Cav1.2 calcium channels modulate the spiking pattern of hippocampal pyramidal cells

Ca(v)1.2 L-type calcium channels support hippocampal synaptic plasticity, likely by facilitating dendritic Ca2+ influx evoked by action potentials (AP) back-propagated from the soma. Ca2+ influx into hippocampal neurons during somatic APs is sufficient to activate signalling pathways associated with late phase LTP. Thus, mechanisms controlling AP firing of hippocampal neurons are of major functional relevance. We examined the excitability of CA1 pyramidal cells using somatic current-clamp recordings in brain slices from control type mice and mice with the Ca(v)1.2 gene inactivated in principal hippocampal neurons. Lack of the Ca(v)1.2 protein did not affect either affect basic characteristics, such as resting membrane potential and input resistance, or parameters of single action potentials (AP) induced by 5 ms depolarising current pulses. However, CA1 hippocampal neurons from control and mutant mice differed in their patterns of AP firing during 500 ms depolarising current pulses: threshold voltage for repetitive firing was shifted significantly by about 5 mV to more depolarised potentials in the mutant mice (p<0.01), and the latency until firing of the first AP was prolonged (73.2+/-6.6 ms versus 48.1+/- 7.8 ms in control; p<0.05). CA1 pyramidal cells from the mutant mice also showed a lowered initial spiking frequency within an AP train. In control cells, isradipine had matching effects, while BayK 8644 facilitated spiking. Our data demonstrate that Ca(v)1.2 channels are involved in regulating the intrinsic excitability of CA1 pyramidal neurons. This cellular mechanism may contribute to the known function of Ca(v)1.2 channels in supporting synaptic plasticity and memory.     

Activation of Nuclear Calcium Dynamics by Synaptic Stimulation in Cultured Cortical Neurons

L-type voltage-sensitive Ca2+ channels (VSCCs) are enriched on the neuronal soma and trigger gene expression during synaptic activity. To understand better how these channels regulate somatic and nuclear Ca2+ dynamics, we have investigated Ca2+ influx through L-type VSCCs following synaptic stimulation, using the long-wavelength Ca2+ indicator fluo-3 combined with laser scanning confocal microscopy. Single synaptic stimuli resulted in rapid Ca2+ transients in somatic cytoplasmic compartments (<5 ms rise time). Nuclear Ca2+ elevations lagged behind cytoplasmic levels by approximately 60 ms, consistent with a dependence on diffusion from a cytoplasmic source. Pharmacological experiments indicated that L-type VSCCs mediated approximately 50% of the nuclear and somatic (cytoplasmic) Ca2+ elevation in response to strong synaptic stimulation. In contrast, relatively weak excitatory postsynaptic potentials (EPSPs; approximately 15 mV) or single action potentials were much less effective at activating L-type VSCCs. Antagonist experiments indicated that activation of the NMDA-type glutamate receptor leads to a long-lasting somatic depolarization necessary to activate L-type VSCCs effectively during synaptic stimuli. Simulation of action potential and somatic EPSP depolarization using voltage-clamp pulses indicated that nuclear Ca2+ transients mediated by L-type VSCCs were produced by sustained depolarization positive to -25 mV. In the absence of synaptic stimulation, action potential stimulation alone led to elevations in nuclear Ca2+ mediated by predominantly non-L-type VSCCs. Our results suggest that action potentials, in combination with long-lived synaptic depolarizations, facilitate the activation of L-type VSCCs. This activity elevates somatic Ca2+ levels that spread to the nucleus.     

Action of hypoxia on different types of calcium channels in hippocampal neurons

Whole-cell patch clamp and polarographic oxygen partial pressure (pO2) measurements were used to establish the sensitivity of high-voltage-activated (HVA) Ca2+ channel subtypes of CA1 hippocampal neurons of rats to hypoxic conditions. Decrease of pO2 to 15-30 mm Hg induced a potentiation of HVA Ca2+ currents by 94%. Using selective blockers of N- and L-types of calcium channels, we found that inhibition of L-type channels decreased the effect by 54%, whereas N-type blocker attenuated the effect by 30%. Taking into account the ratio of currents mediated by these channel subtypes in CA1 hippocampal neurons, we concluded that both types of HVA Ca2+ channels are sensitive to hypoxia, however, L-type was about 3.5 times more sensitive to oxygen reduction.     

The GABAB1b isoform mediates long-lasting inhibition of dendritic Ca2+ spikes in layer 5 somatosensory pyramidal neurons

The apical tuft of layer 5 pyramidal neurons is innervated by a large number of inhibitory inputs with unknown functions. Here, we studied the functional consequences and underlying molecular mechanisms of apical inhibition on dendritic spike activity. Extracellular stimulation of layer 1, during blockade of glutamatergic transmission, inhibited the dendritic Ca2+ spike for up to 400 ms. Activation of metabotropic GABAB receptors was responsible for a gradual and long-lasting inhibitory effect, whereas GABAA receptors mediated a short-lasting (approximately 150 ms) inhibition. Our results suggest that the mechanism underlying the GABAB inhibition of Ca2+ spikes involves direct blockade of dendritic Ca2+ channels. By using knockout mice for the two predominant GABAB1 isoforms, GABAB1a and GABAB1b, we showed that postsynaptic inhibition of Ca2+ spikes is mediated by GABAB1b, whereas presynaptic inhibition of GABA release is mediated by GABAB1a. We conclude that the molecular subtypes of GABAB receptors play strategically different physiological roles in neocortical neurons.     

Autocrine regulation of calcium influx and gonadotropin-releasing hormone secretion in hypothalamic neurons.

Gonadotropin-releasing hormone (GnRH) receptors are expressed in hypothalamic tissues from adult rats, cultured fetal hypothalamic cells, and immortalized GnRH-secreting neurons (GT1 cells). Their activation by GnRH agonists leads to an overall increase in the extracellular Ca2+-dependent pulsatile release of GnRH. Electrophysiological studies showed that GT1 cells exhibit spontaneous, extracellular Ca2+-dependent action potentials, and that their inward currents include Na+, T-type and L-type Ca2+ components. Several types of potassium channels, including apamin-sensitive Ca2+-controlled potassium (SK) channels, are also expressed in GT1 cells. Activation of GnRH receptors leads to biphasic changes in intracellular Ca2+ concentration ([Ca2+]i), with an early and extracellular Ca2+-independent peak and a sustained and extracellular Ca2+-dependent plateau phase. During the peak [Ca2+]i response, electrical activity is abolished due to transient hyperpolarization that is mediated by SK channels. This is followed by sustained depolarization and resumption of firing with increased spike frequency and duration. The agonist-induced depolarization and increased firing are independent of [Ca2+]i and are not mediated by inhibition of K+ currents, but by facilitation of a voltage-insensitive and store depletion-activated Ca2+-conducting inward current. The dual control of pacemaker activity by SK and store depletion-activated Ca2+ channels facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process accounts for the autoregulatory action of GnRH on its release from hypothalamic neurons.     

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels

An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca(2+)-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni(2+)-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca(2+)-independent peak and a sustained, extracellular Ca(2+)-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.     

A post-burst after depolarization is mediated by group i metabotropic glutamate receptor-dependent upregulation of Ca(v)2.3 R-type calcium channels in CA1 pyramidal neurons

Activation of group I metabotropic glutamate receptors (subtypes mGluR1 and mGluR5) regulates neural activity in a variety of ways. In CA1 pyramidal neurons, activation of group I mGluRs eliminates the post-burst afterhyperpolarization (AHP) and produces an afterdepolarization (ADP) in its place. Here we show that upregulation of Ca(v)2.3 R-type calcium channels is responsible for a component of the ADP lasting several hundred milliseconds. This medium-duration ADP is rapidly and reversibly induced by activation of mGluR5 and requires activation of phospholipase C (PLC) and release of calcium from internal stores. Effects of mGluR activation on subthreshold membrane potential changes are negligible but are large following action potential firing. Furthermore, the medium ADP exhibits a biphasic activity dependence consisting of short-term facilitation and longer-term inhibition. These findings suggest that mGluRs may dramatically alter the firing of CA1 pyramidal neurons via a complex, activity-dependent modulation of Ca(v)2.3 R-type channels that are activated during spiking at physiologically relevant rates and patterns.     

Single-cell optogenetic excitation drives homeostatic synaptic depression

Homeostatic processes have been proposed to explain the discrepancy between the dynamics of synaptic plasticity and the stability of brain function. Forms of synaptic plasticity such as long-term potentiation alter synaptic activity in a synapse- and cell-specific fashion. Although network-wide excitation triggers compensatory homeostatic changes, it is unknown whether neurons initiate homeostatic synaptic changes in response to cell-autonomous increases in excitation. Here we employ optogenetic tools to cell-autonomously excite CA1 pyramidal neurons and find that a compensatory postsynaptic depression of both AMPAR and NMDAR function results. Elevated calcium influx through L-type calcium channels leads to activation of a pathway involving CaM kinase kinase and CaM kinase 4 that induces synaptic depression of AMPAR and NMDAR responses. The synaptic depression of AMPARs but not of NMDARs requires protein synthesis and the GluA2 AMPAR subunit, indicating that downstream of CaM kinase activation divergent pathways regulate homeostatic AMPAR and NMDAR depression.     

Dynamics of neuronal activity in the lateral preoptic area of hypothalamus in the course of sleep-waking cycle

Frequency and patterns of activity of 106 neurons in the lateral preoptic area of unanesthetized cats were studied under conditions of indolent head fixation. It was shown that this structure contains two somnogenic neuronal populations with different functions. Neurons increasing their discharge frequency during transition from active to quiet wakefulness and subsequent sleep development to the point of phasic stage of paradoxical sleep development are considered as elements of an anti-waking system, which is involved in the mechanisms of sleep onset and deepening by means of inactivation of the arousal system. Neurons displaying the highest firing rates during light slow-wave sleep and synchronization of discharges with sleep spindles are considered as elements of a slow-wave sleep network.     

CRH-induced electrical activity and calcium signalling in pituitary corticotrophs

Pituitary corticotroph cells generate repetitive action potentials and associated Ca2+ transients in response to the agonist corticotropin releasing hormone (CRH). There is indirect evidence suggesting that the agonist, by way of complex intracellular mechanisms, modulates the voltage sensitivity of the L-type Ca2+ channels embedded in the plasma membrane. We have previously constructed a Hodgkin-Huxley-type model of this process, which indicated that an increase in the L-type Ca2+ current is sufficient to generate repetitive action potentials (LeBeau et al. (1997). Biophys. J.73, 1263-1275). CRH is also believed to inhibit an inwardly rectifying K+ current. In this paper, we have found that a CRH-induced inhibition of the inwardly rectifying K+ current increases the model action potential firing frequency, [Ca2+]i transients and membrane excitability. This dual modulatory action of CRH on inward rectifier and voltage-gated Ca2+ channels better describes the observed CRH-induced effects. This structural alteration to the model along with parameter changes bring the model firing frequency in line with experimental data. We also show that the model exhibits experimentally observed bursting behaviour, where the depolarization spike is followed by small oscillations in the membrane potential.     

Ca2+-induced Ca2+ release supports the relay mode of activity in thalamocortical cells

Ca2+ ions play an important role during rhythmic bursting of thalamocortical neurons within sleep. The function of Ca2+ during the tonic relay mode of these neurons during wakefulness is less clear. Here, we report that tonic activity in thalamocortical cells results in an increase in the intracellular Ca2+ concentration and subsequent release of Ca2+ from intracellular stores mediated via ryanodine receptors (RyRs). Blockade of Ca2+ release shifted the regular firing of single action potentials toward the generation of spike clusters. Regular spike firing and intracellular Ca2+ release thus appear to be functionally coupled in a positive feedback manner, thereby supporting the relay mode of thalamocortical cells during wakefulness. Regulatory influences may be coupled to this system via the cyclic ADP ribose pathway.     

Cholinergic input uncouples Ca2+ changes from K+ conductance activation and amplifies intradendritic Ca2+ changes in hippocampal neurons

Muscarinic synaptic activation is known to be involved in cortical arousal as well as learning. Although simple increases in the electrical responsiveness of neurons might be the basis of arousal, the linkage of muscarinic transmission to the synaptic plasticity that might underlie learning is lacking. Most models of synaptic plasticity involve postsynaptic Ca2+ changes as a trigger for subsequent processes. We imaged muscarinic effects on free Ca2+ accumulation during intracellular recordings from CA3 pyramidal neurons in the guinea pig hippocampal slice. Muscarinic activation, either by repetitive stimulation of cholinergic fibers or by bath-applied carbachol, strongly increased intradendritic Ca2+ accumulation during directly evoked repetitive firing, in part by blocking a Ca(2+)-dependent K+ conductance. The effects of repetitive stimulation of cholinergic fibers were enhanced by the acetylcholine-esterase blocker eserine and blocked by the muscarinic antagonist atropine. These findings demonstrate a novel muscarinic reinforcement of Ca2+ changes during excitation, which are probably significant for synapse modification.     

Specific enhancement of SK channel activity selectively potentiates the afterhyperpolarizing current I(AHP) and modulates the firing properties of hippocampal pyramidal neurons

SK channels are Ca2+-activated K+ channels that underlie after hyperpolarizing (AHP) currents and contribute to the shaping of the firing patterns and regulation of Ca2+ influx in a variety of neurons. The elucidation of SK channel function has recently benefited from the discovery of SK channel enhancers, the prototype of which is 1-EBIO. 1-EBIO exerts profound effects on neuronal excitability but displays a low potency and limited selectivity. This study reports the effects of DCEBIO, an intermediate conductance Ca2+-activated K+ channel modulator, and the effects of the recently identified potent SK channel enhancer NS309 on recombinant SK2 channels, neuronal apamin-sensitive AHP currents, and the excitability of CA1 neurons. NS309 and DCEBIO increased the amplitude and duration of the apamin-sensitive afterhyperpolarizing current without affecting the slow afterhyperpolarizing current in contrast to 1-EBIO. The potentiation by DCEBIO and NS309 was reversed by SK channel blockers. In current clamp experiments, NS309 enhanced the medium afterhyperpolarization (but not the slow afterhyperpolarization sAHP) and profoundly affected excitability by facilitating spike frequency adaptation in a frequency-independent manner. The potent and specific effect of NS309 on the excitability of CA1 pyramidal neurons makes this compound an ideal tool to assess the role of SK channels as possible targets for the treatment of disorders linked to neuronal hyperexcitability.     

Nonlinear regulation of unitary synaptic signals by CaV(2.3) voltage-sensitive calcium channels located in dendritic spines

The roles of voltage-sensitive sodium (Na) and calcium (Ca) channels located on dendrites and spines in regulating synaptic signals are largely unknown. Here we use 2-photon glutamate uncaging to stimulate individual spines while monitoring uncaging-evoked excitatory postsynaptic potentials (uEPSPs) and Ca transients. We find that, in CA1 pyramidal neurons in acute mouse hippocampal slices, CaV(2.3) voltage-sensitive Ca channels (VSCCs) are found selectively on spines and act locally to dampen uncaging-evoked Ca transients and somatic potentials. These effects are mediated by a regulatory loop that requires opening of CaV(2.3) channels, voltage-gated Na channels, small conductance Ca-activated potassium (SK) channels, and NMDA receptors. Ca influx through CaV(2.3) VSCCs selectively activates SK channels, revealing the presence of functional Ca microdomains within the spine. Our results suggest that synaptic strength can be modulated by mechanisms that regulate voltage-gated conductances within the spine but do not alter the properties or numbers of synaptic glutamate receptors.     

Voltage-dependent burst-to-tonic switching of thalamic cell activity: an in vitro study

The electroresponsiveness of mammalian thalamic neurons was studied in a slice preparation of the guinea pig diencephalon. Although the morphology of the cells varied, their electroresponsive properties were the same. Stimulation of thalamic cells at a membrane potential more negative than--60 mV produced burst responses and stimulation of more depolarized levels produced tonic firing of fast spikes. The burst response is generated by an inactivating Ca++-conductance. It is seen as a slow Ca++-spike which in turn triggers fast Na+-spikes. The Ca++-conductance is deinactivated by hyperpolarization beyond--60 mV. The membranes of thalamic neurons contain a number of other conductances including a Ca++-dependent K+-conductance producing spike afterhyperpolarization and a non-inactivating Na+-conductance which plays an important role during tonic activity of the cells. The early part of a response to a long-lasting stimulus given at rest or at a hyperpolarized level is dominated by the burst and thus is is independent of the stimulus amplitude. During the late part of such a response the firing rate is highly dependent of the stimulus intensity. Current-frequency plots for the first inter-spike intervals after the burst during long stimuli are upward convex, but after "steady-state" is reached the plots are almost linear.     

Inositol hexakisphosphate increases L-type Ca2+ channel activity by stimulation of adenylyl cyclase.

Inositol hexakisphosphate (InsP6) is a most abundant inositol polyphosphate that changes simultaneously with inositol 1,4,5-trisphosphate in depolarized neurons. However, the role of InsP6 in neuronal signaling is unknown. Mass assay reveals that the basal levels of InsP6 in several brain regions tested are similar. InsP6 mass is significantly elevated in activated brain neurons and lowered by inhibition of neuronal activity. Furthermore, the hippocampus is most sensitive to electrical challenge with regard to percentage accumulation of InsP6. In hippocampal neurons, InsP6 stimulates adenylyl cyclase (AC) without influencing cAMP phosphodiesterases, resulting in activation of protein kinase A (PKA) and thereby selective enhancement of voltage-gated L-type Ca2+ channel activity. This enhancement was abolished by preincubation with PKA and AC inhibitors. These data suggest that InsP6 increases L-type Ca2+ channel activity by facilitating phosphorylation of PKA phosphorylation sites. Thus, in hippocampal neurons, InsP6 serves as an important signal in modulation of voltage-gated L-type Ca2+ channel activity.     

Long lasting inhibition of adenylyl cyclase selectively mediated by inositol 1,4,5-trisphosphate-evoked calcium release

In A7r5 smooth muscle cells, vasopressin stimulates release of Ca2+ from intracellular stores and Ca2+ entry, and it inhibits adenylyl cyclase (AC) activity. Inhibition of AC is prevented by inhibition of phospholipase C or when the increase in cytosolic [Ca2+] is prevented by the Ca2+ buffer, BAPTA. It is unaffected by pertussis toxin, inhibition of protein kinase C, or L-type Ca2+ channels or by removal of extracellular Ca2+. The independence of extracellular Ca2+ occurs despite inhibition of AC by vasopressin persisting for at least 15 min, whereas the cytosolic [Ca2+] returns to its basal level within 1-2 min in Ca2+-free medium. Although capacitative Ca2+ entry (CCE), activated by emptying stores with thapsigargin, inhibits AC, Ca2+ entry via CCE or L-type Ca2+ channels activated by vasopressin is ineffective. Temporally separating vasopressin-evoked Ca2+ release from the assessment of AC activity revealed that the transient Ca2+ signal resulting from Ca2+ mobilization causes a long lasting inhibition of AC. By contrast, inhibition of AC by thapsigargin-evoked CCE reverses rapidly after removal of extracellular Ca2+. Inhibition of AC by vasopressin is prevented by inhibition of Ca2+-calmodulin-dependent protein kinase II. We conclude that persistent inhibition of AC (probably AC-3) by vasopressin is mediated by inositol trisphosphate-evoked Ca2+ release causing activation of Ca2+-calmodulin-dependent protein kinase II. Our results establish that an important interaction between two ubiquitous signaling pathways is tuned selectively to Ca2+ release via inositol trisphosphate receptors and that the interaction transduces a transient Ca2+ signal into a long lasting inhibition of AC.     

Feedforward excitation and inhibition evoke dual modes of firing in the cat's visual thalamus during naturalistic viewing

Thalamic relay cells transmit information from retina to cortex by firing either rapid bursts or tonic trains of spikes. Bursts occur when the membrane voltage is low, as during sleep, because they depend on channels that cannot respond to excitatory input unless they are primed by strong hyperpolarization. Cells fire tonically when depolarized, as during waking. Thus, mode of firing is usually associated with behavioral state. Growing evidence, however, suggests that sensory processing involves both burst and tonic spikes. To ask if visually evoked synaptic responses induce each type of firing, we recorded intracellular responses to natural movies from relay cells and developed methods to map the receptive fields of the excitation and inhibition that the images evoked. In addition to tonic spikes, the movies routinely elicited lasting inhibition from the center of the receptive field that permitted bursts to fire. Therefore, naturally evoked patterns of synaptic input engage dual modes of firing.     

Impact of intrinsic properties and synaptic factors on the activity of neocortical networks in vivo.

To investigate the relative impact of intrinsic and synaptic factors in the maintenance of the membrane potential of cat neocortical neurons in various states of the network, we performed intracellular recordings in vivo. Experiments were done in the intact cortex and in isolated neocortical slabs of anesthetized animals, and in naturally sleeping and awake cats. There are at least four different electrophysiological cell classes in the neocortex. The responses of different neuronal classes to direct depolarization result in significantly different responses in postsynaptic cells. The activity patterns observed in the intact cortex of anesthetized cats depended mostly on the type of anesthesia. The intracellular activity in small neocortical slabs was composed of silent periods, lasting for tens of seconds, during which only small depolarizing potentials (SDPs, presumed miniature synaptic potentials) were present, and relatively short-lasting (a few hundred milliseconds) active periods. Our data suggest that minis might be amplified by intrinsically-bursting neurons and that the persistent Na+ current brings neurons to firing threshold, thus triggering active periods. The active periods in neurons were composed of the summation of synaptic events and intrinsic depolarizing currents. In chronically-implanted cats, slow-wave sleep was characterized by active (depolarizing) and silent (hyperpolarizing) periods. The silent periods were absent in awake cats. We propose that both intrinsic and synaptic factors are responsible for the transition from silent to active states found in naturally sleeping cats and that synaptic depression might be responsible for the termination of active states during sleep. In view of the unexpected high firing rates of neocortical neurons during the depolarizing epochs in slow-wave sleep, we suggest that cortical neurons are implicated in short-term plasticity processes during this state, in which the brain is disconnected from the outside world, and that memory traces acquired during wakefulness may be consolidated during sleep.     

Sleepy dialogues between cortex and hippocampus: who talks to whom?

During NREM sleep, neocortical neurons undergo near-synchronous transitions, every second or so, between UP states, during which they are depolarized and fire actively, and DOWN states, during which they are hyperpolarized and completely silent. In this issue of Neuron, Isomura et al. report that slow oscillations of membrane potential occur near-synchronously not only in neocortex but also in entorhinal cortex and subiculum. Within the hippocampus proper, pyramidal neurons lack the bistability of UP and DOWN states, but their firing is strongly modulated by cortical activity during the UP state. Intriguingly, many hippocampal neurons fire during the cortical DOWN state. Thus, during sleep UP states, the cortex can talk to the hippocampus, but it is unclear whether the hippocampus talks back.