Suppression of inhibitory GABAergic transmission by cAMP signaling pathway: alterations in learning and memory mutants

The cAMP signaling pathway mediates synaptic plasticity and is essential for memory formation in both vertebrates and invertebrates. In the fruit fly Drosophila melanogaster, mutations in the cAMP pathway lead to impaired olfactory learning. These mutant genes are preferentially expressed in the mushroom body (MB), an anatomical structure essential for learning. While cAMP‐mediated synaptic plasticity is known to be involved in facilitation at the excitatory synapses, little is known about its function in GABAergic synaptic plasticity and learning. In this study, using whole‐cell patch‐clamp techniques on Drosophila primary neuronal cultures, we demonstrate that focal application of an adenylate cyclase activator forskolin (FSK) suppressed inhibitory GABAergic postsynaptic currents (IPSCs). We observed a dual regulatory role of FSK on GABAergic transmission, where it increases overall excitability at GABAergic synapses, while simultaneously acting on postsynaptic GABA receptors to suppress GABAergic IPSCs. Further, we show that cAMP decreased GABAergic IPSCs in a PKA‐dependent manner through a postsynaptic mechanism. PKA acts through the modulation of ionotropic GABA receptor sensitivity to the neurotransmitter GABA. This regulation of GABAergic IPSCs is altered in the cAMP pathway and short‐term memory mutants dunce and rutabaga, with both showing altered GABA receptor sensitivity. Interestingly, this effect is also conserved in the MB neurons of both these mutants. Thus, our study suggests that alterations in cAMP‐mediated GABAergic plasticity, particularly in the MB neurons of cAMP mutants, account for their defects in olfactory learning.     

Adenosine A2A receptors and basal ganglia physiology

Adenosine A2A receptors are highly enriched in the basal ganglia system. They are predominantly expressed in enkephalin-expressing GABAergic striatopallidal neurons and therefore are highly relevant to the function of the indirect efferent pathway of the basal ganglia system. In these GABAergic enkephalinergic neurons, the A2A receptor tightly interacts structurally and functionally with the dopamine D2 receptor. Both by forming receptor heteromers and by targeting common intracellular signaling cascades, A2A and D2 receptors exhibit reciprocal antagonistic interactions that are central to the function of the indirect pathway and hence to basal ganglia control of movement, motor learning, motivation and reward. Consequently, this A2A/D2 receptors antagonistic interaction is also central to basal ganglia dysfunction in Parkinson's disease. However, recent evidence demonstrates that, in addition to this post-synaptic site of action, striatal A2A receptors are also expressed and have physiological relevance on pre-synaptic glutamatergic terminals of the cortico-limbic-striatal and thalamo-striatal pathways, where they form heteromeric receptor complexes with adenosine A1 receptors. Therefore, A2A receptors play an important fine-tuning role, boosting the efficiency of glutamatergic information flow in the indirect pathway by exerting control, either pre- and/or post-synaptically, over other key modulators of glutamatergic synapses, including D2 receptors, group I metabotropic mGlu5 glutamate receptors and cannabinoid CB1 receptors, and by triggering the cAMP-protein kinase A signaling cascade.     

Properties of GABA(A) receptor-mediated transmission at newly formed Golgi-granule cell synapses in the cerebellum

Cerebellar granule cells receive inhibitory synaptic input from Golgi cells, which is mediated by gamma-aminobutyric acid (GABA) acting on GABA(A) receptors. In the present study we examined the properties of GABAergic inhibitory postsynaptic currents (IPSCs) in granule cells of the rat at a time when they first receive synaptic contacts from Golgi cells. Our results demonstrate that granule cells receive functional GABAergic synaptic input as early as postnatal day three (P3). The kinetic properties of these early IPSCs and the single-channel conductance of the synaptic receptors are similar to those seen at the end of the first postnatal week, suggesting a stable subunit composition during this initial period of development. However, at P3, unlike the situation at more mature synapses, two distinct patterns of synaptic activity are evident, with IPSCs occurring either regularly or in bursts. In addition we find that Golgi cells are spontaneously active during early development, and at P7 most IPSCs are action potential-dependent. Moreover, paired Golgi-granule cell recordings suggest a high level of connectivity and a high release probability at these early synapses.     

Lateral Paracapsular GABAergic Synapses in the Basolateral Amygdala Contribute to the Anxiolytic Effects of ��3 Adrenoceptor Activation

Norepinephrine (NE) is known to play an integral role in the neurobiological response to stress. Exposure to stressful stimuli increases NE levels in brain regions that regulate stress and anxiety, like the basolateral amygdala (BLA). NE is thought to increase excitability in these areas through α- and β-adrenoceptors (ARs), leading to increased anxiety. Surprisingly, recent studies have shown that systemic β3-AR agonist administration decreases anxiety-like behaviors, suggesting that β3-ARs may inhibit excitability in anxiety-related brain regions. Therefore, in this study we integrated electrophysiological and behavioral approaches to test the hypothesis that the anxiolytic effects of β3-AR agonists may be mediated by an increase in BLA GABAergic inhibition. We examined the effect of a selective β3-AR agonist, BRL37344 (BRL), on GABAergic synapses arising from local circuit interneurons and inhibitory synapses originating from a recently described population of cells called lateral paracapsular (LPCS) interneurons. Surprisingly, BRL selectively enhanced LPCS-evoked inhibitory postsynaptic currents (eIPSCs) with no effect on local GABAergic inhibition. BRL also had no effect on glutamatergic synaptic excitation within the BLA. BRL potentiation of LPCS eIPSCs was blocked by the selective β3-AR antagonist, SR59230A, or by intracellular dialysis of Rp-CAMPS (cAMP-dependent protein kinase inhibitor), and this enhancement was not associated with any changes in spontaneous IPSCs or LPCS paired-pulse ratio. BRL also increased the amplitude of unitary LPCS IPSCs (uIPSCs) with no effect on uIPSC failure rate. Finally, bilateral BLA microinjection of BRL reduced anxiety-like behaviors in an open-field assay and the elevated plus-maze. Collectively, these data suggest that β3-AR activation selectively enhances LPCS, but not local, BLA GABAergic synapses, and that increases in LPCS-mediated inhibition may contribute to the anxiolytic profile of β3-AR agonists.     

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.     

GABA(B) receptor-mediated presynaptic inhibition of glycinergic transmission onto substantia gelatinosa neurons in the rat spinal cord

The GABA_B receptor-mediated presynaptic inhibition of glycinergic transmission was studied from young rat substantia gelatinosa (SG) neurons using a conventional whole-cell patch clamp technique. Action potential-dependent glycinergic inhibitory postsynaptic currents (IPSCs) were recorded from SG neurons in the presence of 3 mM kynurenic acid and 10 μM SR95531. In these conditions, baclofen (30 μM), a selective GABA_B receptor agonist, greatly reduced the amplitude of glycinergic IPSCs and increased the paired-pulse ratio. Such effects were completely blocked by 3 μM CGP55845, a selective GABA_B receptor antagonist, indicating that the activation of presynaptic GABA_B receptors decreases glycinergic synaptic transmission. Glycinergic IPSCs were largely dependent on Ca²⁺ influxes passing through presynaptic N- and P/Q-type Ca²⁺ channels, and these channels contributed equally to the baclofen-induced inhibition of glycinergic IPSCs. However, the baclofen-induced inhibition of glycinergic IPSCs was not affected by either 100 μM SQ22536, an adenylyl cyclase inhibitor, or 1 mM Ba²⁺, a G-protein coupled inwardly rectifying K⁺ channel blocker. During the train stimulation (10 pulses at 20 Hz), which caused a marked synaptic depression of glycinergic IPSCs, baclofen at a 30 μM concentration completely blocked glycinergic synaptic depression, but at a 3 μM concentration it largely preserved glycinergic synaptic depression. Such GABA_B receptor-mediated dynamic changes in short-term synaptic plasticity of glycinergic transmission onto SG neurons might contribute to the central processing of sensory signals.     

Tuning afferent synapses of hippocampal interneurons by neuropeptide Y

Cholecystokinin (CCK)‐expressing basket cells encompass a subclass of inhibitory GABAergic interneurons that regulate memory‐forming oscillatory network activity of the hippocampal formation in accordance to the emotional and motivational state of the animal, conveyed onto these cells by respective extrahippocampal afferents. Various excitatory and inhibitory afferent and efferent synapses of the hippocampal CCK basket cells express serotoninergic, cholinergic, cannabinoid, and benzodiazepine sensitive receptors, all contributing to their functional plasticity. We explored whether CCK basket cells are modulated by neuropeptide Y (NPY), one of the major local neuropeptides that strongly inhibits hippocampal excitability and has significant effect on its memory function. Here, using GAD65‐GFP transgenic mice for prospective identification of CCK basket cells and whole‐cell patch‐clamp recordings, we show for the first time that excitatory and inhibitory inputs onto CCK basket cells in the dentate gyrus of the hippocampus are modulated by NPY through activation of NPY Y2 receptors. The frequency of spontaneous and miniature EPSCs, as well as the amplitudes of stimulation‐evoked EPSCs were decreased. Similarly, the frequency of both spontaneous and miniature IPSCs, and the amplitudes of stimulation‐evoked IPSCs were decreased after NPY application. Most of the effects of NPY could be attributed to a presynaptic site of action. Our data provide the first evidence that the excitatory and inhibitory inputs onto the CCK basket cells could be modulated by local levels of NPY, and may change the way these cells process extrahippocampal afferent information, influencing hippocampal function and its network excitability during normal and pathological oscillatory activities. © 2009 Wiley‐Liss, Inc.     

Stiripentol, a putative antiepileptic drug, enhances the duration of opening of GABA-A receptor channels

PURPOSE: Stiripentol (STP) is currently an efficient drug for add-on therapy in infantile epilepsies because it improves the efficacy of antiepileptic drugs (AEDs) through its potent inhibition of liver cytochromes P450. In addition, STP directly reduces seizures in several animal models of epilepsy, suggesting that it might also have anticonvulsive effects of its own. However, its underlying mechanisms of action are unknown. METHODS: We examined the interactions of STP with gamma-aminobutyric acid (GABA) transmission by using patch-clamp methods in CA3 pyramidal neurons in the neonatal rat. RESULTS: STP markedly increased miniature inhibitory postsynaptic current (mIPSC) decay-time constant in a concentration-dependent manner. The prolongation of mIPSC duration does not result from an interaction with GABA transporters because it persisted in the presence of GAT-1 inhibitors (SKF-89976A and NO-711). An interaction with benzodiazepine or neurosteroid binding sites also was excluded because STP-mediated increase of decay time was still observed when these sites were initially saturated (by clobazam, zolpidem, or pregnanolone) or blocked (by flumazenil or dehydroepiandrosterone sulfate), respectively. In contrast, saturating barbiturate sites with pentobarbital clearly occluded this effect of STP, suggesting that STP and barbiturates interact at the same locus. This was directly confirmed by using outside-out patches, because STP increased the duration and not the frequency of opening of GABAA channels. CONCLUSIONS: At clinically relevant concentrations, STP enhances central GABA transmission through a barbiturate-like effect, suggesting that STP should possess an antiepileptic effect by itself.     

GABA-activated Chloride Currents of Postnatal Mouse Retinal Ganglion Cells are Blocked by Acetylcholine and Acetylcarnitine: How Specific are Ion Channels in Immature Neurons?

The goal of this study was to clarify pharmacological properties of GABA_A receptors in cells of the mouse retinal ganglion cell layer in situ. Spontaneous synaptic currents and responses to exogenous GABA were recorded from individual neurons in retinal whole mounts (postnatal days 1–3) or retinal stripe preparations (postnatal days 4–6). Drugs were applied by a fast local superfusion system. Current responses were measured with the patch-clamp technique in the whole-cell configuration. All cells responded to exogenous GABA (average EC₅₀ and Hill coefficient: 16.7 μM and 0.95 respectively) and generated GABAergic synaptic currents in response to elevated KCI. GABA-induced currents of retinal ganglion cells were blocked by bicuculline, picrotoxin and Zn²⁺, as well as strychnine, and increased by pentobarbital, clonazepam and 3α-hydroxy-5α-pregnan-20-one. In some retinal ganglion cells GABA caused an increase in the frequency of spontaneous synaptic currents, which points to a partially depolarizing action of this traditionally inhibitory neurotransmitter in the neural retina. Our major observation is that acetylcholine and acetylcarnitine blocked or reduced GABAergic inhibitory postsynaptic currents and responses to exogenous GABA. This effect was seen in only a fraction of retinal ganglion cells and occurred in both the undesensitized and the desensitized state of the GABA_A receptor. The block was voltage-independent and persisted during coapplication with the nicotinic and muscarinic acetylcholine receptor antagonists D-tubocurarine and atropine. In contrast to GABA-activated Cl⁻ currents, glycine-activated Ch currents remained unaffected by acetylcholine and acetylcarnitine. Acetylcarnitine had no effect on voltage-activated Ca²⁺ channel currents and glutamate-activated currents. Similar results were obtained in a dissociated cell culture preparation from the neonatal rat superior colliculus. In these cells acetylcholine induced a rightward shift in the dose - response curve for GABA. Taken together, these results indicate that acetylcholine and acetylcarnitine can act directly at the GABA_A binding site and thereby reduce the action of GABA in the immature retina.