Prolonged Membrane Depolarization Research Articles

Differential response of C9orf72 transcripts following neuronal depolarization

The (G4C2)n nucleotide repeat expansion (NRE) mutation in C9orf72 is the most common genetic cause of ALS and FTD. The biological functions of C9orf72 are becoming understood, but it is unclear if this gene is regulated in a neural-specific manner. Neuronal activity is a crucial modifier of biological processes in health and neurodegenerative disease contexts. Here, we show that prolonged membrane depolarization in healthy human iPSC-cortical neurons leads to a significant downregulation of a transcript variant 3 (V3) of C9orf72, with a concomitant increase in variant 2 (V2), which leads to total C9orf72 RNA transcript levels remaining unchanged. However, the same response is not observed in cortical neurons derived from patients with the C9-NRE mutation. These findings reveal the impact of depolarization on C9orf72 transcripts, and how this response diverges in C9-NRE-carriers, which may have important implications in the underlying unique clinical associations of C9-NRE transcripts and disease pathogenesis.

Effects of Antagonists of Different Types of NMDA Receptor on Evoked Responses of Pyramidal Neurons in Rat Cerebral Cortex Slices

Despite the extensive pharmacology of NMDA receptors, the use of drugs able to decrease their overactivation in CNS pathologies is limited. One reason for this may be lack of knowledge of the effects of NMDA receptor antagonists on the functional properties of neurons, as a significant quantity of data has been obtained in in vitro models, which do not completely reproduce real pathophysiological processes. The present study addressed the effects of NMDA receptor antagonists with different mechanisms of action on the properties of pyramidal neuron responses in the prefrontal cortex of the rat brain. The competitive antagonist APV (50 μM) and the ion channel blocker memantine (100 μM) were inactive against evoked EPSP. In conditions of suppression of inhibitory transmission with picrotoxin (50 μM), extracellular stimulation evoked epileptiform responses with prolonged membrane depolarization and generation of a burst of action potentials. APV limited the duration and amplitude of these responses. Memantine acquired activity only when magnesium was excluded from the extracellular medium. Memantine’s lack of effect can be explained in terms of competition for the magnesium ion binding site in the channel cavity. These studies show that prediction of the systemic effects of blockers on neuron function and excitability requires use of in vitro models in which NMDA receptors are activated in the presence of a physiological magnesium concentration and without clamping of the membrane potential.

Влияние антагонистов NMDA рецепторов разных типов на вызванные ответы пирамидных нейронов в срезах коры мозга крысы

The use of NMDA receptor antagonists for the treatment of CNS diseases is currently limited. This may be due to the lack of knowledge about the effects of antagonists on the functional properties of neurons since in vitro models do not fully reproduce the real pathophysiological processes. We studied the effects of NMDA receptor antagonists on properties of pyramidal neuron responses in the rat prefrontal cortex. A competitive antagonist APV (50 µM) and an ion channel blocker memantine (100 µM) did not produce significant effects on evoked EPSP. When the inhibitory transmission was suppressed by picrotoxin (50 µM), the extracellular stimulation caused prolonged membrane depolarization and generation a series of action potentials. APV decreased duration and amplitude of such responses. Memantine was active only when magnesium was excluded from the extracellular environment. The lack of effects of memantine can be explained by its competition with magnesium for the binding site within the ion channel. This work demonstrates that in order to predict the systemic effects of blockers on the functions of neurons and their excitability, it is necessary to use in vitro models in which the activation of NMDA receptors occurs in the presence of physiological concentrations of magnesium and without clamping of membrane potential.

Long-Lasting Electrophysiological After-Effects of High-Frequency Stimulation in the Globus Pallidus: Human and Rodent Slice Studies.

Deep-brain stimulation (DBS) of the globus pallidus pars interna (GPi) is a highly effective therapy for movement disorders, yet its mechanism of action remains controversial. Inhibition of local neurons because of release of GABA from afferents to the GPi is a proposed mechanism in patients. Yet, high-frequency stimulation (HFS) produces prolonged membrane depolarization mediated by cholinergic neurotransmission in endopeduncular nucleus (EP, GPi equivalent in rodent) neurons. We applied HFS while recording neuronal firing from an adjacent electrode during microelectrode mapping of GPi in awake patients (both male and female) with Parkinson disease (PD) and dystonia. Aside from after-suppression and no change in neuronal firing, high-frequency microstimulation induced after-facilitation in 38% (26/69) of GPi neurons. In neurons displaying after-facilitation, 10 s HFS led to an immediate decrease of bursting in PD, but not dystonia patients. Moreover, the changes of bursting patterns in neurons with after-suppression or no change after HFS, were similar in both patient groups. To explore the mechanisms responsible, we applied HFS in EP brain slices from rats of either sex. As in humans, HFS in EP induced two subtypes of after-excitation: excitation or excitation with late inhibition. Pharmacological experiments determined that the excitation subtype, induced by lower charge density, was dependent on glutamatergic transmission. HFS with higher charge density induced excitation with late inhibition, which involved cholinergic modulation. Therefore HFS with different charge density may affect the local neurons through multiple synaptic mechanisms. The cholinergic system plays a role in mediating the after-facilitatory effects in GPi neurons, and because of their modulatory nature, may provide a basis for both the immediate and delayed effects of GPi-DBS. We propose a new model to explain the mechanisms of DBS in GPi.SIGNIFICANCE STATEMENT Deep-brain stimulation (DBS) in the globus pallidus pars interna (GPi) improves Parkinson disease (PD) and dystonia, yet its mechanisms in GPi remain controversial. Inhibition has been previously described and thought to indicate activation of GABAergic synaptic terminals, which dominate in GPi. Here we report that 10 s high-frequency microstimulation induced after-facilitation of neural firing in a substantial proportion of GPi neurons in humans. The neurons with after-facilitation, also immediately reduced their bursting activities after high-frequency stimulation in PD, but not dystonia patients. Based on these data and further animal experiments, a mechanistic hypothesis involving glutamatergic, GABAergic, and cholinergic synaptic transmission is proposed to explain both short- and longer-term therapeutic effects of DBS in GPi.

Eminence of VSD I in the Voltage-Dependent Inactivation of the Human CaV1.2 Channel

In response to prolonged membrane depolarizations, the L-type calcium channel (CaV1.2) enters a non-conducting inactivated state that diminishes Ca2+ influx. Specifically, CaV1.2 channels undergo two types of inactivation: voltage-dependent inactivation (VDI) and a Ca2+-dependent, calmodulin-mediated inactivation (CDI). What is the structural basis for the voltage dependence of VDI? We hypothesize that at least one of the four CaV1.2 homologous but non-identical voltage-sensing domains (VSDs) should drive or regulate VDI. Using the Voltage-Clamp Fluorometry technique, we optically tracked the movement of the individual VSDs in conducting human CaV1.2 channels (α1C/α2δ-1) expressed in Xenopus oocytes under conditions that show a different extent of VDI (300ms depolarizations to 10 mV, using Ba2+ as the charge carrier to prevent CDI): β3 co-expression, VDI∼35%; β2a co-expression, VDI∼15%; Timothy-Syndrome-causing mutation G406R with β3, VDI∼8%. We found that, in all channels, upon repolarization to −90mV, VSD I deactivated with a bi-exponential decay (τ1∼1ms, τ2∼35ms). Channels with attenuated VDI (Timothy-Syndrome-causing mutation and β2a co-expression) exhibited mostly fast VSD I deactivation (Amp2∼6% and 12%, respectively), while a more pronounced VDI (β3 co-expression) correlated with an increase in the fraction of VSD I returning slowly to their resting state (Amp2∼55%). This feature is reminiscent of “charge immobilization”, whereby the inactivation of voltage-gated channels slows down VSD deactivation and produces an apparent reduction of the off gating charge. Our data support the view that VSD I undergoes “immobilization” following CaV1.2 inactivation. On the other hand, VSDs III and IV kinetics were not affected by the time-course of VDI. In conclusion, our findings reveal a strong correlation between the degree of inactivation and VSD I kinetics, suggesting the involvement of VSD I in setting the rate of VDI.

A non-inactivating high-voltage-activated two-pore Na⁺ channel that supports ultra-long action potentials and membrane bistability.

Action potentials (APs) are fundamental cellular electrical signals. The genesis of short APs lasting milliseconds is well understood. Ultra-long APs (ulAPs) lasting seconds to minutes also occur in eukaryotic organisms, but their biological functions and mechanisms of generation are largely unknown. Here, we identify TPC3, a previously uncharacterized member of the two-pore channel protein family, as a new voltage-gated Na+ channel (NaV) that generates ulAPs, and that establishes membrane potential bistability. Unlike the rapidly inactivating NaVs that generate short APs in neurons, TPC3 has a high activation threshold, activates slowly, and does not inactivate—three properties that help generate long-lasting APs and guard the membrane against unintended perturbation. In amphibian oocytes, TPC3 forms a channel similar to channels induced by depolarization and sperm entry into eggs. TPC3 homologs are present in plants and animals, and they may be important for cellular processes and behaviors associated with prolonged membrane depolarization.

HIV-1-Tat excites cardiac parasympathetic neurons of nucleus ambiguus and triggers prolonged bradycardia in conscious rats

The mechanisms of autonomic imbalance and subsequent cardiovascular manifestations in HIV-1-infected patients are poorly understood. We report here that HIV-1 transactivator of transcription (Tat, fragment 1-86) produced a concentration-dependent increase in cytosolic Ca(2+) in cardiac-projecting parasympathetic neurons of nucleus ambiguus retrogradely labeled with rhodamine. Using store-specific pharmacological agents, we identified several mechanisms of the Tat-induced Ca(2+) elevation: 1) lysosomal Ca(2+) mobilization, 2) Ca(2+) release via inositol 1,4,5-trisphosphate-sensitive endoplasmic reticulum pools, and 3) Ca(2+) influx via transient receptor potential vanilloid type 2 (TRPV2) channels. Activation of TRPV2, nonselective cation channels, induced a robust and prolonged neuronal membrane depolarization, thus triggering an additional P/Q-mediated Ca(2+) entry. In vivo microinjection studies indicate a dose-dependent, prolonged bradycardic effect of Tat administration into the nucleus ambiguus of conscious rats, in which neuronal TRPV2 played a major role. Our results support previous studies, indicating that Tat promotes bradycardia and, consequently, may be involved in the QT interval prolongation reported in HIV-infected patients. In the context of an overall HIV-dependent autonomic dysfunction, these Tat-mediated mechanisms may account for the higher prevalence of sudden cardiac death in HIV-1-infected patients compared with general population with similar risk factors. Our results may be particularly relevant in view of the recent findings that significant Tat levels can still be identified in the cerebrospinal fluid of HIV-infected patients with viral load suppression due to efficient antiretroviral therapy.

A novel calmodulin site in the Cav1.2 N-terminus regulates calcium-dependent inactivation

The L-type voltage-gated calcium channel Cav1.2 is important for excitation-contraction coupling in the heart, as well as CREB-mediated transcription in the brain. The ubiquitous calcium-binding protein calmodulin (CaM) is known to modulate calcium-dependent inactivation (CDI) of these channels, thus limiting the amount of calcium entering via Cav1.2 during prolonged or repetitive membrane depolarizations. The proximal N-terminus of Cav1.2 contains a CaM-binding site at residue W52 that is critical for a type of CDI that is mediated by the N-terminal lobe of CaM. Here, we identify a second CaM interaction site in the Cav1.2 N-terminus downstream of the W52 site that is formed by residue C106. We show by site-directed mutagenesis coupled with electrophysiological measurements that this region of the channel functionally partakes in N-lobe CDI, likely by acting as a gating transduction motif. Thus, our data indicate that calcium regulation of Cav1.2 channels is more complex than previously thought, and involves more than one region within the channel's N-terminal domain.

Regulation of ERK1/2 mitogen-activated protein kinase by NMDA-receptor-induced seizure activity in cortical slices

Extracellular signal-regulated kinase 1/2 (ERK1/2) that belongs to a subfamily of mitogen-activated protein kinases (MAPKs) plays diverse roles in the central nervous system. Activation of ERK1/2 has been observed in various types of neuronal excitation, including seizure activity in vivo and in vitro, as well as in NMDA-receptor (NMDA-R)-dependent long-term potentiation in the hippocampus. On the other hand, recent studies in cultured neurons have shown that NMDA-R stimulation could result in either ERK1/2 activation or non-activation, depending on the pharmacological manipulations. To assess NMDA-R-dependent regulation of ERK1/2 activity in vivo, here we examined the effect of NMDA-R-induced seizure activity on ERK1/2 activation by using rat cortical slice preparations. NMDA-R-dependent seizure activity introduced by Mg2+-free condition did not cause ERK1/2 activation. On the other hand, when picrotoxin was added to concurrently suppress GABAA-receptor-mediated inhibition, profound ERK1/2 activation occurred, which was accompanied by strong phospho-ERK1/2-staining in the superficial and deep cortical layer neurons. In this case, prolonged membrane depolarization and enhanced burst action potential firings, both of which were much greater than those in Mg2+-free condition alone, were observed. Differential ERK1/2 activation was supported by the concurrent selective increase in phosphorylation of a substrate protein, phospho-site 4/5 of synapsin I. These results indicate that NMDA-R activation through a release from Mg2+-blockade, which accompanies enhancement of both excitatory and inhibitory synaptic transmission, was not enough, but concurrent suppression of GABAergic inhibition, which leads to a selective increase in excitatory synaptic transmission, was necessary for robust ERK1/2 activation to occur within the cortical network.

The Late Na+ Current - Origin and Pathophysiological Relevance

In excitable tissues, voltage-dependent Na+ current (INa) is best known for supporting autoregenerative depolarization and impulse propagation. Its transient component (INaT), which is large and terminated within several milliseconds by channel inactivation, fulfils this role. Nevertheless, INa also includes a smaller sustained component, i.e. one persisting during prolonged membrane depolarization, which contributes to repolarization course. Sustained INa implies slow or incomplete inactivation of a proportion of the Na+ channels activated during the action potential upstroke. Several mechanisms may underlie this phenomenon and contribute to arrhythmogenesis in different conditions.

Molecular mechanism for depolarization-induced modulation of Kv channel closure

Voltage-dependent potassium (Kv) channels provide the repolarizing power that shapes the action potential duration and helps control the firing frequency of neurons. The K+ permeation through the channel pore is controlled by an intracellularly located bundle-crossing (BC) gate that communicates with the voltage-sensing domains (VSDs). During prolonged membrane depolarizations, most Kv channels display C-type inactivation that halts K+ conduction through constriction of the K+ selectivity filter. Besides triggering C-type inactivation, we show that in Shaker and Kv1.2 channels (expressed in Xenopus laevis oocytes), prolonged membrane depolarizations also slow down the kinetics of VSD deactivation and BC gate closure during the subsequent membrane repolarization. Measurements of deactivating gating currents (reporting VSD movement) and ionic currents (BC gate status) showed that the kinetics of both slowed down in two distinct phases with increasing duration of the depolarizing prepulse. The biphasic slowing in VSD deactivation and BC gate closure was strongly correlated in time and magnitude. Simultaneous recordings of ionic currents and fluorescence from a probe tracking VSD movement in Shaker directly demonstrated that both processes were synchronized. Whereas the first slowing originates from a stabilization imposed by BC gate opening, the subsequent slowing reflects the rearrangement of the VSD toward its relaxed state (relaxation). The VSD relaxation was observed in the Ciona intestinalis voltage-sensitive phosphatase and in its isolated VSD. Collectively, our results show that the VSD relaxation is not kinetically related to C-type inactivation and is an intrinsic property of the VSD. We propose VSD relaxation as a general mechanism for depolarization-induced slowing of BC gate closure that may enable Kv1.2 channels to modulate the firing frequency of neurons based on the depolarization history.

Prolonged Membrane Depolarization Enhances Midbrain Dopamine Neuron Differentiation via Epigenetic Histone Modifications

Understanding midbrain dopamine (DA) neuron differentiation is of importance, because of physiological and clinical implications of this neuronal subtype. We show that prolonged membrane depolarization induced by KCl treatment promotes DA neuron differentiation from neural precursor cells (NPCs) derived from embryonic ventral midbrain (VM). Interestingly, the depolarization-induced increase of DA neuron yields was not abolished by L-type calcium channel blockers, along with no depolarization-mediated change of intracellular calcium level in the VM-derived NPCs (VM-NPCs), suggesting that the depolarization effect is due to a calcium-independent mechanism. Experiments with labeled DA neuron progenitors indicate that membrane depolarization acts at the differentiation fate determination stage and promotes the expression of DA phenotype genes (tyrosine hydroxylase [TH] and DA transporter [DAT]). Recruitment of Nurr1, a transcription factor crucial for midbrain DA neuron development, to the promoter of TH gene was enhanced by depolarization, along with increases of histone 3 acetylation (H3Ac) and trimethylation of histone3 on lysine 4 (H3K4m3), and decreases of H3K9m3 and H3K27m3 in the consensus Nurr1 binding regions of TH promoter. Depolarization stimuli on differentiating VM-NPCs also induced dissociation of methyl CpG binding protein 2 and related repressor complex molecules (repressor element-1 silencing transcription factor corepressor and histone deacetylase 1) from the CpG sites of TH and DAT promoters. Based on these findings, we suggest that membrane depolarization promotes DA neuron differentiation by opening chromatin structures surrounding DA phenotype genes and inhibiting the binding of corepressors, thus allowing transcriptional activators such as Nurr1 to access DA neuron differentiation gene promoter regions.

Calcium entry in skeletal muscle

Calcium entry in skeletal muscle

Cold‐induced disruption of Na+ channel slow inactivation underlies paralysis in highly thermosensitive paramyotonia

The Q270K mutation of the skeletal muscle Na(+) channel alpha subunit (Nav1.4) causes atypical paramyotonia with a striking sensitivity to cold. Attacks of paralysis and a drop in the compound muscle action potential (CMAP) are exclusively observed at cold. To understand the pathogenic process, we studied the consequences of this mutation on channel gating at different temperatures. WT or Q270K recombinant Nav1.4 channels fused at their C-terminal end to the enhanced green fluorescent protein (EGFP) were expressed in HEK-293 cells. Whole-cell Na(+) currents were recorded using the patch clamp technique to examine channel gating at 30 degrees C and after cooling the bathing solution to 20 degrees C. Mutant channel fast inactivation was impaired at both temperatures. Cooling slowed the kinetics and enhanced steady-state fast inactivation of both mutant and WT channels. Mutant channel slow inactivation was fairly comparable to that of the WT at 30 degrees C, but became clearly abnormal at 20 degrees C. Cooling enhanced slow inactivation in the WT by shifting the voltage dependence toward hyperpolarization, but induced the opposite effect in the mutant. Destabilization of mutant channel slow inactivation in combination with defective fast inactivation is expected to increase the susceptibility to prolonged membrane depolarization, and can ultimately lead to membrane inexcitability and paralysis at cold. Thus, abnormal temperature sensitivity of slow inactivation can be a determinant pathogenic factor, and should therefore be more widely considered in thermosensitive Na(+) channelopathies.

Mechanism of the Modulation of Kv4:KChIP-1 Channels by External K +

In response to a prolonged membrane depolarization, inactivation autoregulates the activity of voltage-gated ion channels. Slow inactivation involving a localized constriction of the selectivity filter (P/C-type mechanism) is prevalent in many voltage-gated K + channels of the Kv1 subfamily. However, the generalization of this mechanism to other Kv channel subfamilies has remained uncertain and controversial. In agreement with a “foot-in-the-door” mechanism and the presence of ion-ion interactions in the pore, elevated external K + slows the development of P/C-type inactivation and accelerates its recovery. In sharp contrast and resembling the regulation of the hippocampal A-type K + current, we found that Kv4.x channels associated with KChIP-1 (an auxiliary subunit) exhibit accelerated inactivation and unaffected recovery from inactivation when exposed to elevated external K +. This regulation depends on the ability of a permeant ion to enter the selectivity filter (K + = Rb + = NH4 + > Cs + > Na +); and the apparent equilibrium dissociation constant of a single regulatory site is 8 mM for K +. By applying a robust quantitative global kinetic modeling approach to all macroscopic properties over a 210-mV range of membrane potentials, we determined that elevated external K + inhibits unstable closed states outside the main activation pathway and thereby promotes preferential closed-state inactivation. These results suggest the presence of a vestigial and unstable P/C-type mechanism of inactivation in Kv4 channels and strengthen the concept of novel mechanisms of closed-state inactivation. Regulation of Kv4 channel inactivation by hyperkalemia may help to explain the pathophysiology of electrolyte imbalances in excitable tissues.

Time-Dependent Molecular Memory in Single Voltage-Gated Sodium Channel

Excitability in neurons is associated with firing of action potentials and requires the opening of voltage-gated sodium channels with membrane depolarization. Sustained membrane depolarization, as seen in pathophysiological conditions like epilepsy, can have profound implications on the biophysical properties of voltage-gated ion channels. Therefore, we sought to characterize the effect of sustained membrane depolarization on single voltage-gated Na+ channels. Single-channel activity was recorded in the cell-attached patch-clamp mode from the rNa(v)1.2 alpha channels expressed in CHO cells. Classical statistical analysis revealed complex nonlinear changes in channel dwell times and unitary conductance of single Na+ channels as a function of conditioning membrane depolarization. Signal processing tools like weighted wavelet Z (WWZ) and discrete Fourier transform analyses attributed a "pseudo-oscillatory" nature to the observed nonlinear variation in the kinetic parameters. Modeling studies using the hidden Markov model (HMM) illustrated significant changes in kinetic states and underlying state transition rate constants upon conditioning depolarization. Our results suggest that sustained membrane depolarization induces novel nonlinear properties in voltage-gated Na+ channels. Prolonged membrane depolarization also induced a "molecular memory" phenomenon, characterized by clusters of dwell time events and strong autocorrelation in the dwell time series similar to that reported recently for single enzyme molecules. The persistence of such molecular memory was found to be dependent on the duration of depolarization. Voltage-gated Na+ channel with the observed time-dependent nonlinear properties and the molecular memory phenomenon may determine the functional state of the channel and, in turn, the excitability of a neuron.

Orexin-induced modulation of state-dependent intrinsic properties in thalamic paraventricular nucleus neurons attenuates action potential patterning and frequency

The thalamic paraventricular nucleus (PVT) receives a dense innervation from orexin-synthesizing lateral hypothalamic neurons. Since PVT neurons display state-dependent tonic or low threshold spike-driven burst firing patterns, we examined how the response to exogenously applied orexins might modulate these features. Data were obtained with whole-cell patch clamp recording techniques in rat brain slices prepared during the subjective lights-on period. PVT neurons displayed a mean resting membrane potential of −61±6 mV and input conductance of 1.3±0.1 nS (n=60). The majority (90/107) of cells tested responded to orexin A and/or orexin B peptides (100–1000 nM), each inducing similar slowly rising and prolonged membrane depolarizations. We next evaluated associated changes in firing patterns and action potential frequency. Of 17 spontaneously silent neurons, 5 were induced into tonic firing and 4 into burst firing modes. Of nine spontaneously bursting neurons, three displayed an increase in burst frequency and in the number of action potentials within a burst. By contrast, another six cells were induced into tonic firing mode, with a marked decrease in instantaneous firing frequency and a shift in their excitatory postsynaptic potential-evoked responses from burst firing patterns to single action potentials. Under voltage clamp, orexins induced inward current (−21.8±2.4 pA at −60 mV) in 20/22 cells. In 13 cells, current–voltage (I–V) plots revealed a decrease in net conductance and reversal at −110±9 mV, while 3 cells displayed an increase in net conductance that reversed at −26±8 mV. These observations imply suppression of potassium and/or induction of nonselective cationic conductances in orexin-induced depolarization in PVT neurons, permitting these peptides to modulate intrinsic state-dependent properties. In vivo, such changes in firing patterns and frequency of action potential discharges could influence neurotransmission through PVT and activity-dependent synaptic plasticity at target sites of these neurons.

Cholecystokinin B-Type Receptors Mediate a G-Protein-Dependent Depolarizing Action of Sulphated Cholecystokinin Ocatapeptide (CCK-8s) on Rodent Neonatal Spinal Ventral Horn Neurons

Reports of cholecystokinin (CCK) binding and expression of CCK receptors in neonatal rodent spinal cord suggest that CCK may influence neuronal excitability. In patch-clamp recordings from 19/21 ventral horn motoneurons in neonatal (PN 5-12 days) rat spinal cord slices, we noted a slowly rising and prolonged membrane depolarization induced by bath-applied sulfated CCK octapeptide (CCK-8s; 1 microM), blockable by the CCK B receptor antagonist L-365,260 (1 microM). Responses to nonsulfated CCK-8 or CCK-4 were significantly weaker. Under voltage clamp (V H -65 mV), 22/24 motoneurons displayed a CCK-8s-induced tetrodotoxin-resistant inward current [peak: -136 +/- 28 pA] with a similar time course, mediated via reduction in a potassium conductance. In 29/31 unidentified neurons, CCK-8s induced a significantly smaller inward current (peak: -42.8 +/- 5.6 pA), and I-V plots revealed either membrane conductance decrease with net inward current reversal at 101.3 +/- 4.4 mV (n = 16), membrane conductance increase with net current reversing at 36.1 +/- 3.8 mV (n = 4), or parallel shift (n = 9). Intracellular GTP-gamma-S significantly prolonged the effect of CCK-8s (n = 6), whereas GDP-beta-S significantly reduced the CCK-8s response (n = 6). Peak inward currents were significantly reduced after 5-min perfusion with N-ethylmaleimide. In isolated neonatal mouse spinal cord preparations, CCK-8s (30-300 nM) increased the amplitude and discharge of spontaneous depolarizations recorded from lumbosacral ventral roots. These observations imply functional postsynaptic G-protein-coupled CCK B receptors are prevalent in neonatal rodent spinal cord.

AMPA/kainate receptor-mediated up-regulation of GABA A receptor δ subunit mRNA expression in cultured rat cerebellar granule cells is dependent on NMDA receptor activation

We have studied the effects of AMPA/kainate receptor agonists on GABA A receptor subunit mRNA expression in vitro in cultured rat cerebellar granule cells (CGCs). Kainate (KA) (100 μM) and high K + (25 mM) dramatically up-regulated δ subunit mRNA expression to 500–700% of that in control cells grown in low K + (5 mM). KA or high K + had no effect on the expression of the other major GABA A receptor subunits α1, α6, β2, β3 or γ2. Up-regulation of δ mRNA was also detected with the AMPA receptor-selective agonist CPW-399 and to a lesser extent with the KA receptor-selective agonist ATPA. AMPA/kainate receptor-selective antagonist DNQX completely inhibited KA-, CPW-399- and ATPA-induced δ mRNA up-regulation indicating that the effects were mediated via AMPA and KA receptor activation. NMDA receptor-selective antagonist MK-801 inhibited 76% of the KA- and 57% of the CPW-399-induced δ up-regulation suggesting that KA and CPW-399 treatments may induce glutamate release resulting in NMDA receptor activation, and subsequently to δ mRNA up-regulation. In CGCs, δ subunit is a component of extrasynaptic α6βδ receptors that mediate tonic inhibition. Up-regulation of δ during prolonged glutamate receptor activation or cell membrane depolarization may be a mechanism to increase tonic inhibition to counteract excessive excitation.

Functional Analysis of Cav3.2 T‐type Calcium Channel Mutations Linked to Childhood Absence Epilepsy

Childhood absence epilepsy (CAE) is an idiopathic form of seizure disorder that is believed to have a genetic basis. We examined the biophysical consequences of seven mutations in the Ca(v)3.2 T-type calcium channel gene linked to CAE. Of the channel variants examined, one of the mutants, a replacement of glycine 848 in the domain II-S2 region with serine, resulted in significant slowing of the time courses of both activation and inactivation across a wide range of membrane potentials. These changes are consistent with increased channel activity in response to prolonged membrane depolarizations. Taken together, these findings suggest that such little changes in channel gating may contribute to the etiology of CAE.