Distinct modes of modulation of GABAergic transmission by Group I metabotropic glutamate receptors in rat entorhinal cortex

Activation of metabotropic glutamate receptors (mGluRs) modulates synaptic transmission, whereas the roles of mGluRs in GABAergic transmission in the entorhinal cortex (EC) are elusive. Here, we examined the effects of mGluRs on GABAergic transmission onto the principal neurons in the superficial layers of the EC. Bath application of DHPG, a selective Group I mGluR agonist, increased the frequency and amplitude of spontaneous IPSCs (sIPSCs) whereas application of DCG‐IV, an agonist for Group II mGluRs or L‐AP4, an agonist for Group III mGluRs failed to change significantly sIPSC frequency and amplitude. Bath application of DHPG failed to change significantly the frequency and amplitude of miniature IPSCs (mIPSCs) recorded in the presence of tetradotoxin but significantly reduced the amplitude of IPSCs evoked by extracellular field stimulation or in synaptically connected interneuron‐pyramidal neuron pairs in layer III of the EC. DHPG increased the frequency but reduced the amplitude of APs recorded from entorhinal interneurons. Bath application of DHPG generated membrane depolarization and increased the input resistance of GABAergic interneurons. DHPG‐mediated depolarization of GABAergic interneurons was mediated by inhibition of background K⁺ channels which are insensitive to extracellular Cs⁺, TEA, 4‐AP, and Ba²⁺. DHPG‐induced facilitation of sIPSCs was mediated by mGluR₅ and required the function of Gαq but was independent of phospholipase C activity. Elevation of synaptic glutamate concentration by bath application of glutamate transporter inhibitors significantly increased sIPSC frequency and amplitude demonstrating a physiological role of mGluRs in GABAergic transmission. Our results provide a cellular and molecular mechanism to explain the physiological and pathological roles of mGluRs in the EC. © 2009 Wiley‐Liss, Inc.     

Impaired striatal GABA transmission in experimental autoimmune encephalomyelitis

Synaptic dysfunction triggers neuronal damage in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). While excessive glutamate signaling has been reported in the striatum of EAE, it is still uncertain whether GABA synapses are altered. Electrophysiological recordings showed a reduction of spontaneous GABAergic synaptic currents (sIPSCs) recorded from striatal projection neurons of mice with MOG_(35−55)-induced EAE. GABAergic sIPSC deficits started in the acute phase of the disease (20-25 days post immunization, dpi), and were exacerbated at later time-points (35, 50, 70 and 90 dpi). Of note, in slices they were independent of microglial activation and of release of TNF-α. Indeed, sIPSC inhibition likely involved synaptic inputs arising from GABAergic interneurons, because EAE preferentially reduced sIPSCs of high amplitude, and was associated with a selective loss of striatal parvalbumin (PV)-positive GABAergic interneurons, which contact striatal projection neurons in their somatic region, giving rise to more efficient synaptic inhibition. Furthermore, we found also that the chronic persistence of pro-inflammatory cytokines were able, per se, to produce profound alterations of electrophysiological network properties, that were reverted by GABA administration.The results of the present investigation indicate defective GABA transmission in MS models depending from alteration of PV cells number and, in part, deriving from the effects of a chronic inflammation, and suggest that pharmacological agents potentiating GABA signaling might be considered to limit neuronal damage in MS patients.     

Histamine facilitates GABAergic transmission in the rat entorhinal cortex: Roles of H1 and H2 receptors,Na+‐permeable cation channels,and inward rectifier K+ channels

In the brain, histamine (HA) serves as a neuromodulator and a neurotransmitter released from the tuberomammillary nucleus (TMN). HA is involved in wakefulness, thermoregulation, energy homeostasis, nociception, and learning and memory. The medial entorhinal cortex (MEC) receives inputs from the TMN and expresses HA receptors (H₁, H₂, and H₃). We investigated the effects of HA on GABAergic transmission in the MEC and found that HA significantly increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) with an EC₅₀ of 1.3 µM, but failed to significantly alter sIPSC amplitude. HA‐induced increases in sIPSC frequency were sensitive to tetrodotoxin (TTX), required extracellular Ca²⁺, and persisted when GDP‐β‐S, a G‐protein inactivator, was applied postsynaptically via the recording pipettes, indicating that HA increased GABA release by facilitating the excitability of GABAergic interneurons in the MEC. Recordings from local MEC interneurons revealed that HA significantly increased their excitability as determined by membrane depolarization, generation of an inward current at ?65 mV, and augmentation of action potential firing frequency. Both H₁ and H₂ receptors were involved in HA‐induced increases in sIPSCs and interneuron excitability. Immunohistochemical staining showed that both H₁ and H₂ receptors are expressed on GABAergic interneurons in the MEC. HA‐induced depolarization of interneurons involved a mixed ionic mechanism including activation of a Na⁺‐permeable cation channel and inhibition of a cesium‐sensitive inward rectifier K⁺ channel, although HA also inhibited the delayed rectifier K⁺ channels. Our results may provide a cellular mechanism, at least partially, to explain the roles of HA in the brain. © 2017 Wiley Periodicals, Inc.     

Deletion of Cav2.1(α1A) subunit of Ca2+‐channels impairs synaptic GABA and glutamate release in the mouse cerebellar cortex in cultured slices

Deletion of both alleles of the P/Q‐type Ca²⁺‐channel Ca_v2.1(α_1A) subunit gene in mouse leads to severe ataxia and early death. Using cerebellar slices obtained from 10 to 15 postnatal days mice and cultured for at least 3 weeks in vitro, we have analysed the synaptic alterations produced by genetically ablating the P/Q‐type Ca²⁺‐channels, and compared them with the effect of pharmacological inhibition of the P/Q‐ or N‐type channels on wild‐type littermate mice. Analysis of spontaneous synaptic currents recorded in Purkinje cells (PCs) indicated that the P/Q‐type channels play a prominent role at the inhibitory synapses afferent onto the PCs, with the effect of deleting Ca_v2.1(α_1A) partially compensated. At the granule cell (GC) to PC synapses, both N‐ and P/Q‐type Ca²⁺‐channels were found playing a role in glutamate exocytosis, but with no significant phenotypic compensation of the Ca_v2.1(α_1A) deletion. We also found that the P/Q‐ but not N‐type Ca²⁺‐channel is indispensable at the autaptic contacts between PCs. Tuning of the GC activity implicates both synaptic and sustained extrasynaptic γ‐aminobutyric acid (GABA) release, only the former was greatly impaired in the absence of P/Q‐type Ca²⁺‐channels. Overall, our data demonstrate that both P/Q‐ and N‐type Ca²⁺‐channels play a role in glutamate release, while the P/Q‐type is essential in GABA exocytosis in the cerebellum. Contrary to the other regions of the CNS, the effect of deleting the Ca_v2.1(α_1A) subunit is partially or not compensated at the inhibitory synapses. This may explain why cerebellar ataxia is observed at the mice lacking functional P/Q‐type channels.     

Enhanced Synaptic Inhibition in the Cerebellar Cortex of the Ataxic PMCA2−/− Knockout Mouse

Mice with genetic deletion of a calcium extrusion pump, the plasma membrane calcium ATPase isoform 2, PMCA2, exhibit overt cerebellar ataxia, but the cellular mechanisms are only partially understood. Here, we report an enhanced synaptic GABAergic inhibition within the molecular layer of cerebellar cortex slices from PMCA2 knockout (PMCA2^?/?) mice, a finding that could contribute to the observed ataxia. Purkinje neurons from PMCA2^?/? mice exhibited an increased frequency and amplitude of spontaneous inhibitory post-synaptic currents that was accompanied by an enhanced spontaneous firing frequency of molecular layer interneurons (both basket cells and stellate cells). The elevated inhibition was sufficient to reduce the frequency and regularity of spike firing by PMCA2^?/? Purkinje neurons. Acute pharmacological inhibition of PMCA recapitulated some of these features in wild-type mice indicating that the changes were in part a direct result of PMCA2 removal. However, additional compensatory mechanisms within the PMCA2^?/? mouse were also a major factor. Indeed, morphological studies revealed an abnormally large number of molecular layer interneurons (basket cells and stellate cells) and GABAergic synapses within the PMCA2^?/? cerebellar cortex. We conclude that loss of PMCA2 adversely influences the function and organisation of Purkinje neuron synaptic inhibition as a major contributory mechanism to the ataxic phenotype of the PMCA2^?/? mouse.     

Adenosine modulates the excitability of layer II stellate neurons in entorhinal cortex through A1 receptors

Stellate neurons in layer II entorhinal cortex (EC) provide the main output from the EC to the hippocampus. It is believed that adenosine plays a crucial role in neuronal excitability and synaptic transmission in the CNS, however, the function of adenosine in the EC is still elusive. Here, the data reported showed that adenosine hyperpolarized stellate neurons in a concentration‐dependent manner, accompanied by a decrease in firing frequency. This effect corresponded to the inhibition of the hyperpolarization‐activated, cation nonselective (HCN) channels. Surprisingly, the adenosine‐induced inhibition was blocked by 3 μM 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), a selective A₁ receptor antagonists, but not by 10 μM 3,7‐dimethyl‐1‐propargylxanthine (DMPX), a selective A₂ receptor antagonists, indicating that activation of adenosine A₁ receptors were responsible for the direct inhibition. In addition, adenosine reduced the frequency but not the amplitude of miniature EPSCs and IPSCs, suggesting that the global depression of glutamatergic and GABAergic transmission is mediated by a decrease in glutamate and GABA release, respectively. Again the presynaptic site of action was mediated by adenosine A₁ receptors. Furthermore, inhibition of spontaneous glutamate and GABA release by adenosine A₁ receptor activation was mediated by voltage‐dependent Ca²⁺ channels and extracellular Ca²⁺. Therefore, these findings revealed direct and indirect mechanisms by which activation of adenosine A₁ receptors on the cell bodies of stellate neurons and on the presynaptic terminals could regulate the excitability of these neurons. © 2010 Wiley‐Liss, Inc.     

GABAB receptor-mediated inhibition of spontaneous action potential discharge in rat supraoptic neurons in vitro

To elucidate the role of GABA_B receptors in the regulation of the electrical activity of magnocellular neurons of the supraoptic nucleus (SON), the effects of GABA_B agonist and antagonist on the firing rate of spontaneous action potentials were studied in SON slice preparations of rats by extracellular recordings. In the presence of the γ-amino butyric acid (GABA)-gated chloride channel blocker, picrotoxin, the selective GABA_B agonist, baclofen, reduced the firing rate of action potentials in both phasic and non-phasic neurons in a dose-dependent manner. The reduction in the firing rate induced by baclofen was reversed by the selective GABA_B antagonist, 2-hydroxy saclofen (2OH-saclofen), also in a dose-dependent manner. In non-phasic neurons, 2OH-saclofen significantly increased the firing rate and the effect was additive to the effect of picrotoxin. In phasic neurons, 2OH-saclofen alone did not increase the firing rate, but it reversed suppression of the firing induced by increasing extracellular Ca²⁺ concentration to 2.1 mM. Baclofen also reduced the firing rate of non-phasic neurons of virgin and lactating female rats, indicating that the GABA_B receptor-mediated inhibition is not confined to SON neurons of male rats. The evidence indicates that activation of GABA_B receptors inhibits electrical activity of SON neurons of both male and female rats and that GABA_B receptors may play an important role in the inhibitory regulation of the electrical activity of SON neurons by GABA.     

Arginine vasopressin differentially modulates GABAergic synaptic transmission onto temperature‐sensitive and temperature‐insensitive neurons in the rat preoptic area

The preoptic area (POA) of the hypothalamus, containing temperature‐sensitive and temperature‐insensitive neurons, plays a key role in specific thermoregulatory responses. Although arginine vasopressin (AVP) has been shown to induce hypothermia by increasing the firing activities of warm‐sensitive neurons and decreasing those of cold‐sensitive and temperature‐insensitive neurons, the effects of AVP on POA GABAergic transmission remain unknown. Herein, inhibitory postsynaptic currents (IPSCs) of temperature‐sensitive and temperature‐insensitive neurons in POA slices were recorded using whole‐cell patch clamp. By monitoring changes in GABAergic transmission during AVP treatment, we showed that AVP decreased the amplitudes and frequencies of spontaneous IPSCs in mostly warm‐sensitive neurons and in some temperature‐insensitive neurons but increased these parameters in other temperature‐insensitive neurons. The IPSC amplitude was reduced for only cold‐sensitive neurons. RT‐PCR and Western blot analyses further confirmed the POA expression of V1a receptors and GABA_A receptors, including the subunits α1, α2, α3, β2, β3 and γ2. The effects of AVP on IPSCs in temperature‐sensitive and temperature‐insensitive neurons were dependent on G proteins and intracellular Ca²⁺. AVP‐mediated modulation was associated with changes in the kinetic parameters (decay time, 10–90% rise time, half‐width). Together, these results suggest that AVP, acting via V1a receptors but not V1b receptors, differentially modulates GABAergic synaptic transmission and fine‐tunes the firing activities of temperature‐sensitive and temperature‐insensitive neurons in the rat POA.     

Effect of Cocaine on Ion Channels and Glutamatergic EPSCs in Noradrenergic Locus Coeruleus Neurons

The locus coeruleus (LC) is an important brainstem area involved in cocaine addiction. However, evidence to elucidate how cocaine modulates the activity of LC neurons remains incomplete. Here, we performed whole recordings in brain slices to evaluate the effects of cocaine on the sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺) channels, and glutamatergic synaptic transmission in the locus coeruleus neurons. Local application of cocaine significantly and reversibly reduced the spontaneous firing rate but did not affect action potential amplitude, rising time, decay time, or half width of noradrenergic locus coeruleus neurons. Moreover, cocaine attenuated the sodium current but did not affect potassium and calcium currents. The N-methyl-d-aspartate receptor mediated excitatory postsynaptic currents were reduced by neuropeptide galanin but not cocaine. All those data demonstrate that cocaine has inhibitory effect on the spontaneous activities and sodium current in locus coeruleus neurons. Therefore, neuromodulation of sodium channel in locus coeruleus neurons may play an important role in drug addiction.     

Synaptic Multivesicular Release in the Cerebellar Cortex: Its Mechanism and Role in Neural Encoding and Processing

The number of synaptic vesicles released during fast release plays a major role in determining the strength of postsynaptic response. However, it remains unresolved how the number of vesicles released in response to action potentials is controlled at a single synapse. Recent findings suggest that the Ca_v2.1 subtype (P/Q-type) of voltage-gated calcium channels is responsible for inducing presynaptic multivesicular release (MVR) at rat cerebellar glutamatergic synapses from granule cells to molecular layer interneurons. The topographical distance from Ca_v2.1 channels to exocytotic Ca²⁺ sensors is a critical determinant of MVR. In physiological trains of presynaptic neurons, MVR significantly impacts the excitability of postsynaptic neurons, not only by increasing peak amplitude but also by prolonging decay time of the postsynaptic currents. Therefore, MVR contributes additional complexity to neural encoding and processing in the cerebellar cortex.     

Different effects of α-chloralose on spontaneous and evoked GABA release in rat hippocampal CA1 neurons

The effects of α-chloralose on presynaptic GABA(A) receptors were investigated with respect to spontaneous and evoked GABAergic transmission (sIPSCs and eIPSCs) in rat hippocampal CA1 pyramidal neurons. sIPSCs were recorded in mechanically dissociated CA1 neurons with intact GABAergic terminals, namely the "synaptic bouton preparation." eIPSCs were elicited by focal electrical stimuli of a single GABAergic bouton on an isolated CA1 neuron using the whole-cell patch recording configurations under voltage-clamp condition. We found that α-chloralose potentiated the exogenous GABA-induced Cl(-) response in a concentration dependent manner, and the drug itself induced Cl(-) response at high concentrations (>100 μM). α-Chloralose at low concentrations (3-10 μM) increased sIPSC frequency without affecting the current amplitude and kinetics, but prolonged the slow current decay time constant (τ(s)) at concentrations greater than 30 μM without changing either current amplitude or frequency. α-Chloralose at 10 μM enhanced amplitude of eIPSCs and decreased the failure rate (Rf), but at 30 μM decreased the amplitude and increased the Rf. Pretreatment with bumetanide, a blocker of NKCC-1, completely prevented the 30 μM α-chloralose-induced inhibition on eIPSC amplitude and Rf. These results suggest that α-chloralose activates GABA(A) receptors on GABAergic presynaptic nerve terminals and depolarizes the terminals, mediating presynaptic inhibition or autoregulation, in a concentration-dependent manner. In addition, α-chloralose at high concentrations activates not only extrasynaptic GABA(A) receptors on the postsynaptic soma membrane but also synaptic GABA(A) receptors resulting in prolongation of current decay phase. Thus α-chloralose induces complex and differential modulation of sIPSCs and eIPSCs in a concentration dependent manner.     

Basic fibroblast growth factor modulates synaptic transmission in cultured rat hippocampal neurons

Basic fibroblast growth factor (bFGF) is one of the effective growth factors that protect neurons against excitotoxic/ischemic injury and promote neuronal survival. In the present study, we examined the acute modulative effect of bFGF on synaptic transmission by monitoring spontaneous intracellular Ca²⁺ ([Ca²⁺]i]) oscillation, the amplitudes of which reflect excitatory and inhibitory inputs. The hippocampal cells from embryonic day 18 rats were cultured for 11–14 days, and changes in [Ca²⁺]i of single neurons were measured by a microfluometrical technique with fura-2. The amplitude of spontaneous oscillation was decreased by 10 ng/ml bFGF, but not by nerve growth factor (10–1000 ng/ml). Acidic FGF (1000 ng/ml) had a weaker depressant effect. The effect of bFGF was counteracted by suramin. bFGF did not affect the increase in [Ca²⁺]i evoked by glutamate agonists, NMDA or kainate, indicating that glutamate receptors are not involved in the mechanism. This is supported by similar results that kainate-evoked current was not affected by bFGF. On the other hand, bicuculline masked the effect of bFGF on the Ca²⁺ oscillation. But GABA-evoked current was slightly decreased by bFGF. These results suggest the possible role of bFGF in modulating GABAergic rather than glutamatergic neurotransmission.     

Inhibition of spontaneous GABAergic transmission by trimethylolpropane phosphate

Trimethylolpropane phosphate (TMPP) is a neuroactive organophosphate generated during partial pyrolysis of a synthetic ester turbine engine lubricant. While TMPP had been shown to have little affinity for acetylcholinesterase, previous binding studies and 6Cl- flux measurements have implicated TMPP as an antagonist of GABA, receptor/Cl- channels. Using the whole-cell patch clamp method, spontaneous inhibitory postsynaptic currents (sIPSCs) mediated by bicuculline-sensitive GABA(A) receptors were measured in neurons cultured from the rat embryonic hippocampus for 13-21 days. Experiments were conducted in the presence of tetrodotoxin and 6-cyano-7-nitroquinoxaline to inhibit spontaneous presynaptic action potentials and glutamate transmission, respectively, thus isolating GABAergic sIPSCs for study. TMPP induced a concentration-dependent inhibition of sIPSC amplitude and frequency suggesting both postsynaptic and presynaptic actions. Administration of 5 microM TMPP reversibly diminished sIPSC amplitude by 23 +/- 8% (mean SEM, n=5 cells) while markedly decreasing the mean sIPSC frequency by 40 +/- 2% (n=5). The mean time constant of sIPSC decay was reversibly decreased by 20 +/- 4% (n=3) in the presence of 20 microM TMPP, suggesting an increase in the rate of inactivation. To directly verify the blockade of ionotropic GABA receptors by TMPP, the effects of TMPP were examined on whole-cell Cl- current responses activated by exogenous GABA. Administration of TMPP (5 microM) depressed peak whole-cell GABA-induced currents to 73 1% (n=4) of control levels, consistent with the results on sIPSC amplitude. Our data directly demonstrate that TMPP directly inhibits GABA(A) receptor function, as indicated by the blockade of whole-cell GABA-mediated Cl- current and the reduction in sIPSC amplitude. Furthermore, TMPP exerts a presynaptic effect on GABAergic transmission, as evidenced by the reduction in sIPSC frequency, which may be independent of a GABA(A) receptor. The molecular basis for the presynaptic action of TMPP remains to be elucidated.     

Familial hemiplegic migraine

Familial hemiplegic migraine (FHM) is a rare and genetically heterogeneous autosomal dominant subtype of migraine with aura. Mutations in the genes CACNA1A and SCNA1A, encoding the pore-forming α₁ subunits of the neuronal voltage-gated Ca²⁺ channels Ca_v2.1 and Na⁺ channels Na_v1.1, are responsible for FHM1 and FHM3, respectively, whereas mutations in ATP1A2, encoding the α₂ subunit of the Na⁺, K⁺ adenosinetriphosphatase (ATPase), are responsible for FHM2. This review discusses the functional studies of two FHM1 knockin mice and of several FHM mutants in heterologous expression systems (12 FHM1, 8 FHM2, and 1 FHM3). These studies show the following: (1) FHM1 mutations produce gain-of-function of the Ca_v2.1 channel and, as a consequence, increased Ca_v2.1-dependent neurotransmitter release from cortical neurons and facilitation of in vivo induction and propagation of cortical spreading depression (CSD: the phenomenon underlying migraine aura); (2) FHM2 mutations produce loss-of-function of the α₂ Na⁺,K⁺-ATPase; and (3) the FHM3 mutation accelerates recovery from fast inactivation of Na_v1.5 (and presumably Na_v1.1) channels. These findings are consistent with the hypothesis that FHM mutations share the ability of rendering the brain more susceptible to CSD by causing either excessive synaptic glutamate release (FHM1) or decreased removal of K⁺ and glutamate from the synaptic cleft (FHM2) or excessive extracellular K⁺ (FHM3). The FHM data support a key role of CSD in migraine pathogenesis and point to cortical hyperexcitability as the basis for vulnerability to CSD and to migraine attacks. Hence, they support novel therapeutic strategics that consider CSD and cortical hyperexcitability as key targets for preventive migraine treatment.     

Differential sensitivity of cerebellar purkinje neurons to ethanol in selectivity outbred lines of mice: maintenance in vitro independent of synaptic transmission

The effects of ethanol on spontaneous firing of cerebellar Purkinje neurons were examined in outbred lines of mice (short-sleep, SS; and long-sleep, LS) which exhibit differential behavioral sensitivity to ethanol. In order to determine whether the differences in Purkinje cell ethanol sensitivity which are observed in situ reflect differences in intrinsic properties of Purkinje neurons, we developed an isolated in vitro preparation of mouse cerebellum. Even when synaptic transmission was largely inhibited by elevating Mg²⁺ and decreasing Ca²⁺ concentrations, Purkinje cells demonstrated stable long-term firing rates quite similar to those observed in vivo.Purkinje cells responded to superfusion of ethanol with both increases and decreases in firing rate. Inhibition of rate was more commonly observed, and was the only response which was demonstrably dose-dependent. The differential sensitivity to ethanol which we have previously reported in vivo was maintained even under these condtions, with the LS mice being approximately 5 times more sensitive to the depressant effects of ethanol. In addition, it was shown that ethanol, at the concentrations used in these experiments, decreased the amplitude and increased the duration of single action potentials.Thus, taken together, these results suggest that the differential sensitivity of outbred lines to the soporific effects of ethanol are paralleled by differences in the sensitivity of Purkinje neurons in vitro to superfusion with ethanol. Because these differences can be observed even when synaptic transmission is largely suppressed, it would appear that these differences are intrinsic to the Purkinje neurons themselves.     

Roscovitine differentially facilitates cerebellar glutamatergic and GABAergic neurotransmission by enhancing Cav2.1 channel‐mediated multivesicular release

Synaptic vesicle exocytosis is triggered by Ca²⁺ influx through several subtypes of voltage‐gated calcium channels in the presynaptic terminal. We previously reported that paired‐pulse stimulation at brief intervals increases Ca_v2.1 (P/Q‐type) channel‐mediated multivesicular release (MVR) at glutamatergic synapses between granule cells (GCs) and molecular layer interneurons (MLIs) in rat cerebellar slices. However, it has yet to be determined how Ca_v2 channel subtypes take part in MVR in single axon terminal. This study therefore aimed at examining the effects of roscovitine on different types of cerebellar synapses that make contacts with Purkinje cells (PCs), because this compound has been shown to enhance Ca_v2.1 channel‐mediated MVR at GC‐MLI synapses. Bath application of roscovitine profoundly increased the amplitude of excitatory postsynaptic currents (EPSCs) at GC‐PC synapses by a presynaptic mechanism as previously observed at GC‐MLI synapses, whereas it caused a marginal effect on climbing fiber‐mediated EPSCs in PCs. At MLI‐PC synapses, roscovitine increased both the amplitude and decay time of inhibitory postsynaptic currents (IPSCs) by enhancing multivesicular GABA release. When extracellular Ca²⁺ concentration ([Ca²⁺]_e) decreased, roscovitine became less effective in increasing GC‐PC EPSCs. By contrast, roscovitine was able to augment MLI‐PC IPSCs in the low [Ca²⁺]_e. The Ca_v2.1 channel blocker ω‐agatoxin IVA suppressed the roscovitine‐induced facilitatory actions on both GC‐PC EPSCs and MLI‐PC IPSCs. These results demonstrate that roscovitine enhances MVR at the GC‐PC excitatory synapses in a manner dependent on the driving force of Ca_v2.1 channel‐mediated Ca²⁺ influx into the nerve terminal, while it also facilitates MLI‐PC inhibitory transmission via Ca²⁺‐insensitive mechanisms.     

Differences in synaptic integration between direct and indirect striatal projection neurons: Role of CaV3 channels

Different corticostriatal suprathreshold responses in direct and indirect striatal projection neurons (SPNs) of rodents have been reported. Responses consist in prolonged synaptic potentials of polysynaptic and intrinsic origin, in which voltage‐gated Ca²? currents play a role. Recording simultaneous Ca²? imaging and voltage responses at the soma, while activating the corticostriatal pathway, we show that encoding of synaptic responses into trains of action potentials (APs) is different in SPNs: firing of APs in D1‐SPNs increase gradually, in parallel with Ca²? entry, as a function of stimulus intensity. In contrast, D2‐SPNs attain a maximum number of evoked spikes at low stimulus intensities, Ca²? entry is limited, and both remain the same in spite of increasing stimulus strength. Stimulus needs to reach certain intensity, to have propagated Ca²? potentials to the soma plus a sudden step in Ca²? entry, without changing the number of fired APs, phenomena never seen in D1‐SPNs. Constant firing in spite of changing stimulus, suggested the involvement of underlying inactivating potentials. We found that Ca?3 currents contribute to Ca²⁺ entry in both classes of SPNs, but have a more notable effect in D2‐SPNs, where a low‐threshold spike was disclosed. Blockade of Ca_V3 channels retarded the steep rise in firing in D2‐SPNs. Inhibition block increased the number of spikes fired by D2‐SPNs, without changing firing in D1‐SPNs. These differences in synaptic integration enable a biophysical dissimilarity: dendritic inhibition appears to be more relevant for D2‐SPNs. This may imply distinctions in the set of interneurons affecting each SPN class.     

Fentanyl inhibits GABAergic neurotransmission to cardiac vagal neurons in the nucleus ambiguus

Fentanyl citrate is a synthetic opiate analgesic often used clinically for neonatal anesthesia. Although fentanyl significantly depresses heart rate, the mechanism of inducing bradycardia remains unclear. One possible site of action is the cardioinhibitory parasympathetic vagal neurons in the nucleus ambiguus (NA), from which originates control of heart rate and cardiac function. Inhibitory synaptic activity to cardiac vagal neurons is a major determinant of their activity. Therefore, the effect of fentanyl on GABAergic neurotransmission to parasympathetic cardiac vagal neurons was studied using whole-cell patch clamp electrophysiology. Application of fentanyl induced a reduction in both the frequency and amplitude of GABAergic IPSCs in cardiac vagal neurons. This inhibition was mediated at both pre- and postsynaptic sites as evidenced by a dual decrease in the frequency and amplitude of spontaneous miniature IPSCs. Application of the selective micro-antagonist CTOP abolished the fentanyl-mediated inhibition of GABAergic IPSCs. These results demonstrate that fentanyl acts on micro-opioid receptors on cardiac vagal neurons and neurons preceding them to reduce GABAergic neurotransmission and increase parasympathetic activity. The inhibition of GABAergic effects may be one mechanism by which fentanyl induces bradycardia.     

Inhibitory effects of levetiracetam on the high-voltage-activated L-type Ca2+ channels in hippocampal CA3 neurons of spontaneously epileptic rat (SER)

Levetiracetam (LEV) is a widely used antiepileptic agent for partial refractory epilepsy in humans. LEV has unique antiepileptic effects in that it does not inhibit electroshock- or pentylenetetrazol-induced convulsion, but does inhibit seizures in kindling animal and spontaneously epileptic rat (SER: zi/zi, tm/tm) that shows both tonic convulsion and absence-like seizures. LEV also has unique characteristics in terms of its antiepileptic mechanism; it has no activity on Na⁺ and K⁺ channels or on glutamate and GABA_A receptors. Recently, we found that LEV inhibits the depolarization shift and accompanying repetitive firing induced by mossy fiber stimulation in CA3 neurons of SER hippocampal slices. Therefore, this study was performed to determine whether LEV could inhibit the voltage-activated L-type Ca²⁺ current of hippocampal CA3 neurons obtained from SER and the non-epileptic Wistar rat. As previously reported, SER CA3 neurons were classified into type 1 and type 2 neurons. The application of LEV (100 μM) elevated the threshold for activation of the Ca²⁺ current, which was lowered in SER type 1 neurons and reduced the current size. Type 2 neurons of SER have a similar current–voltage relationship to Wistar rat neurons and the decay component of Ca²⁺ current during depolarization pulse in type 2 neurons was found to be smaller than that in Wistar rat neurons. LEV (100 μM) also reduced Ca²⁺ current in SER type 2 neurons. The effects of LEV were examined on such type 2 SER hippocampal CA3 neurons, compared with those on Wistar rat CA3 neurons. Application of LEV (10 μM) produced a significant decrease of amplitude of the Ca²⁺ current in SER neurons, although at this concentration of LEV there was no statistically significant decrease in the amplitude of Ca²⁺ current in Wistar rat neurons. Furthermore, LEV (100 nM–1 mM) reduced the Ca²⁺ current in a concentration-dependent manner in both SER and Wistar rat neurons, but the inhibition was much more potent in the former neurons than in the latter. Under the condition that the Ca²⁺ current had already been inhibited by LEV (10 μM), the addition of nifedipine (10 μM) did not cause further inhibition. Conversely, LEV had no effects on the current that had already been decreased by nifedipine (10 μM) given before LEV treatment (10 μM), indicating that LEV could act on the L-type Ca²⁺channel. LEV elevated the threshold potential level for activation of the Ca²⁺ current and reduced the L-type Ca²⁺ current in type 1 neurons of SER, and the inhibitory action in type 2 neurons was much more potent than that in Wistar rat neurons, suggesting that these effects contribute, at least partly, to the antiepileptic action of LEV.     

Ryanodine‐sensitive intracellular Ca2+ channels are involved in the output from the SCN circadian clock

The suprachiasmatic nuclei (SCN) contain the major circadian clock responsible for generation of circadian rhythms in mammals. The time measured by the molecular circadian clock must eventually be translated into a neuronal firing rate pattern to transmit a meaningful signal to other tissues and organs in the animal. Previous observations suggest that circadian modulation of ryanodine receptors (RyR) is a key element of the output pathway from the molecular circadian clock. To directly test this hypothesis, we studied the effects of RyR activation and inhibition on real time expression of PERIOD2::LUCIFERASE, intracellular calcium levels and spontaneous firing frequency in mouse SCN neurons. Furthermore, we determined whether the RyR‐2 mRNA is expressed with a daily variation in SCN neurons. We provide evidence that pharmacological manipulation of RyR in mice SCN neurons alters the free [Ca²⁺]_i in the cytoplasm and the spontaneous firing without affecting the molecular clock mechanism. Our data also show a daily variation in RyR‐2 mRNA from single mouse SCN neurons with highest levels during the day. Together, these results confirm the hypothesis that RyR‐2 is a key element of the circadian clock output from SCN neurons.