mIPSC Frequency Research Articles

YTHDF1 in periaqueductal gray inhibitory neurons contributes to morphine withdrawal responses in mice

BackgroundPhysical symptoms and aversion induced by opioid withdrawal strongly affect the management of opioid addiction. YTH N6-methyladenosine (m6A) RNA binding protein 1 (YTHDF1), an m6A-binding protein, from the periaqueductal gray (PAG) reportedly contributes to morphine tolerance and hyperalgesia. However, the role of YTHDF1 in morphine withdrawal remains unclear.MethodsA naloxone-precipitated morphine withdrawal model was established in C57/BL6 mice or transgenic mice. YTHDF1 was knocked down via adeno-associated virus transfection. Combined with the results of the single-cell RNA sequencing analysis, the changes in morphine withdrawal somatic signs and conditioned place aversion (CPA) scores were compared when YTHDF1 originating from different neurons in the ventrolateral periaqueductal gray (vlPAG) was knocked down. We further explored the role of inflammatory factors and transcription factors related to inflammatory response in morphine withdrawal.ResultsOur results revealed that YTHDF1 expression was upregulated in the vlPAG of mice with morphine withdrawal and that the knockdown of vlPAG YTHDF1 attenuated morphine withdrawal-related somatic signs and aversion. The levels of NF-κB and p-NF-κB were reduced after the inhibition of YTHDF1 in the vlPAG. YTHDF1 from vlPAG inhibitory neurons, rather than excitatory neurons, facilitated morphine withdrawal responses. The inhibition of YTHDF1 in vlPAG somatostatin (Sst)-expressing neurons relieved somatic signs of morphine withdrawal and aversion, whereas the knockdown of YTHDF1 in cholecystokinin (Cck)-expressing or parvalbumin (PV)-expressing neurons did not change morphine withdrawal-induced responses. The activity of c-fos + neurons, the intensity of the calcium signal, the density of dendritic spines, and the frequency of mIPSCs in the vlPAG, which were increased in mice with morphine withdrawal, were decreased with the inhibition of YTHDF1 from vlPAG inhibitory neurons or Sst-expressing neurons. Knockdown of NF-κB in Sst-expressing neurons also alleviated morphine withdrawal-induced responses.ConclusionsYTHDF1 originating from Sst-expressing neurons in the vlPAG is crucial for the modulation of morphine withdrawal responses, and the underlying mechanism might be related to the regulation of the expression and phosphorylation of NF-κB.

A brain-body feedback loop driving HPA-axis dysfunction in breast cancer.

Breast cancer patients often exhibit disrupted circadian rhythms in circulating glucocorticoids (GCs), such as cortisol. This disruption correlates with reduced quality of life and higher cancer mortality 1-3 . The exact cause of this phenomenon - whether due to treatments, stress, age, co-morbidities, lifestyle factors, or the cancer itself remains unclear. Here, we demonstrate that primary breast cancer alone blunts host GC rhythms by disinhibiting neurons in the hypothalamus, and that circadian phase-specific neuromodulation of these neurons can attenuate tumor growth by enhancing anti-tumor immunity. We find that mice with mammary tumors exhibit blunted GC rhythms before tumors are palpable, alongside increased activity in paraventricular hypothalamic neurons expressing corticotropin-releasing hormone (i.e., PVN CRH neurons). Tumor-bearing mice have fewer inhibitory synapses contacting PVN CRH neurons and reduced miniature inhibitory post-synaptic current (mIPSC) frequency, leading to net excitation. Tumor-bearing mice experience impaired negative feedback on GC production, but adrenal and pituitary gland functions are largely unaffected, indicating that alterations in PVN CRH neuronal activity are likely a primary cause of hypothalamic-pituitary-adrenal (HPA) axis dysfunction in breast cancer. Using chemogenetics (hM3Dq) to stimulate PVN CRH neurons at different circadian phases, we show that stimulation just before the light-to-dark transition restores normal GC rhythms and reduces tumor progression. These mice have significantly more effector T cells (CD8+) within the tumor than non-stimulated controls, and the anti-tumor effect of PVN CRH neuronal stimulation is absent in mice lacking CD8+ T cells. Our findings demonstrate that breast cancer distally regulates neurons in the hypothalamus that control output of the HPA axis and provide evidence that therapeutic targeting of these neurons could mitigate tumor progression.

Complex housing in adulthood state-dependently affects the excitation-inhibition balance in the infralimbic prefrontal cortex of male C57Bl/6 mice

The prefrontal cortex (PFC) plays an important role in social behavior and is sensitive to stressful circumstances. Challenging life conditions might change PFC function and put individuals at risk for maladaptive social behavior. The excitation-inhibition (EI) balance of prefrontal neurons appears to play a crucial role in this process. Here, we examined how a challenging life condition in C57BL/6JolaHsd mice, i.e. group-housing 6 mice in a complex environment for 10 days in adulthood, changes the EI-balance of infralimbic prefrontal neurons in layer 2/3, compared to standard pair-housing. Slices were prepared from “undisturbed” mice, i.e. the first mouse taken from the cage, or mice taken ∼15 min later, who were mildly aroused after removal of the first mouse. We observed a housing-condition by arousal-state interaction, with in the complex housing group an elevated EI-balance in undisturbed and reduced EI-balance in mildly aroused animals, while no differences were observed in standard housed animals. The change was explained by a shift in mIPSC and mEPSC frequency, while amplitudes remained unaffected. Female mice showed no housing-by-state interaction, but a main effect of housing was found for mIPSCs, with a higher frequency in complex- versus standard-housed females. No effects were observed in males who were complex-housed from a young age onwards. Explorative investigations support a potential mediating role of corticosterone in housing effects on the EI-balance of males. We argue that taking the arousal state of individuals into account is necessary to better understand the consequences of exposure to challenging life conditions for prefrontal function.

Topical administration of GLP-1 eyedrops improves retinal ganglion cell function by facilitating presynaptic GABA release in early experimental diabetes.

Diabetic retinopathy is a prominent cause of blindness in adults, with early retinal ganglion cell (RGC) loss contributing to visual dysfunction or blindness. In the brain, defects in y-aminobutyric acid (GABA) synaptic transmission are associated with pathophysiological and neurodegenerative disorders, whereas glucagon-like peptide-1 (GLP-1) has demonstrated neuroprotective effects. However, it is not yet clear whether diabetes causes alterations in inhibitory input to RGCs and whether and how GLP-1 protects against neurodegeneration in the diabetic retina through regulating inhibitory synaptic transmission to RGCs. In the present study, we used the patch-clamp technique to record GABA subtype A receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) in RGCs from streptozotocin-induced diabetes model rats. We found that early diabetes (4 weeks of hyperglycemia) decreased the frequency of GABAergic mIPSCs in RGCs without altering their amplitude, suggesting a reduction in the spontaneous release of GABA to RGCs. Topical administration of GLP-1 eyedrops over a period of 2 weeks effectively countered the hyperglycemia-induced downregulation of GABAergic mIPSC frequency, subsequently enhancing the survival of RGCs. Concurrently, the protective effects of GLP-1 on RGCs in diabetic rats were eliminated by topical administration of exendin-9-39, a specific GLP-1 receptor antagonist, or SR95531, a specific antagonist of the GABA subtype A receptor. Furthermore, extracellular perfusion of GLP-1 was found to elevate the frequencies of GABAergic mIPSCs in both ON- and OFF-type RGCs. This elevation was shown to be mediated by activation of the phosphatidylinositol-phospholipase C/inositol 1,4,5-trisphosphate receptor/Ca2+/protein kinase C signaling pathway downstream of GLP-1 receptor activation. Moreover, multielectrode array recordings revealed that GLP-1 functionally augmented the photoresponses of ON-type RGCs. Optomotor response tests demonstrated that diabetic rats exhibited reductions in visual acuity and contrast sensitivity that were significantly ameliorated by topical administration of GLP-1. These results suggest that GLP-1 facilitates the release of GABA onto RGCs through the activation of GLP-1 receptor, leading to the de-excitation of RGC circuits and the inhibition of excitotoxic processes associated with diabetic retinopathy. Collectively, our findings indicate that the GABA system has potential as a therapeutic target for mitigating early-stage diabetic retinopathy. Furthermore, the topical administration of GLP-1 eyedrops represents a non-invasive and effective treatment approach for managing early-stage diabetic retinopathy.

A Reduction in the Readily Releasable Vesicle Pool Impairs GABAergic Inhibition in the Hippocampus after Blood-Brain Barrier Dysfunction.

Major burdens for patients suffering from stroke are cognitive co-morbidities and epileptogenesis. Neural network disinhibition and deficient inhibitive pulses for fast network activities may result from impaired presynaptic release of the inhibitory neurotransmitter GABA. To test this hypothesis, a cortical photothrombotic stroke was induced in Sprague Dawley rats, and inhibitory currents were recorded seven days later in the peri-infarct blood-brain barrier disrupted (BBBd) hippocampus via patch-clamp electrophysiology in CA1 pyramidal cells (PC). Miniature inhibitory postsynaptic current (mIPSC) frequency was reduced to about half, and mIPSCs decayed faster in the BBBd hippocampus. Furthermore, the paired-pulse ratio of evoked GABA release was increased at 100 Hz, and train stimulations with 100 Hz revealed that the readily releasable pool (RRP), usually assumed to correspond to the number of tightly docked presynaptic vesicles, is reduced by about half in the BBBd hippocampus. These pathophysiologic changes are likely to contribute significantly to disturbed fast oscillatory activity, like cognition-associated gamma oscillations or sharp wave ripples and epileptogenesis in the BBBd hippocampus.

Chronic Intermittent Hypoxia Alters GABAergic Inhibition in Median Preoptic Nucleus (MnPO) Neurons of Rats

Chronic intermittent hypoxia (CIH) is an animal model simulating the hypoxemia associated with obstructive sleep apnea (OSA) in humans. Male rats exposed to CIH exhibit increased sympathetic tone and persistent elevations in blood pressure similar to that observed in patients with. In MnPO neurons, CIH reduces GABAa inhibition and, in some neurons, GABAa activation produces excitation. Here, loose patch and perforated patch recordings were conducted in MnPO neurons in response to optogenetic stimulation of GABAergic SFO (efferent) and MnPO (local) neurons to assess the role of GABAergic signaling in these nuclei on MnPO excitability. Our working hypothesis was that CIH would diminish the inhibitory effects of optogenetic stimulation of GABA neurons. This hypothesis was tested using an AAV vector that selectively expresses channel rhodopsin in GABA neurons Using isoflurane (2-3%) anesthesia, male Sprague-Dawley rats (250-350g) received infusions (0.2 μL) of pAAV-mDlx-ChR2-mCherry-Fishell-3 in the SFO or MnPO. After recovery, rats were subjected to 7 days of CIH (0800-1600 hr) or normoxia (Norm). CIH consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10x/hr for 8 hours (during the normal inactive/sleep phase) on 7 consecutive days. For in vitro recordings, rats were anesthetized with isoflurane (2-3%), and sagittal or coronal slices (300 μm) containing MnPO were cut using standard slice procedures. Voltage-clamp loose patch or perforated patch recordings using gramicidin (100 ug/ml in 140 mM K-gluconate) were collected in MnPO neurons in response to optogenetic stimulation of GABAergic neurons (2 ms pulse, 20 Hz, 1 sec). MnPO neurons from CIH-treated rats showed differing responses to GABAergic stimulation while MnPO neurons from Norm-treated rats predominantly exhibited GABAergic inhibition. MnPO neurons from Norm rats exhibited mIPSCs and outward currents in response to muscimol. MnPO neurons from CIH-treated rats show variable mIPSC frequencies and heterogeneous responses to GABAergic neuronal activation in the SFO and MnPO. The results demonstrate that CIH alters the Cl− homeostasis in some MnPO neurons altering their response to GABAa stimulation. These changes may contribute to the increased sympathetic tone and reactivity, as well as the persistent hypertension associated with CIH. Supported by R01 HL155977. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

ADAM17 in glutamatergic neurons promote pre-sympathetic neuronal activation through microglia-mediated blockade of GABAergic inputs

The brain renin-angiotensin system (RAS) plays an important role in regulating autonomic function. We have demonstrated that A Disintegrin and Metalloproteinase 17 (ADAM17) in glutamatergic neurons facilitate activation of presympathetic neurons (PSN) upon angiotensin II (Ang-II) stimulation and promote development of salt-sensitive hypertension. However, in PVN, glutamatergic neurons express limited angiotensin-converting enzyme 2, a substrate of ADAM17 and crucial member of regulatory RAS, thus bringing question regarding how ADAM17 supports PSN activation in the PVN. Using whole-cell patch clamp, the PVN-RVLM-projecting PSN in wildtype (WT) mice exhibited a dramatical reduction in the frequency of miniature inhibitory postsynaptic currents (mIPSC), when Ang-II was bath-supplied (2 μM). By contrast, Ang-II-associated suppression of mIPSC frequency in RVLM-projecting PSN, from mice lacking ADAM17 in glutamatergic neurons (A17GKO), was significantly smaller (suppression ratio: WT vs. A17GKO = 46.39±10.59 vs. 19.27±15.03, P<0.0001). Meanwhile, no difference was detected in the amplitude of mIPSC in either case of baseline or Ang-II bath-incubation, indicating that only presynaptic GABAergic inputs, rather than postsynaptic receptors of GABA, was affected by Ang-II. Considering the sheddase role of ADAM17, we then hypothesized that ADAM17 in glutamatergic neurons mediated blockade of GABAergic inputs via extracellular mechanism. Via immunoelectron microscopy, we observed co-localization of PSN, microglia and GABA. Using DOCA-salt hypertension model (1mg/g body weight, 1%NaCl, 21 days), we observed that DOCA-salt-induced reduction of GABA (LC-MS), together with increase of TNFα, IL-6, and TGFβ (ELISA), which were closely related to microglial activation, were abolished in the PVN of A17GKO mice. These findings all suggested an involvement of microglial activation in blockade of GABAergic inputs during hypertension development. Accordingly, TRAM34, a small molecule that freezes microglia, was introduced in whole-cell patch clamp recording of mIPSC. With pre-incubation of TRAM34, Ang-II-associated suppression of mIPSC frequency in the PVN-RVLM projecting PSN was blunted, in a similar manner as A17GKO (suppression ratio: WT vs. WT+ TRAM34 = 46.39±10.59 vs. 15.38±6.28, P<0.0001). In addition, contacts between microglia and PSN were increased in WT mice during the early stage of hypertension development (1st week of DOCA-salt treatment), and they were significantly less in A17GKO (percentage of neurons being touched: WT+DOCA vs. A17GKO+DOCA = 28.59±1.9% vs. 11.83±2.27%, P<0.0001). Together, our data provided preliminary evidence that ADAM17 in glutamatergic neurons mediates the blockade of tonic GABAergic inputs via facilitating chemotaxis of PSN, therefore supporting PSN activation and development of hypertension. National Natural Science Foundation of China, 82100454 to JX. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Regulation of anxiety-like behaviors by S-palmitoylation and S-nitrosylation in basolateral amygdala

Protein posttranslational modification regulates synaptic protein stability, sorting and trafficking, and is involved in emotional disorders. Yet the molecular mechanisms regulating emotional disorders remain unelucidated. Here we report unknown roles of protein palmitoylation/nitrosylation crosstalk in regulating anxiety-like behaviors in rats. According to the percentages of open arm duration in the elevated plus maze test, the rats were divided into high-, intermediate- and low-anxiety groups. The palmitoylation and nitrosylation levels were detected by acyl-biotin exchange assay, and we found low palmitoylation and high nitrosylation levels in the basolateral amygdala (BLA) of high-anxiety rats. Furthermore, we observed that 2-bromopalmitate (2-BP), a palmitoylation inhibitor, induced anxiety-like behaviors, accompanied with decreased amplitude and frequency of mEPSCs and mIPSCs in the BLA. Additionally, we also found that inhibiting nNOS activity with 7-nitroindazole (7-NI) in the BLA caused anxiolytic effects and reduced the synaptic transmission. Interestingly, diazepam (DZP) rapidly elevated the protein palmitoylation level and attenuated the protein nitrosylation level in the BLA. Specifically, similar to DZP, the voluntary wheel running exerted DZP-like anxiolytic action, and induced high palmitoylation and low nitrosylation levels in the BLA. Lastly, blocking the protein palmitoylation with 2-BP induced an increase in protein nitrosylation level, and attenuating the nNOS activity by 7-NI elevated the protein palmitoylation level. Collectively, these results show a critical role of protein palmitoylation/nitrosylation crosstalk in orchestrating anxiety behavior in rats, and it may serve as a potential target for anxiolytic intervention.

Synapse-Specific Defects in Synaptic Transmission in the Cerebellum of W246G Mutant ELOVL4 Rats-a Model of Human SCA34.

Elongation of very long fatty acids-4 (ELOVL4) mediates biosynthesis of very long chain-fatty acids (VLC-FA; ≥28 carbons). Various mutations in this enzyme result in spinocerebellar ataxia-34 (SCA34). We generated a rat model of human SCA34 by knock-in of a naturally occurring c.736T>G, p.W246G mutation in the Elovl4 gene. Our previous analysis of homozygous W246G mutant ELOVL4 rats (MUT) revealed early-onset gait disturbance and impaired synaptic transmission and plasticity at parallel fiber-Purkinje cell (PF-PC) and climbing fiber-Purkinje cell (CF-PC) synapses. However, the underlying mechanisms that caused these defects remained unknown. Here, we report detailed patch-clamp recordings from Purkinje cells that identify impaired synaptic mechanisms. Our results show that miniature EPSC (mEPSC) frequency is reduced in MUT rats with no change in mEPSC amplitude, suggesting a presynaptic defect of excitatory synaptic transmission on Purkinje cells. We also find alterations in inhibitory synaptic transmission as miniature IPSC (mIPSC) frequency and amplitude are increased in MUT Purkinje cells. Paired-pulse ratio is reduced at PF-PC synapses but increased at CF-PC synapses in MUT rats, which along with results from high-frequency stimulation suggest opposite changes in the release probability at these two synapses. In contrast, we identify exaggerated persistence of EPSC amplitude at CF-PC and PF-PC synapses in MUT cerebellum, suggesting a larger readily releasable pool (RRP) at both synapses. Furthermore, the dendritic spine density is reduced in MUT Purkinje cells. Thus, our results uncover novel mechanisms of action of VLC-FA at cerebellar synapses, and elucidate the synaptic dysfunction underlying SCA34 pathology.SIGNIFICANCE STATEMENT Very long chain-fatty acids (VLC-FA) are an understudied class of fatty acids that are present in the brain. They are critical for brain function as their deficiency caused by mutations in elongation of very long fatty acids-4 (ELOVL4), the enzyme that mediates their biosynthesis, results in neurologic diseases including spinocerebellar ataxia-34 (SCA34), neuroichthyosis, and Stargardt-like macular dystrophy. In this study, we investigated the synaptic defects present in a rat model of SCA34 and identified defects in presynaptic neurotransmitter release and dendritic spine density at synapses in the cerebellum, a brain region involved in motor coordination. These results advance our understanding of the synaptic mechanisms regulated by VLC-FA and describe the synaptic dysfunction that leads to motor incoordination in SCA34.

Experimental study on developmental changes of synaptic currents in V1B cortical layer Ⅳ pyramidal neurons in C57BL/6J mice

Objective: To investigate the developmental changes of miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) of layer Ⅳ pyramidal neurons in the primary visual cortex binocular zone (V1B) of C57BL/6J wild-type mice at different developmental stages. Methods: Sixteen male C57BL/6J mice of specific-pathogen-free grade were selected and divided into 4 groups according to their postnatal age: P14 group (before and after eye opening), P28 group (the peak of the critical period), P35 group (the end of the critical period), and P130 group (fully adult). Whole-cell patch-clamp technique was used to record the frequency and amplitude of mEPSCs and mIPSCs of layer Ⅳ pyramidal neurons in V1B of each group, and to analyze their differences and changes. Results: The frequency of mEPSCs of layer Ⅳ pyramidal neurons in V1B of mice in the four groups was statistically different (F=9.46, P<0.001), with the P35 group being higher than the P28 group [P28 and P35 groups were (8.72±1.34) and (13.28±4.05) Hz, t=3.39, P=0.012], and the P130 group being lower than the P35 group [P35 and P130 groups were (13.28±4.05) and (5.82±1.98) Hz, t=5.21, P<0.001]; the amplitude of mEPSCs of layer Ⅳ pyramidal neurons in V1B of mice in the four groups was not statistically different (F=2.84, P=0.055). The frequency of mIPSCs of layer Ⅳ pyramidal neurons in V1B of mice in the four groups was statistically different (F=8.14, P<0.001), with the P130 group being higher than the P14 group [P14 and P130 groups were (5.22±1.33) and (12.03±3.94) Hz, t=4.678, P<0.001]; the amplitude of mIPSCs of layer Ⅳ pyramidal neurons in V1B of mice in the four groups was statistically different (F=7.06, P=0.001), with the P35 group being higher than the P28 group [P28 and P35 groups were (20.07±3.56) and (28.47±5.98) pA, t=3.66, P=0.006], and the P130 group being lower than the P35 group [P35 and P130 groups were (28.47±5.98) and (20.32±3.55) pA, t=3.33, P=0.014]. Conclusions: The excitatory synaptic development of layer Ⅳ pyramidal neurons in V1B of mice is in a vigorous growth state during development and gradually weakens with age, while the inhibitory synaptic development gradually strengthens with increasing postnatal age, and both of them rapidly develop during the critical period.

Cell-type-specific synaptic modulation of mAChR on SST and PV interneurons.

The muscarinic acetylcholine receptor (mAChR) antagonist, scopolamine, has been shown to have a rapid antidepressant effect. And it is believed that GABAergic interneurons play a crucial role in this action. Therefore, characterizing the modulation effects of mAChR on GABAergic interneurons is crucial for understanding the mechanisms underlying scopolamine's antidepressant effects. In this study, we examined the effect of mAChR activation on the excitatory synaptic transmissions in two major subtypes of GABAergic interneurons, somatostatin (SST)- and parvalbumin (PV)-expressing interneurons, in the anterior cingulate cortex (ACC). We found that muscarine, a mAChR agonist, non-specifically facilitated the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in both SST and PV interneurons. Scopolamine completely blocked the effects of muscarine, as demonstrated by recovery of sESPCs and mEPSCs in these two types of interneurons. Additionally, individual application of scopolamine did not affect the EPSCs of these interneurons. In inhibitory transmission, we further observed that muscarine suppressed the frequency of both spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) in SST interneurons, but not PV interneurons. Interestingly, scopolamine directly enhanced the frequency of both sIPSCs and mIPSCs mainly in SST interneurons, but not PV interneurons. Overall, our results indicate that mAChR modulates excitatory and inhibitory synaptic transmission to SST and PV interneurons within the ACC in a cell-type-specific manner, which may contribute to its role in the antidepressant effects of scopolamine.

GABAergic synaptic transmission and cortical oscillation patterns in the primary somatosensory area of a valproic acid rat model of autism spectrum disorder.

Autism spectrum disorder (ASD) is characterized by impaired social communication and interaction associated with repetitive or stereotyped behaviour. Prenatal valproic acid (VPA) exposure in rodents is a commonly used model of ASD. Resveratrol (RSV) has been shown to prevent interneuronal and behavioural impairments in the VPA model. We investigated the effects of prenatal VPA exposure and RSV on the GABAergic synaptic transmission, brain oscillations and on the genic expression of interneuron-associated transcription factor LHX6 in the primary somatosensory area (PSSA). Prenatal VPA exposure decreased the sIPSC and mIPSC frequencies and the sIPSC decay kinetics onto layers 4/5 pyramidal cells of PSSA. About 40% of VPA animals exhibited absence-like spike-wave discharge (SWD) events associated with behaviour arrest and increased power spectrum density of delta, beta and gamma cortical oscillations. VPA animals had reduced LHX6 expression in PSSA, but VPA animals treated with RSV had no changes on synaptic inhibition or LHX6 expression in the PSSA. SWD events associated with behaviour arrest and the abnormal increment of cortical oscillations were also absent in VPA animals treated with RSV. These findings provide new venues to investigate the role of both RSV and VPA in the pathophysiology of ASD and highlight the VPA animal model as an interesting tool to investigate pathways related to the aetiology and possible future therapies to this neuropsychiatric disorder.

Motor training promotes both synaptic and intrinsic plasticity of layer V pyramidal neurons in the primary motor cortex.

Here we examined motor training-induced synaptic and intrinsic plasticity of layer V pyramidal neurons in primary motor cortex. The training reduced presynaptic GABA release in early phase, but strengthened AMPA receptor-mediated excitatory synapses in later phase: acquired motor performance after training correlated with the strength of excitatory synapses rather than inhibitory synapses. As to the intrinsic property, the training transiently decreased firing rate in early phase, but returned to pre-training levels in later phase. Western-blot analysis detected decreased phosphorylation of Ser408-409 in GABAA receptor β3 subunits in acute phase, and increased phosphorylation of Ser831 in AMPA receptor GluA1 subunits in later phase. Live-imaging analysis of Thy1-YFP transgenic mice showed rapid and long-term spine plasticity in M1 layer V neurons, suggesting training-induced increases in self-entropy per spine. Layer V neurons in primary motor cortex (M1) are important for motor skill learning. Since pretreatment of either CNQX or APV in rats M1 layer V impaired rotor rod learning, we analyzed training-induced synaptic plasticity by whole-cell patch-clamp technique in acute brain slices. One-day trained rats showed a decrease in small inhibitory postsynaptic current (mIPSC) frequency and an increase in the paired-pulse ratio of evoked IPSCs, suggesting a transient decrease in presynaptic GABA release in early phase. Two-days trained rats showed an increase in miniature excitatory postsynaptic current (mEPSC) amplitudes/frequency and elevated AMPA/NMDA ratios, suggesting a long-term strengthening of AMPA receptor-mediated excitatory synapses. Importantly, rotor rod performance in trained rats was correlated with the mean mEPSC amplitude and the frequency obtained from that animal. In current-clamp analysis, 1-day-trained rats transiently decreased current-induced firing rate, while 2-day-trained rats returned to pre-training levels, suggesting a dynamic changes in intrinsic properties. Furthermore, western-blot analysis of layer V detected decreased phosphorylation of Ser408-409 in GABAA receptor β3 subunits in 1-day trained rats, and increased phosphorylation of Ser831 in AMPA receptor GluA1 subunits in 2-days trained rats. Finally, live-imaging analysis of Thy1-YFP transgenic mice showed that the training rapidly recruited a substantial number of spines for long-term plasticity in M1 layer V neurons. Taken together, these results indicate that motor training induces complex and diverse plasticity in M1 layer V pyramidal neurons. Abstract figure legend Schematic of motor training and rapid spine dynamics in M1 layer V neurons. Exercise training promotes physiological and morphological spine diversity. Red: glutamate and AMPA receptors. Blue: GABA and GABAA receptors. This article is protected by copyright. All rights reserved.

Dynamic regulation of excitatory and inhibitory synaptic transmission by growth hormone in the developing mouse brain.

Normal sensory and cognitive function of the brain relies on its intricate and complex neural network. Synaptogenesis and synaptic plasticity are critical to neural circuit formation and maintenance, which are regulated by coordinated intracellular and extracellular signaling. Growth hormone (GH) is the most abundant anterior pituitary hormone. Its deficiencies could alter brain development and impair learning and memory, while GH replacement therapy in human patients and animal models has been shown to ameliorate cognitive deficits caused by GH deficiency. However, the underlying mechanism remains largely unknown. In this study, we investigated the neuromodulatory function of GH in young (pre-weaning) mice at two developmental time points and in two different brain regions. Neonatal mice were subcutaneously injected with recombinant human growth hormone (rhGH) on postnatal day (P) 14 or 21. Excitatory and inhibitory synaptic transmission was measured using whole-cell recordings in acute cortical slices 2 h after the injection. We showed that injection of rhGH (2 mg/kg) in P14 mice significantly increased the frequency of mEPSCs, but not that of mIPSCs, in both hippocampal CA1 pyramidal neurons and L2/3 pyramidal neurons of the barrel field of the primary somatosensory cortex (S1BF). Injection of rhGH (2 mg/kg) in P21 mice significantly increased the frequency of mEPSCs and mIPSCs in both brain regions. Perfusion of rhGH (1 μM) onto acute brain slices in P14 mice had similar effects. Consistent with the electrophysiological results, the dendritic spine density of CA1 pyramidal neurons and S1BF L2/3 pyramidal neurons increased following in vivo injection of rhGH. Furthermore, NMDA receptors and postsynaptic calcium-dependent signaling contributed to rhGH-dependent regulation of both excitatory and inhibitory synaptic transmission. Together, these results demonstrate that regulation of excitatory and inhibitory synaptic transmission by rhGH occurs in a developmentally dynamic manner, and have important implication for identifying GH treatment strategies without disturbing excitation/inhibition balance.

Sevoflurane Ameliorates Schizophrenia in a Mouse Model and Patients: A Pre-Clinical and Clinical Feasibility Study.

GABAergic deficits have been considered to be associated with the pathophysiology of schizophrenia, and hence, GABA receptors subtype A (GABAARs) modulators, such as commonly used volatile anesthetic sevoflurane, may have therapeutic values for schizophrenia. The present study investigates the therapeutic effectiveness of low-concentration sevoflurane in MK801-induced schizophrenia-like mice and schizophrenia patients. Three weeks after MK801 administration (0.5 mg kg-1, i.p. twice a day for 5 days), mice were exposed to 1% sevoflurane 1hr/day for 5 days. Behavioral tests, immunohistochemical analysis, western blot assay, and electrophysiology assessments were performed 1-week post-exposure. Ten schizophrenia patients received 1% sevoflurane 5 hrs per day for 6 days and were assessed with the Positive and Negative Syndrome Scale (PANSS) and the 18-item Brief Psychiatric Rating Scale (BPRS-18) at week 1 and week 2. MK801 induced hypolocomotion and social deficits, downregulated expression of NMDARs subunits and postsynaptic density protein 95 (PSD95), reduced parvalbumin - and GAD67-positive neurons, altered amplitude and frequency of mEPSCs and mIPSCs, and increased the excitation/inhibition ratio. All these changes induced by MK-801 were attenuated by sevoflurane administration. Six and eight patients achieved a response defined as a reduction of at least 30% in the PANSS total score at 1st and 2nd week after treatments. The BPRS-18 total score was found to be significantly decreased by 38% at the 2nd week (p < 0.01). Low-concentration sevoflurane effectively reversed MK801-induced schizophrenialike disease in mice and alleviated schizophrenia patients' symptoms. Our work suggests sevoflurane to be a valuable therapeutic strategy for treating schizophrenia patients.

Sex-dependent changes of hippocampal synaptic plasticity and cognitive performance in C57BL/6J mice exposed to neonatal repeated maternal separation

The repeated maternal separation (RMS) is a useful experimental model useful in rodents to study the long-term influence of early-life stress on brain neurophysiology. We here investigated the influence of RMS exposure on hippocampal inhibitory and excitatory synaptic transmission, long-term synaptic plasticity and the related potential alterations in learning and memory performance in adult male and female C57Bl/6J mice. Mice were separated daily from their dam for 360 min, from postnatal day 2 (PND2) to PND17, and experiments were performed at PND 60. Patch-clamp recordings in hippocampal CA1 pyramidal neurons revealed a significant enhancement of GABAergic miniature IPSC (mIPSC) frequency, and a decrease in the amplitude of glutamatergic mEPSCs in male mice exposed to RMS. Only a slight but significant reduction in the amplitude of GABAergic mIPSCs was observed in females exposed to RMS compared to the relative controls. A marked increase in long-term depression (LTD) at CA3-CA1 glutamatergic synapses and in the response to the CB1r agonist win55,212 were detected in RMS male, but not female mice. An impaired spatial memory and a reduced preference for novelty was observed in males exposed to RMS but not in females. A single injection of β-ethynyl estradiol at PND2, prevented the changes observed in RMS male mice, suggesting that estrogens may play a protective role early in life against the exposure to stressful conditions. Our findings strengthen the idea of a sex-dependent influence of RMS on long-lasting modifications in synaptic transmission, effects that may be relevant for cognitive performance.

Chronic high-fat diet induces overeating and impairs synaptic transmission in feeding-related brain regions.

Obesity is linked to overeating, which can exacerbate unhealthy weight gain. However, the mechanisms for mediating such linkages are elusive. In the current study, we hypothesized that synaptic remodeling occurs in feeding-related brain regions of obese mice. To investigate this, we established a high-fat diet (HFD)-induced obese mouse model and observed that these mice consumed excessive calories. The effect of chronic HFD feeding on lipid droplet accumulation in different brain structures was also investigated. We found that lipid droplets accumulated on the ependyma of the third ventricle (3V), which is surrounded by key areas of the hypothalamus that are involved in feeding. Then, the spontaneous synaptic activity of miniature excitatory postsynaptic current (mEPSC) and miniature inhibitory postsynaptic current (mIPSC) was recorded in these hypothalamic areas. HFD induced a decreased amplitude of mEPSC in the arcuate nucleus (ARC) and the ventromedial hypothalamus (VMH), meanwhile, increased the frequency in the VMH. In addition, HFD reduced the frequency of mIPSC in the lateral hypothalamus (LH) and increased the amplitude of mIPSC in the paraventricular nucleus of the hypothalamus (PVH). Subsequently, we also measured the synaptic activity of nucleus accumbens (NAc) neurons, which play a vital role in the hedonic aspect of eating, and discovered that HFD diminished the frequency of both mEPSC and mIPSC in the NAc. These findings suggest that chronic HFD feeding leads to lipid accumulation and synaptic dysfunction in specific brain regions, which are associated with energy homeostasis and reward regulation, and these impairments may lead to the overeating of obesity.

Epigenetic regulation of GABAergic differentiation in the developing brain.

In the vertebrate brain, GABAergic cell development and neurotransmission are important for the establishment of neural circuits. Various intrinsic and extrinsic factors have been identified to affect GABAergic neurogenesis. However, little is known about the epigenetic control of GABAergic differentiation in the developing brain. Here, we report that the number of GABAergic neurons dynamically changes during the early tectal development in the Xenopus brain. The percentage of GABAergic neurons is relatively unchanged during the early stages from stage 40 to 46 but significantly decreased from stage 46 to 48 tadpoles. Interestingly, the histone acetylation of H3K9 is developmentally decreased from stage 42 to 48 (about 3.5 days). Chronic application of valproate acid (VPA), a broad-spectrum histone deacetylase (HDAC) inhibitor, at stage 46 for 48 h increases the acetylation of H3K9 and the number of GABAergic cells in the optic tectum. VPA treatment also reduces apoptotic cells. Electrophysiological recordings show that a VPA induces an increase in the frequency of mIPSCs and no changes in the amplitude. Behavioral studies reveal that VPA decreases swimming activity and visually guided avoidance behavior. These findings extend our understanding of histone modification in the GABAergic differentiation and neurotransmission during early brain development.

Gabra5 plays a sexually dimorphic role in POMC neuron activity and glucose balance.

Pro-opiomelanocortin (POMC) neurons are important for the regulation of body weight and glucose balance. The inhibitory tone to POMC neurons is mediated primarily by the GABA receptors. However, the detailed mechanisms and functions of GABA receptors are not well understood. The α5 subunit of GABAA receptor, Gabra5, is reported to regulate feeding, and we found that Gabra5 is highly expressed in POMC neurons. To explore the function of Gabra5 in POMC neurons, we knocked down Gabra5 specifically from mature hypothalamic POMC neurons using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 strategy. This POMC-specific knock-down of Gabra5 did not affect body weight or food intake in either male or female mice. Interestingly, the loss of Gabra5 caused significant increases in the firing frequency and resting membrane potential, and a decrease in the amplitude of the miniature inhibitory postsynaptic current (mIPSC) in male POMC neurons. However, the loss of Gabra5 only modestly decreased the frequency of mIPSC in female POMC neurons. Consistently, POMC-specific knock-down of Gabra5 significantly improved glucose tolerance in male mice but not in female mice. These results revealed a sexually dimorphic role of Gabra5 in POMC neuron activity and glucose balance, independent of body weight control.

Integrins Bidirectionally Regulate the Efficacy of Inhibitory Synaptic Transmission and Control GABAergic Plasticity.

For many decades, synaptic plasticity was believed to be restricted to excitatory transmission. However, in recent years, this view started to change, and now it is recognized that GABAergic synapses show distinct forms of activity-dependent long-term plasticity, but the underlying mechanisms remain obscure. Herein, we asked whether signaling mediated by β1 or β3 subunit-containing integrins might be involved in regulating the efficacy of GABAergic synapses, including the NMDA receptor-dependent inhibitory long-term potentiation (iLTP) in the hippocampus. We found that activation of β3 integrin with fibrinogen induced a stable depression, whereas inhibition of β1 integrin potentiated GABAergic synapses at CA1 pyramidal neurons in male mice. Additionally, compounds that interfere with the interaction of β1 or β3 integrins with extracellular matrix blocked the induction of NMDA-iLTP. In conclusion, we provide the first evidence that integrins are key players in regulating the endogenous modulatory mechanisms of GABAergic inhibition and plasticity in the hippocampus.SIGNIFICANCE STATEMENT Epilepsy, schizophrenia, and anxiety are just a few medical conditions associated with dysfunctional inhibitory synaptic transmission. GABAergic synapses are known for their extraordinary susceptibility to modulation by endogenous factors and exogenous pharmacological agents. We describe here that integrins, adhesion proteins, play a key role in the modulation of inhibitory synaptic transmission. Specifically, we show that interference with integrin-dependent adhesion results in a variety of effects on the amplitude and frequency of GABAergic mIPSCs. Activation of β3 subunit-containing integrins induces inhibitory long-term depression, whereas the inhibition of β1 subunit-containing integrins induces iLTP. Our results unveil an important mechanism controlling synaptic inhibition, which opens new avenues into the usage of integrin-aimed pharmaceuticals as modulators of GABAergic synapses.