TCF4 Mutations Disrupt Synaptic Function Through Dysregulation of RIMBP2 in Patient-Derived Cortical Neurons.

BACKGROUND: Genetic variation in the TCF4 (transcription factor 4) gene is associated with risk for a variety of developmental and psychiatric conditions, which includes a syndromic form of autism spectrum disorder called Pitt-Hopkins syndrome (PTHS). TCF4 encodes an activity-dependent transcription factor that is highly expressed during cortical development and in animal models has been shown to regulate various aspects of neuronal development and function. However, our understanding of how disease-causing mutations in TCF4 confer pathophysiology in a human context is lacking. METHODS: To model PTHS, we differentiated human cortical neurons from human induced pluripotent stem cells that were derived from patients with PTHS and neurotypical individuals. To identify pathophysiology and disease mechanisms, we assayed cortical neurons with whole-cell electrophysiology, Ca2+ imaging, multielectrode arrays, immunocytochemistry, and RNA sequencing. RESULTS: Cortical neurons derived from patients with TCF4 mutations showed deficits in spontaneous synaptic transmission, network excitability, and homeostatic plasticity. Transcriptomic analysis indicated that these phenotypes resulted in part from altered expression of genes involved in presynaptic neurotransmission and identified the presynaptic binding protein RIMBP2 as the most differentially expressed gene in PTHS neurons. Remarkably, TCF4-dependent deficits in spontaneous synaptic transmission and network excitability were rescued by increasing RIMBP2 expression in presynaptic neurons. CONCLUSIONS: Taken together, these results identify TCF4 as a critical transcriptional regulator of human synaptic development and plasticity and specifically identifies dysregulation of presynaptic function as an early pathophysiology in PTHS.

TCF4 mutations disrupt synaptic function through dysregulation of RIMBP2 in patient-derived cortical neurons.

Genetic variation in the transcription factor 4 ( TCF4) gene is associated with risk for a variety of developmental and psychiatric conditions, which includes a syndromic form of ASD called Pitt Hopkins Syndrome (PTHS). TCF4 encodes an activity-dependent transcription factor that is highly expressed during cortical development and in animal models is shown to regulate various aspects of neuronal development and function. However, our understanding of how disease-causing mutations in TCF4 confer pathophysiology in a human context is lacking. Here we show that cortical neurons derived from patients with TCF4 mutations have deficits in spontaneous synaptic transmission, network excitability and homeostatic plasticity. Transcriptomic analysis indicates these phenotypes result from altered expression of genes involved in presynaptic neurotransmission and identifies the presynaptic binding protein, RIMBP2 as the most differentially expressed gene in PTHS neurons. Remarkably, TCF4-dependent deficits in spontaneous synaptic transmission and network excitability were rescued by increasing RIMBP2 expression in presynaptic neurons. Together, these results identify TCF4 as a critical transcriptional regulator of human synaptic development and plasticity and specifically identifies dysregulation of presynaptic function as an early pathophysiology in PTHS.

Electrophysiological measures from human iPSC-derived neurons are associated with schizophrenia clinical status and predict individual cognitive performance.

Neurons derived from human induced pluripotent stem cells (hiPSCs) have been used to model basic cellular aspects of neuropsychiatric disorders, but the relationship between the emergent phenotypes and the clinical characteristics of donor individuals has been unclear. We analyzed RNA expression and indices of cellular function in hiPSC-derived neural progenitors and cortical neurons generated from 13 individuals with high polygenic risk scores (PRSs) for schizophrenia (SCZ) and a clinical diagnosis of SCZ, along with 15 neurotypical individuals with low PRS. We identified electrophysiological measures in the patient-derived neurons that implicated altered Na+ channel function, action potential interspike interval, and gamma-aminobutyric acid-ergic neurotransmission. Importantly, electrophysiological measures predicted cardinal clinical and cognitive features found in these SCZ patients. The identification of basic neuronal physiological properties related to core clinical characteristics of illness is a potentially critical step in generating leads for novel therapeutics.

Resveratrol noncompetitively inhibits glycine receptor-mediated currents in neurons of rat central auditory neurons.

Resveratrol, a naturally occurring stilbene found in red wine, is known to modulate the activity of several types of ion channels and membrane receptors, including Ca2+, K+, and Na+ ion channels. However, little is known about the effects of resveratrol on some important receptors, such as glycine receptors and GABAA receptors, in the central nervous system (CNS). In the present study, the effects of resveratrol on glycine receptor or GABAA receptor-mediated currents in cultured rat inferior colliculus (IC) and auditory cortex (AC) neurons were studied using whole-cell voltage-clamp recordings. Resveratrol itself did not evoke any currents in IC neurons but it reversibly decreased the amplitude of glycine-induced current (IGly) in a concentration-dependent manner. Resveratrol did not change the reversal potential of IGly but it shifted the concentration-response relationship to the right without changing the Hill coefficient and with decreasing the maximum response of IGly. Interestingly, resveratrol inhibited the amplitude of IGly but not that of GABA-induced current (IGABA) in AC neurons. More importantly, resveratrol inhibited GlyR-mediated but not GABAAR-mediated inhibitory postsynaptic currents in IC neurons using brain slice recordings. Together, these results demonstrate that resveratrol noncompetitively inhibits IGly in auditory neurons by decreasing the affinity of glycine to its receptor. These findings suggest that the native glycine receptors but not GABAA receptors in central neurons are targets of resveratrol during clinical administrations.

A myelin-related transcriptomic profile is shared by Pitt-Hopkins syndrome models and human autism spectrum disorder.

Autism spectrum disorder (ASD) is genetically heterogeneous with convergent symptomatology, suggesting common dysregulated pathways. In this study, we analyzed brain transcriptional changes in five mouse models of Pitt-Hopkins syndrome (PTHS), a syndromic form of ASD caused by mutations in the TCF4 gene, but not the TCF7L2 gene. Analyses of differentially expressed genes (DEGs) highlighted oligodendrocyte (OL) dysregulation, which we confirmed in two additional mouse models of syndromic ASD (Ptenm3m4/m3m4 and Mecp2tm1.1Bird). The PTHS mouse models showed cell-autonomous reductions in OL numbers and myelination, functionally confirming OL transcriptional signatures. We also integrated PTHS mouse model DEGs with human idiopathic ASD postmortem brain RNA-sequencing data and found significant enrichment of overlapping DEGs and common myelination-associated pathways. Notably, DEGs from syndromic ASD mouse models and reduced deconvoluted OL numbers distinguished human idiopathic ASD cases from controls across three postmortem brain data sets. These results implicate disruptions in OL biology as a cellular mechanism in ASD pathology.

Arc Oligomerization Is Regulated by CaMKII Phosphorylation of the GAG Domain: An Essential Mechanism for Plasticity and Memory Formation.

Arc is a synaptic protein essential for memory consolidation. Recent studies indicate that Arc originates in evolution from a Ty3-Gypsy retrotransposon GAG domain. The N-lobe of Arc GAG domain acquired a hydrophobic binding pocket in higher vertebrates that is essential for Arc's canonical function to weaken excitatory synapses. Here, we report that Arc GAG also acquired phosphorylation sites that can acutely regulate its synaptic function. CaMKII phosphorylates the N-lobe of the Arc GAG domain and disrupts an interaction surface essential for high-order oligomerization. In Purkinje neurons, CaMKII phosphorylation acutely reverses Arc's synaptic action. Mutant Arc that cannot be phosphorylated by CaMKII enhances metabotropic receptor-dependent depression in the hippocampus but does not alter baseline synaptic transmission or long-term potentiation. Behavioral studies indicate that hippocampus- and amygdala-dependent learning requires Arc GAG domain phosphorylation. These studies provide an atomic model for dynamic and local control of Arc function underlying synaptic plasticity and memory.

Estrogen-Dependent Functional Spine Dynamics in Neocortical Pyramidal Neurons of the Mouse.

Surgical ovariectomy has been shown to reduce spine density in hippocampal CA1 pyramidal cells of rodents, and this reduction is reversed by 17ß-estradiol (E2) treatment in a model of human estrogen replacement therapy. Here, we report reduction of spine density in apical dendrites of layer 5 pyramidal neurons of several neocortical regions that is reversed by subsequent E2 treatment in ovariectomized (OVX) female Thy1M-EGFP mice. We also found that OVX-associated reduction of spine density in somatosensory cortex was accompanied by a reduction in miniature EPSC (mEPSC) frequency (but not mIPSC frequency), indicating a change in functional synapses. OVX-associated spine loss in somatosensory cortex was also rescued by an agonist of the G-protein-linked estrogen receptor (GPER) but not by agonists of the classic estrogen receptors ERα/ERß, whereas the opposite selectivity was found in area CA1. Acute treatment of neocortical slices with E2 also rescued the OVX-associated reduction in mEPSC frequency, which could be mimicked by a GPER agonist and abolished by a GPER antagonist. Time-lapse in vivo two-photon imaging showed that OVX-associated reduction in spine density is achieved by both an increase in spine loss rate and a decrease in spine gain rate and that subsequent rescue by E2 reversed both of these processes. Crucially, the spines added after E2 rescue were no more likely to reappear at or nearby the sites of pre-OVX spines than those in control mice treated with vehicle. Thus, a model of estrogen replacement therapy, although restoring spine density and dynamics, does not entirely restore functional connectivity.SIGNIFICANCE STATEMENT Estrogen replacement therapy following menopause or surgical removal of the ovaries is a widespread medical practice, yet little is known about the consequences of such treatment for cells in the brain. Here, we show that estrogen replacement reverses some of the effects of surgical removal of the ovaries on the structure and function of brain cells in the mouse. Yet, importantly, the fine wiring of the brain is not returned to the presurgery state by estrogen treatment, suggesting lasting functional consequences.

Increased heat shock transcription factor 1 in the cerebellum reverses the deficiency of Purkinje cells in Alzheimer's disease.

Alzheimer's disease (AD) is one of the most debilitating neurodegenerative nerve diseases, seriously affecting one's ability to carry out daily activities. AD is both progressive and incurable, but molecular studies have begun to shed light on the mechanisms that underlie it. Immunochemical staining showed that cell bodies of Purkinje cells in the cerebellum were significantly reduced in AD rats compared with normal rats. Heat shock protein 70 (HSP70) was found to prevent polyglutamine aggregation in Huntington's disease and spinocerebellar ataxias (SCAs) and to relieve symptoms in SCAs and Parkinson's disease. Recently, AD-related phenotypes were found to be suppressed in HSP70 transgenic rats. However, the effects of other HSPs and the mechanisms of HSP-triggered changes in AD are unknown. In this study, we found that expression levels of HSP60, -70, and -90 were downregulated in the cerebella of rats with AD. Furthermore, heat shock factor 1 (HSF1), a key transcription factor for the expression of HSP genes, was found to be greatly decreased in the cerebella of AD rats. Even more interesting, injection of lentivirus vector-HSF1 into the cerebella of AD rats significantly increased HSF1 and HSP expression levels and induced an increase in the number of Purkinje cell bodies. Our findings provide novel evidence that low expression of HSPs in AD rats is dependent on the low expression of HSF1, and increased expression of HSF1 contributes to the reversal of cerebellar Purkinje cell deficiency in AD. Therefore, increasing HSF1 expression is a potential new strategy for the treatment of AD.

Regulation of Hypothalamic Presympathetic Neurons and Sympathetic Outflow by Group II Metabotropic Glutamate Receptors in Spontaneously Hypertensive Rats.

Increased glutamatergic input in the hypothalamic paraventricular nucleus (PVN) plays an important role in the development of hypertension. Group II metabotropic glutamate receptors are expressed in the PVN, but their involvement in regulating synaptic transmission and sympathetic outflow in hypertension is unclear. Here, we show that the group II metabotropic glutamate receptors agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) produced a significantly greater reduction in the frequency of spontaneous and miniature excitatory postsynaptic currents and in the amplitude of electrically evoked excitatory postsynaptic currents in retrogradely labeled spinally projecting PVN neurons in spontaneously hypertensive rats (SHRs) than in normotensive control rats. DCG-IV similarly decreased the frequency of GABAergic inhibitory postsynaptic currents of labeled PVN neurons in the 2 groups of rats. Strikingly, DCG-IV suppressed the firing of labeled PVN neurons only in SHRs. DCG-IV failed to inhibit the firing of PVN neurons of SHRs in the presence of ionotropic glutamate receptor antagonists. Lowering blood pressure with celiac ganglionectomy in SHRs normalized the DCG-IV effect on excitatory postsynaptic currents to the same level seen in control rats. Furthermore, microinjection of DCG-IV into the PVN significantly reduced blood pressure and sympathetic nerve activity in SHRs. Our findings provide new information that presynaptic group II metabotropic glutamate receptor activity at the glutamatergic terminals increases in the PVN in SHRs. Activation of group II metabotropic glutamate receptors in the PVN inhibits sympathetic vasomotor tone through attenuation of increased glutamatergic input and neuronal hyperactivity in SHRs.

Melatonin modulates the GABAergic response in cultured rat hippocampal neurons.

In the present study, we investigated the effect of melatonin on the GABA-induced current (I(GABA) and GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in cultured rat hippocampal neurons using the whole-cell patch-clamp technique. We found that melatonin rapidly and reversibly enhanced I(GABA) in a dose-dependent manner, with an EC50 of 949 µM. Melatonin markedly enhanced the peak amplitude of a subsaturating I(GABA) but not that of a saturating I(GABA). Interestingly, melatonin was effective only when GABA and melatonin were applied together. Furthermore, the effect of melatonin on I(GABA) was voltage-independent and did not change the ion selectivity of the GABA(A) receptor. The melatonin enhancement on I(GABA) can not be blocked by luzindole, a melatonin receptor antagonist, indicating that melatonin-induced I(GABA) enhancement was not via activation of its own membrane receptors. However, this enhancement may be mediated via high-affinity benzodiazepine sites as it was inhibited by the classical benzodiazepine antagonist flumazenil, suggesting an allosteric modulation of melatonin by binding to the sites of GABA(A) receptors. In addition, melatonin increased both amplitude and frequency of GABAergic mIPSCs, indicating that melatonin enhances GABAergic inhibitory transmission. Hence, our observation that melatonin has an enhancing effect on the GABAergic system may implicate a potential pathway for the neuroprotective effects of melatonin.

NKCC1 upregulation disrupts chloride homeostasis in the hypothalamus and increases neuronal activity-sympathetic drive in hypertension.

Hypertension is a major risk factor for coronary artery disease, stroke, and kidney failure. However, the etiology of hypertension in most patients is poorly understood. Increased sympathetic drive emanating from the hypothalamic paraventricular nucleus (PVN) plays a major role in the development of hypertension. Na(+)-K(+)-2Cl(-) cotransporter-1 (NKCC1) in the brain is critically involved in maintaining chloride homeostasis and in neuronal responses mediated by GABA(A) receptors. Here we present novel evidence that the GABA reversal potential (E(GABA)) of PVN presympathetic neurons undergoes a depolarizing shift that diminishes GABA inhibition in spontaneously hypertensive rats (SHRs). Inhibition of NKCC1, but not KCC2, normalizes E(GABA) and restores GABA inhibition of PVN neurons in SHRs. The mRNA and protein levels of NKCC1, but not KCC2, in the PVN are significantly increased in SHRs, and the NKCC1 proteins on the plasma membrane are highly glycosylated. Inhibiting NKCC1 N-glycosylation restores E(GABA) and GABAergic inhibition of PVN presympathetic neurons in SHRs. Furthermore, NKCC1 inhibition significantly reduces the sympathetic vasomotor tone and augments the sympathoinhibitory responses to GABA(A) receptor activation in the PVN in SHRs. These findings suggest that increased NKCC1 activity and glycosylation disrupt chloride homeostasis and impair synaptic inhibition in the PVN to augment the sympathetic drive in hypertension. This information greatly improves our understanding of the pathogenesis of hypertension and helps to design better treatment strategies for neurogenic hypertension.

Casein kinase 2-mediated synaptic GluN2A up-regulation increases N-methyl-D-aspartate receptor activity and excitability of hypothalamic neurons in hypertension.

Increased glutamatergic input, particularly N-methyl-D-aspartate receptor (NMDAR) activity, in the paraventricular nucleus (PVN) of the hypothalamus is closely associated with high sympathetic outflow in essential hypertension. The molecular mechanisms underlying augmented NMDAR activity in hypertension are unclear. GluN2 subunit composition at the synaptic site critically determines NMDAR functional properties. Here, we found that evoked NMDAR-excitatory postsynaptic currents (EPSCs) of retrogradely labeled spinally projecting PVN neurons displayed a larger amplitude and shorter decay time in spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto (WKY) rats. Blocking GluN2B caused a smaller decrease in NMDAR-EPSCs of PVN neurons in SHRs than in WKY rats. In contrast, GluN2A blockade resulted in a larger reduction in evoked NMDAR-EPSCs and puff NMDA-elicited currents of PVN neurons in SHRs than in WKY rats. Blocking presynaptic GluN2A, but not GluN2B, significantly reduced the frequency of miniature EPSCs and the firing activity of PVN neurons in SHRs. The mRNA and total protein levels of GluN2A and GluN2B in the PVN were greater in SHRs than in WKY rats. Furthermore, the GluN2B Ser(1480) phosphorylation level and the synaptosomal GluN2A protein level in the PVN were significantly higher in SHRs than in WKY rats. Inhibition of protein kinase CK2 normalized the GluN2B Ser(1480) phosphorylation level and the contribution of GluN2A to NMDAR-EPSCs and miniature EPSCs of PVN neurons in SHRs. Collectively, our findings suggest that CK2-mediated GluN2B phosphorylation contributes to increased synaptic GluN2A, which potentiates pre- and postsynaptic NMDAR activity and the excitability of PVN presympathetic neurons in hypertension.

Complex regulation of capsaicin on intracellular second messengers by calcium dependent and independent mechanisms in primary sensory neurons.

Intracellular second messengers play an important role in capsaicin- and analogous-induced sensitization and desensitization in pain. Fluorescence Ca²âº imaging, enzyme immunoassay and PKC assay kit were used to determine a novel mechanism of different Ca²âº dependency in the signal transduction of capsaicin-induced desensitization. On the average, capsaicin increased cAMP, cGMP concentration and SP release in bell-shaped concentration-dependent manner, with the maximal responses at concentrations around 1 µM, suggesting acute desensitization of TRPV1 receptor activation. Capsaicin-induced intracellular Ca²âº concentration ([Ca²âº](i)) increase depended on extracellular Ca²âº influx as an initial trigger. The Ca²âº influx by capsaicin increased PKC activation and SP release. These increases were completely abolished in Ca²âº-free solution, suggesting that the modulation of capsaicin on PKC and SP are Ca²âº-dependent. Interestingly, the maximal cAMP increase by TRPV1 activation was not blocked Ca²âº removal, suggesting at least in part a Ca²âº-independent pathway is involved. Further study showed that cAMP increase was totally abolished by G-protein and adenylate cyclase (AC) antagonist, suggesting a G-protein-dependent pathway in cAMP increase. However, SP release was blocked by inhibiting PKC, but not G-protein or AC, suggesting a G-protein independent pathway in SP release. These results suggest that both Ca²âº-dependent and independent mechanisms are involved in the regulation of capsaicin on second messengers systems, which could be a novel mechanism underlying distinct desensitization of capsaicin and might provide additional opportunities in the development of effective analgesics in pain treatment.

Identification of diverse modulators of central and peripheral circadian clocks by high-throughput chemical screening.

The circadian clock coordinates daily oscillations of essential physiological and behavioral processes. Conversely, aberrant clocks with damped amplitude and/or abnormal period have been associated with chronic diseases and aging. To search for small molecules that perturb or enhance circadian rhythms, we conducted a high-throughput screen of approximately 200,000 synthetic compounds using Per2lucSV reporter fibroblast cells and validated 11 independent classes of molecules with Bmal1:luciferase reporter cells as well as with suprachiasmatic nucleus and peripheral tissue explants. Four compounds were found to lengthen the period in both central and peripheral clocks, including three compounds that inhibited casein kinase Iε in vitro and a unique benzodiazepine derivative acting through a non-GABA(A) receptor target. In addition, two compounds acutely induced Per2lucSV reporter bioluminescence, delayed the rhythm, and increased intracellular cAMP levels, but caused rhythm damping. Importantly, five compounds shortened the period of peripheral clocks; among them, four compounds also enhanced the amplitude of central and/or peripheral reporter rhythms. Taken together, these studies highlight diverse activities of drug-like small molecules in manipulating the central and peripheral clocks. These small molecules constitute a toolbox for probing clock regulatory mechanisms and may provide putative lead compounds for treatment of clock-associated diseases.

Protein kinase CK2 increases glutamatergic input in the hypothalamus and sympathetic vasomotor tone in hypertension.

Increased glutamatergic input in the paraventricular nucleus (PVN) is important for high sympathetic outflow in hypertension, but the associated molecular mechanisms remain unclear. Here, we determined the role of protein kinase CK2 (formerly casein kinase II) in increased N-methyl-d-aspartate receptor (NMDAR) activity in spinally projecting PVN neurons and sympathetic vasomotor tone in spontaneously hypertensive rats (SHRs). The selective CK2 inhibitors 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole (DRB) or 4,5,6,7-tetrabromobenzotriazole (TBB) significantly decreased the frequency of miniature EPSCs (mEPSCs) of labeled PVN neurons in SHRs but not in Wistar-Kyoto (WKY) normotensive rats. Also, DRB abolished the inhibitory effect of the NMDAR antagonist AP5 on the frequency of mEPSCs in SHRs. Treatment with DRB or TBB significantly reduced the amplitude of evoked NMDA-EPSCs but not AMPA-EPSCs in SHRs. Furthermore, DRB significantly decreased the firing activity of PVN neurons in SHRs but not in WKY rats. The membrane protein level of CK2α in the PVN, but not brainstem and prefrontal cortex, was significantly higher in SHRs than in WKY rats. Lowering blood pressure with celiac ganglionectomy in SHRs did not alter the increased CK2α level and the effects of DRB on mEPSCs and NMDA-EPSCs. In addition, intracerebroventricular injection of DRB not only significantly reduced blood pressure and lumbar sympathetic nerve discharges but also eliminated the inhibitory effect of AP5 microinjected into the PVN on sympathetic nerve activity in SHRs. Our findings suggest that augmented CK2 activity critically contributes to increased presynaptic and postsynaptic NMDAR activity in the PVN and elevated sympathetic vasomotor tone in essential hypertension.

Activation of the melanocortin-4 receptor causes enhanced excitation in presympathetic paraventricular neurons in obese Zucker rats.

Sympathetic nerve activity is increased in obesity-related hypertension. However, the central mechanisms involved in the increased sympathetic outflow remain unclear. The hypothalamic melanocortin system is important for regulating energy balance and sympathetic outflow. To understand the mechanisms by which the melanocortin systems regulates sympathetic outflow, we investigated the role of melanocortin 4 receptors (MC4R) in regulating presympathetic paraventricular nucleus (PVN) neurons. We performed whole-cell patch-clamp recordings on retrogradely labeled PVN neurons projecting to the rostral ventrolateral medulla in brain slices from obese zucker rats (OZRs) and lean zucker rats (LZRs). The MC4R agonists melanotan II (MTII) and α-melanocyte-stimulating hormone (α-MSH) increased the firing activity and depolarized the labeled PVN neurons from both LZRs and OZRs in a concentration-dependent manner. MTII produced significant greater increase in the firing activity in OZRs than in LZRs. Blocking MC4R with the specific antagonist SHU9119 had no effect on the basal firing rate but abolished the MTII-induced increase in the firing rate in both OZRs and LZRs. Furthermore, intracellular dialysis of guanosine 5'-O-(2-thodiphosphate), but not bath application of kynurenic acid and bicuculline, eliminated the MTII-induced increase in firing activity. In addition, MTII had no effect on the frequency and amplitude of glutamatergic excitatory postsynaptic currents and GABAergic inhibitory postsynaptic currents in labeled PVN neurons. Collectively, our findings suggest that MC4R contributes to the elevated excitability of PVN presympathetic neurons, which may be involved in obesity-related hypertension.

Fluoxetine inhibition of glycine receptor activity in rat hippocampal neurons.

Fluoxetine is a selective serotonin reuptake inhibitor widely used for treating depression. However, fluoxetine treatment may lead to seizures at higher doses, which underlying mechanism remains largely unknown. In this study, we examined the effects of fluoxetine on glycine receptor (GlyR) activity. Using the whole-cell patch-clamp recording method, we found that fluoxetine and its metabolite norfluoxetine inhibited glycine-induced currents in cultured rat hippocampal neurons. This inhibition was dose-dependent, and voltage-independent. Fluoxetine shifted the glycine concentration-response curve to the right without altering the maximal current. Both Lineweaver-Burk and Schild plots suggest competitive inhibition. The amount of fluoxetine inhibition significantly increased when homomeric GlyRs were selectively inhibited with picrotoxin. Moreover, fluoxetine inhibited the current mediated by heteromeric alpha2beta- but not homomeric alpha2-GlyRs transiently expressed in HEK293T cells. These results suggest that fluoxetine is a competitive and subtype-selective GlyR inhibitor, which may explain its capacity to induce seizures.

Fluoxetine potentiates GABAergic IPSCs in rat hippocampal neurons.

The GABA system is highly involved in the pathophysiology of mood disorders such as depression. Altered GABAergic function is evident in depressed patients and animal models of depression. Currently, the most widely used antidepressants are selective 5-HT reuptake inhibitors, such as fluoxetine. However, the effects of fluoxetine on GABAergic synaptic neurotransmission remain poorly investigated. Whole-cell patch-clamp recordings from cultured rat hippocampal neurons were therefore conducted to investigate the effects of fluoxetine on GABAergic neurotransmission. The spontaneous inhibitory postsynaptic current (sIPSC) was completely blocked by 10 microM bicuculline and reversibly potentiated by 30 microM fluoxetine. The fluoxetine potentiation on either amplitude or frequency of sIPSCs was dose-dependent, with the EC(50) values of 10.96 and 14.26 microM, respectively. This potentiation was also TTX-insensitive, suggesting independence of presynaptic action potentials. The ritanserin (5 microM), a selective 5-HT(2) receptor antagonist, did not alter the fluoxetine potentiation on miniature inhibitory postsynaptic currents. Taken together, our data suggest that fluoxetine can potentiate GABAergic neurotransmission without depending on presynaptic firing of action potentials and its elevating of 5-HT receptor activities. This potentiation by fluoxetine may normalize the hippocampal GABA deficit during depression and in part exert its antidepressant activity.