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.     

Serotonin (5‐HT) regulates neurite outgrowth through 5‐HT1A and 5‐HT7 receptors in cultured hippocampal neurons

Serotonin (5‐HT) production and expression of 5‐HT receptors (5‐HTRs) occur early during prenatal development. Recent evidence suggests that, in addition to its classical role as a neurotransmitter, 5‐HT regulates neuronal connectivity during mammalian development by modulating cell migration and neuronal cytoarchitecture. Given the variety of 5‐HTRs, researchers have had difficulty clarifying the specific role of each receptor subtype in brain development. Signalling mediated by the G‐protein‐coupled 5‐HT_1AR and 5‐HT₇R, however, has been associated with neuronal plasticity. Thus, we hypothesized that 5‐HT promotes neurite outgrowth through 5‐HT_1AR and 5‐HT₇R. The involvement of 5‐HT_1AR and 5‐HT₇R in the morphology of rat hippocampal neurons was evaluated by treating primary cultures at 2 days in vitro with 5‐HT and specific antagonists for 5‐HT_1AR and 5‐HT₇R (WAY‐100635 and SB269970, respectively). The stimulation of hippocampal neurons with 100 nM 5‐HT for 24 hr produced no effect on either the number or the length of primary neurites. Nonetheless, after 5HT₇R was blocked, the addition of 5‐HT increased the number of primary neurites, suggesting that 5HT₇R could inhibit neuritogenesis. In contrast, 5‐HT induced secondary neurite outgrowth, an effect inhibited by 1 μM WAY‐100635 or SB269970. These results suggest that both serotonergic receptors participate in secondary neurite outgrowth. We conclude that 5‐HT_1AR and 5‐HT₇R regulate neuronal morphology in primary hippocampal cultures by promoting secondary neurite outgrowth. © 2014 Wiley Periodicals, Inc.     

Experimental early‐life febrile seizures induce changes in GABAAR‐mediated neurotransmission in the dentate gyrus

Purpose: Febrile seizures (FS), the most frequent seizure type during childhood, have been linked to temporal lobe epilepsy (TLE) in adulthood. Yet, underlying mechanisms are still largely unknown. Altered γ‐aminobutyric acid (GABA)ergic neurotransmission in the dentate gyrus (DG) circuit has been hypothesized to be involved. This study aims at analyzing whether experimental FS change inhibitory synaptic input and postsynaptic GABA_AR function in dentate granule cells. Methods: We applied an immature rat model of hyperthermia (HT)–induced FS. GABA_AR‐mediated neurotransmission was studied using whole‐cell patch‐clamp recordings from dentate granule neurons in hippocampal slices within 6–9 days post‐HT. Key Findings: Frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs) were reduced in HT rats that had experienced seizures, whereas sIPSC amplitudes were enhanced. Whole‐cell GABA responses revealed a doubled GABA_AR sensitivity in dentate granule cells from HT animals, compared to that of normothermic (NT) controls. Analysis of sIPSCs and whole‐cell GABA responses showed similar kinetics in postsynaptic GABA_ARs of HT and NT rats. quantitative real‐time polymerase chain reaction (qPCR) experiments indicated changes in DG GABA_AR subunit expression, which was most pronounced for the α3 subunit. Significance: The data support the hypothesis that FS persistently alter neuronal excitability.     

5‐hydroxytryptamine (5‐HT,serotonin) and Parkinson's disease – opportunities for novel therapeutics to reduce the problems of levodopa therapy

While Parkinson's disease is undoubtedly a disorder with a primary pathology of dopamine neuronal loss, that loss of dopamine and subsequent dopamine replacement therapy leads to imbalances in many non‐dopaminergic transmitter systems, including 5‐hydroxytryptamine (5‐HT). Recent advances in understanding the role of 5‐HT in parkinsonism and the generation of side‐effects of dopamine replacement therapy (e.g. wearing‐off and levodopa‐induced dyskinesia) have identified 5‐HT_1A, 5‐HT_1B and 5‐HT_2C receptors as potential therapeutic targets in Parkinson's disease.     

Heterogeneous distribution of the serotonin 5‐HT1A receptor mRNA in chemically identified neurons of the mouse rostral brainstem: Implications for the role of serotonin in the regulation of wakefulness and REM sleep

The 5‐HT_1A receptor (5‐HT_1AR) plays a key role in the inhibitory influence of serotonin (5‐HT) on rapid eye movement (REM) sleep in rodents. However, the neuronal networks mediating such influence are mostly unknown, notably in the mouse. This led us to map 5‐HT_1AR mRNA, by in situ hybridization histochemistry (ISHH), and to characterize the neuronal phenotype of 5‐HT_1AR mRNA‐positive neurons by dual ISHH and ISHH combined with immunohistochemistry, throughout the mouse rostral brainstem, a pivotal region for the generation of REM sleep and cortical activation. 5‐HT_1AR mRNA was found in most 5‐HT neurons in the dorsal raphe (DR), the median raphe (MnR), the B9, and the interpeduncular (IP) nuclei. 5‐HT_1AR mRNA‐positive neurons were also identified in individualized clusters of γ‐aminobutyric acid (GABA)ergic neurons in the DR and in neurons of an undetermined phenotype in the MnR. In addition, 1) GABAergic neurons of the ventral portion of Gudden's dorsal tegmental nucleus (DTg), the IP, and the caudal portion of the deep mesencephalic nucleus (DpMe), and 2) glutamatergic neurons scattered in the caudal pontine reticular nucleus (PnC) and densely packed in the internal lateral parabrachial subnucleus (PBil) also expressed 5‐HT_1AR mRNA. In contrast, no specific 5‐HT_1AR‐related ISHH signal was generally detected in brainstem cholinergic and catecholaminergic neurons. These results emphasize the role of 5‐HT_1AR as an autoreceptor and the phenotypical heterogeneity of 5‐HT_1AR‐expressing neurons within the DR and the MnR in the mouse brain. They also provide a neuroanatomical basis for understanding the influence of 5‐HT_1AR on REM sleep and wakefulness. J. Comp. Neurol. 518:2744–2770, 2010. © 2010 Wiley‐Liss, Inc.     

Roles for γ‐aminobutyric acid in the development of the paraventricular nucleus of the hypothalamus

The development of the hypothalamic paraventricular nucleus (PVN) involves several factors that work together to establish a cell group that regulates neuroendocrine functions and behaviors. Several molecular markers were noted within the developing PVN, including estrogen receptors (ER), neuronal nitric oxide synthase (nNOS), and brain‐derived neurotrophic factor (BDNF). By contrast, immunoreactive γ‐aminobutyric acid (GABA) was found in cells and fibers surrounding the PVN. Two animal models were used to test the hypothesis that GABA works through GABA_A and GABA_B receptors to influence the development of the PVN. Treatment with bicuculline to decrease GABA_A receptor signaling from embryonic day (E) 10 to E17 resulted in fewer cells containing immunoreactive (ir) ERα in the region of the PVN vs. control. GABA_BR1 receptor subunit knockout mice were used to examine the PVN at P0 without GABA_B signaling. In female but not male GABA_BR1 subunit knockout mice, the positions of cells containing ir ERα shifted from medial to lateral compared with wild‐type controls, whereas the total number of ir ERα‐containing cells was unchanged. In E17 knockout mice, ir nNOS cells and fibers were spread over a greater area. There was also a significant decrease in ir BDNF in the knockout mice in a region‐dependent manner. Changes in cell position and protein expression subsequent to disruption of GABA signaling may be due, in part, to changes in nNOS and BDNF signaling. Based on the current study, the PVN can be added as another site where GABA exerts morphogenetic actions in development. J. Comp. Neurol. 518:2710–2728, 2010. © 2010 Wiley‐Liss, Inc.     

Activation of 5‐HT receptors inhibits GABAergic transmission by pre‐and post‐synaptic mechanisms in layer II/III of the juvenile rat auditory cortex

The specific mechanisms by which serotonin (5‐HT) modulates synaptic transmission in the auditory cortex are still unknown. In this work, we used whole‐cell recordings from layer II/III of pyramidal neurons in rat brain slices to characterize the influence of 5‐HT on inhibitory synaptic activity in the auditory cortex after pharmacological blockade of excitatory glutamatergic transmission. We found that bath application of 5‐HT (5 µM) reduced the frequency and amplitude of both spontaneous and miniature inhibitory postsynaptic currents (IPSCs), reduced the amplitude of evoked IPSCs, and enhanced facilitation of paired pulse ratio (PPR), suggesting presynaptic inhibition. To determine which the serotonin receptors were involved in this effect, we studied the influence of specific 5‐HT receptor agonists and antagonists on ?‐aminobutyric acid (GABA)ergic synaptic transmission. The inhibiting influence of 5‐HT in the GABAergic synaptic activity was mimicked by using the selective agonists of the 5‐HT_1A and 5‐HT_2A receptors, 8(OH)‐DPAT (10 µM) and DOI (10 µM), respectively; and it was prevented by their respective antagonists NAN‐190 (1 µM) and ritanserin (1 μM). Furthermore, the application of the selective agonist of 5‐HT_1A receptors, 8‐(OH)‐DPAT (10 µM), produced PPR facilitation, while DOI application (5‐HT_2A agonist) did not change the PPR. Moreover, the 5‐HT_2A agonist reduced the amplitude of the IPSCs evoked by application of the selective GABA agonist, muscimol. These results suggest a presynaptic and postsynaptic reduction of GABAergic transmission mediated by 5‐HT_1A and 5‐HT_2A serotonergic receptors, respectively. Synapse 69:115–127, 2015. © 2014 Wiley Periodicals, Inc.     

An immunocapture/scintillation proximity analysis of Gαq/11 activation by native serotonin (5‐HT)2A receptors in rat cortex: Blockade by clozapine and mirtazapine

Though transduction mechanisms recruited by heterologously expressed 5‐HT_2A receptors have been extensively studied, their interaction with specific subtypes of G‐protein remains to be directly evaluated in cerebral tissue. Herein, as shown by an immunocapture/scintillation proximity analysis, 5‐HT, the prototypical 5‐HT_2A agonist, DOI, and Ro60,0175 all enhanced [³⁵S]GTPγS binding to Gαq/11 in rat cortex with pEC₅₀ values of 6.22, 7.24 and 6.35, respectively. No activation of Go or Gs/olf was seen at equivalent concentrations of DOI. Stimulation of Gαq/11 by 5‐HT (30 μM) and DOI (30 μM) was abolished by the selective 5‐HT_2A vs. 5‐HT_2C/5‐HT_2B antagonists, ketanserin (pK_B values of 9.11 and 8.88, respectively) and MDL100,907 (9.82 and 9.68). By contrast, 5‐HT‐induced [³⁵S]GTPγS binding to Gαq/11 was only weakly inhibited by the preferential 5‐HT_2C receptor antagonists, RS102,221 (6.94) and SB242,084 (7.39), and the preferential 5‐HT_2B receptor antagonist, LY266,097 (6.66). The antipsychotic, clozapine, which had marked affinity for 5‐HT_2A receptors, blocked the recruitment of Gαq/11 by 5‐HT and DOI with pK_B values of 8.54 and 8.14, respectively. Its actions were mimicked by the “atypical” antidepressant and 5‐HT_2A receptor antagonist, mirtazapine, which likewise blocked 5‐HT and DOI‐induced Gαq/11 protein activation with pK_B values of 7.90 and 7.76, respectively. In conclusion, by use of an immunocapture/scintillation proximity strategy, this study shows that native 5‐HT_2A receptors in rat frontal cortex specifically recruit Gαq/11 and that this action is blocked by clozapine and mirtazapine. Quantification of 5‐HT_2A receptor‐mediated Gαq/11 activation in frontal cortex should prove instructive in characterizing the actions of diverse classes of psychotropic agent. Synapse 63:95–105, 2009. © 2008 Wiley‐Liss, Inc.     

Behavioral and electrophysiological effects of 5‐HT in globus pallidus of 6‐hydroxydopamine lesioned rats

Anatomical studies have shown that the globus pallidus receives abundant 5‐hydroxytryptamine (5‐HT) innervations from raphe nuclei. 5‐HT may occupy an important position in the modulation of motor function through its affect on the activity of globus pallidus. In the present study, intrapallidal microinjection of 5‐HT (0.1 mM) alone did not induce any motor behavior or postural asymmetry in the unilateral 6‐hydroxydopamine (6‐OHDA)‐lesioned rats. However, when infused concomitantly with a low dose of 3, 4‐dihydroxyphenylalanine (L‐DOPA, 3 mg/kg i.p.), which itself can induce modest contralateral rotational behavior, 5‐HT significantly potentiated the number of contralateral rotations. To elucidate the cellular mechanism, in vivo extracellular recordings were performed to examine the effects of 5‐HT on globus pallidus neurons. In normal rats, the predominant effect of micropressure ejection of 5‐HT on pallidal neurons was excitation. In 6‐OHDA‐lesioned rats, although 5‐HT increased the firing rate in most pallidal neurons, 5‐HT‐induced inhibitory effects was stronger than that on the unlesioned side as well as normal rats. Furthermore, 5‐HT_1B receptors are mainly involved in 5‐HT‐induced excitation while 5‐HT_1A receptors are involved in 5‐HT‐induced inhibition. The results suggest that 5‐HT may potentiate the antiparkinsonian effect of L‐DOPA through modulating the activity of globus pallidus. © 2009 Wiley‐Liss, Inc.     

5‐HT1B autoreceptor regulation of serotonin transporter activity in synaptosomes

Serotonin‐1B (5‐HT_1B) autoreceptors are located in serotonin (5‐HT) terminals, along with serotonin transporters (SERT), and play a critical role in autoregulation of serotonergic neurotransmission and are implicated in disorders of serotonergic function, particularly emotional regulation. SERT modulates serotonergic neurotransmission by high‐affinity reuptake of 5‐HT. Alterations in SERT activity are associated with increased risk for depression and anxiety. Several neurotransmitter receptors are known to regulate SERT K_m and V_max, and previous work suggests that 5‐HT_1B autoreceptors may regulate 5‐HT reuptake, in addition to modulating 5‐HT release and synthesis. We used rotating disk electrode voltammetry to investigate 5‐HT_1B autoreceptor regulation of SERT‐mediated 5‐HT uptake into synaptosomes. The selective 5‐HT_1B antagonist SB224289 decreased SERT activity in synaptosomes prepared from wild‐type but not 5‐HT_1B knockout mice, whereas SERT uptake was enhanced after pretreatment with the selective 5‐HT_1B agonist CP94253. Furthermore, SERT activity varies as a function of 5‐HT_1B receptor expression—specifically, genetic deletion of 5‐HT_1B decreased SERT function, while viral‐mediated overexpression of 5‐HT_1B autoreceptors in rat raphe neurons increased SERT activity in rat hippocampal synaptosomes. Considered collectively, these results provide evidence that 5‐HT_1B autoreceptors regulate SERT activity. Because SERT clearance rate varies as a function of 5‐HT_1B autoreceptor expression levels and is modulated by both activation and inhibition of 5‐HT_1B autoreceptors, this dynamic interaction may be an important mechanism of serotonin autoregulation with therapeutic implications. Synapse, 2012. © 2012 Wiley Periodicals, Inc.     

Adaptations in 5‐HT receptor expression and function: Implications for treatment of cognitive impairment in aging

Malfunction of the serotonin system may contribute to memory deficits during aging. We evaluated the 5‐HT₆ antagonist RO4368554 in two models of learning and memory in aged rats. Male rats (18 months) were assigned to two groups of equal cognitive performance. After 2 weeks of 5‐HT₆ antagonist RO4368554 (5 mg/kg, i.p.) treatment, rats showed significant improvement in object recognition and social discrimination compared with rats given chronic vehicle. Brains from these animals were examined for changes in plasticity‐associated proteins Ki‐67 and PCNA. No differences were seen between groups in any of these markers. We also measured mRNA expression of 5‐HT₆, along with 5‐HT_1A, 5‐HT_1B, and tryptophan hydroxylase‐2 mRNAs in 4‐month‐old and 24‐month‐old F344 rats. Decreases in 5‐HT_1B expression were observed in several forebrain regions in the old rats. These results demonstrate that 5‐HT₆ and 5‐HT_1B receptors are potential targets for treatment of age‐related memory disorders. © 2009 Wiley‐Liss, Inc.     

A 5‐HT4‐receptor activation‐induced neural plasticity enhances in vivo reconstructs of enteric nerve circuit insult

Background It was recently reported that some 5‐HT₄‐receptor agonists increased neuronal numbers and length of neurites in enteric neurons developing in vitro from immunoselected neural crest‐derived precursors. We aimed to explore a novel approach in vivo to reconstruct the enteric neural circuitry that mediates a fundamental distal gut reflex. Methods The neural circuit insult was performed in guinea pigs by rectal transection and subsequent end‐to‐end one layer anastomosis. A 5‐HT₄‐receptor agonist, mosapride citrate (10–100 μmol L^?1) (applied for a patent) was applied locally at the anastomotic site. Key Results Mosapride promoted the regeneration of the neural circuit in the impaired myenteric plexus and the recovery of the defecation reflex in the distal gut. Furthermore, mosapride generated neurofilament (NF)‐, 5‐HT₄‐receptor‐ and 5‐bromo‐2′‐deoxyuridine (BrdU)‐positive cells and surprisingly formed neural network in the newly formed granulation tissue at the anastomotic site 2 weeks after enteric nerve circuit insult. Possible neural stem cell markers, anti‐distal less homeobox 2 (DLX2)‐ and p75‐positive and NF‐positive cells increased during the same time period. All actions by mosapride were inhibited by the specific 5‐HT₄‐receptor antagonist, GR113808 (10 μmol L^?1). Conclusions & Inferences These results indicate that activation of enteric neural 5‐HT₄‐receptors promotes reconstruction of an enteric neural circuit leading to the recovery of the defecation reflex in the distal gut, and that this reconstruction involves possibly neural stem cells. These findings indicate that treatment with 5‐HT₄ agonists could be a novel therapy for generating new enteric neurons to rescue aganglionic gut disorders.     

Effects of temperature elevation on neuronal inhibition in hippocampal neurons of immature and mature rats

Febrile seizures are the most common seizure type in children, and hyperthermia may contribute to seizure generation during fever. We have previously demonstrated that hyperthermia suppressed γ‐aminobutyric acid (GABA)‐ergic synaptic transmission in CA1 neurons of immature rats. However, whether this suppression is age‐dependent is unknown. Moreover, it is unclear whether hyperthermia has differential effects on neuronal inhibition in CA1 pyramidal cells (PCs) and dentate gyrus granule cells (GCs). In this study, we investigated the effects of hyperthermia on GABA_A and GABA_B receptor‐mediated inhibitory postsynaptic currents (IPSCs) in CA1 and DG neurons from immature (11–17 days old) and mature (6–8 weeks old) rats using whole‐cell recordings in vitro. In immature rats, hyperthermia decreased the peak amplitude of GABA_A receptor‐mediated IPSCs (GABA_A IPSCs) in PCs but not in GCs. However, hyperthermia decreased the decay time constant of GABA_A IPSCs to a similar extent in both PCs and GCs. In mature rats, hyperthermia decreased the peak amplitude but not the decay time constant of GABA_A IPSCs in both PCs and GCs. Hyperthermia decreased charge transfer (area) of the GABA_A IPSC of PCs more in immature than in mature rats. In contrast, hyperthermia decreased the GABA_B receptor‐mediated IPSCs to the same degree in immature and mature rats, for either CA1 or DG neurons. Because the hippocampus has been found to be involved in hyperthermia‐induced behavioral seizures in immature rats, we suggest that the higher sensitivity of CA1 inhibitory synaptic function to hyperthermia in immature compared with mature rats might partially explain the higher susceptibility for febrile seizures in immature animals. © 2009 Wiley‐Liss, Inc.     

Tripchlorolide protects neuronal cells from microglia‐mediated β‐amyloid neurotoxicity through inhibiting NF‐κB and JNK signaling

Recent research has focused on soluble oligomeric assemblies of β‐amyloid peptides (Aβ) as the proximate cause of neuroinflammation, synaptic loss, and the eventual dementia associated with Alzheimer's disease (AD). In this study, tripchlorolide (T₄), an extract of Tripterygium wilfordii Hook. F (TWHF), was studied as a novel agent to suppress neuroinflammatory process in microglial cells and to protect neuronal cells against microglia‐mediated oligomeric Aβ toxicity. T₄ significantly attenuated oligomeric Aβ(1‐42)‐induced release of inflammatory productions such as tumor necrosis factor‐α, interleukin‐1β, nitric oxide (NO), and prostaglandin E₂. It also downregulated the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) in microglial cells. Further molecular mechanism study demonstrated that T₄ inhibited the nuclear translocation of nuclear factor‐κB (NF‐κB) without affecting I‐κBα phosphorylation. It repressed Aβ‐induced JNK phosphorylation but not ERK or p38 MAPK. The inhibition of NF‐κB and JNK by T₄ is correlated with the suppression of inflammatory mediators in Aβ‐stimulated microglial cells. These results suggest that T₄ protects neuronal cells by blocking inflammatory responses of microglial cells to oligomeric Aβ(1‐42) and that T₄ acts on the signaling of NF‐κB and JNK, which are involved in the modulation of inflammatory response. Therefore, T₄ may be an effective agent in modulating neuroinflammatory process in AD. © 2009 Wiley‐Liss, Inc.     

Serotonin‐2C receptor agonists decrease potassium‐stimulated GABA release in the nucleus accumbens

The serotonin 5‐HT_2C receptor has shown promise in vivo as a pharmacotherapeutic target for alcoholism. For example, recently, a novel 4‐phenyl‐2‐N,N‐dimethylaminotetralin (PAT) drug candidate, that demonstrates 5‐HT_2C receptor agonist activity together with 5‐HT_2A/2B receptor inverse agonist activity, was shown to reduce operant responding for ethanol after peripheral administration to rats. Previous studies have shown that the 5‐HT_2C receptor is found throughout the mesoaccumbens pathway and that 5‐HT_2C receptor agonism causes activation of ventral tegmental area (VTA) GABA neurons. It is unknown what effect 5‐HT_2C receptor modulation has on GABA release in the nucleus accumbens core (NAcc). To this end, microdialysis coupled to capillary electrophoresis with laser‐induced fluorescence was used to quantify extracellular neurotransmitter concentrations in the NAcc under basal and after potassium stimulation conditions, in response to PAT analogs and other 5‐HT_2C receptor modulators administered by reverse dialysis to rats. 5‐HT_2C receptor agonists specifically attenuated stimulated GABA release in the NAcc while 5‐HT_2C antagonists or inverse agonists had no effect. Agents with activity at 5‐HT_2A receptors had no effect on GABA release. Thus, in contrast to results reported for the VTA, current results suggest 5‐HT_2C receptor agonists decrease stimulated GABA release in the NAcc, and provide a possible mechanism of action for 5HT_2C‐mediated negative modulation of ethanol self‐administration. Synapse 69:78–85, 2015. © 2014 Wiley Periodicals, Inc.     

γ‐Aminobutyric acid‐ρ expression in ependymal glial cells of the mouse cerebellum

The ependymal glial cells (EGCs) from the periventricular zone of the cerebellum were studied to determine their distribution and the functional properties of their γ‐aminobutyric acid type A (GABA_A) receptors. EGCs were identified by the presence of ciliated structures on their ventricular surface and their expression of glial fibrillary acidic protein (GFAP). Interestingly, diverse cell types, including neurons, astrocytes, and other types of glia, were identified in the subventricular zone by their current profiles. Electron microscopy showed ciliated cells and myelinated axons in this zone, but we found no collateral connections to suggest the presence of functional synapses. GABA‐mediated currents were recorded from EGCs in cerebellar slices from postnatal days 13 to 35 (PN13–PN35). These currents were blocked by TPMPA (a highly specific GABA_Aρ subunit antagonist) and bicuculline (a selective antagonist for classic GABA_A receptors). Pentobarbital failed to modulate GABA_A‐mediated currents despite the expression of GABAα1 and GABAγ2 subunits. In situ hybridization, RT‐PCR, and immunofluorescence studies confirmed GABAρ1 expression in EGCs of the cerebellum. We conclude that cerebellar EGCs express GABAρ1, which is functionally involved in GABA_A receptor‐mediated responses that are unique among glial cells of the brain. © 2013 Wiley Periodicals, Inc.     

GABAB receptor‐dependent modulation of network activity in the rat prefrontal cortex in vitro

GABA (γ‐aminobutyric acid) can mediate inhibition via pre‐ and post/extrasynaptic GABA receptors. In this paper we demonstrate potentially post/extrasynaptic GABA_B receptor‐dependent tonic inhibition in L2/3 pyramidal cells of rat medial prefrontal cortex (mPFC) in vitro. First, we show via voltage‐clamp experiments the presence of a tonic GABA_B receptor‐dependent outward current in these neurons. This GABA_Bergic current could be induced by ambient GABA when present at sufficient concentrations. To increase ambient GABA levels in the usually silent slice preparation, we amplified network activity and hence synaptic GABA release with a modified artificial cerebrospinal fluid. The amplitude of tonic GABA_B current was similar at different temperatures. In addition to the tonic GABA_B current, we found presynaptic GABA_B effects, GABA_B‐mediated inhibitory postsynaptic currents and tonic GABA_A currents. Second, we performed current‐clamp experiments to evaluate the functional impact of GABA_B receptor‐mediated inhibition in the mPFC. Activating or inactivating GABA_B receptors led to rightward (reduction of excitability) or leftward (increase of excitability) shifts, respectively, of the input–output function of mPFC L2/3 pyramidal cells without effects on the slope. Finally, we showed in electrophysiological recordings and epifluorescence Ca²⁺‐imaging that GABA_B receptor‐mediated tonic inhibition is capable of regulating network activity. Blocking GABA_B receptors increased the frequency of excitatory postsynaptic currents impinging on a neuron and prolonged network upstates. These results show that ambient GABA via GABA_B receptors is powerful enough to modulate neuronal excitability and the activity of neural networks.     

Cocaine withdrawal reduces GABABR transmission at entopeduncular nucleus – lateral habenula synapses

Lateral habenula (LHb) hyperactivity plays a pivotal role in the emergence of negative emotional states, including those occurring during withdrawal from addictive drugs. We have previously implicated cocaine‐driven adaptations at synapses from the entopeduncular nucleus (EPN) to the LHb in this process. Specifically, ionotropic GABA_A receptor (R)‐mediated neurotransmission at EPN‐to‐LHb synapses is reduced during cocaine withdrawal, due to impaired vesicle filling. Recent studies have shown that metabotropic GABA_BR signaling also controls LHb activity, although its role at EPN‐to‐LHb synapses during drug withdrawal is unknown. Here, we predicted that cocaine treatment would reduce GABA_BR‐mediated neurotransmission at EPN‐to‐LHb synapses. We chronically treated mice with saline or cocaine, prepared brain slices after two days of withdrawal and performed voltage‐clamp recordings from LHb neurons whilst optogenetically stimulating EPN terminals. Compared with controls, mice in cocaine withdrawal exhibited reduced GABA_AR‐mediated input to LHb neurons, and a reduced occurrence of GABA_BR‐signaling at EPN‐to‐LHb synapses. We then assessed the underlying mechanism of this decrease. Application of GABA_BR agonist baclofen evoked similar postsynaptic responses in EPN‐innervated LHb neurons in saline‐ and cocaine‐treated mice. Release probability at EPN‐to‐LHb GABAergic synapses was also comparable between groups. However, incubating brain slices in glutamine to facilitate GABA vesicle filling, normalized GABA_BR‐currents at EPN‐to‐LHb synapses in cocaine‐treated mice. Overall, we show that during cocaine withdrawal, together with reduced GABA_AR transmission, also GABA_BR‐mediated inhibitory signaling is diminished at EPN‐to‐LHb synapses, likely via the same presynaptic deficit. In concert, these alterations are predicted to contribute to the emergence of drug withdrawal symptoms, facilitating drug relapse.     

Anti‐dyskinetic mechanisms of amantadine and dextromethorphan in the 6‐OHDA rat model of Parkinson’s disease: role of NMDA vs. 5‐HT1A receptors

Amantadine and dextromethorphan suppress levodopa (L‐DOPA)‐induced dyskinesia (LID) in patients with Parkinson’s disease (PD) and abnormal involuntary movements (AIMs) in the unilateral 6‐hydroxydopamine (6‐OHDA) rat model. These effects have been attributed to N‐methyl‐d ‐aspartate (NMDA) antagonism. However, amantadine and dextromethorphan are also thought to block serotonin (5‐HT) uptake and cause 5‐HT overflow, leading to stimulation of 5‐HT_1A receptors, which has been shown to reduce LID. We undertook a study in 6‐OHDA rats to determine whether the anti‐dyskinetic effects of these two compounds are mediated by NMDA antagonism and/or 5‐HT_1A agonism. In addition, we assessed the sensorimotor effects of these drugs using the Vibrissae‐Stimulated Forelimb Placement and Cylinder tests. Our data show that the AIM‐suppressing effect of amantadine was not affected by the 5‐HT_1A antagonist WAY‐100635, but was partially reversed by the NMDA agonist d ‐cycloserine. Conversely, the AIM‐suppressing effect of dextromethorphan was prevented by WAY‐100635 but not by d ‐cycloserine. Neither amantadine nor dextromethorphan affected the therapeutic effects of L‐DOPA in sensorimotor tests. We conclude that the anti‐dyskinetic effect of amantadine is partially dependent on NMDA antagonism, while dextromethorphan suppresses AIMs via indirect 5‐HT_1A agonism. Combined with previous work from our group, our results support the investigation of 5‐HT_1A agonists as pharmacotherapies for LID in PD patients.     

Attenuated Benzodiazepine‐Sensitive Tonic GABAA Currents of Supraoptic Magnocellular Neuroendocrine Cells in 24‐h Water‐Deprived Rats

In supraoptic nucleus (SON) magnocellular neurosecretory cells (MNCs), γ‐GABA, via activation of GABA_A receptors (GABA_ARs), mediates persistent tonic inhibitory currents (I_tonic), as well as conventional inhibitory postsynaptic currents (IPSCs, I_phasic). In the present study, we examined the functional significance of I_tonic in SON MNCs challenged by 24‐h water deprivation (24WD). Although the main characteristics of spontaneous IPSCs were similar in 24WD compared to euhydrated (EU) rats, I_tonic, measured by bicuculline (BIC)‐induced I_holding shifts, was significantly smaller in 24WD compared to EU rats (P < 0.05). Propofol and diazepam prolonged IPSC decay time to a similar extent in both groups but induced less I_tonic in 24WD compared to EU rats, suggesting a selective decrease in GABA_A receptors mediating I_tonic over I_phasic in 24WD rats. THIP (4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol), a preferential δ subunit agonist, and L‐655,708, a GABA_A receptor α₅ subunit selective imidazobenzodiazepine, caused a significantly smaller inward and outward shift in I_holding, respectively, in 24WD compared to EU rats (P < 0.05 in both cases), suggesting an overall decrease in the α₅ subunit‐containing GABA_ARs and the δ subunit‐containing receptors mediating I_tonic in 24WD animals. Consistent with a decrease in 24WD I_tonic, bath application of GABA induced significantly less inhibition of the neuronal firing activity in 24WD compared to EU SON MNCs (P < 0.05). Taken together, the results of the present study indicate a selective decrease in GABA_ARs functions mediating I_tonic as opposed to those mediating I_phasic in SON MNCs, demonstrating the functional significance of I_tonic with respect to increasing neuronal excitability and hormone secretion in 24WD rats.