synaptic isoform of GlyRs (heteromeric α1/β GlyRs). In 'fast' bursts with superimposed events, a correlation was found between the amplitude of mIPSCs and the basal current level measured at their onset, which could suggest that the same group of GlyRs is activated during such bursts. Altogether, our results indicate that glycine synapses can display different release modes in the presence of α-latrotoxin. They also indicate that, in our model, postsynaptic GlyRs cannot be saturated by the release of a single vesicle.     

Development of presynaptic inhibition onto retinal bipolar cell axon terminals is subclass-specific.

Synaptic integration is modulated by inhibition onto the dendrites of postsynaptic cells. However, presynaptic inhibition at axonal terminals also plays a critical role in the regulation of neurotransmission. In contrast to the development of inhibitory synapses onto dendrites, GABAergic/glycinergic synaptogenesis onto axon terminals has not been widely studied. Because retinal bipolar cells receive subclass-specific patterns of GABAergic and glycinergic presynaptic inhibition, they are a good model for studying the development of inhibition at axon terminals. Here, using whole cell recording methods and transgenic mice in which subclasses of retinal bipolar cells are labeled, we determined the temporal sequence and patterning of functional GABAergic and glycinergic input onto the major subclasses of bipolar cells. We found that the maturation of GABAergic and glycinergic synapses onto the axons of rod bipolar cells (RBCs), on-cone bipolar cells (ON-CBCs) and off-cone bipolar cells (OFF-CBCs) were temporally distinct: spontaneous chloride-mediated currents are present in RBCs earlier in development compared with ON- and OFF-CBC, and RBCs receive GABAergic and glycinergic input simultaneously, whereas in OFF-CBCs, glycinergic transmission emerges before GABAergic transmission. Because on-CBCs show little inhibitory activity, GABAergic and glycinergic events could not be pharmacologically distinguished for these bipolar cells. The balance of GABAergic and glycinergic input that is unique to RBCs and OFF-CBCs is established shortly after the onset of synapse formation and precedes visual experience. Our data suggest that presynaptic modulation of glutamate transmission from bipolar cells matures rapidly and is differentially coordinated for GABAergic and glycinergic synapses onto distinct bipolar cell subclasses.     

Adenosine inhibition via A1 receptor of N-type Ca2+ current and peptide release from isolated neurohypophysial terminals of the rat

Effects of adenosine on voltage-gated Ca²⁺ channel currents and on arginine vasopressin (AVP) and oxytocin (OT) release from isolated neurohypophysial (NH) terminals of the rat were investigated using perforated-patch clamp recordings and hormone-specific radioimmunoassays. Adenosine, but not adenosine 5'-triphosphate (ATP), dose-dependently and reversibly inhibited the transient component of the whole-terminal Ba²⁺ currents, with an IC₅₀ of 0.875 μ m. Adenosine strongly inhibited, in a dose-dependent manner (IC₅₀= 2.67 μ m ), depolarization-triggered AVP and OT release from isolated NH terminals. Adenosine and the N-type Ca²⁺ channel blocker ω-conotoxin GVIA, but not other Ca²⁺ channel-type antagonists, inhibited the same transient component of the Ba²⁺ current. Other components such as the L-, Q- and R-type channels, however, were insensitive to adenosine. Similarly, only adenosine and ω-conotoxin GVIA were able to inhibit the same component of AVP release. A₁ receptor agonists, but not other purinoceptor-type agonists, inhibited the same transient component of the Ba²⁺ current as adenosine. Furthermore, the A₁ receptor antagonist 8-cyclopentyltheophylline (CPT), but not the A₂ receptor antagonist 3, 7-dimethyl-1-propargylxanthine (DMPGX), reversed inhibition of this current component by adenosine. The inhibition of AVP and OT release also appeared to be via the A₁ receptor, since it was reversed by CPT. We therefore conclude that adenosine, acting via A₁ receptors, specifically blocks the terminal N-type Ca²⁺ channel thus leading to inhibition of the release of both AVP and OT.     

Mechanisms underlying short-term modulation of transmitter release by presynaptic depolarization

Presynaptic terminal depolarization modulates the efficacy of transmitter release. Residual Ca²⁺ remaining after presynaptic depolarization is thought to play a critical role in facilitation of transmitter release, but its downstream mechanism remains unclear. By making simultaneous pre- and postsynaptic recordings at the rodent calyx of Held synapse, we have investigated mechanisms involved in the facilitation and depression of postsynaptic currents induced by presynaptic depolarization. In voltage-clamp experiments, cancellation of the Ca²⁺-dependent presynaptic Ca²⁺ current ( I _pCa) facilitation revealed that this mechanism can account for 50% of postsynaptic current facilitation, irrespective of intraterminal EGTA concentrations. Intraterminal EGTA, loaded at 10 m m , failed to block postsynaptic current facilitation, but additional BAPTA at 1 m m abolished it. Potassium-induced sustained depolarization of non-dialysed presynaptic terminals caused a facilitation of postsynaptic currents, superimposed on a depression, with the latter resulting from reductions in presynaptic action potential amplitude and number of releasable vesicles. We conclude that presynaptic depolarization bidirectionally modulates transmitter release, and that the residual Ca²⁺ mechanism for synaptic facilitation operates in the immediate vicinity of voltage-gated Ca²⁺ channels in the nerve terminal.     

Dual and opposing roles of presynaptic Ca2+ influx for spontaneous GABA release from rat medial preoptic nerve terminals

Calcium influx into the presynaptic nerve terminal is well established as a trigger signal for transmitter release by exocytosis. By studying dissociated preoptic neurons with functional adhering nerve terminals, we here show that presynaptic Ca²⁺ influx plays dual and opposing roles in the control of spontaneous transmitter release. Thus, application of various Ca²⁺ channel blockers paradoxically increased the frequency of spontaneous (miniature) inhibitory GABA-mediated postsynaptic currents (mIPSCs). Similar effects on mIPSC frequency were recorded upon washout of Cd²⁺ or EGTA from the external solution. The results are explained by a model with parallel Ca²⁺ influx through channels coupled to the exocytotic machinery and through channels coupled to Ca²⁺-activated K⁺ channels at a distance from the release site.     

Voltage-dependent inactivation of l-type ca2+ Currents in Guinea-Pig Ventricular Myocytes

The objective of this study was to describe the kinetics of voltage-dependent inactivation of native cardiac L-type Ca²⁺ currents. Whole-cell currents were recorded from guinea-pig isolated ventricular myocytes. Voltage-dependent inactivation was separated from Ca²⁺-dependent inactivation by replacing extracellular Ca²⁺ with Mg²⁺ and recording outward currents through Ca²⁺ channels. Voltage-dependent inactivation accelerated from slow monophasic decay at −30 mV to maximal rapid biphasic decay at +20 mV. Maximal voltage-dependent inactivation occurred with τ_f≈30 ms and τ_s≈300 ms, the fast component of decay accounted for 70 % of the current amplitude. In basal conditions Ca²⁺ current availability was sigmoid. Isoproterenol (isoprenaline) evoked a large increase in a time-independent component of the Ca²⁺ current which also increased with depolarisation. This was responsible for the apparent recovery of Ca²⁺ channel current availability at positive membrane potentials and thus a U-shaped availability-voltage ( A-V) relationship. It is concluded that β-adrenergic stimulation altered the reaction of native cardiac L-type Ca²⁺ channels to membrane voltage. In basal conditions, voltage accelerated inactivation. In isoproterenol, voltage could also reduce inactivation.     

Calcium oscillations in interstitial cells of the rabbit urethra

Measurements were made (using fast confocal microscopy) of intracellular Ca²⁺ levels in fluo-4 loaded interstitial cells isolated from the rabbit urethra. These cells exhibited regular Ca²⁺ oscillations which were associated with spontaneous transient inward currents recorded under voltage clamp. Interference with d - myo -inositol 1,4,5-trisphosphate (IP₃) induced Ca²⁺ release using 100 μ m 2-aminoethoxydiphenyl borate, and the phospholipase C (PLC) inhibitors 2-nitro-4-carboxyphenyl N , N -diphenylcarbamate and U73122 decreased the amplitude of spontaneous oscillations but did not abolish them. However, oscillations were abolished when ryanodine receptors were blocked with tetracaine or ryanodine. Oscillations ceased in the absence of external Ca²⁺, and frequency was directly proportional to the external Ca²⁺ concentration. Frequency of Ca²⁺ oscillation was reduced by SKF-96365, but not by nifedipine. Lanthanum and cadmium completely blocked oscillations. These results suggest that Ca²⁺ oscillations in isolated rabbit urethral interstitial cells are initiated by Ca²⁺ release from ryanodine-sensitive intracellular stores, that oscillation frequency is very sensitive to the external Ca²⁺ concentration and that conversion of the primary oscillation to a propagated Ca²⁺ wave depends upon IP₃-induced Ca²⁺ release.     

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.     

Astrocytic control of synaptic NMDA receptors

Astrocytes express a wide range of G-protein coupled receptors that trigger release of intracellular Ca²⁺, including P2Y, bradykinin and protease activated receptors (PARs). By using the highly sensitive sniffer-patch technique, we demonstrate that the activation of P2Y receptors, bradykinin receptors and protease activated receptors all stimulate glutamate release from cultured or acutely dissociated astrocytes. Of these receptors, we have utilized PAR1 as a model system because of favourable pharmacological and molecular tools, its prominent expression in astrocytes and its high relevance to neuropathological processes. Astrocytic PAR1-mediated glutamate release in vitro is Ca²⁺ dependent and activates NMDA receptors on adjacent neurones in culture. Activation of astrocytic PAR1 in hippocampal slices induces an APV-sensitive inward current in CA1 neurones and causes APV-sensitive neuronal depolarization in CA1 neurones, consistent with release of glutamate from astrocytes. PAR1 activation enhances the NMDA receptor-mediated component of synaptic miniature EPSCs, evoked EPSCs and evoked EPSPs in a Mg²⁺-dependent manner, which may reflect spine head depolarization and consequent reduction of NMDA receptor Mg²⁺ block during subsequent synaptic currents. The release of glutamate from astrocytes following PAR1 activation may also lead to glutamate occupancy of some perisynaptic NMDA receptors, which pass current following relief of tonic Mg²⁺ block during synaptic depolarization. These results suggest that astrocytic G-protein coupled receptors that increase intracellular Ca²⁺ can tune synaptic NMDA receptor responses.     

Dendritic signals from rat hippocampal CA1 pyramidal neurons during coincident pre- and post-synaptic activity: a combined voltage- and calcium-imaging study

The non-linear and spatially inhomogeneous interactions of dendritic membrane potential signals that represent the first step in the induction of activity-dependent long-term synaptic plasticity are not fully understood, particularly in dendritic regions which are beyond the reach of electrode measurements. We combined voltage-sensitive-dye recordings and Ca²⁺ imaging of hippocampal CA1 pyramidal neurons to study large regions of the dendritic arbor, including branches of small diameter (distal apical and oblique dendrites). Dendritic membrane potential transients were monitored at high spatial resolution and correlated with supra-linear [Ca²⁺]_i changes during one cycle of a repetitive patterned stimulation protocol that typically results in the induction of long-term potentiation (LTP). While the increase in the peak membrane depolarization during coincident pre- and post-synaptic activity was required for the induction of supra-linear [Ca²⁺]_i signals shown to be necessary for LTP, the change in the baseline-to-peak amplitude of the backpropagating dendritic action potential (bAP) was not critical in this process. At different dendritic locations, the baseline-to-peak amplitude of the bAP could be either increased, decreased or unaltered at sites where EPSP–AP pairing evoked supra-linear summation of [Ca²⁺]_i transients. We suggest that modulations in the bAP baseline-to-peak amplitude by local EPSPs act as a mechanism that brings the membrane potential into the optimal range for Ca²⁺ influx through NMDA receptors (0 to −15 mV); this may require either boosting or the reduction of the bAP, depending on the initial size of both signals.     

Microheterogeneity of calcium signalling in dendrites

Transient changes in the intracellular concentration of free Ca²⁺ ([Ca²⁺]_i) originating from voltage- or ligand-gated influx and by ligand- or Ca²⁺-gated release from intracellular stores, trigger or modulate many fundamental neuronal processes, including neurotransmitter release and synaptic plasticity. Of the intracellular compartments involved in Ca²⁺ clearance, the endoplasmic reticulum (ER) has received the most attention because it expresses Ca²⁺ pumps and Ca²⁺ channels, thus endowing it with the potential to act as both an intracellular calcium sink and store. We review here our ongoing work on the role of calcium sequestration into, and release from, ER cisterns and the role that this plays in the generation and termination of free [Ca²⁺]_i transients in dendrites of pyramidal neurons in hippocampal slices during and after synaptic activity. These studies have been approached by combining parallel microfluorometric measurements of free cytosolic [Ca²⁺]_i transients with energy-dispersive X-ray microanalytical measurements of total Ca content within specific dendritic compartments at the electron microscopy level. Our observations support the emerging realization that specific subsets of dendritic ER cisterns provide spatial and temporal microheterogeneity of Ca²⁺ signalling, acting not only as a major intracellular Ca sink involved in active clearance mechanisms after voltage- and ligand-gated Ca²⁺ influx, but also as an intracellular Ca²⁺ source that can be mobilized by a signal cascade originating at activated synapses.