Serotonin 5‐HT1B receptor‐mediated calcium influx‐independent presynaptic inhibition of GABA release onto rat basal forebrain cholinergic neurons

Modulatory roles of serotonin (5‐HT) in GABAergic transmission onto basal forebrain cholinergic neurons were investigated, using whole‐cell patch‐clamp technique in the rat brain slices. GABA_A receptor‐mediated inhibitory postsynaptic currents (IPSCs) were evoked by focal stimulation. Bath application of 5‐HT (0.1–300 μm ) reversibly suppressed the amplitude of evoked IPSCs in a concentration‐dependent manner. Application of a 5‐HT_1B receptor agonist, CP93129, also suppressed the evoked IPSCs, whereas a 5‐HT_1A receptor agonist, 8‐OH‐DPAT had little effect on the evoked IPSCs amplitude. In the presence of NAS‐181, a 5‐HT_1B receptor antagonist, 5‐HT‐induced suppression of evoked IPSCs was antagonised, whereas NAN‐190, a 5‐HT_1A receptor antagonist did not antagonise the 5‐HT‐induced suppression of evoked IPSCs. Bath application of 5‐HT reduced the frequency of spontaneous miniature IPSCs without changing their amplitude distribution. The effect of 5‐HT on miniature IPSCs remained unchanged when extracellular Ca²⁺ was replaced by Mg²⁺. The paired‐pulse ratio was increased by CP93129. In the presence of ω‐CgTX, the N‐type Ca²⁺ channel blocker, ω‐Aga‐TK, the P/Q‐type Ca²⁺ channel blocker, or SNX‐482, the R‐type Ca²⁺ channel blocker, 5‐HT could still inhibit the evoked IPSCs. 4‐AP, a K⁺ channel blocker, enhanced the evoked IPSCs, and CP93129 had no longer inhibitory effect in the presence of 4‐AP. CP93129 increased the number of action potentials elicited by depolarising current pulses. These results suggest that activation of presynaptic 5‐HT_1B receptors on the terminals of GABAergic afferents to basal forebrain cholinergic neurons inhibits GABA release in Ca²⁺ influx‐independent manner by modulation of K⁺ channels, leading to enhancement of neuronal activities.     

Impaired function of GABAB receptors in tissues from pharmacoresistant epilepsy patients

Purpose: Effects of pre‐ and postsynaptic γ‐aminobutyric acid B (GABA_B) receptor activation were characterized in human tissue from epilepsy surgery. Methods: Slices of human cortical tissue were investigated in a submerged‐type chamber with intracellular recordings in layers II/III. Parallel experiments were performed in rat neocortical slices with identical methods. Synaptic responses were elicited with single or paired stimulations of incrementing intervals. Results: Neurons in human epileptogenic tissue exhibited usually small inhibitory postsynaptic potentials (IPSP) mediated by GABA_B receptor, verified by the sensitivity to the selective antagonist CGP 55845A. The IPSP_B conductance averaged 5.8 nS in neurons from epileptogenic tissues and 15.9 nS in neurons from nonepileptogenic tissues (p < 0.0001). Application of baclofen caused small conductance increases in human neurons, which were linearly related to IPSP_B conductances. Paired‐pulse stimulation revealed constant synaptic responses in human temporal lobe epilepsy (TLE) slices at all interstimulus intervals (ISIs). Pharmacologically isolated IPSP_A in the human tissue exhibited a small paired‐pulse depression (average 10% at 500 ms ISI). Bicuculline‐induced paroxysmal depolarization shifts (PDSs) were transiently depressed by 24% in human TLE tissue; and by 74% in rat neocortical slices (200 ms ISI; p = 0.015). The depressions of bicuculline‐induced PDSs were antagonized by CGP 55845A in both species. Staining for GABA_B receptors revealed significantly smaller numbers of immunopositive dots in human epileptogenic neurons versus human control neurons. Discussion: The small IPSP_B, baclofen‐conductances, and paired‐pulse depression of PDSs and IPSPs in human TLE tissue indicate a reduced density of post‐ and presynaptic GABA_B receptors. The reduced efficacy of presynaptic GABA_B receptors facilitates the occurrence of repetitive synaptic activity.     

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.     

GABAB receptors increase intracellular calcium concentrations in chromaffin cells through two different pathways: Their role in catecholamine secretion

The activation of GABA_B receptors of adrenal chomaffin cells produces an increase of [Ca²⁺]_i measured by fura-2 AM techniques. GABA_B agonists 3-aminopropylphosphinic acid or (-)baclofen, at concentrations of 0.5 mM, increased basal Ca²⁺, values 332 ± 60.9 and 306 ± 40.5 nM, respectively, in cells suspended in a 2.5 mM Ca²⁺ buffer. The GABA_B-induced increase of [Ca²⁺]_i seemed to have two different components. The first was due to an entry from the extracellular medium mainly through L-type voltage-dependent Ca²⁺ channels as the dihydropiridine nifedipine 50 μM was able to decrease it more than 60%, while ω-conotoxin, which blocks N-type channels, did not produce any change in the GABA_B-evoked Ca²⁺ increment. The second component was due to a release of Ca²⁺ from intracellular pools and was about one-third of the total GABAB-induced increase of [Ca²⁺]_i. GABA_B receptors stimulated inositol 1,4,5-trisphosphate-sensitive and not the caffeine-sensitive Ca²⁺ store. In a low Ca²⁺ buffer after treatment with 2 μM angiotensin II, neither 0.5 mM 3-APPA nor baclofen were able to produce an additional increase of [Ca²⁺]_i, whereas 4 mM caffeine had no effect on GABA_B response. This intracellular Ca²⁺ mobilization could be due to inositol 1,4,5-trisphosphate accumulation produced by the activation of GABA_B receptors. In fact, the specific agonists after 10 minutes incubation produced a dosedependent increase of inositol 1,4,5-trisphosphate. The maximal effect was obtained at 100 μM baclofen and 3-APPA, and it was 3.63 ± 0.75 and 3.2 ± 1.5 times the basal levels (7.3 ± 0.3 pmol/10⁶ cells), respectively. In the absence of extracellular Ca²⁺, GABA_B-evoked catecholamine secretion and cyclic AMP formation were reduced more than 70%, suggesting an important role of extracellular Ca²⁺ in GABA_B mechanisms in adrenal chromaffin cells. © 1995 Wiley-Liss, Inc.     

Mechanism of the medium‐duration afterhyperpolarization in rat serotonergic neurons

Most serotonergic neurons display a prominent medium‐duration afterhyperpolarization (mAHP), which is mediated by small‐conductance Ca²⁺‐activated K⁺ (SK) channels. Recent ex vivo and in vivo experiments have suggested that SK channel blockade increases the firing rate and/or bursting in these neurons. The purpose of this study was therefore to characterize the source of Ca²⁺ which activates the mAHP channels in serotonergic neurons. In voltage‐clamp experiments, an outward current was recorded at ?60 mV after a depolarizing pulse to +100 mV. A supramaximal concentration of the SK channel blockers apamin or (‐)‐bicuculline methiodide blocked this outward current. This current was also sensitive to the broad Ca²⁺ channel blocker Co²⁺ and was partially blocked by both ω‐conotoxin and mibefradil, which are blockers of N‐type and T‐type Ca²⁺ channels, respectively. Neither blockers of other voltage‐gated Ca²⁺ channels nor DBHQ, an inhibitor of Ca²⁺‐induced Ca²⁺ release, had any effect on the SK current. In current‐clamp experiments, mAHPs following action potentials were only blocked by ω‐conotoxin and were unaffected by mibefradil. This was observed in slices from both juvenile and adult rats. Finally, when these neurons were induced to fire in an in vivo‐like pacemaker rate, only ω‐conotoxin was able to increase their firing rate (by ~30%), an effect identical to the one previously reported for apamin. Our results demonstrate that N‐type Ca²⁺ channels are the only source of Ca²⁺ which activates the SK channels underlying the mAHP. T‐type Ca²⁺ channels may also activate SK channels under different circumstances.     

Developmental increase in D1‐like dopamine receptor‐mediated inhibition of glutamatergic transmission through P/Q‐type channel regulation in the basal forebrain of rats

Whole‐cell patch‐clamp recordings of non‐N‐methyl‐d ‐aspartate glutamatergic excitatory postsynaptic currents (EPSCs) were carried out from cholinergic neurons in slices of basal forebrain (BF) of developing rats aged 21–42 postnatal days to elucidate postnatal developmental change in Ca²⁺ channel subtypes involved in the transmission as well as that in dopamine D₁‐like receptor‐mediated presynaptic inhibition. The amplitude of EPSCs was inhibited by bath application of ω‐conotoxin GVIA (ω‐CgTX; 3 μm ) or ω‐agatoxin‐TK (ω‐Aga‐TK; 200 nm ) throughout the age range examined, suggesting that multiple types of Ca²⁺ channel are involved in the transmission. The EPSC fraction reduced by ω‐CgTX decreased with age, whereas that reduced by ω‐Aga‐TK increased. Inhibition of the EPSCs by a D₁‐like receptor agonist, SKF 81297 (SKF; 30 μm ) increased with age in parallel with the increase in ω‐Aga‐TK‐induced inhibition. An activator of the adenylyl cyclase (AC) pathway, forskolin (FK; 10 μm ) inhibited the EPSCs, and FK‐induced inhibition also increased with age in parallel with the increase in SKF‐induced inhibition. Throughout the age range examined, SKF showed no further inhibitory effect on the EPSCs after ω‐Aga‐TK‐ or FK‐induced effect had reached steady‐state. These findings suggest that D₁‐like receptor‐mediated presynaptic inhibition of glutamate release onto cholinergic BF neurons increases with age, and that the change is coupled with a developmental increase in the contribution of P/Q‐type Ca²⁺ channels as well as a developmental increase in AC pathway contribution.     

Neurosteroid allopregnanolone inhibits glutamate release from rat cerebrocortical nerve terminals

Allopregnanolone, an active metabolite of progesterone, has been reported to exhibit neuroprotective activity in several preclinical models. Considering that the excitotoxicity caused by excessive glutamate is implicated in many brain disorders, the effect of allopregnanolone on glutamate release in rat cerebrocortical nerve terminals and possible underlying mechanism were investigated. We observed that allopregnanolone inhibited 4‐aminopyridine (4‐AP)‐evoked glutamate release, and this inhibition was prevented by chelating the extracellular Ca²⁺ ions and the vesicular transporter inhibitor. Allopregnanolone reduced the elevation of 4‐AP‐evoked intrasynaptosomal Ca²⁺ levels, but did not affect the synaptosomal membrane potential. In the presence of N‐, P/Q‐, and R‐type channel blockers, allopregnanolone‐mediated inhibition of 4‐AP‐evoked glutamate release was markedly reduced; however, the intracellular Ca²⁺‐release inhibitors did not affect the allopregnanolone effect. Furthermore, allopregnanolone‐mediated inhibition of 4‐AP‐evoked glutamate release was completely abolished in the synaptosomes pretreated with inhibitors of Ca²⁺/calmodulin, adenylate cyclase, and protein kinase A (PKA), namely calmidazolium, MDL12330A, and H89, respectively. Additionally, the allopregnanolone effect on evoked glutamate release was antagonized by the GABA_A receptor antagonist SR95531. Our data are the first to suggest that allopregnanolone reduce the Ca²⁺ influx through N‐, P/Q‐, and R‐type Ca²⁺ channels, through the activation of GABA_A receptors present on cerebrocortical nerve terminals, subsequently suppressing the Ca²⁺‐calmodulin/PKA cascade and decreasing 4‐AP‐evoked glutamate release.     

Role of GABAB receptors in intracellular Ca2+ homeostasis and possible interaction between GABAA and GABAB receptors in regulation of transmitter release in cerebellar granule neurons

The expression of GABA_B receptors in cultured mouse cerebellar granule cells was investigated in binding experiments using [³H](S, R)-baclofen as well as in functional assessment of the ability of (R)-baclofen to interact with depolarization (15–40 mM KCI) coupled changes in intracellular Ca²⁺ homeostasis and neurotransmitter release. In the latter case a possible functional coupling between GABA_A and GABA_B receptors was investigated. The binding studies showed that the granule cells express specific binding sites for (R)-baclofen. The number of binding sites could be increased by exposure of the cells to the GABA_A receptor agonist THIP (4,5,6,7-tetrahy-droisoxazolo[5,4-c]pyridin-3-ol) during the culture period. Pretreatment of the neurons with pertussis toxin showed that the GABA_B receptors are coupled to G-proteins. This coupling was, however, less pronounced when the cells had been cultured in the presence of THIP. When ⁴⁵Ca²⁺ uptake was measured or the intracellular Ca²⁺ concentration ([Ca²⁺]_i) determined using the fluorescent Ca²⁺ chelator Fluo-3 it could be demonstrated that culturing the neurons in THIP influences intracellular Ca²⁺ homeostasis. Moreover, this homeostasis was found to be functionally coupled to the GABA_B receptors as (R)-baclofen inhibited depolarization-induced increases in ⁴⁵Ca²⁺ uptake and [Ca²⁺]_i. (R)-Baclofen also inhibited K⁺-induced transmitter release from the neurons as monitored by the use of [³H]D -aspartate which labels the neurotransmitter pool of glutamate. Using the selective GABA_A receptor agonist isoguvacine it could be demonstrated that the GABA_B receptors are functionally coupled to GABA_A receptors in the neurons leading to a disinhibitory action of GABA_B receptor agonists. © 1994 Wiley-Liss, Inc.     

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.     

R‐type Ca2+ channels contribute to fast synaptic excitation and action potentials in subsets of myenteric neurons in the guinea pig intestine

Background R‐type Ca²⁺ channels are expressed by myenteric neurons in the guinea pig ileum but the specific function of these channels is unknown. Methods In the present study, we used intracellular electrophysiological techniques to determine the function of R‐type Ca²⁺ channels in myenteric neurons in the acutely isolated longitudinal muscle‐myenteric plexus. We used immunohistochemical methods to localize the Ca_V2.3 subunit of the R‐type Ca²⁺ channel in myenteric neurons. We also studied the effects of the non‐selective Ca²⁺ channel antagonist, CdCl₂ (100 μmol L^?1), the R‐type Ca²⁺ channel blockers NiCl₂ (50 μmol L^?1) and SNX‐482 (0.1 μmol L^?1), and the N‐type Ca²⁺ channel blocker ω‐conotoxin GVIA (CTX 0.1 μmol L^?1) on action potentials and fast and slow excitatory postsynaptic potentials (fEPSPs and sEPSPs) in S and AH neurons in vitro. Key Results Ca_V2.3 co‐localized with calretinin and calbindin in myenteric neurons. NiCl₂ and SNX‐482 reduced the duration and amplitude of action potentials in AH but not S neurons. NiCl₂ inhibited the afterhyperpolarization in AH neurons. ω‐conotoxin GVIA, but not NiCl₂, blocked sEPSPs in AH neurons. NiCl₂ and SNX‐482 inhibited cholinergic, but not cholinergic/purinergic, fEPSPs in S neurons. Conclusions and Inferences These data show that R‐type Ca²⁺ channels contribute to action potentials, but not slow synaptic transmission, in AH neurons. R‐type Ca²⁺ channels contribute to release of acetylcholine as the mediator of fEPSPs in some S neurons. These data indicate that R‐type Ca²⁺ channels may be a target for drugs that selectively modulate activity of AH neurons or could alter fast synaptic excitation in specific pathways in the myenteric plexus.     

GABAB-mediated modulation of ionic conductances in type I hair cells isolated from guinea-pig semicircular canals

Mammalian vestibular type I hair cells (VIHCs) are innervated by an afferent synaptic calyx which contains vesicles and is immunoreactive for GABA. We describe here the effects of GABA on electrophysiological properties and on cytosolic free-calcium levels ([Ca²⁺]_i) of VIHCs isolated from guinea-pig ampullae. Whole-cell tight-seal macroscopic currents recorded from VIHCs showed that 100 μM GABA induced a decrease in the outward currents elicited by depolarizing membrane potentials. These are known to comprise potassium calcium-dependent currents. This effect was mimicked by baclofen, a GABA_B agonist, and was not affected by picrotoxin, a GABA_A antagonist. GABA also induced an increase in the inward current elicited at hyperpolarized membrane potentials in 50% of the tested cells. Single channel recording in cell-attached patches revealed that externally applied GABA produced a decrease and an increase in the open probability of 170 pS and 45 pS and of 15 pS channels, respectively. In imaging experiments using the dye Fura-2 to measure [Ca²⁺]_i, the only reversible modulation of [Ca²⁺]_i observed in response to GABA application was a decrease. These results demonstrate a modulation of calcium and potassium conductances by GABA, via GABA_B receptors, in guinea-pig VIHCs.     

Electrophysiology of GABAA and GABAB receptor subtypes

The participation of GABA receptors in the inhibitory transmission at mammalian central synapses was demonstrated experimentally two decades ago. Whilst the ‘classical’ action of GABA involves the opening of Cl⁻ channels, pharmacologically distinct effects of GABA on cation channels were detected later. This led to the notion of GABA_A and GABA_B receptor subtypes. The GABA_A receptor complex contains an integral Cl⁻ ionophore, whereas GABA_B receptors couple to Ca²⁺ and K⁺ channels via GTP-binding proteins. The physiological and pharmacological properties of GABA_A and GABA_B receptors will be discussed below in terms of ion channels that are activated by the two receptor subtypes.     

Postnatal development of GABAB receptor-mediated modulation of voltage-activated Ca2+ currents in mouse brain-stem neurons.

GABA_B receptors modulate respiratory rhythm generation in adult mammals. However, little is currently known of their functional significance during postnatal development. In the present investigation, the effects of GABA_B receptor activation on voltage-activated Ca²⁺ currents were examined in rhythmically active neurons of the pre-Bötzinger complex (PBC). Both low- (LVA) and high-voltage-activated (HVA) Ca²⁺ currents were present from the first postnatal day (P1). The density of LVA Ca²⁺ currents increased during the first week, whilst the density of HVA Ca²⁺ currents increased after the first week. In the second postnatal week, the HVA Ca²⁺ currents were composed of L- (47 ± 10%) and N-type (21 ± 8%) currents plus a ‘residual’ current, whilst there were no N-type currents detectable in the first few days. The GABA_B receptor agonist baclofen (30 μm ) increased LVA Ca²⁺ currents (30 ± 11%) at P1–P3, but it decreased the currents (35 ± 11%) at P7–P15 without changing its time course. At all ages, baclofen (30 μm ) decreased the HVA Ca²⁺ currents by ≈ 54%. Threshold of baclofen effects on both LVA and HVA Ca²⁺ currents was 5 μm at P1–P3 and lower than 1 μm at P7–P15. The effect of baclofen was abolished in the presence of the GABA_B receptor antagonist CGP 55845A (50 n m ). We conclude that both LVA and HVA Ca²⁺ currents increased postnatally. The GABA_B receptor-mediated modulation of these currents undergo marked developmental changes during the first two postnatal weeks, which may contribute essentially to modulation of respiratory rhythm generation.     

Seletracetam (ucb 44212) inhibits high-voltage–activated Ca2+ currents and intracellular Ca2+ increase in rat cortical neurons in vitro

Purpose: We analyzed the effects of seletracetam (ucb 44212; SEL), a new antiepileptic drug candidate, in an in vitro model of epileptic activity. The activity of SEL was compared to the effects of levetiracetam (LEV; Keppra), in the same assays. Methods: Combined electrophysiologic and microfluorometric recordings were performed from layer V pyramidal neurons in rat cortical slices to study the effects of SEL on the paroxysmal depolarization shifts (PDSs), and the simultaneous elevations of intracellular Ca²⁺ concentration [Ca²⁺]_i. Moreover, the involvement of high‐voltage activated Ca²⁺ currents (HVACCs) was investigated by means of patch‐clamp recordings from acutely dissociated pyramidal neurons. Results: SEL significantly reduced both the duration of PDSs (IC₅₀ = 241.0 ± 21.7 nm ) as well as the number of action potentials per PDS (IC₅₀ = 82.7 ± 9.7 nm ). In addition, SEL largely decreased the [Ca²⁺]_i rise accompanying PDSs (up to 75% of control values, IC₅₀ = 345.0 ± 15.0 nm ). Furthermore, SEL significantly reduced HVACCs in pyramidal neurons. This effect was mimicked by ω‐conotoxin GVIA and, to a lesser extent, by ω‐conotoxin MVIIC, blockers of N‐ and Q‐type HVACC, respectively. The combination of these two toxins occluded the action of SEL, suggesting that N‐type Ca²⁺ channels, and partly Q‐type subtypes are preferentially targeted. Conclusions: These results demonstrate a powerful inhibitory effect of SEL on epileptiform events in vitro. SEL showed a higher potency than LEV. The effective limitation of [Ca²⁺]_i influx might be relevant for its antiepileptic efficacy and, more broadly, for pathologic processes involving neuronal [Ca²⁺]_i overload.     

γ‐Aminobutyric acid‐mediated regulation of the activity‐dependent olfactory bulb dopaminergic phenotype

γ‐Aminobutyric acid (GABA) regulates the proliferation and migration of olfactory bulb (OB) interneuron progenitors derived from the subventricular zone (SVZ), but the role of GABA in the differentiation of these progenitors has been largely unexplored. This study examines the role of GABA in the differentiation of OB dopaminergic interneurons using neonatal forebrain organotypic slice cultures prepared from transgenic mice expressing green fluorescent protein (GFP) under the control of the tyrosine hydroxylase (Th) gene promoter (ThGFP). KCl‐mediated depolarization of the slices induced ThGFP expression. The addition of GABA to the depolarized slices further increased GFP fluorescence by inducing ThGFP expression in an additional set of periglomerular cells. These findings show that GABA promoted differentiation of SVZ‐derived OB dopaminergic interneurons and suggest that GABA indirectly regulated Th expression and OB dopaminergic neuron differentiation through an acceleration of the maturation rate for the dopaminergic progenitors. Additional studies revealed that the effect of GABA on ThGFP expression required activation of L‐ and P/Q‐type Ca²⁺ channels as well as GABA_A and GABA_B receptors. These voltage‐gated Ca²⁺ channels and GABA receptors have previously been shown to be required for the coexpressed GABAergic phenotype in the OB interneurons. Together, these findings suggest that Th expression and the differentiation of OB dopaminergic interneurons are coupled to the coexpressed GABAergic phenotype and demonstrate a novel role for GABA in neurogenesis. © 2009 Wiley‐Liss, Inc.     

Effects of GABA on spontaneous [Ca]c dynamics and electrical properties of rat adrenal chromaffin cells

A large fraction of rat adrenal chromaffin cells (about 60%) shows spontaneous [Ca²⁺]_c oscillations and spontaneous action potentials. In the present study the effects of γ-aminobutyric acid (GABA) on the spontaneous [Ca²⁺]_c oscillations and electrical properties of rat adrenal chromaffin cells were investigated using Fura-2 [Ca²⁺]_c imaging and patch clamp techniques. GABA inhibited the spontaneous [Ca²⁺]_c oscillations in a reversible manner. The effect of GABA was mimicked by the GABA_A and GABA_C receptor agonist, muscimol, but not by the GABA_B receptor agonist, baclofen. Moreover, the effect was antagonized by the selective GABA_A receptor antagonist, bicuculline. The mode of the inhibition was all-or-none, and the threshold concentration at which the inhibition occurred varied widely (50 μM to over 1 μM) from cell to cell. GABA (100 μM) elicited a transient burst of action potentials of diminished amplitude, which was followed by arrest of action potentials. Further analysis showed that GABA (100 μM) induced inward whole-cell currents in voltage-clamp experiments and produced depolarization and membrane conductance increase in current-clamp experiments. The effects appear to be due to an increase in chloride ion conductance since the degree of GABA-induced depolarization depended on the pipette [Cl⁻]. These results suggest that GABA, acting through GABA_A receptor, may play a role in the physiological regulation of rat adrenal chromaffin cells by directly modifying the discharge of spontaneous action potentials and spontaneous [Ca²⁺]_c oscillations.     

Depression of glutamatergic and gabaergic synaptic responses in striatal spiny neurons by stimulation of presynaptic GABAB receptors

The influence of γ-aminobutyric acid_B (GABA_B) receptor stimulation on the excitatory and inhibitory synaptic potentials and membrane properties of identified striatal spiny neurons was examined in a corticostriatal slice preparation. Stimulation of the subcortical white matter evoked a monosynaptic, excitatory postsynaptic potential (EPSP) and a polysynaptic, inhibitory postsynaptic potential (IPSP) in spiny neurons. The EPSP had two components: a large amplitude response which could be blocked by the kainate/quisqualate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10μM), and a small amplitue, long-duration depolarization which could be blocked by the N-methyl-D-aspartate receptor antagonist, d-(-)-2-amino-5-phosphonovaleric acid (APV, 100 μM). The IPSP was observed as a membrane depolarization when recorded from neurons at resting membrane potential. However, when neurons were injected with the Na⁺-channel blocker, QX-314, allowing cells to be depolarized above their spike thresholds, a prominent hyperpolarizing IPSP was readily observed which could be blocked by the GABA_A antagonist, bicuculline (10-50 μM). This bicuculline-sensitive IPSP was responsible for the inhibition of EPSP amplitude and probability of spike discharge revealed using paired stimulation of the subcortical white matter. The amplitude of both the EPSP and the IPSP were depressed by the GABA_B receptor agonist, p-chlorophenyl-GABA (baclofen, 0.5-100 μM) in a concentration-dependent manner. Baclofen also blocked paired stimulus inhibition of spike discharge. These effects of baclofen persisted in slices in which the cortex was removed and were reversed by the GABA_B receptor antagonist, 3-amino-3-hydroxy-2-(4-chlorophenyl)-propanesulphonic acid (saclofen, 100-500 μM). In contrast to its profound influence on synaptic input, baclofen did not alter resting membrane potential, input resistance, membrane current-voltage relationship, or spike threshold of the cells recorded, and therefore did not appear to exert direct postsynaptic effects on the striatal spiny neurons. Taken together, these data indicate that the depressant effects of baclofen on EPSPs are mediated through GABA_B receptors located on the terminals of glutamatergic afferents within the striatum. Moreover, the results suggest that the actions of baclofen on IPSPs and paired stimulus inhibition are produced by activation of GABA_B receptors within the striatum at a site presynaptic to spiny neurons, either on the terminals of GABAergic afferents or on an interposed non-spiny GABAergic cell. Thus, GABA_B hetero- and auto-receptors have the capacity to provide a negative feedback mechanism through which the major excitatory and inhibitory inputs to striatal spiny neurons are regulated. © 1993 Wiley-Liss, Inc.     

Involvement of R‐type Ca2+ channels in neurotransmitter release from spinal dorsolateral funiculus terminals synapsing motoneurons

Molecular studies have revealed the presence of R‐type voltage‐gated Ca²⁺ channels at pre‐ and postsynaptic regions; however, no evidence for the participation of these channels in transmitter release has been presented for the spinal cord. Here we characterize the effects of SNX‐482, a selective R channel blocker, on the monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in motoneurons by stimulation of dorsolateral funiculus (DLF) terminals in a slice preparation from the adult turtle spinal cord. SNX‐482 inhibited neurotransmission in a dose‐dependent manner, with an IC₅₀ of ～9 ± 1 nM. The EPSP time course and membrane time constant of the motoneurons were not altered, suggesting a presynaptic mechanism. The toxin inhibited the residual component of the EPSPs recorded in the presence of N‐ and P/Q‐type Ca²⁺ channel blockers, strongly suggesting a role for the R channels in neurotransmission at the spinal cord DLF terminals. Consistently with this, RT‐PCR analysis of turtle spinal cord segments revealed the expression of the Ca_V2.3 pore‐forming (α_1E) subunit of R channels, whereas the use of anti‐α_1E‐specific antibodies resulted in its localization in the DLF fibers as demonstrated by immunohistochemistry coupled with laser confocal microscopy. J. Comp. Neurol. 513:188–196, 2009. © 2009 Wiley‐Liss, Inc.     

siRNA‐mediated GABAB receptor at early fetal rat brain upon acute and chronic ethanol exposure: Down regulation of PKA and p‐CREB expression

To observe the modulatory role of GABA_B1R upon ethanol's effect during early brain development, we studied the effects of chronic maternal (10% ethanol during pregnancy) and acute (in vitro) ethanol exposure on the neuronal protein kinase A (PKA‐α) and phosphorylation of cAMP‐response element binding protein (p‐CREB), using a system where GABA_B1R were specifically knocked down in the primary cells cultured at gestational day (GD) 12.5. The results showed that upon acute and chronic ethanol treatment the GABA_B1R expression was decreased and further decreased when GABA_B1R was transfection with siRNA, while increased upon exposure of baclofen, and baclofen plus phaclofen treatment. PKA expression was also decreased with acute and chronic ethanol treatment, whereas it showed increase upon exposure of baclofen and baclofen with phaclofen. Furthermore, intracellular Ca²⁺ concentration was increased upon ethanol, baclofen, phaclofen exposure but showed decrease in GABA_B1R siRNA group. Finally, these effects could lead to changes of p‐CREB expression, which showed same expression pattern as PKA. We speculate that GABA_BR activity upon ethanol exposure could modulate intracellular calcium homeostasis and the expressional changes of PKA and p‐CREB, which cause various negative effects on fetal brain development and modulation of GABA_BR upon ethanol exposure may underlying cause of ethanol's effects. Synapse, 2011. © 2010 Wiley‐Liss, Inc.