Pregabalin (PGB) exerts its therapeutic effects by binding to the α2δ auxiliary subunits of voltage-gated calcium channels and modulates synaptic transmission in the brain. However, its influence on cerebellar parallel fiber-Purkinje cell (PF-PC) synaptic transmission remains unclear. In the present study, we investigated the effects of PGB on PF-PC synaptic transmission using whole-cell patch-clamp recording, glutamate fluorescence imaging, immunohistochemistry, co-immunoprecipitation, Western blotting, and pharmacological approaches. Micro-application of PGB to the cerebellar molecular layer induced a concentration-dependent inhibition of PF-PC excitatory postsynaptic currents (EPSCs), accompanied by an increased paired-pulse ratio. The inhibitory effect of PGB on PF-PC EPSCs was abolished by extracellular blockade of N-methyl-D-aspartate receptors (NMDAR) or their GluN2A subtype, as well as by disruption of α2δ-1-NMDAR complexes, but not by intracellular NMDAR inhibition. Glutamate sensor imaging further showed that PGB markedly reduced the fluorescence intensity of glutamate release evoked by PF stimulation. In the presence of tetrodotoxin (TTX) and a gamma-aminobutyric acid type A (GABAA) receptor antagonist, PGB reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting their amplitude. The PGB-induced reduction in mEPSC frequency was fully abolished by extracellular blockade of GluN2A-containing NMDARs or disruption of α2δ-1-NMDAR complexes. Similarly, the inhibitory effects of PGB on PF-PC EPSCs and mEPSCs were eliminated by extracellular PKA inhibition, but not by intracellular protein kinase A (PKA) inhibition. Western blot analysis showed that PGB significantly increased PKA phosphorylation in the molecular layer of the cerebellar cortex. Immunoreactivity for GluN2A and α2δ-1 subunits was colocalized within the molecular layer and abundantly distributed around the dendrites and somata of PCs. Co-immunoprecipitation further verified that α2δ-1 was co-precipitated with GluN1 in cerebellar molecular layer tissue samples. The results indicate that PGB depresses glutamate release from parallel-fiber terminals in the mouse cerebellar cortex through the presynaptic α2δ-1-coupled GluN2A-containing NMDAR/PKA signaling pathway, thereby attenuating PF-PC synaptic transmission.

Glucagon-like peptide-1 (GLP-1) in the brain is synthesized by preproglucagon neurons and released through axonal projections to extensive regions such as the arcuate nucleus, hypothalamic paraventricular nucleus (PVN), rostral ventrolateral medulla, and dorsomedial medulla. It is involved in functional activities including substance metabolism, neuroendocrine regulation, and autonomic nerve control. The GLP-1 receptor (GLP-1R) is highly expressed in the PVN and plays an important role in stress responses and cardiovascular activities. However, the mechanism by which GLP-1 influences the activity of magnocellular neurosecretory cells (MNCs) in the hypothalamic PVN remains unclear. In this study, we used whole-cell patch-clamp recording in acute hypothalamic slices, biotin staining, double immunofluorescence staining, and neuropharmacology methods to investigate the regulatory mechanism of GLP-1 on the activity of the hypothalamic PVN MNCs in vitro in rats. Under current-clamp conditions, perfusion with GLP-1 (100 nmol/L) resulted in a significant decrease in the spontaneous firing frequency of PVN MNCs accompanied with a membrane potential hyperpolarization. Blocking ionotropic glutamate receptors and GABAA receptors either alone or simultaneously did not affect the inhibitory effect of GLP-1 on the firing frequency of PVN MNCs. GLP-1 had no significant impact on the half-width, rise constant, or decay constant of action potentials, but it significantly increased the after-hyperpolarization (AHP) and inwardly rectifying potassium currents of PVN MNCs. The results of double immunofluorescence showed that GLP-1 significantly reduced the expression of c-fos in arginine vasopressin (AVP)-ergic neurons. These results indicate that GLP-1 activates postsynaptic inwardly rectifying potassium channels, reduces the excitability of PVN MNCs, and decreases their firing frequency and c-fos expression. These findings suggest that GLP-1 reduces the secretion and release of AVP by inhibiting the functional activity of PVN MNCs in rats.

Locus coeruleus (LC) noradrenergic neurons project their axons to the cerebellar cortex and modulate cerebellar circuit function via distinct adrenergic receptor (AR) subtypes. The present study investigated the mechanism by which optogenetic activation of LC noradrenergic neurons modulates facial stimulation-evoked long-term synaptic plasticity at cerebellar mossy fiber-granule cell (MF-GrC) synapses in urethane-anesthetized DBH-Cre mice. Blockade of GABAA receptors, 20 Hz facial stimulation induced MF-GrC long-term potentiation (LTP) in the control group, and this LTP was impaired by optogenetic activation of LC noradrenergic neurons via α2-ARs. Meanwhile, facial stimulation induced LTP of glutamate sensor fluorescence in the granular layer, which was abolished by chemogenetic activation of LC noradrenergic neurons. Following NMDA receptor blockade, optogenetic activation of LC noradrenergic neurons triggered facial stimulation-induced MF-GrC long-term depression (LTD) via α2A-ARs. Optogenetically activated LC noradrenergic neuron-induced MF-GrC LTD was abolished by protein kinase A (PKA) inhibition but not by protein kinase C inhibition. Immunofluorescence results revealed abundant α2A-AR expression in the granular layer, with particularly high levels in glomeruli, and no colocalization with the glutamate sensor. These results indicate that optogenetic activation of LC noradrenergic neurons impairs facial stimulation-induced MF-GrC LTP by triggering presynaptic LTD via the α2A-AR/PKA signaling cascade.

Cerebellar climbing fiber-Purkinje cell (CF-PC) long-term depression (LTD) plays a critical role in motor learning and is modulated by locus coeruleus (LC) noradrenergic afferents via distinct adrenergic receptor (AR) subtypes. Nevertheless, the mechanisms underlying LC noradrenergic neuron-mediated regulation of CF-PC LTD remain poorly understood. Here, we investigated the effects of chemogenetic activation of LC noradrenergic afferents on CF-PC LTD in cerebellar slices from dopamine β-hydroxylase (DBH)-Cre mice using electrophysiology, glutamate sensor imaging, immunofluorescence and pharmacological approaches. Tetanic stimulation (5 Hz) of CFs induced CF-PC LTD under control conditions, and this LTD was enhanced by chemogenetic activation of LC noradrenergic afferents. Blockade of group I metabotropic glutamate receptors (mGluR1) abolished LTD under control conditions, whereas chemogenetic activation of LC noradrenergic afferents triggered a novel form of CF-PC LTD accompanied by an increased N2/N1 ratio. With mGluR1 blocked, chemogenetic activation of LC noradrenergic afferents failed to trigger the novel CF-PC LTD following blockade of α2-AR or α2A-AR, but not α2B-AR or α2C-AR. Importantly, chemogenetic activation of LC noradrenergic afferents triggered LTD of glutamate fluorescence at CF terminals, which was abolished by blockade of α2-AR or α2A-AR, but not α2B-AR or α2C-AR. Notably, inhibition of either cyclin-dependent kinase 5 (CDK5) or presynaptic, but not postsynaptic, protein kinase A (PKA) completely abolished the CF-PC LTD triggered by chemogenetic activation of LC noradrenergic afferents in mouse cerebellar slices. Immunofluorescence results showed robust α2A-AR expression throughout the cerebellar molecular layer, with intense signals along PC dendrites and clear colocalization with vesicular glutamate transporter 2 (vGluT2) at cerebellar CF terminals. These results indicate that activation of LC noradrenergic afferents potentiates CF-PC LTD by triggering Glu-LTD at CF terminals through the α2A-AR/CDK5/PKA signaling cascade in the mouse cerebellar cortex.

OBJECTIVE:The central neuromedin U receptor 2 (NMUR2) participates in regulating neuronal activity and synaptic transmission. This study aimed to explore the functional role of NMUR2 in climbing fiber-Purkinje cell synaptic transmission in mouse cerebellar slices. METHODS:Climbing fiber-Purkinje cell excitatory postsynaptic currents (EPSCs) and miniature excitatory postsynaptic currents (mEPSCs) were recorded from Purkinje cells using an Axopatch 700B patch-clamp amplifier. Western blot, glutamate sensor fluorescence imaging, and immunohistochemistry were applied to measure phosphorylated protein kinase A (PKA) levels, climbing fiber terminal glutamate release, and NMUR2 expression, respectively. RESULTS:Bath application of the selective NMUR2 agonist CPN-219 reduced the amplitude and area under the curve of climbing fiber-Purkinje cell EPSCs and increased the paired-pulse ratio ( N 2/ N 1). The CPN-219-induced suppression of EPSCs was reversed by an adenylyl cyclase activator and occluded by an AC inhibitor. The CPN-219-mediated inhibitory effect on EPSCs was completely abolished by bath perfusion of the PKA inhibitor, but not by intracellular PKA inhibition. Western blot analysis revealed that CPN-219 decreased phosphorylated PKA levels in cerebellar molecular layer tissue. Furthermore, CPN-219 markedly attenuated Ca 2+ transients and complex spike activity in Purkinje cells, suppressed climbing fiber-evoked glutamate fluorescent signals, and decreased mEPSC frequency, with no significant alteration in mEPSC amplitude. Moreover, immunohistochemical staining showed NMUR2 immunoreactivity distributed throughout the somata and dendritic arbors of Purkinje cells. CONCLUSION:These results indicate that NMUR2 activation suppresses climbing fiber-Purkinje cell synaptic transmission in mouse cerebellar slices predominantly via a mechanism consistent with a presynaptic adenylyl cyclase-PKA signaling cascade.

The abnormalities in cerebellar circuit function within the Crus I/II lobules in autism spectrum disorder (ASD) are widely recognized as critical contributors to impairments in motor behavior and social interaction. We here investigated the impact of prenatal valproic acid (VPA) exposure on facial stimulation-evoked synaptic plasticity at cerebellar mossy fiber-granule cell (MF-GC) synapses in urethane-anesthetized offspring mice in vivo. When the GABAA receptor was blocked, 20 Hz facial stimulation induced MF-GC LTP in control mice, whereas the same stimulation paradigm triggered MF-GC LTD in VPA-treated offspring. Blockade of N -methyl-D-aspartate receptors abolished LTP in control mice while unmasking LTD in VPA-treated offspring. Notably, the facial stimulation-induced LTD observed in VPA-treated offspring was abolished by blocking group I metabotropic glutamate receptors, but not by the selective blockade of either mGluR1 or mGluR5 alone. Blockade of cannabinoid receptor 1 (CB1) or inhibition of diacylglycerol lipase (DGL) prevented the induction of LTD. Pharmacologically activating mGluR1/5 or CB1 produced MF-GC LTD and overwhelmed the facial stimulation-induced LTD in VPA-treated offspring mice. Conversely, mGluR1/5 activation failed to induce LTD in control mice. Moreover, 20 Hz facial stimulation induced MF-GC LTP in the absence of mGluR1/5 or CB1 receptor activity in VPA-treated offspring mice. Immunohistochemical analyses revealed that the expression levels of mGluR1/5 were significantly higher in the cerebellar GCs of VPA-treated offspring compared to those of control mice. These results indicate that 20 Hz facial stimulation induces MF-GC LTD by enhancing the mGluR1/5-DGL-CB1 receptor signaling cascade, thereby resulting in impairment of MF-GC LTP in VPA-treated offspring mice.

The α2-adrenergic receptor (α2-AR) is involved in various forms of information transmission in the cerebellar cortex, but its role in modulating sensory stimulation-induced synaptic plasticity remains unclear. We investigated the role of the α2-AR in facial stimulation-evoked long-term synaptic plasticity within molecular layer interneuron–Purkinje cell (MLI–PC) circuitry in the cerebellum by electrophysiological, pharmacological and immunohistochemical methods, and used alongside the rotarod test to assess the impact of receptor activity on motor learning in mice. Facial stimulation at 1 Hz induced MLI–PC long-term depression (LTD), which was significantly enhanced by microinjection of noradrenaline (NA) into the cerebellar molecular layer. Blockade of the NMDA receptor abolished facial stimulation-induced MLI–PC LTD; However, the effect could be triggered in the absence of NMDA activity through NA or UK14304 α2-AR activation with concurrent stimulation. In the absence of NMDA receptor activity, α2-AR-mediated facial stimulation-induced MLI–PC LTD was abolished by blockade of α2A-, but not α2B- or α2C-ARs. Facial stimulation-induced MLI–PC LTD was triggered by a selective α2A-AR agonist, guanfacine, but it was completely prevented by inhibition of protein kinase A (PKA) activity with KT5720. The rotarod test indicated that microinjection of guanfacine into the cerebellar cortex to activate α2A-ARs significantly improved early motor learning. Immunochemistry revealed α2A-AR immunoreactivity throughout the mouse cerebellar cortex, mostly in the PC layer and MLIs. These results suggest that NA facilitates facial stimulation-induced, α2A-AR/PKA signaling cascade-dependent MLI–PC LTD and promotes the acquisition of motor learning in mice.

17β-estradiol (E2) enhances the cerebellar molecular layer interneurons (MLIs)—Purkinje cells (PCs) synaptic transmission via activation of the Erβ in vivo in mice. Whether E2 regulates cerebellar MLI-PC synaptic plasticity is unknown. To investigate the mechanism of E2, we evaluated the modulation of facial stimulation-evoked MLI-PC long-term plasticity in mice. Cell-attached recordings from PCs of Crus II were performed using an Axopatch-700B patch-clamp amplifier. The MLI-PC synaptic transmission was evoked by facial stimulation. Immunohistochemistry was used to detect the expression of ERβ. Under control conditions, 1 Hz facial stimuli induced long-term depression (LTD) at MLI-PC synapses, characterized by a sustained reduction in P1 amplitude and a simple spike (SS) pause. The facial stimulus-induced MLI-PC LTD was completely prevented by E2, but this effect was reversed by a selective ERα/ERβ antagonist, ICI182780. Blockade of cannabinoid receptor 1 (CB1R) eliminated the MLI-PC LTD under control conditions, but revealed an E2-triggered long-term potentiation (LTP). The E2-triggered MLI-PC LTP persisted in the presence of an ERα antagonist but was absent in the presence of an ERβ antagonist PHTPP. The E2-triggered MLI-PC LTP remained unaffected by protein kinase A inhibition but was abolished by inhibition of protein kinase C (PKC) and intracellular Ca2+ depletion. Moreover, ERβ immunoreactivity was abundantly distributed around dendrites and somas of PCs in the Crus II region of the mouse cerebellar cortex. The present results suggest that E2 activates ERβ, thereby triggering facial stimulation-induced MLI-PC LTP via the PKC signaling cascade, which occludes CB1R-dependent MLI-PC LTD in the cerebellar cortex of mice in vivo.

Spinal cord injury (SCI) causes interruption of external information input from the spinal cord to the cerebellum. We here investigated the effect of SCI on mouse cerebellar climbing fiber-Purkinje cell (CF-PC) synaptic transmission. The SCI was caused at T10 using 6-week-old ICR mice. Mice recovered 4 weeks after surgery, the spontaneous complex spike (CS) activity of PC was recorded using cell-attached recording and whole-cell recording method in urethane-anesthetized mice cerebellar Crus II. The CF-PC excitatory postsynaptic currents (EPSCs) were evoked by paired electrical stimulation of CF in cerebellar slices to evaluate the changes of CF-PC synaptic transmission and paired-pulse ratio (PPR). The results showed that the number of spikelets, duration of spontaneous CS, and pause of simple spike firing were significantly increased in SCI than that in sham group. Application of a nonselective corticotropin-releasing factor receptor (CRF-R) antagonist significantly decreased spontaneous CS activity in SCI group but not in sham group. The enhanced CS activity in SCI mice was significantly decreased by a selective CRF-R2 antagonist but not a specific CRF-R1 antagonist. The amplitude of CF-PC EPSC1 was large accompanied by a lower PPR in SCI group than that in sham group. Blockade of CRF-R2 antagonist significantly decreased the amplitude of EPSC1 and increased PPR in SCI group. SCI induces enhancement of the spontaneous CS activity and CF-PC synaptic transmission via CRF-R2 in mouse cerebellar cortex, which suggests that remodeling of CF-PC synaptic transmission occurred in cerebellar cortex after SCI.

OBJECTIVE:Acute stress enhances the activity of magnocellular neurons (MNs) by inducing long-term changes in excitatory inputs. We aim to investigate the mechanism underlying long-term potentiation (LTP) of glutamatergic inputs to paraventricular nucleus (PVN) MNs in stressed rats. METHODS:Rats were subjected to multiple stressors and randomly assigned to control and stress groups. In some experiments, stressed rats received intracerebroventricular (i.c.v.) injections of the β-adrenergic receptor (AR) antagonist or the β1-AR antagonist. Excitatory postsynaptic currents evoked by electrical stimulation in hypothalamic slices were recorded from PVN MNs using an Axopatch 200B amplifier. LTP of glutamatergic inputs to MNs was induced by electrical stimulation trains (100 Hz, 100 pulses, three times). Biocytin staining and immunohistochemistry were used to characterize the morphology of recorded neurons and detect β1-AR expression. RESULTS:Blockade of gamma-aminobutyric acid receptor, tetanic stimulation-induced glutamatergic LTP in MNs of nonstressed rats, which was significantly augmented in stressed rats. Blocking N-methy-D-aspartate receptors abolished LTP in nonstressed rats but revealed a novel LTP in stressed rats. I.c.v. administration of propranolol, or CGP 20712, before the stress procedure abolished this novel LTP in stressed rats. In contrast, administration of norepinephrine or a selective β1-AR agonist, dobutamine triggered the novel LTP in nonstressed rats. The novel LTP in stressed rats was abolished by intracellular inhibition of protein kinase A (PKA). β1-AR immunoreactivity was detected in PVN MN areas. CONCLUSION:Acute stress enhances the β1-AR/PKA signaling, leading to long-term modifications of glutamatergic inputs in the hypothalamic PVN MNs in rats in vivo.

Dose-dependent neuromodulation has been well established; however, the molecular mechanisms underlying astrocytic involvement in this process remain largely unexplored. Using the autoregulation of supraoptic oxytocin (OT) neurons (OTNs) as a model, we investigated the role of distinct astrocytic G proteins and their targets in the dose-dependent effects of OT on OTN activity. The results showed that OT in a low concentration (10 pmol/L, L-OT) excited OTN activity, whereas a high concentration (1 nmol/L, H-OT) inhibited it in brain slices. These effects were abolished upon disruption of astrocytic plasticity using L-aminoadipic acid, a gliotoxin. In primary astrocyte cultures, L-OT slightly reduced the current through astrocyte-specific inwardly rectifying K⁺ channel 4.1 (Kir4.1) while H-OT strongly enhanced it. Selectively blocking Kir4.1 with BaCl₂ (100 µmol/L) did not affect the basal activity but blocked the excitatory effect of L-OT in brain slices. In cultured astrocytes, L-OT mobilized Gαq subunit expression, increased glial fibrillary acidic protein (GFAP) filaments, and quickly expanded astrocytic volume, predominantly visible at the somata. Conversely, H-OT released Gαi subunits and induced progressive volume expansion. Pretreatment of brain slices with U73122 (a Gq inhibitor) or SQ22536 (a Gs inhibitor) suppressed L-OT-induced excitation. Conversely, activation of adenylyl cyclase with forskolin reversed the inhibitory effect of H-OT, and inhibition of Gi with pertussis toxin blocked H-OT-induced inhibition. These findings imply that the dose-dependent effects of OT on OTN activity are mediated, at least partially, by different receptor-coupled G proteins and their subsequent modulation of astrocytic Kir4.1 currents, GFAP expression, and volume dynamics. This mechanism underlying the autoregulation of OTN activity provides an important reference for understanding the concentration-dependent neuromodulation.

Atrial natriuretic peptide (ANP) and its receptor are abundantly distributed in hypothalamic paraventricular nucleus (PVN), and contribute to regulation of corticotropin releasing factor (CRF) release in the brain. However, the mechanism by which ANP regulates the activity of PVN CRFergic neurons remains unclear. Here, we investigated the mechanism by which ANP modulates the neuronal activity related to PVN CRF-mRNA expression in vitro in mice using whole-cell patch-clamp recording, single-cell RT-PCR, immunofluorescence, and pharmacological methods. Our data demonstrated that ANP significantly reduced the membrane excitability of PVN neurons expressing CRF-mRNA. This effect was abolished by an antagonist of ANP receptor A (NPR - A), namely A71915. Furthermore, ANP significantly suppressed the hyperpolarization - activated cationic currents (Ih) of neurons expressing CRF-mRNA. Blockade of either Ih or NPR - A completely prevented this inhibitory effect. Immunohistochemistry results indicated that NPR - A immunoreactivity was present in PVN CRF-positive neurons. These findings suggest that ANP decreases the excitability of PVN CRFergic neurons via NPR - A - mediated inhibition of Ih channel activity in vitro in mice.

In the cerebellar cortex, 17β-estradiol (E2) binds to estrogen receptors (ERs) and plays a role in regulating cerebellar synaptic plasticity and motor learning behaviors. However, the underlying mechanisms remain unclear. In this study, we investigated the effects of E2 on synaptic transmission between cerebellar molecular layer interneurons (MLIs) and Purkinje cells (PCs) in urethane-anesthetized mice. Using in vivo cell-attached and whole-cell recordings combined with immunohistochemistry, we examined MLI-PC synaptic responses elicited by facial air-puff stimulation. Cell-attached recordings from PCs demonstrated that air-puff stimulation of the ipsilateral whisker pad elicited MLI-PC synaptic currents (P1), which were significantly enhanced by local micro-application of E2 to the cerebellar molecular layer. The E2-induced potentiation of P1 amplitude exhibited dose dependency, with a 50

The mechanism by which α2-adrenergic receptors (ARs) modulate the cerebellar parallel fiber-Purkinje cell (PF-PC) synaptic transmission is unclear. We investigated this issue using electrophysiological and neuropharmacological methods. Six- to eight-week-old ICR mice were used in the study. Under in vivo conditions, PF-PC synaptic transmission was evoked by facial stimulation of ipsilateral whisker pad, and recorded using cell-attached patch from PCs. Under in-vitro conditions, PF-PC synaptic transmission was evoked by electrical stimulation of the molecular layer in cerebellar slices, and was recorded using whole-cell recording from PCs. SR95531 (20 µM) was added to the ACSF during all recordings to prevent GABAA receptor-mediated inhibition. Air-puff stimulation of the ipsilateral whisker pad in-vivo evoked simple spike (eSS) firing of cerebellar PCs. Microapplication of noradrenaline (15 µM) to the molecular layer significantly decreased the numbers and frequency of eSS, an effect abolished by the α2-AR antagonist. Microapplication of an α2-AR agonist, UK14304 (1 µM), significantly decreased the numbers of eSS in PCs, which was abolished by either α2A- or α2B-AR antagonist, but not by α2C-AR antagonist. Under in-vitro conditions, application of UK 14304 significantly decreased the amplitude of PF-PC EPSCs and increased the paired-pulse ratio, which were abolished by either α2A- or α2B-AR antagonist. The present results indicate that activation of presynaptic α2A- and α2B-AR downregulates PF-PC synaptic transmission in mouse cerebellar cortex.

Norepinephrine (NE) modulates synaptic transmission and long-term plasticity through distinct subtype adrenergic receptor (AR)-mediated-intracellular signaling cascades. However, the role of NE modulates glutamatergic long-term potentiation (LTP) in the hypothalamic paraventricular nucleus (PVN) magnocellular neuroendocrine cells (MNCs) is unclear. We here investigate the effect of NE on high frequency stimulation (HFS)-induced glutamatergic LTP in rat hypothalamic PVN MNCs in vitro, by whole-cell patch-clamp recording, biocytin staining and pharmacological methods. Delivery of HFS induced glutamatergic LTP with a decrease in N2/N1 ratio in the PVN MNCs, which was enhanced by application of NE (100 nM). HFS-induced LTP was abolished by the blockade of N-methyl-D-aspartate receptors (NMDAR) with D-APV, but it was rescued by the application of NE. NE failed to rescue HFS-induced LTP of MNCs in the presence of a selective β1-AR antagonist, CGP 20712. However, application of β1-AR agonist, dobutamine HCl rescued HFS-induced LTP of MNCs in the absence of NMDAR activity. In the absence of NMDAR activity, NE failed to rescue HFS-induced MNC LTP when protein kinase A (PKA) was inhibited by extracellular applying KT5720 or intracellular administration of PKI. These results indicate that NE activates β1-AR and triggers HFS to induce a novel glutamatergic LTP of hypothalamic PVN NMCs via the postsynaptic PKA signaling pathway in vitro in rats.

Etomidate (ET) is a widely used intravenous imidazole general anesthetic, which depresses the cerebellar neuronal activity by modulating various receptors activity and synaptic transmission. In this study, we investigated the effects of ET on the cerebellar climbing fiber-Purkinje cells (CF -PC) plasticity in vitro in mice using whole-cell recording technique and pharmacological methods. Our results demonstrated that CF tetanic stimulation produced a mGluR1-dependent long -term depression (LTD) of CF -PC excitatory postsynaptic currents (EPSCs), which was enhanced by bath application of ET (10 mu M). Blockade of mGluR1 receptor with JNJ16259685, ET triggered the tetanic stimulation to induce a CF -PC LTD accompanied with an increase in paired-pulse ratio (PPR). The ET-triggered CF -PC LTD was abolished by extracellular administration of an Nmethyl-(D)-aspartate (NMDA) receptor antagonist, D-APV, as well as by intracellular blockade of NMDA receptors activity with MK801. Furthermore, blocking cannabinoids 1 (CB1) receptor with AM251 or chelating intracellular Ca 2 + with BAPTA, ET failed to trigger the CF -PC LTD. Moreover, the ET-triggered CF -PC LTD was abolished by inhibition of protein kinase A (PKA), but not by inhibition of protein kinase C inhibiter. The present results suggest that ET acts on postsynaptic NMDA receptor resulting in an enhancement of the cerebellar CF -PC LTD through CB1 receptor/PKA cascade in vitro in mice. These results provide new evidence and possible mechanism for ET anesthesia to affect motor learning and motor coordination by regulating cerebellar CF -PC LTD.

Abstract Acute stress alternates the hypothalamic paraventricular nucleus (PVN) magnocellular neuronal activity through modulation of excitatory and inhibitory synaptic inputs, leading to abnormal secretion of oxytocin (OT) and vasopressin (VP). However, mechanism of acute stress modulates the glutamatergic long-term potentiation (LTP) in PVN magnocellular neuroendocrine cells (MNCs) is unclear. We here investigated the effect of acute stress on the glutamatergic LTP of PVN MNCs, by whole-cell patch-clamp recording with biocytin staining and pharmacological methods. Delivery of high frequency stimulation (HFS) induced a glutamatergic LTP accompanied with a decrease in paired-pulse ratio in PVN MNCs, which was significantly enhanced in acute stress rats. Blockade of N-methyl-D-aspartate receptors (NMDAR) activity abolished the LTP of PVN MNCs in control group, but reveal a NMDAR-independent LTP in acute stress group. The NMDAR-independent LTP of PVN MNCs in stress rats was abolished by a β-AR inhibitor, propranolol, but not by an α-AR inhibitor, Phentolamine. The NMDAR-independent LTP of PVN MNCs in stress rats was abolished by bath application of a potent protein kinase A (PKA) inhibitor, KT5720 (200 nM), but not by a PKC inhibitor. Moreover, postsynaptic blockade of PKA completely prevented the HFS-induced glutamatergic LTP in PVN MNCs of stress rats. These results indicate that acute stress triggers an NMDAR-independent glutamatergic LTP of the PVN MNCs through a postsynaptic β-AR/PKA signaling pathway, resulting in an enhancement of an NMDAR-dependent presynaptic LTP in vitro in rats. The results suggest that acute stress upregulates OT and VP secretion by enhancing the excitatory glutamatergic LTP of PVN MNCs.

Valproic acid (VPA) is one of the most effective antiepileptic drugs, and exposing animals to VPA during gestation has been used as a model for autism spectrum disorder (ASD). Numerous studies have shown that impaired synaptic transmission in the cerebellar cortical circuits is one of the reasons for the social deficits and repetitive behavior seen in ASD. In this study, we investigated the effect of VPA exposure during pregnancy on tactile stimulation-evoked cerebellar mossy fiber-granule cell (MF-GC) synaptic transmission in mice anesthetized with urethane. Three-chamber testing showed that mice exposed to VPA mice exhibited a significant reduction in social interaction compared with the control group. In vivo electrophysiological recordings revealed that a pair of air-puff stimulation on ipsilateral whisker pad evoked MF-GC synaptic transmission, N1, and N2. The evoked MF-GC synaptic responses in VPA-exposed mice exhibited a significant increase in the area under the curve (AUC) of N1 and the amplitude and AUC of N2 compared with untreated mice. Cerebellar surface application of the selective N-methyl-D-aspartate (NMDA) receptor blocker D-APV significantly inhibited facial stimulation-evoked MF-GC synaptic transmission. In the presence of D-APV, there were no significant differences between the AUC of N1 and the amplitude and AUC of N2 in the VPA-exposed mice and those of the untreated mice. Notably, blockade of the GluN2A subunit-containing, but not the GluN2B subunit-containing, NMDA receptor, significantly inhibited MF-GC synaptic transmission and decreased the AUC of N1 and the amplitude and AUC of N2 in VPA-exposed mice to levels similar to those seen in untreated mice. In addition, the GluN2A subunit-containing NMDA receptor was expressed at higher levels in the GC layer of VPA-treated mice than in control mice. These results indicate that gestational VPA exposure in mice produces ASD-like behaviors, accompanied by increased cerebellar MF-GC synaptic transmission and an increase in GluN2A subunit-containing NMDA receptor expression in the offspring.

Norepinephrine (NE) modulates synaptic transmission and long-term plasticity through distinct subtype adrenergic receptor (AR)-mediated-intracellular signaling cascades. However, the role of NE modulates glutamatergic long-term potentiation (LTP) in the hypothalamic paraventricular nucleus (PVN) magnocellular neuroendocrine cells (MNCs) is unclear. We here investigate the effect of NE on high frequency stimulation (HFS)-induced glutamatergic LTP in rat hypothalamic PVN MNCs in vitro, by whole-cell patch-clamp recording, biocytin staining and pharmacological methods. Delivery of HFS induced glutamatergic LTP with a decrease in N2/N1 ratio in the PVN MNCs, which was enhanced by application of NE (100 nM). HFS-induced LTP was abolished by the blockade of N-methyl-D-aspartate receptors (NMDAR) with D-APV, but it was rescued by the application of NE. NE failed to rescue HFS-induced LTP of MNCs in the presence of a selective (31-AR antagonist, CGP 20712. However, application of (31-AR agonist, dobutamine HCl rescued HFS-induced LTP of MNCs in the absence of NMDAR activity. In the absence of NMDAR activity, NE failed to rescue HFS-induced MNC LTP when protein kinase A (PKA) was inhibited by extracellular applying KT5720 or intracellular administration of PKI. These results indicate that NE activates (31-AR and triggers HFS to induce a novel glutamatergic LTP of hypothalamic PVN NMCs via the postsynaptic PKA signaling pathway in vitro in rats.

依托咪酯(etomidate,ET)是一种作用强,且在临床麻醉中得到广泛应用的短效非巴比妥类静脉麻醉药,其麻醉机制与抑制多个脑区神经元活动与突触传递环路功能有关.ET抑制在体小鼠小脑浦肯野细胞放电活动,下调颗粒细胞感觉信息传递,提示ET影响小鼠小脑皮层神经环路突触传递长时程可塑性,但其影响机制尚不清楚.