Phasic and tonic attenuation of EPSPs by inward rectifier K+ channels in rat hippocampal pyramidal cells

We made whole-cell recordings from CA1 pyramidal cells of hippocampal slices in combination with brief dendritic glutamate pulses to study the role of constitutive inwardly rectifying K⁺ channels (IRK, Kir2.0) and G-protein-activated inwardly rectifying K⁺ channels (GIRK, Kir3.0) in the processing of excitatory inputs. Phasic activation of GIRK channels by baclofen (20 μ m ) produced a reversible reduction of glutamate-evoked postsynaptic potentials (GPSPs), our equivalent of EPSPs, by about one-third. Conversely, tertiapin (30 n m ), a selective inhibitor of GIRK channels, and Ba²⁺ (200 μ m ), a non-selective blocker of inwardly rectifying K⁺ channels, enhanced GPSPs and, in voltage-clamp experiments, reduced the underlying K⁺ conductances, indicating a functionally significant background GIRK conductance, in addition to constitutive IRK channel activity. When examined after suppression of endogenous adenosinergic inhibition, using either adenosine deaminase or the selective A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine, tertiapin failed to influence either the GPSPs or the inwardly rectifying K⁺ conductance. Voltage-clamp recordings from acutely isolated CA1 pyramidal cells not exposed to ambient adenosine exhibited no response to tertiapin, whereas Ba²⁺ was still capable of reducing hyperpolarizing inward rectification. Our data indicate that in hippocampal pyramidal cells, two components of the inwardly rectifying K⁺ conductance can be identified, which together exert a tonic modulation of excitatory synaptic input: one arises from constitutive putative IRK channels, the other is mediated by the background activity of GIRK channels that results from the tonic activation of A1 receptors by ambient adenosine.     

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.     

Spike Ca2+ influx upmodulates the spike afterdepolarization and bursting via intracellular inhibition of KV7/M channels

In principal brain neurons, activation of Ca²⁺ channels during an action potential, or spike, causes Ca²⁺ entry into the cytosol within a millisecond. This in turn causes rapid activation of large conductance Ca²⁺-gated channels, which enhances repolarization and abbreviates the spike. Here we describe another remarkable consequence of spike Ca²⁺ entry: enhancement of the spike afterdepolarization. This action is also mediated by intracellular modulation of a particular class of K⁺ channels, namely by inhibition of K_V7 (KCNQ) channels. These channels generate the subthreshold, non-inactivating M-type K⁺ current, whose activation curtails the spike afterdepolarization. Inhibition of K_V7/M by spike Ca²⁺ entry allows the spike afterdepolarization to grow and can convert solitary spikes into high-frequency bursts of action potentials. Through this novel intracellular modulatory action, Ca²⁺ spike entry regulates the discharge mode and the signalling capacity of principal brain neurons.     

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.     

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.     

Indirect coupling between Cav1.2 channels and ryanodine receptors to generate Ca2+ sparks in murine arterial smooth muscle cells

In arterial vascular smooth muscle cells (VSMCs), Ca²⁺ sparks stimulate nearby Ca²⁺-activated K⁺ (BK) channels that hyperpolarize the membrane and close L-type Ca²⁺ channels. We tested the contribution of L-type Ca_v1.2 channels to Ca²⁺ spark regulation in tibial and cerebral artery VSMCs using VSMC-specific Ca_v1.2 channel gene disruption in (SMAKO) mice and an approach based on Poisson statistical analysis of activation frequency and first latency of elementary events. Ca_v1.2 channel gene inactivation reduced Ca²⁺ spark frequency and amplitude by ∼50% and ∼80%, respectively. These effects were associated with lower global cytosolic Ca²⁺ levels and reduced sarcoplasmic reticulum (SR) Ca²⁺ load. Elevating cytosolic Ca²⁺ levels reversed the effects completely. The activation frequency and first latency of elementary events in both wild-type and SMAKO VSMCs weakly reflected the voltage dependency of L-type channels. This study provides evidence that local and tight coupling between the Ca_v1.2 channels and ryanodine receptors (RyRs) is not required to initiate Ca²⁺ sparks. Instead, Ca_v1.2 channels contribute to global cytosolic [Ca²⁺], which in turn influences luminal SR calcium and thus Ca²⁺ sparks.     

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.     

Parallel decrease in ω-conotoxin-sensitive transmission and dopamine-induced inhibition at the striatal synapse of developing rats

Whole-cell patch-clamp recordings of GABAergic IPSCs were made from cholinergic interneurones in slices of striatum from developing rats aged 21-60 days postnatal. In addition, the Ca²⁺ channel subtypes involved in synaptic transmission, as well as dopamine (DA)-induced presynaptic inhibition, were investigated pharmacologically with development by bath application of Ca²⁺ channel blockers and DA receptor agonists. The IPSC amplitude was reduced by ω-conotoxin GVIA (ω-CgTX) or ω-agatoxin TK (ω-Aga-TK) across the whole age range, suggesting that multiple types of Ca²⁺ channels mediate transmission of the synapse. The IPSC fraction reduced by ω-CgTX significantly decreased, whereas that reduced by ω-Aga-TK remained unchanged with development. DA or quinpirole, a D₂-like receptor agonist, presynaptically reduced the IPSC amplitude throughout development. The DA-induced inhibition decreased with age in parallel with the decrease in N-type Ca²⁺ channels. DA showed no further inhibition of IPSCs after the inhibitory effect of ω-CgTX had reached steady state throughout development. These results demonstrate that there is a functional link between presynaptic N-type Ca²⁺ channels and D₂-like DA receptors at inhibitory synapses in the striatum. They also demonstrate that the suppression of GABAergic transmission by D₂-like receptors is mediated by modulation of N-type Ca²⁺ channels and decreases in parallel with the developmental decline in the contribution of N-type Ca²⁺ channels to exocytosis.     

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.     

Spike-independent release of ATP from Xenopus spinal neurons evoked by activation of glutamate receptors

As the release of ATP from neurons has only been directly studied in a few cases, we have used patch sniffing to examine ATP release from Xenopus spinal neurons. ATP release was detected following intracellular current injection to evoke spikes. However, spiking was not essential as both glutamate and NMDA could evoke release of ATP in the presence of TTX. Neither acetylcholine nor high K⁺ was effective at inducing ATP release in the presence of TTX. Although Cd²⁺ blocked glutamate-evoked release of ATP suggesting a dependence on Ca²⁺ entry, neither ω-conotoxin-GVIA nor nifedipine prevented ATP release. N-type and L-type channels are thus not essential for glutamate-evoked ATP release. That glutamate receptors can elicit release in the absence of spiking suggests a close physical relationship between these receptors, the Ca²⁺ channels and release sites. As the dependence of ATP release on the influx of Ca²⁺ through Ca²⁺ channel subtypes differs from that of synaptic transmitter release, ATP may be released from sites that are distinct from those of the principal transmitter. In addition to its role as a fast transmitter, ATP may thus be released as a consequence of the activation of excitatory glutamatergic synapses and act to signal information about activity patterns in the nervous system.     

Subthreshold inactivation of voltage-gated K+ channels modulates action potentials in neocortical bitufted interneurones from rats

Voltage-gated K⁺ channels perform many functions in integration of synaptic input and action potential (AP) generation. In this study we show that in bitufted interneurones from layer 2/3 of the somatosensory cortex, the height and width of APs recorded at the soma are sensitive to changes in the resting membrane potential, suggesting subthreshold activity of voltage-gated conductances. Attributes of K⁺ currents examined in nucleated patches revealed a fast subthreshold-inactivating K⁺ conductance (K_f) and a slow suprathreshold-inactivating K⁺ conductance (K_s). Simulations of these K⁺ conductances, incorporated into a Hodgkin–Huxley-type model, suggested that during a single AP or during low frequency trains of APs, subthreshold inactivation of K_f was the primary modulator of AP shape, whereas during trains of APs the shape was governed to a larger degree by K_s resulting in the generation of smaller and broader APs. Utilizing the facilitating function of unitary pyramidal-to-bitufted cell synaptic transmission, single back-propagating APs were initiated in a bitufted interneurone by repeated stimulation of a presynaptic pyramidal cell. Ca²⁺ imaging and dendritic whole-cell recordings revealed that modulation of APs, which also affect the shape of back-propagating APs, resulted in a change in dendritic Ca²⁺ influx. Compartmental simulation of the back-propagating AP suggested a mechanism for the modulation of the back-propagating AP height and width by subthreshold activation of K_f. We speculate that this signal may modulate retrograde GABA release and consequently depression of synaptic efficacy of excitatory input from neighbouring pyramidal neurones.     

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.     

Enhanced G protein-dependent modulation of excitatory synaptic transmission in the cerebellum of the Ca2+ channel-mutant mouse, tottering

Tottering , a mouse model for absence epilepsy and cerebellar ataxia, carries a mutation in the gene encoding class A (P/Q-type) Ca²⁺ channels, the dominant exocytotic Ca²⁺ channel at most synapses in the mammalian central nervous system. Comparing tottering to wild-type mice, we have studied glutamatergic transmission between parallel fibres and Purkinje cells in cerebellar slices. Results from biochemical assays and electrical field recordings demonstrate that glutamate release from parallel fibre terminals of the tottering mouse is controlled largely by class B Ca²⁺ channels (N-type), in contrast to the P/Q-channels that dominate release from wild-type terminals. Since N-channels, in a variety of assays, are more effectively inhibited by G proteins than are P/Q-channels, we tested whether synaptic transmission between parallel fibres and Purkinje cells in tottering mice was more susceptible to inhibitory modulation by G protein-coupled receptors than in their wild-type counterparts. GABA_B receptors and α₂-adrenergic receptors (activated by bath application of transmitters) produced a three- to fivefold more potent inhibition of transmission in tottering than in wild-type synapses. This increased modulation is likely to be important for cerebellar transmission in vivo , since heterosynaptic depression, produced by activating GABAergic interneurones, greatly prolonged GABA_B receptor-mediated presynaptic inhibition in tottering as compared to wild-type slices. We propose that this enhanced modulation shifts the balance of synaptic input to Purkinje cells in favour of inhibition, reducing Purkinje cell output from the cerebellum, and may contribute to the aberrant motor phenotype that is characteristic of this mutant animal.     

mGluR1/5 subtype-specific calcium signalling and induction of long-term potentiation in rat hippocampal oriens/alveus interneurones

Hippocampal inhibitory interneurones demonstrate pathway- and synapse-specific rules of transmission and plasticity, which are key determinants of their role in controlling pyramidal cell excitability. Mechanisms underlying long-term changes at interneurone excitatory synapses, despite their importance, remain largely unknown. We use two-photon calcium imaging and whole-cell recordings to determine the Ca²⁺ signalling mechanisms linked specifically to group I metabotropic glutamate receptors (mGluR1α and mGluR5) and their role in hebbian long-term potentiation (LTP) in oriens/alveus (O/A) interneurones. We demonstrate that mGluR1α activation elicits dendritic Ca²⁺ signals resulting from Ca²⁺ influx via transient receptor potential (TRP) channels and Ca²⁺ release from intracellular stores. By contrast, mGluR5 activation produces dendritic Ca²⁺ transients mediated exclusively by intracellular Ca²⁺ release. Using Western blot analysis and immunocytochemistry, we show mGluR1α-specific extracellular signal-regulated kinase (ERK1/2) activation via Src in CA1 hippocampus and, in particular, in O/A interneurones. Moreover, we find that mGluR1α/TRP Ca²⁺ signals in interneurone dendrites are dependent on activation of the Src/ERK cascade. Finally, this mGluR1α-specific Ca²⁺ signalling controls LTP at interneurone synapses since blocking either TRP channels or Src/ERK and intracellular Ca²⁺ release prevents LTP induction. Thus, our findings uncover a novel molecular mechanism of interneurone-specific Ca²⁺ signalling, critical in regulating synaptic excitability in hippocampal networks.     

Group I metabotropic glutamate receptors activate a calcium-sensitive transient receptor potential-like conductance in rat hippocampus

In CA3 pyramidal neurons from organotypic slice cultures, activation of G_q-coupled group I metabotropic glutamate receptors (mGluRs) induces a non-selective cationic conductance that enhances excitability. We have found that this response shares several properties with conductances that are mediated by the transient receptor potential (TRP) family of ion channels, including inhibition by La³⁺, 2-aminoethoxydiphenylborane (2APB), cis - N- (2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL 12,330A) and a doubly rectifying current-voltage relationship. Stimulation of mGluR1 and mGluR5 converged to activate the TRP-like conductance in a synergistic manner, and activation of either subtype alone produced only a fraction of the normal response. Activation of the cationic current required elevated intracellular Ca²⁺. Chelating intracellular Ca²⁺ or blocking Ca²⁺ entry through voltage-gated Ca²⁺ channels attenuated responses to the activation of mGluRs. Conversely, raising intracellular Ca²⁺ potentiated mGluR activation of the TRP-like conductance. Under control conditions, blocking G protein activation using intracellular GDPβS with or without N -(2, 6-dimethylphenylcarbamoylmethyl) triethylammonium chloride (QX-314) prevented mGluR-mediated activation of the TRP-like conductance. Following G protein blockade, however, the coupling between mGluRs 1 and/or 5 and the TRP-like conductance was rescued by increasing intracellular Ca²⁺. This suggests that a G protein-independent signalling pathway is also activated by group I mGluRs. Such a pathway may represent an alternative transduction mechanism to maintain metabotropic responses under conditions where G proteins are functionally uncoupled from their cognate receptors.     

Short-term potentiation of mEPSCs requires N-, P/Q- and L-type Ca2+ channels and mitochondria in the supraoptic nucleus

The glutamatergic synapses of the supraoptic nucleus display a unique activity-dependent plasticity characterized by a barrage of tetrodotoxin-resistant miniature EPSCs (mEPSCs) persisting for 5–20 min, causing postsynaptic excitation. We investigated how this short-term synaptic potentiation (STP) induced by a brief high-frequency stimulation (HFS) of afferents was initiated and maintained without lingering presynaptic firing, using in vitro patch-clamp recording on rat brain slices. We found that following the immediate rise in mEPSC frequency, STP decayed with two-exponential functions indicative of two discrete phases. STP depends entirely on extracellular Ca²⁺ which enters the presynaptic terminals through voltage-gated Ca²⁺ channels but also, to a much lesser degree, through a pathway independent of these channels or reverse mode of the plasma membrane Na⁺–Ca²⁺ exchanger. Initiation of STP is largely mediated by any of the N-, P/Q- or L-type channels, and only a simultaneous application of specific blockers for all these channels attenuates STP. Furthermore, the second phase of STP is curtailed by the inhibition of mitochondrial Ca²⁺ uptake or mitochondrial Na⁺–Ca²⁺ exchanger. mEPSCs amplitude is also potentiated by HFS which requires extracellular Ca²⁺. In conclusion, induction of mEPSC-STP is redundantly mediated by presynaptic N-, P/Q- and L-type Ca²⁺ channels while the second phase depends on mitochondrial Ca²⁺ sequestration and release. Since glutamate influences unique firing patterns that optimize hormone release by supraoptic magnocellular neurons, a prolonged barrage of spontaneous excitatory transmission may aid in the induction of respective firing activities.     

Regulation of synaptic signalling by postsynaptic, non-glutamate receptor ion channels

Activation of glutamatergic synapses onto pyramidal neurons produces a synaptic depolarization as well as a buildup of intracellular calcium (Ca²⁺). The synaptic depolarization propagates through the dendritic arbor and can be detected at the soma with a recording electrode. Current influx through AMPA-type glutamate receptors (AMPARs) provides the depolarizing drive, and the amplitudes of synaptic potentials are generally thought to reflect the number and properties of these receptors at each synapse. In contrast, synaptically evoked Ca²⁺ transients are limited to the spine containing the active synapse and result primarily from Ca²⁺ influx through NMDA-type glutamate receptors (NMDARs). Here we review recent studies that reveal that both synaptic depolarizations and spine head Ca²⁺ transients are strongly regulated by the activity of postsynaptic, non-glutamate receptor ion channels. In hippocampal pyramidal neurons, voltage- and Ca²⁺-gated ion channels located in dendritic spines open as downstream consequences of glutamate receptor activation and act within a complex signalling loop that feeds back to regulate synaptic signals. Dynamic regulation of these ion channels offers a powerful mechanism of synaptic plasticity that is independent of direct modulation of glutamate receptors.     

Discrete store-operated calcium influx into an intracellular compartment in rabbit arteriolar smooth muscle

This study tested the hypothesis that store-operated channels (SOCs) exist as a discrete population of Ca²⁺ channels activated by depletion of intracellular Ca²⁺ stores in cerebral arteriolar smooth muscle cells and explored their direct contractile function. Using the Ca²⁺ indicator fura-PE3 it was observed that depletion of sarcoplasmic reticulum (SR) Ca²⁺ by inhibition of SR Ca²⁺-ATPase (SERCA) led to sustained elevation of [Ca²⁺]_i that depended on extracellular Ca²⁺ and slightly enhanced Mn²⁺ entry. Enhanced background Ca²⁺ influx did not explain the raised [Ca²⁺]_i in response to SERCA inhibitors because it had marked gadolinium (Gd³⁺) sensitivity, which background pathways did not. Effects were not secondary to changes in membrane potential. Thus SR Ca²⁺ depletion activated SOCs. Strikingly, SOC-mediated Ca²⁺ influx did not evoke constriction of the arterioles, which were in a resting state. This was despite the fura-PE3-indicated [Ca²⁺]_i rise being greater than that evoked by 20 m m [K⁺]_o (which did cause constriction). Release of endothelial vasodilators did not explain the absence of SOC-mediated constriction, nor did a change in Ca²⁺ sensitivity of the contractile proteins. We suggest SOCs are a discrete subset of Ca²⁺ channels allowing Ca²⁺ influx into a 'non-contractile' compartment in cerebral arteriolar smooth muscle cells.