Synaptic alterations in pyramidal cells following genetic manipulation of neuronal excitability in monkey prefrontal cortex.

In schizophrenia, layer 3 pyramidal neurons (L3PNs) in the dorsolateral prefrontal cortex (DLPFC) are thought to receive fewer excitatory synaptic inputs and to have lower expression levels of activity-dependent genes and of genes involved in mitochondrial energy production. In concert, these findings from previous studies suggest that DLPFC L3PNs are hypoactive in schizophrenia, disrupting the patterns of activity that are crucial for working memory, which is impaired in the illness. However, whether lower PN activity produces alterations in inhibitory and/or excitatory synaptic strength has not been tested in the primate DLPFC. Here, we decreased PN excitability in rhesus monkey DLPFC in vivo using adeno-associated viral vectors (AAVs) to produce Cre recombinase-mediated overexpression of Kir2.1 channels, a genetic silencing tool that efficiently decreases neuronal excitability. In acute slices prepared from DLPFC 7-12 weeks post-AAV microinjections, Kir2.1-overexpressing PNs had a significantly reduced excitability largely attributable to highly specific effects of the AAV-encoded Kir2.1 channels. Moreover, recordings of synaptic currents showed that Kir2.1-overexpressing DLPFC PNs had reduced strength of excitatory synapses whereas inhibitory synaptic inputs were not affected. The decrease in excitatory synaptic strength was not associated with changes in dendritic spine number, suggesting that excitatory synapse quantity was unaltered in Kir2.1-overexpressing DLPFC PNs. These findings suggest that, in schizophrenia, the excitatory synapses on hypoactive L3PNs are weaker and thus might represent a substrate for novel therapeutic interventions. Significance Statement: In schizophrenia, dorsolateral prefrontal cortex (DLPFC) pyramidal neurons (PNs) have both transcriptional and structural alterations that suggest they are hypoactive. PN hypoactivity is thought to produce synaptic alterations in schizophrenia, however the effects of lower neuronal activity on synaptic function in primate DLPFC have not been examined. Here, we used, for the first time in primate neocortex, adeno-associated viral vectors (AAVs) to reduce PN excitability with Kir2.1 channel overexpression and tested if this manipulation altered the strength of synaptic inputs onto the Kir2.1-overexpressing PNs. Recordings in DLPFC slices showed that Kir2.1 overexpression depressed excitatory (but not inhibitory), synaptic currents, suggesting that, in schizophrenia, the hypoactivity of PNs might be exacerbated by reduced strength of the excitatory synapses they receive.

Anoctamin 4 channel currents activate glucose-inhibited neurons in the mouse ventromedial hypothalamus during hypoglycemia.

Glucose is the basic fuel essential for maintenance of viability and functionality of all cells. However, some neurons - namely, glucose-inhibited (GI) neurons - paradoxically increase their firing activity in low-glucose conditions and decrease that activity in high-glucose conditions. The ionic mechanisms mediating electric responses of GI neurons to glucose fluctuations remain unclear. Here, we showed that currents mediated by the anoctamin 4 (Ano4) channel are only detected in GI neurons in the ventromedial hypothalamic nucleus (VMH) and are functionally required for their activation in response to low glucose. Genetic disruption of the Ano4 gene in VMH neurons reduced blood glucose and impaired counterregulatory responses during hypoglycemia in mice. Activation of VMHAno4 neurons increased food intake and blood glucose, while chronic inhibition of VMHAno4 neurons ameliorated hyperglycemia in a type 1 diabetic mouse model. Finally, we showed that VMHAno4 neurons represent a unique orexigenic VMH population and transmit a positive valence, while stimulation of neurons that do not express Ano4 in the VMH (VMHnon-Ano4) suppress feeding and transmit a negative valence. Together, our results indicate that the Ano4 channel and VMHAno4 neurons are potential therapeutic targets for human diseases with abnormal feeding behavior or glucose imbalance.

Cooperative synaptic and intrinsic plasticity in a disynaptic limbic circuit drive stress-induced anhedonia and passive coping in mice.

Stress promotes negative affective states, which include anhedonia and passive coping. While these features are in part mediated by neuroadaptations in brain reward circuitry, a comprehensive framework of how stress-induced negative affect may be encoded within key nodes of this circuit is lacking. Here, we show in a mouse model for stress-induced anhedonia and passive coping that these phenomena are associated with increased synaptic strength of ventral hippocampus (VH) excitatory synapses onto D1 medium spiny neurons (D1-MSNs) in the nucleus accumbens medial shell (NAcmSh), and with lateral hypothalamus (LH)-projecting D1-MSN hyperexcitability mediated by decreased inwardly rectifying potassium channel (IRK) function. Stress-induced negative affective states are prevented by depotentiation of VH to NAcmSh synapses, restoring Kir2.1 function in D1R-MSNs, or disrupting co-participation of these synaptic and intrinsic adaptations in D1-MSNs. In conclusion, our data provide strong evidence for a disynaptic pathway controlling maladaptive emotional behavior.

Paraventricular hypothalamus mediates diurnal rhythm of metabolism.

Defective rhythmic metabolism is associated with high-fat high-caloric diet (HFD) feeding, ageing and obesity; however, the neural basis underlying HFD effects on diurnal metabolism remains elusive. Here we show that deletion of BMAL1, a core clock gene, in paraventricular hypothalamic (PVH) neurons reduces diurnal rhythmicity in metabolism, causes obesity and diminishes PVH neuron activation in response to fast-refeeding. Animal models mimicking deficiency in PVH neuron responsiveness, achieved through clamping PVH neuron activity at high or low levels, both show obesity and reduced diurnal rhythmicity in metabolism. Interestingly, the PVH exhibits BMAL1-controlled rhythmic expression of GABA-A receptor γ2 subunit, and dampening rhythmicity of GABAergic input to the PVH reduces diurnal rhythmicity in metabolism and causes obesity. Finally, BMAL1 deletion blunts PVH neuron responses to external stressors, an effect mimicked by HFD feeding. Thus, BMAL1-driven PVH neuron responsiveness in dynamic activity changes involving rhythmic GABAergic neurotransmission mediates diurnal rhythmicity in metabolism and is implicated in diet-induced obesity.

Profound and redundant functions of arcuate neurons in obesity development.

The current obesity epidemic faces a lack of mechanistic insights. It is known that the acute activity changes of a growing number of brain neurons rapidly alter feeding behaviour; however, how these changes translate to obesity development and the fundamental mechanism underlying brain neurons in controlling body weight remain elusive. Here, we show that chronic activation of hypothalamic arcuate GABAergic (GABA+), agouti-related protein (AgRP) neurons or arcuate non-AgRP GABA+ neurons leads to obesity, which is similar to the obese phenotype observed in ob/ob mice. Conversely, chronic inhibition of arcuate GABA+, but not AgRP, neurons reduces ageing-related weight gain and corrects ob/ob obesity. These results demonstrate that the modulation of Arc GABA+ neuron activity is a fundamental mechanism of body-weight regulation, and that arcuate GABA+ neurons are the major mediator of leptin action, with a profound and redundant role in obesity development.

Disrupted hypothalamic CRH neuron responsiveness contributes to diet-induced obesity.

The current obesity epidemic mainly results from high-fat high-caloric diet (HFD) feeding and may also be contributed by chronic stress; however, the neural basis underlying stress-related diet-induced obesity remains unknown. Corticotropin-releasing hormone (CRH) neurons in the paraventricular hypothalamus (PVH), a known body weight-regulating region, represent one key group of stress-responsive neurons. Here, we found that HFD feeding blunted PVH CRH neuron response to nutritional challenges as well as stress stimuli and dexamethesone, which normally produce rapid activation and inhibition on these neurons, respectively. We generated mouse models with the activity of these neurons clamped at high or low levels, both of which showed HFD-mimicking, blunted PVH CRH neuron responsiveness. Strikingly, both models developed rapid HFD-induced obesity, associated with HFD-mimicking, reduced diurnal rhythmicity in feeding and energy expenditure. Thus, blunted responsiveness of PVH CRH neurons, but not their absolute activity levels, underlies HFD-induced obesity and may also contribute to stress-induced obesity.

Stxbp1/Munc18-1 haploinsufficiency impairs inhibition and mediates key neurological features of STXBP1 encephalopathy.

Mutations in genes encoding synaptic proteins cause many neurodevelopmental disorders, with the majority affecting postsynaptic apparatuses and much fewer in presynaptic proteins. Syntaxin-binding protein 1 (STXBP1, also known as MUNC18-1) is an essential component of the presynaptic neurotransmitter release machinery. De novo heterozygous pathogenic variants in STXBP1 are among the most frequent causes of neurodevelopmental disorders including intellectual disabilities and epilepsies. These disorders, collectively referred to as STXBP1 encephalopathy, encompass a broad spectrum of neurologic and psychiatric features, but the pathogenesis remains elusive. Here we modeled STXBP1 encephalopathy in mice and found that Stxbp1 haploinsufficiency caused cognitive, psychiatric, and motor dysfunctions, as well as cortical hyperexcitability and seizures. Furthermore, Stxbp1 haploinsufficiency reduced cortical inhibitory neurotransmission via distinct mechanisms from parvalbumin-expressing and somatostatin-expressing interneurons. These results demonstrate that Stxbp1 haploinsufficient mice recapitulate cardinal features of STXBP1 encephalopathy and indicate that GABAergic synaptic dysfunction is likely a crucial contributor to disease pathogenesis.

Targeting light-gated chloride channels to neuronal somatodendritic domain reduces their excitatory effect in the axon.

Light-gated chloride channels are emerging as promising optogenetic tools for inhibition of neural activity. However, their effects depend on the transmembrane chloride electrochemical gradient and may be complex due to the heterogeneity of this gradient in different developmental stages, neuronal types, and subcellular compartments. Here we characterized a light-gated chloride channel, GtACR2, in mouse cortical neurons. We found that GtACR2 activation inhibited the soma, but unexpectedly depolarized the presynaptic terminals resulting in neurotransmitter release. Other light-gated chloride channels had similar effects. Reducing the chloride concentrations in the axon and presynaptic terminals diminished the GtACR2-induced neurotransmitter release, indicating an excitatory effect of chloride channels in these compartments. A novel hybrid somatodendritic targeting motif reduced the GtACR2-induced neurotransmitter release while enhancing the somatic photocurrents. Our results highlight the necessity of precisely determining the effects of light-gated chloride channels under specific experimental conditions and provide a much-improved light-gated chloride channel for optogenetic inhibition.

Corrigendum: Quantitative real-time imaging of glutathione.

This corrects the article DOI: 10.1038/ncomms16087.

Quantitative real-time imaging of glutathione.

Glutathione plays many important roles in biological processes; however, the dynamic changes of glutathione concentrations in living cells remain largely unknown. Here, we report a reversible reaction-based fluorescent probe-designated as RealThiol (RT)-that can quantitatively monitor the real-time glutathione dynamics in living cells. Using RT, we observe enhanced antioxidant capability of activated neurons and dynamic glutathione changes during ferroptosis. RT is thus a versatile tool that can be used for both confocal microscopy and flow cytometry based high-throughput quantification of glutathione levels in single cells. We envision that this new glutathione probe will enable opportunities to study glutathione dynamics and transportation and expand our understanding of the physiological and pathological roles of glutathione in living cells.

Both pre- and post-synaptic alterations contribute to aberrant cholinergic transmission in superior cervical ganglia of APP(-/-) mice.

Though amyloid precursor protein (APP) can potentially be cleaved to generate the pathological amyloid ß peptide (Aß), APP itself plays an important role in regulating neuronal activity. APP deficiency causes functional impairment in cholinergic synaptic transmission and cognitive performance. However, the mechanisms underlying altered cholinergic synaptic transmission in APP knock-out mice (APP(-/-)) are poorly understood. In this study, we conducted in vivo extracellular recording to investigate cholinergic compound action potentials (CAPs) of the superior cervical ganglion (SCG) in APP(-/-) and littermate wild-type (WT) mice. Our results demonstrate that APP not only regulates presynaptic activity, but also affects postsynaptic function at cholinergic synapses in SCG. APP deficiency reduces the number of vesicles in presynaptic terminalsand attenuatesthe amplitude of CAPs, likely due to dysfunction of high-affinity choline transporters. Pharmacological and biochemical examination showed that postsynaptic responsesmediated by α4ß2 and α7 nicotinic acetylcholine receptors are reduced in the absence of APP. Our research provides evidences on how APP regulates cholinergic function and therefore may help to identify potential therapeutic targets to treat cholinergic dysfunction associated with Alzheimer's disease pathogenesis.

Effects of intrahippocampal GABAB receptor antagonist treatment on the behavioral long-term potentiation and Y-maze learning performance.

GABAB receptor is present at pre- and post-synaptic sites and participates in many brain functions including cognition, reward and anxiety. Although a lot of research has shown that activation or blockade of GABAB receptor may produce different even opposing effects on long-term potentiation (LTP) and cognitive function, there is little information available concerning the effect of GABAB receptor on behavioral LTP, a learning-induced LTP model. Herein, we firstly examined the effects of 2-OH saclofen, a GABAB receptor antagonist, on the induction of behavioral LTP and Y-maze learning performance. In addition, GABAB receptor has been reported to be present on cholinergic terminals and to regulate the ACh release. Therefore, we also investigated the effect of 2-OH saclofen on the impairments in behavioral LTP and cognitive function induced by scopolamine, an acetylcholine receptor antagonist. We found that intrahippocampal application of 2-OH saclofen could significantly enhance the population spike (PS) amplitude with a dose-response relationship, and 20 µM 2-OH saclofen evidently facilitated the formation of behavioral LTP in the perforant pathway to the dentate gyrus (PP-DG) and led to an obvious improvement in maze learning performance. Furthermore, intrahippocampal 20 µM 2-OH saclofen administration could markedly reverse the scopolamine-induced impairments in behavioral LTP and maze performance. Our data demonstrate that blockade of GABAB receptor displays a facilitatory role in the induction of behavioral LTP and maze learning task, and the antagonist of GABAB receptor seems to exert the potentially therapeutic value in the cognitive defect induced by cholinergic dysfunction.

Effects of cordycepin on Y-maze learning task in mice.

Cordycepin (3'-deoxyadenosine) is the major bioactive component of Cordyceps militaris that has been widely used in oriental countries as a Traditional Chinese Medicine and healthy food for preventing early aging, improving physical performance and increasing lifespan. Cordyceps militaris extracts other than cordycepin have been reported to improve cognitive function. Although cordycepin is one of the most utilized Cordyceps militaris components, it remains unknown whether cordycepin could improve learning and memory. Here we investigated effects of cordycepin on learning and memory in healthy and ischemic mice using Y-maze test. We found that oral cordycepin administration at dose of 10 mg/kg significantly improved Y-maze learning performance both in healthy and ischemic mice. However, cordycepin at dose of 5 mg/kg enhanced Y-maze learning only in ischemic mice but not healthy mice. In this study, simultaneously, we found that orally administrated cordycepin significantly decreased the neuronal loss induced by ischemia in hippocampal CA1 and CA3 regions. Collectively, our results can provide valuable evidence that cordycepin may act as a nootropic product or potential clinical application in improving cognitive function of patients with ischemic stroke in the future.

Tenuigenin ameliorates learning and memory impairments induced by ovariectomy.

Estrogen deficiency is associated with cognitive impairment. Hormone replacement therapy (HRT) has proven to be effective in preventing and reversing the memory and learning deficiencies. However, conventional estrogenic treatment could increase the risks of breast cancer and venous thromboembolism. Tenuigenin (TEN) is putatively believed as the active component extracted from a Chinese herb Polygala tenuifolia root. Although TEN has been shown to enhance learning and memory in healthy mice, it remains unknown whether or not TEN could ameliorate learning and memory impairments. In the present study, mice were divided into four groups: sham-operated (sham), ovariectomized (OVX), OVX+estradiol benzoate (EB) and OVX+TEN groups. Step-through passive avoidance and Y-maze tests were used to assess learning and memory abilities, and the number of nitric oxide synthase (NOS) positive neurons and the synaptic measurement of hippocampal CA1 area were examined. The results showed that TEN was given orally to OVX mice, leading to the improvement of learning and memory in step-through passive avoidance and Y-maze tests. TEN could reduce the loss of NOS positive neurons and prevent the synaptic morphological changes induced by ovariectomy. Our results suggest that TEN may exert a potential therapeutic value for menopause cognitive dysfunction.

Cordycepin suppresses excitatory synaptic transmission in rat hippocampal slices via a presynaptic mechanism.

AIMS: Cordycepin plays an important role in modulating the function of central nervous system (CNS). However, the modulating mechanism is poorly understood. Excitatory synaptic transmission, the essential process in brain physiology and pathology, is critical in the signal integration activities of the CNS. To further understand the effects of cordycepin on CNS, we investigated the effects of cordycepin on excitatory synaptic transmission in the CA1 region of rat hippocampal slices. METHODS: The effects of cordycepin on excitatory synaptic transmission were investigated by using in vitro field potential electrophysiology and whole-cell patch clamp techniques. RESULTS: Cordycepin significantly decreased the amplitudes of field excitatory postsynaptic potentials (fEPSPs) elicited in the CA1 by stimulation of the Schaffer-commissural fibers. And the reduction in fEPSPs amplitude was associated with an increase in the paired-pulse facilitation. Cordycepin also suppressed α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and N-methyl-d-aspartic acid (NMDA) receptor-mediated responses but did not directly affect AMPA receptors and NMDA receptors. Furthermore, quantal analysis revealed that cordycepin decreased the frequency but not amplitude of miniature spontaneous excitatory postsynaptic currents. CONCLUSIONS: These results demonstrate that cordycepin suppresses excitatory synaptic transmission by decreasing the excitatory neurotransmitter release presynaptically, which provides an evidence for the novel potential mechanism of cordycepin in modulating the function of CNS.

Effects of intrahippocampal L-NAME treatment on the behavioral long-term potentiation in dentate gyrus.

Using a combination of electrophysiological recordings, behavioral tests and local pharmacological administration in hippocampus, we investigated in the present study the effects of nitric oxide (NO) synthase inhibitor N-nitro-l-arginine methyl ester (l-NAME) on the behavioral long-term potentiation (LTP) and maze learning performance in freely moving rats. The results showed as follows: (1) intrahippocampal l-NAME administration led to a defect in maze learning performance of the animals; (2) l-NAME treatment also substantially impaired the induction of the behavioral LTP in perforant pathway to dentate gyrus (PP-DG) pathway induced by maze learning task, while it had no significant effects on basic synaptic transmission in PP-DG pathway; Collectively, these results indicate that NO synthesis may be critical for the behavioral LTP in PP-DG pathway and maze learning performance.