Synapsin II Regulation of GABAergic Synaptic Transmission Is Dependent on Interneuron Subtype.

Synapsins are epilepsy susceptibility genes that encode phosphoproteins reversibly associated with synaptic vesicles. Synapsin II (SynII) gene deletion produces a deficit in inhibitory synaptic transmission, and this defect is thought to cause epileptic activity. We systematically investigated how SynII affects synchronous and asynchronous release components of inhibitory transmission in the CA1 region of the mouse hippocampus. We found that the asynchronous GABAergic release component is diminished in SynII-deleted (SynII(-)) slices. To investigate this defect at different interneuron subtypes, we selectively blocked either N-type or P/Q-type Ca2+ channels. SynII deletion suppressed the asynchronous release component at synapses dependent on N-type Ca2+ channels but not at synapses dependent on P/Q-type Ca2+ channels. We then performed paired double-patch recordings from inhibitory basket interneurons connected to pyramidal neurons and used cluster analysis to classify interneurons according to their spiking and synaptic parameters. We identified two cell subtypes, presumably parvalbumin (PV) and cholecystokinin (CCK) expressing basket interneurons. To validate our interneuron classification, we took advantage of transgenic animals with fluorescently labeled PV interneurons and confirmed that their spiking and synaptic parameters matched the parameters of presumed PV cells identified by the cluster analysis. The analysis of the release time course at the two interneuron subtypes demonstrated that the asynchronous release component was selectively reduced at SynII(-) CCK interneurons. In contrast, the transmission was desynchronized at SynII(-) PV interneurons. Together, our results demonstrate that SynII regulates the time course of GABAergic release, and that this SynII function is dependent on the interneuron subtype.SIGNIFICANCE STATEMENT Deletion of the neuronal protein synapsin II (SynII) leads to the development of epilepsy, probably due to impairments in inhibitory synaptic transmission. We systematically investigated SynII function at different subtypes of inhibitory neurons in the hippocampus. We discovered that SynII affects the time course of GABA release, and that this effect is interneuron subtype specific. Within one of the subtypes, SynII deficiency synchronizes the release and suppresses the asynchronous release component, while at the other subtype SynII deficiency suppresses the synchronous release component. These results reveal a new SynII function in the regulation of the time course of GABA release and demonstrate that this function is dependent on the interneuron subtype.

Inefficient constitutive inhibition of P2X3 receptors by brain natriuretic peptide system contributes to sensitization of trigeminal sensory neurons in a genetic mouse model of familial hemiplegic migraine.

BACKGROUND: On trigeminal ganglion neurons, pain-sensing P2X3 receptors are constitutively inhibited by brain natriuretic peptide via its natriuretic peptide receptor-A. This inhibition is associated with increased P2X3 serine phosphorylation and receptor redistribution to non-lipid raft membrane compartments. The natriuretic peptide receptor-A antagonist anantin reverses these effects. We studied whether P2X3 inhibition is dysfunctional in a genetic familial hemiplegic migraine type-1 model produced by introduction of the human pathogenic R192Q missense mutation into the mouse CACNA1A gene (knock-in phenotype). This model faithfully replicates several properties of familial hemiplegic migraine type-1, with gain-of-function of CaV2.1 Ca(2+) channels, raised levels of the algogenic peptide calcitonin gene-related peptide, and enhanced activity of P2X3 receptors in trigeminal ganglia. RESULTS: In knock-in neurons, anantin did not affect P2X3 receptor activity, membrane distribution, or serine phosphorylation level, implying ineffective inhibition by the constitutive brain natriuretic peptide/natriuretic peptide receptor-A pathway. However, expression and functional properties of this pathway remained intact together with its ability to downregulate TRPV1 channels. Reversing the familial hemiplegic migraine type-1 phenotype with the CaV2.1-specific antagonist, ω-agatoxin IVA restored P2X3 activity to wild-type level and enabled the potentiating effects of anantin again. After blocking calcitonin gene-related peptide receptors, P2X3 receptors exhibited wild-type properties and were again potentiated by anantin. CONCLUSIONS: P2X3 receptors on mouse trigeminal ganglion neurons are subjected to contrasting modulation by inhibitory brain natriuretic peptide and facilitatory calcitonin gene-related peptide that both operate via complex intracellular signaling. In the familial hemiplegic migraine type-1 migraine model, the action of calcitonin gene-related peptide appears to prevail over brain natriuretic peptide, thus suggesting that peripheral inhibition of P2X3 receptors becomes insufficient and contributes to trigeminal pain sensitization.

Interdependent Conductances Drive Infraslow Intrinsic Rhythmogenesis in a Subset of Accessory Olfactory Bulb Projection Neurons.

The accessory olfactory system controls social and sexual behavior. However, key aspects of sensory signaling along the accessory olfactory pathway remain largely unknown. Here, we investigate patterns of spontaneous neuronal activity in mouse accessory olfactory bulb mitral cells, the direct neural link between vomeronasal sensory input and limbic output. Both in vitro and in vivo, we identify a subpopulation of mitral cells that exhibit slow stereotypical rhythmic discharge. In intrinsically rhythmogenic neurons, these periodic activity patterns are maintained in absence of fast synaptic drive. The physiological mechanism underlying mitral cell autorhythmicity involves cyclic activation of three interdependent ionic conductances: subthreshold persistent Na(+) current, R-type Ca(2+) current, and Ca(2+)-activated big conductance K(+) current. Together, the interplay of these distinct conductances triggers infraslow intrinsic oscillations with remarkable periodicity, a default output state likely to affect sensory processing in limbic circuits. SIGNIFICANCE STATEMENT: We show for the first time that some rodent accessory olfactory bulb mitral cells-the direct link between vomeronasal sensory input and limbic output-are intrinsically rhythmogenic. Driven by ≥ 3 distinct interdependent ionic conductances, infraslow intrinsic oscillations show remarkable periodicity both in vitro and in vivo. As a novel default state, infraslow autorhythmicity is likely to affect limbic processing of pheromonal information.

Functional Maturation of GABA Synapses During Postnatal Development of the Monkey Dorsolateral Prefrontal Cortex.

Development of inhibition onto pyramidal cells may be crucial for the emergence of cortical network activity, including gamma oscillations. In primate dorsolateral prefrontal cortex (DLPFC), inhibitory synaptogenesis starts in utero and inhibitory synapse density reaches adult levels before birth. However, in DLPFC, the expression levels of γ-aminobutyric acid (GABA) synapse-related gene products changes markedly during development until young adult age, suggesting a highly protracted maturation of GABA synapse function. Therefore, we examined the development of GABA synapses by recording GABAAR-mediated inhibitory postsynaptic currents (GABAAR-IPSCs) from pyramidal cells in the DLPFC of neonatal, prepubertal, peripubertal, and adult macaque monkeys. We found that the decay of GABAAR-IPSCs, possibly including those from parvalbumin-positive GABA neurons, shortened by prepubertal age, while their amplitude increased until the peripubertal period. Interestingly, both GABAAR-mediated quantal response size, estimated by miniature GABAAR-IPSCs, and the density of GABAAR synaptic appositions, measured with immunofluorescence microscopy, were stable with age. Simulations in a computational model network with constant GABA synapse density showed that the developmental changes in GABAAR-IPSC properties had a significant impact on oscillatory activity and predicted that, whereas DLPFC circuits can generate gamma frequency oscillations by prepubertal age, mature levels of gamma band power are attained at late stages of development.

Presynaptic NCAM is required for motor neurons to functionally expand their peripheral field of innervation in partially denervated muscles.

The function of neural cell adhesion molecule (NCAM) expression in motor neurons during axonal sprouting and compensatory reinnervation was explored by partially denervating soleus muscles in mice lacking presynaptic NCAM (Hb9(cre)NCAM(flx)). In agreement with previous studies, the contractile force of muscles in wild-type (NCAM(+/+)) mice recovered completely 2 weeks after 75% of the motor innervation was removed because motor unit size increased by 2.5 times. In contrast, similarly denervated muscles in Hb9(cre)NCAM(flx) mice failed to recover the force lost due to the partial denervation because motor unit size did not change. Anatomical analysis indicated that 50% of soleus end plates were completely denervated 1-4 weeks post-partial denervation in Hb9(cre)NCAM(flx) mice, while another 25% were partially reinnervated. Synaptic vesicles (SVs) remained at extrasynaptic regions in Hb9(cre)NCAM(flx) mice rather than being distributed, as occurs normally, to newly reinnervated neuromuscular junctions (NMJs). Electrophysiological analysis revealed two populations of NMJs in partially denervated Hb9(cre)NCAM(flx) soleus muscles, one with high (mature) quantal content, and another with low (immature) quantal content. Extrasynaptic SVs in Hb9(cre)NCAM(flx) sprouts were associated with L-type voltage-dependent calcium channel (L-VDCC) immunoreactivity and maintained an immature, L-VDCC-dependent recycling phenotype. Moreover, acute nifedipine treatment potentiated neurotransmission at newly sprouted NMJs, while chronic intraperitoneal treatment with nifedipine during a period of synaptic consolidation enhanced functional motor unit expansion in the absence of presynaptic NCAM. We propose that presynaptic NCAM bridges a critical link between the SV cycle and the functional expansion of synaptic territory through the regulation of L-VDCCs.

Blockade of Cav2.1-mediated NMDA receptor signaling disrupts conditioned fear extinction.

Although fear extinction requires N-methyl-d-aspartate (NMDA) receptor signaling, Cav2.1-regulated synaptic function in extinction remains unknown. This study examined whether Cav2.1-mediated signaling plays role in consolidation of extinction. Wild-type mice received intracerebroventricular injection of Cav2.1 blocker (ω-agatoxin IVA, 4.0 pg/side) showed impaired extinction behavior and increased expression of CREB-dependent gene Arc in medial prefrontal cortex (mPFC). Intra-mPFC injections of NMDA receptor antagonist (MK-801, 0.5 µg/midline), which was ineffective in wild-type controls, blocked extinction in heterozygous rolling Nagoya (rol/+) mice carrying Cav2.1α1 gene mutation rol/+ mice. These results indicate that Cav2.1-mediated NMDA receptor signaling is functional pathway in mPFC-dependent fear extinction. Our results also indicate that the combination of pharmacological and genetic approaches can be used to study functional signaling pathways in neuronal circuits.

Dominance of P/Q-type calcium channels in depolarization-induced presynaptic FM dye release in cultured hippocampal neurons.

Neurotransmitter release probability is related by high power to the local concentration of calcium in presynaptic terminals, which in turn is controlled by voltage-gated calcium channels. P/Q- and N-type channels trigger synaptic transmission in the majority of neurons of the central nervous system. However, whether and under which conditions both channel types act cooperatively or independently is still insufficiently understood. Previous studies suggested either a dominance of N- or P/Q-type channels, or a synergistic action of both channels, depending on the experimental paradigms. Thus, to provide insight into the properties of neurotransmitter release in cultured mouse hippocampal neurons, we used quantitative analysis of FM dye release from presynaptic boutons induced by high potassium membrane depolarization. Increasing extracellular potassium concentrations revealed a sigmoid dependence of FM dye release to the stimulation strength. Individual and combined application of the P/Q- and N-type channel-specific blockers ω-agatoxin-IVA and ω-conotoxin-GVIA, respectively, allowed us to specifically isolate the contribution of both channel types to release triggered with 40 mM KCl. Analysis of the release kinetics and the fractional release amplitude demonstrate that, whereas in only 15% of the synapses release depended exclusively on P/Q-type channels, the majority of synapses (85%) contained both N- and P/Q-type channels. Nevertheless, the kinetics of FM dye release in synapses containing both channel types was determined by the P/Q-type channels. Together, our data suggest a more direct coupling of P/Q-type channels to synaptic release compared to N-type channels, which may explain the high prevalence of neurological P/Q-type channelopathies.

Dystonia-associated protein torsinA is not detectable at the nerve terminals of central neurons.

Presynaptic functions of the mammalian central neurons are regulated by a network of protein interactions. Synaptic vesicle recycling in and neurotransmitter release from the presynaptic nerve terminals are altered when a glutamate-deleting mutation is present in the torsinA protein (ΔE-torsinA). This mutation is linked with a hereditary form of the movement disorder dystonia known as DYT1 dystonia. Although torsinA expression is prevalent throughout the central nervous system, its subcellular localization - in particular with respect to presynaptic nerve terminals - remains unclear. This information would be useful in narrowing down possible models for how wild-type torsinA affects presynaptic function, as well as the nature of the presynaptic dysfunction that arises in the context of ΔE-torsinA mutation. Here we report on an analysis of the presynaptic localization of torsinA in cultured neurons obtained from a knock-in mouse model of DYT1 dystonia. Primary cultures of neurons were established from heterozygous and homozygous ΔE-torsinA knock-in mice, as well as from their wild-type littermates. Neurons were obtained from the striatum, cerebral cortex and hippocampus of these mice, and were subjected to immunocytochemistry. This analysis revealed the expression of both proteins in the somata and dendrites. However, neither the nerve terminals nor axonal shafts were immunoreactive. These results were confirmed by fluorogram-based quantitation. Our findings indicate that neither the wild-type nor the ΔE-torsinA mutant protein is present at substantial levels in the presynaptic structures of cultured neurons. Thus, the effects of torsinA, in wild-type and mutant forms, appear to influence presynaptic function indirectly, without residing in presynaptic structures.

Methylmercury decreases cellular excitability by a direct blockade of sodium and calcium channels in bovine chromaffin cells: an integrative study.

Methylmercury, a potent environmental pollutant responsible for fatal food poisoning, blocked calcium channels of bovine chromaffin cells in a time- and concentration-dependent manner with an IC50 of 0.93 µM. This blockade was not reversed upon wash-out and was greater at more depolarising holding potentials (i.e. 21 % at -110 mV and 60 % at -50 mV, after 3 min perfusion with methylmercury). In ω-toxins-sensitive calcium channels, methylmercury caused a higher blockade of I Ba than in ω-toxins-resistant ones, in which a lower blockade was detected. The sodium current was also blocked by acute application of methylmercury in a time- and concentration-dependent manner with an IC50 of 1.05 µM. The blockade was not reversed upon wash-out of the drug. The drug inhibited sodium current at all test potentials and shows a shift of the I-V curve to the left of about 10 mV. Intracellular dialysis with methylmercury caused no blockade of calcium or sodium channels. Voltage-dependent potassium current was not affected by methylmercury. Calcium- and voltage-dependent potassium current was also drastically depressed. This blockade was related to the prevention of Ca(2+) influx through voltage-dependent calcium channels coupled to BK channels. Under current-clamp conditions, the blockade of ionic current present during the generation and termination of action potentials led to a drastic alteration of cellular excitability. The application of methylmercury greatly reduced the shape and the number of electrically evoked action potentials. Taken together, these results point out that the neurotoxic action evoked by methylmercury may be associated to alteration of cellular excitability by blocking ionic currents responsible for the generation and termination of action potentials.

Intrathecal P/Q- and R-type calcium channel blockade of spinal substance P release and c-Fos expression.

Intrathecal (IT) studies have shown that several voltage sensitive calcium channels (VSCCs), such as the L-, N- and T-type may play roles in nociception and that of these only the N-type regulates primary afferent substance P (SP) release. However, the actions of other VSCCs at the spinal level are not well known. We investigated the roles of spinal P/Q- and R-type VSCCs, by IT administration of R-type (SNX-482) and P/Q-type (ω-agatoxin IVA) VSCC blockers on intraplantar formalin-evoked flinching, SP release from primary afferents and c-Fos expression in spinal dorsal horn. Intraplantar injection of formalin (2.5%, 50 µL) produced an intense, characteristic biphasic paw flinching response. In rats with IT catheters, IT SNX-482 (0.5 µg) reduced formalin-evoked paw flinching in both phase 1 and 2 compared with vehicle. Intraplantar formalin caused robust neurokinin 1 receptor (NK1r) internalization (indicating SP release) and c-Fos expression in the ipsilateral dorsal horn, which were blocked by IT SNX-482. IT ω-agatoxin IVA (0.03, 0.125 and 0.5 µg) did not reduce formalin-evoked paw flinching or c-Fos expression at any doses, with higher doses resulting in motor dysfunction. Thus, we demonstrated that blockade of spinal R-type, but not P/Q type VSCCs attenuated formalin-induced pain behavior, NK1r internalization and c-Fos expression in the superficial dorsal horn. This study supports a role for Cav2.3 in presynaptic neurotransmitter release from peptidergic nociceptive afferents and pain behaviors.

Balance of calcineurin Aα and CDK5 activities sets release probability at nerve terminals.

The control of neurotransmitter release at nerve terminals is of profound importance for neurological function and provides a powerful control system in neural networks. We show that the balance of enzymatic activities of the α isoform of the phosphatase calcineurin (CNAα) and the kinase cyclin-dependent kinase 5 (CDK5) has a dramatic influence over single action potential (AP)-driven exocytosis at nerve terminals. Acute or chronic loss of these enzymatic activities results in a sevenfold impact on single AP-driven exocytosis. We demonstrate that this control is mediated almost entirely through Cav2.2 (N-type) voltage-gated calcium channels as blocking these channels with a peptide toxin eliminates modulation by these enzymes. We found that a fraction of nerve terminals are kept in a presynaptically silent state with no measurable Ca(2+) influx driven by single AP stimuli attributable to the balance of CNAα and CDK5 activities because blockade of either CNAα or CDK5 activity changes the proportion of presynaptically silent nerve terminals. Thus, CNAα and CDK5 enzymatic activities are key determinants of release probability.

Presynaptic calcium influx controls neurotransmitter release in part by regulating the effective size of the readily releasable pool.

The steep calcium dependence of synaptic strength that has been observed at many synapses is thought to reflect a calcium dependence of the probability of vesicular exocytosis (p), with the cooperativity of three to six corresponding to the multiple calcium ion binding sites on the calcium sensor responsible for exocytosis. Here we test the hypothesis that the calcium dependence of the effective size of the readily releasable pool (RRP) also contributes to the calcium dependence of release at the calyx of Held synapse in mice. Using two established methods of quantifying neurotransmitter release evoked by action potentials (effective RRP), we find that when calcium influx is changed by altering the external calcium concentration, the calcium cooperativity of p is insufficient to account for the full calcium dependence of EPSC size; the calcium dependence of the RRP size also contributes. Reducing calcium influx by blocking R-type voltage-gated calcium channels (VGCCs) with Ni(2+), or by blocking P/Q-type VGCCs with ω-agatoxin IVA also changes EPSC amplitude by reducing both p and the effective RRP size. This suggests that the effective RRP size is dependent on calcium influx through VGCCs. Furthermore, activation of GABAB receptors, which reduces presynaptic calcium through VGCCs without other significant effects on release, also reduces the effective RRP size in addition to reducing p. These findings indicate that calcium influx regulates the RRP size along with p, which contributes to the calcium dependence of synaptic strength, and it influences the manner in which presynaptic modulation of presynaptic calcium channels affects neurotransmitter release.

Synthetic Aß oligomers (Aß(1-42) globulomer) modulate presynaptic calcium currents: prevention of Aß-induced synaptic deficits by calcium channel blockers.

Alzheimer's disease is accompanied by increased brain levels of soluble amyloid-ß (Aß) oligomers. It has been suggested that oligomers directly impair synaptic function, thereby causing cognitive deficits in Alzheimer's disease patients. Recently, it has been shown that synthetic Aß oligomers directly modulate P/Q-type calcium channels, possibly leading to excitotoxic cascades and subsequent synaptic decline. Using whole-cell recordings we studied the modulation of recombinant presynaptic calcium channels in HEK293 cells after application of a stable Aß oligomer preparation (Aß1-42 globulomer). Aß globulomer shifted the half-activation voltage of P/Q-type and N-type calcium channels to more hyperpolarized values (by 11.5 and 7.5 mV). Application of non-aggregated Aß peptides had no effect. We then analyzed the potential of calcium channel blockers to prevent Aß globulomer-induced synaptic decline in hippocampal slice cultures. Specific block of P/Q-type or N-type calcium channels with peptide toxins completely reversed Aß globulomer-induced deficits in glutamatergic neurotransmission. Two state-dependent low molecular weight P/Q-type and N-type calcium channel blockers also protected neurons from Aß-induced alterations. On the contrary, inhibition of L-type calcium channels failed to reverse the deficit. Our data show that Aß globulomer directly modulates recombinant P/Q-type and N-type calcium channels in HEK293 cells. Block of presynaptic calcium channels with both state-dependent and state-independent modulators can reverse Aß-induced functional deficits in synaptic transmission. These findings indicate that presynaptic calcium channel blockers may be a therapeutic strategy for the treatment of Alzheimer's disease.

Over-expression of N-type calcium channels in cortical neurons from a mouse model of Amyotrophic Lateral Sclerosis.

Voltage-gated Ca(2+) channels (VGCCs) mediate calcium entry into neuronal cells in response to membrane depolarisation and play an essential role in a variety of physiological processes. In Amyotrophic Lateral Sclerosis (ALS), a fatal neurodegenerative disease caused by motor neuron degeneration in the brain and spinal cord, intracellular calcium dysregulation has been shown, while no studies have been carried out on VGCCs. Here we show that the subtype N-type Ca(2+) channels are over expressed in G93A cultured cortical neurons and in motor cortex of G93A mice compared to Controls. In fact, by western blotting, immunocytochemical and electrophysiological experiments, we observe higher membrane expression of N-type Ca(2+) channels in G93A neurons compared to Controls. G93A cortical neurons filled with calcium-sensitive dye Fura-2, show a net calcium entry during membrane depolarization that is significantly higher compared to Control. Analysis of neuronal vitality following the exposure of neurons to a high K(+) concentration (25 mM, 5h), shows a significant reduction of G93A cellular survival compared to Controls. N-type channels are involved in the G93A higher mortality because ω-conotoxin GVIA (1 µM), which selectively blocks these channels, is able to abolish the higher G93A mortality when added to the external medium. These data provide robust evidence for an excess of N-type Ca(2+) expression in G93A cortical neurons which induces a higher mortality following membrane depolarization. These results may be central to the understanding of pathogenic pathways in ALS and provide novel molecular targets for the design of rational therapies for the ALS disorder.

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>50pF) 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.

Gamma band activity in the developing parafascicular nucleus.

The parafascicular nucleus (Pf) receives cholinergic input from the pedunculopontine nucleus, part of the reticular activating system involved in waking and rapid eye movement (REM) sleep, and sends projections to the cortex. We tested the hypothesis that Pf neurons fire maximally at gamma band frequency (30-90 Hz), that this mechanism involves high-threshold voltage-dependent P/Q- and N-type calcium channels, and that this activity is enhanced by the cholinergic agonist carbachol (CAR). Patch-clamped 9- to 25-day-old rat Pf neurons (n = 299) manifested a firing frequency plateau at gamma band when maximally activated (31.5 ± 1.5 Hz) and showed gamma oscillations when voltage-clamped at holding potentials above -20 mV, and the frequency of the oscillations increased significantly with age (24.6 ± 3.8 vs. 51.6 ± 4.4 Hz, P < 0.001) but plateaued at gamma frequencies. Cells exposed to CAR showed significantly higher frequencies early in development compared with those without CAR (24.6 ± 3.8 vs. 41.7 ± 4.3 Hz, P < 0.001) but plateaued with age. The P/Q-type calcium channel blocker ω-agatoxin-IVA (ω-Aga) blocked gamma oscillations, whereas the N-type blocker ω-conotoxin-GVIA (ω-CgTx) only partially decreased the power spectrum amplitude of gamma oscillations. The blocking effect of ω-Aga on P/Q-type currents and ω-CgTx on N-type currents was consistent over age. We conclude that P/Q- and N-type calcium channels appear to mediate Pf gamma oscillations during development. We hypothesize that the cholinergic input to the Pf could activate these cells to oscillate at gamma frequency, and perhaps relay these rhythms to cortical areas, thus providing a stable high-frequency state for "nonspecific" thalamocortical processing.

Activity-dependent increases in local oxygen consumption correlate with postsynaptic currents in the mouse cerebellum in vivo.

Evoked neural activity correlates strongly with rises in cerebral metabolic rate of oxygen (CMRO(2)) and cerebral blood flow (CBF). Activity-dependent rises in CMRO(2) fluctuate with ATP turnover due to ion pumping. In vitro studies suggest that increases in cytosolic Ca(2+) stimulate oxidative metabolism via mitochondrial signaling, but whether this also occurs in the intact brain is unknown. Here we applied a pharmacological approach to dissect the effects of ionic currents and cytosolic Ca(2+) rises of neuronal origin on activity-dependent rises in CMRO(2). We used two-photon microscopy and current source density analysis to study real-time Ca(2+) dynamics and transmembrane ionic currents in relation to CMRO(2) in the mouse cerebellar cortex in vivo. We report a direct correlation between CMRO(2) and summed (i.e., the sum of excitatory, negative currents during the whole stimulation period) field EPSCs (∑fEPSCs) in Purkinje cells (PCs) in response to stimulation of the climbing fiber (CF) pathway. Blocking stimulus-evoked rises in cytosolic Ca(2+) in PCs with the P/Q-type channel blocker ω-agatoxin-IVA (ω-AGA), or the GABA(A) receptor agonist muscimol, did not lead to a time-locked reduction in CMRO(2), and excitatory synaptic or action potential currents. During stimulation, neither ω-AGA or (µ-oxo)-bis-(trans-formatotetramine-ruthenium) (Ru360), a mitochondrial Ca(2+) uniporter inhibitor, affected the ratio of CMRO(2) to fEPSCs or evoked local field potentials. However, baseline CBF and CMRO(2) decreased gradually with Ru360. Our data suggest that in vivo activity-dependent rises in CMRO(2) are correlated with synaptic currents and postsynaptic spiking in PCs. Our study did not reveal a unique role of neuronal cytosolic Ca(2+) signals in controlling CMRO(2) increases during CF stimulation.

Mechanism behind gamma band activity in the pedunculopontine nucleus.

The pedunculopontine nucleus (PPN), part of the reticular activating system, modulates waking and paradoxical sleep. During waking and paradoxical sleep, EEG responses are characterized by low-amplitude, high-frequency oscillatory activity in the beta-gamma band range (~20-80 Hz). We have previously reported that gamma band activity may be intrinsically generated by the membrane electroresponsiveness of PPN neurons, and that the neuronal ensemble generates different patterns of gamma activity in response to specific transmitters. This study attempted to identify the voltage-gated calcium and potassium channels involved in the rising and falling phases of gamma oscillations in PPN neurons. We found that all rat (8-14 day) PPN cell types showed gamma oscillations in the presence of TTX and synaptic blockers when membrane potential was depolarized using current ramps. PPN neurons showed gamma oscillations when voltage-clamped at holding potentials above -30 mV, suggesting that their origin may be spatially located beyond voltage-clamp control. The average frequency for all PPN cell types was 23 ± 1 Hz and this increased under carbachol (47 ± 2 Hz; anova df = 64, t = 12.5, P < 0.001). The N-type calcium channel blocker ω-conotoxin-GVIA partially reduced gamma oscillations, while the P/Q-type blocker ω-agatoxin-IVA abolished them. Both ω-CgTX and ω-Aga blocked voltage-dependent calcium currents, by 56 and 52% respectively. The delayed rectifier-like potassium channel blocker α-dendrotoxin also abolished gamma oscillations. In carbachol-induced PPN population responses, ω-agatoxin-IVA reduced higher, and ω-CgTx mostly lower, frequencies. These results suggest that voltage-dependent P/Q- and, to a lesser extent, N-type calcium channels mediate gamma oscillations in PPN.

Functional importance of L- and P/Q-type voltage-gated calcium channels in human renal vasculature.

Calcium channel blockers are widely used for treatment of hypertension, because they decrease peripheral vascular resistance through inhibition of voltage-gated calcium channels. Animal studies of renal vasculature have shown expression of several types of calcium channels that are involved in kidney function. It was hypothesized that human renal vascular excitation-contraction coupling involves different subtypes of channels. In human renal artery and dissected intrarenal blood vessels from nephrectomies, PCR analysis showed expression of L-type (Ca(v) 1.2), P/Q-type (Ca(v) 2.1), and T-type subtype (Ca(v) 3.1 and Ca(v) 3.2) voltage-gated calcium channels (Ca(v)s), and quantitative PCR showed highest expression of L-type channels in renal arteries and variable expression between patients of subtypes of calcium channels in intrarenal vessels. Immunohistochemical labeling of kidney sections revealed signals for Ca(v) 2.1 and Ca(v) 3.1 associated with smooth muscle cells of preglomerular and postglomerular vessels. In human intrarenal arteries, depolarization with potassium induced a contraction inhibited by the L-type antagonist nifedipine, EC(50) 1.2×10(-8) mol/L. The T-type antagonist mibefradil inhibited the potassium-induced constriction with large variations between patients. Interestingly, the P/Q-type antagonist, ω-agatoxin IVA, inhibited significantly the contraction with 24% at 10(-9) mol/L. In conclusion L-, P/Q, and T-type channels are expressed in human renal blood vessels, and L- and P/Q-type channels are of functional importance for the depolarization-induced vasoconstriction. The contribution of P/Q-type channels to contraction in the human vasculature is a novel mechanism for the regulation of renal blood flow and suggests that clinical treatment with calcium blockers might affect vascular reactivity also through P/Q-type channel inhibition.

Calcium microdomains near R-type calcium channels control the induction of presynaptic long-term potentiation at parallel fiber to purkinje cell synapses.

R-type calcium channels in postsynaptic spines signal through functional calcium microdomains to regulate a calcium/calmodulin-sensitive potassium channel that in turn regulates postsynaptic hippocampal long-term potentiation (LTP). Here, we ask whether R-type calcium channels in presynaptic terminals also signal through calcium microdomains to control presynaptic LTP. We focus on presynaptic LTP at parallel fiber to Purkinje cell synapses in the cerebellum (PF-LTP), which is mediated by calcium/calmodulin-stimulated adenylyl cyclases. Although most presynaptic calcium influx is through N-type and P/Q-type calcium channels, blocking these channels does not disrupt PF-LTP, but blocking R-type calcium channels does. Moreover, global calcium signaling cannot account for the calcium dependence of PF-LTP because R-type channels contribute modestly to overall calcium entry. These findings indicate that, within presynaptic terminals, R-type calcium channels produce calcium microdomains that evoke presynaptic LTP at moderate frequencies that do not greatly increase global calcium levels.