Hippocampal excitation-inhibition balance underlies the 5-HT2C receptor in modulating depressive behaviours.

The implication of 5-hydroxytryptamine 2C receptor (5-HT2CR) in depression is a topic of debate, and the underlying mechanisms remain largely unclear. We now elucidate hippocampal excitation-inhibition (E/I) balance underlies the regulatory effects of 5-HT2CR in depression. Molecular biological analyses showed that chronic mild stress (CMS) reduced the expression of 5-HT2CR in hippocampus. We revealed that inhibition of 5-HT2CR induced depressive-like behaviors, reduced GABA release and shifted the E/I balance towards excitation in CA3 pyramidal neurons by using behavioral analyses, microdialysis coupled with mass spectrum, and electrophysiological recording. Moreover, 5-HT2CR modulated neuronal nitric oxide synthase (nNOS)-carboxy-terminal PDZ ligand of nNOS (CAPON) interaction through influencing intracellular Ca2+ release, as determined by fiber photometry and coimmunoprecipitation. Notably, disruption of nNOS-CAPON by specific small molecule compound ZLc-002 or AAV-CMV-CAPON-125C-GFP, abolished 5-HT2CR inhibition-induced depressive-like behaviors, as well as the impairment in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly-mediated GABA vesicle release and a consequent E/I imbalance. Importantly, optogenetic inhibition of CA3 GABAergic neurons prevented the effects of AAV-CMV-CAPON-125C-GFP on depressive behaviors in the presence of 5-HT2CR antagonist. Conclusively, our findings disclose the regulatory role of 5-HT2CR in depressive-like behaviors and highlight the hippocampal nNOS-CAPON coupling-triggered E/I imbalance as a pivotal cellular event underpinning the behavioral consequences of 5-HT2CR inhibition.

A kidney-brain neural circuit drives progressive kidney damage and heart failure.

Chronic kidney disease (CKD) and heart failure (HF) are highly prevalent, aggravate each other, and account for substantial mortality. However, the mechanisms underlying cardiorenal interaction and the role of kidney afferent nerves and their precise central pathway remain limited. Here, we combined virus tracing techniques with optogenetic techniques to map a polysynaptic central pathway linking kidney afferent nerves to subfornical organ (SFO) and thereby to paraventricular nucleus (PVN) and rostral ventrolateral medulla that modulates sympathetic outflow. This kidney-brain neural circuit was overactivated in mouse models of CKD or HF and subsequently enhanced the sympathetic discharge to both the kidney and the heart in each model. Interruption of the pathway by kidney deafferentation, selective deletion of angiotensin II type 1a receptor (AT1a) in SFO, or optogenetic silence of the kidney-SFO or SFO-PVN projection decreased the sympathetic discharge and lessened structural damage and dysfunction of both kidney and heart in models of CKD and HF. Thus, kidney afferent nerves activate a kidney-brain neural circuit in CKD and HF that drives the sympathetic nervous system to accelerate disease progression in both organs. These results demonstrate the crucial role of kidney afferent nerves and their central connections in engaging cardiorenal interactions under both physiological and disease conditions. This suggests novel therapies for CKD or HF targeting this kidney-brain neural circuit.

Electroacupuncture alleviated depression-like behaviors in ventromedial prefrontal cortex of chronic unpredictable mild stress-induced rats: Increasing synaptic transmission and phosphorylating dopamine transporter.

AIMS: Electroacupuncture (EA) shows advantages in both clinical practice and depression animal models. Dopaminergic-related dysfunction in the prefrontal cortex (PFC) may be a hidden antidepressant mechanism of EA, where dopamine transporter (DAT) plays an essential role. This study aimed to investigate the synaptic transmission and DAT-related changes of EA in depression. METHODS: Male Sprague-Dawley rats were subjected to 3-week chronic unpredictable mild stress (CUMS). The successfully modeled rats were then randomly and equally assigned to CUMS, selective serotonin reuptake inhibitor (SSRI), and EA or SSRI + EA groups, followed by a 2-week treatment respectively. After monitoring body weight and behavioral tests of all rats, the ventromedial PFC (vmPFC) tissue was collected for electrophysiology and the expression detection of DAT, phosphorylated DAT (p-DAT), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and trace amine-associated receptor 1 (TAAR1). RESULTS: Depressive-like behaviors induced by CUMS were alleviated by EA, SSRI, and SSRI + EA treatments through behavioral tests. Compared with CUMS group, EA improved synaptic transmission in vmPFC by upregulating spontaneous excitatory postsynaptic currents amplitude. Molecularly, EA reversed the increased total DAT and p-DAT expression as well as the decreased ratio of p-DAT/total DAT along with the activation of TAAR1, cAMP, and PKA in vmPFC. CONCLUSION: We speculated that the antidepressant effect of EA was associated with enhanced synaptic transmission in vmPFC, and the upregulated phosphorylation of DAT relevant to TAAR1, cAMP, and PKA may be the potential mechanism.

Functional investigation of SLC1A2 variants associated with epilepsy.

Epilepsy is a common neurological disorder and glutamate excitotoxicity plays a key role in epileptic pathogenesis. Astrocytic glutamate transporter GLT-1 is responsible for preventing excitotoxicity via clearing extracellular accumulated glutamate. Previously, three variants (G82R, L85P, and P289R) in SLC1A2 (encoding GLT-1) have been clinically reported to be associated with epilepsy. However, the functional validation and underlying mechanism of these GLT-1 variants in epilepsy remain undetermined. In this study, we reported that these disease-linked mutants significantly decrease glutamate uptake, cell membrane expression of the glutamate transporter, and glutamate-elicited current. Additionally, we found that these variants may disturbed stromal-interacting molecule 1 (STIM1)/Orai1-mediated store-operated Ca2+ entry (SOCE) machinery in the endoplasmic reticulum (ER), in which GLT-1 may be a new partner of SOCE. Furthermore, knock-in mice with disease-associated variants showed a hyperactive phenotype accompanied by reduced glutamate transporter expression. Therefore, GLT-1 is a promising and reliable therapeutic target for epilepsy interventions.

TNF-α-mediated reduction in inhibitory neurotransmission precedes sporadic Alzheimer's disease pathology in young Trem2R47H rats.

Alzheimer's disease (AD) is a neurodegenerative dementia associated with deposition of amyloid plaques and neurofibrillary tangles, formed by amyloid ß (Aß) peptides and phosphor-tau, respectively, in the central nervous system. Approximately 2% of AD cases are due to familial AD (FAD); ∼98% of cases are sporadic AD (SAD). Animal models with FAD are commonly used to study SAD pathogenesis. Because mechanisms leading to FAD and SAD may be distinct, to study SAD pathogenesis, we generated Trem2R47H knock-in rats, which carry the SAD risk factor p.R47H variant of the microglia gene triggering receptor expressed on myeloid cells 2 (TREM2). Trem2R47H rats produce human-Aß from a humanized-App rat allele because human-Aß is more toxic than rodent-Aß and the pathogenic role of the p.R47H TREM2 variant has been linked to human-Aß-clearing deficits. Using periadolescent Trem2R47H rats, we previously demonstrated that supraphysiological tumor necrosis factor-α (TNF-α) boosts glutamatergic transmission, which is excitatory, and suppresses long-term potentiation, a surrogate of learning and memory. Here, we tested the effect of the p.R47H variant on the inhibitory neurotransmitter γ-aminobutyric acid. We report that GABAergic transmission is decreased in Trem2R47H/R47H rats. This decrease is due to acute and reversible action of TNF-α and is not associated with increased human-Aß levels and AD pathology. Thus, the p.R47H variant changes the excitatory/inhibitory balance, favoring excitation. This imbalance could potentiate glutamate excitotoxicity and contribute to neuronal dysfunction, enhanced neuronal death, and neurodegeneration. Future studies will determine whether this imbalance represents an early, Aß-independent pathway leading to dementia and may reveal the AD-modifying therapeutic potential of TNF-α inhibition in the central nervous system.

Resveratrol mediates mechanical allodynia through modulating inflammatory response via the TREM2-autophagy axis in SNI rat model.

BACKGROUND: Neuropathic pain (NeuP) is a chronic and challenging clinical problem, with little effective treatment. Resveratrol has shown neuroprotection by inhibiting inflammatory response in NeuP. Recently, the triggering receptor expressed on myeloid cells 2 (TREM2) expressed by microglia was identified as a critical factor of inflammation in nervous system diseases. In this study, we explored whether resveratrol could ameliorate neuroinflammation and produce anti-mechanical allodynia effects via regulating TREM2 in spared nerve injury rats, as well as investigated the underlying mechanisms. METHODS: A spared nerve injury (SNI) rat model was performed to investigate whether resveratrol could exert anti-mechanical allodynia effects via inhibiting neuroinflammation. To evaluate the role of TREM2 in anti-neuroinflammatory function of resveratrol, lentivirus coding TREM2 was intrathecally injected into SNI rats to activate TREM2, and the pain behavior was detected by the von Frey test. Furthermore, 3-methyladenine (3-MA, an autophagy inhibitor) was applied to study the molecular mechanisms of resveratrol-mediated anti-neuroinflammation using Western blot, qPCR, and immunofluorescence. RESULTS: The TREM2 expression and number of the microglial cells were significantly increased in the ipsilateral spinal dorsal horn after SNI. We found that intrathecal administration of resveratrol (300ug/day) alleviated mechanical allodynia; obviously enhanced autophagy; and markedly reduced the levels of interleukin-1ß, interleukin-6, and tumor necrosis factor-α in the ipsilateral spinal dorsal horn after SNI. Moreover, the number of Iba-1+ microglial cells and TREM2 expression were downregulated after resveratrol treatment. Intrathecal administration of lentivirus coding TREM2 and/or 3-MA in those rats induced deficiencies in resveratrol-mediated anti-inflammation, leading to mechanical allodynia that could be rescued via administration of Res. Furthermore, 3-MA treatment contributed to TREM2-mediated mechanical allodynia. CONCLUSIONS: Taken together, these data reveal that resveratrol relieves neuropathic pain through suppressing microglia-mediated neuroinflammation via regulating the TREM2-autophagy axis in SNI rats.

Microglia TREM2R47H Alzheimer-linked variant enhances excitatory transmission and reduces LTP via increased TNF-α levels.

To study the mechanisms by which the p.R47H variant of the microglia gene and Alzheimer's disease (AD) risk factor TREM2 increases dementia risk, we created Trem2R47H KI rats. Trem2R47H rats were engineered to produce human Aß to define human-Aß-dependent and -independent pathogenic mechanisms triggered by this variant. Interestingly, pre- and peri-adolescent Trem2R47H rats present increased brain concentrations of TNF-α, augmented glutamatergic transmission, suppression of Long-term-Potentiation (LTP), an electrophysiological surrogate of learning and memory, but normal Aß levels. Acute reduction of TNF-α activity with a neutralizing anti-TNF-α antibody occludes the boost in amplitude of glutamatergic transmission and LTP suppression observed in young Trem2R47H/R47H rats. Thus, the microglia-specific pathogenic Trem2 variant boosts glutamatergic neuronal transmission and suppresses LTP by increasing brain TNF-α concentrations, directly linking microglia to neuronal dysfunction. Future studies will determine whether this phenomenon represents an early, Aß-independent pathway that facilitates dementia pathogenesis in humans.

TBR2 coordinates neurogenesis expansion and precise microcircuit organization via Protocadherin 19 in the mammalian cortex.

Cerebral cortex expansion is a hallmark of mammalian brain evolution; yet, how increased neurogenesis is coordinated with structural and functional development remains largely unclear. The T-box protein TBR2/EOMES is preferentially enriched in intermediate progenitors and supports cortical neurogenesis expansion. Here we show that TBR2 regulates fine-scale spatial and circuit organization of excitatory neurons in addition to enhancing neurogenesis in the mouse cortex. TBR2 removal leads to a significant reduction in neuronal, but not glial, output of individual radial glial progenitors as revealed by mosaic analysis with double markers. Moreover, in the absence of TBR2, clonally related excitatory neurons become more laterally dispersed and their preferential synapse development is impaired. Interestingly, TBR2 directly regulates the expression of Protocadherin 19 (PCDH19), and simultaneous PCDH19 expression rescues neurogenesis and neuronal organization defects caused by TBR2 removal. Together, these results suggest that TBR2 coordinates neurogenesis expansion and precise microcircuit assembly via PCDH19 in the mammalian cortex.

Precise Long-Range Microcircuit-to-Microcircuit Communication Connects the Frontal and Sensory Cortices in the Mammalian Brain.

The frontal area of the cerebral cortex provides long-range inputs to sensory areas to modulate neuronal activity and information processing. These long-range circuits are crucial for accurate sensory perception and complex behavioral control; however, little is known about their precise circuit organization. Here we specifically identified the presynaptic input neurons to individual excitatory neuron clones as a unit that constitutes functional microcircuits in the mouse sensory cortex. Interestingly, the long-range input neurons in the frontal but not contralateral sensory area are spatially organized into discrete vertical clusters and preferentially form synapses with each other over nearby non-input neurons. Moreover, the assembly of distant presynaptic microcircuits in the frontal area depends on the selective synaptic communication of excitatory neuron clones in the sensory area that provide inputs to the frontal area. These findings suggest that highly precise long-range reciprocal microcircuit-to-microcircuit communication mediates frontal-sensory area interactions in the mammalian cortex.

Amyloid ß causes excitation/inhibition imbalance through dopamine receptor 1-dependent disruption of fast-spiking GABAergic input in anterior cingulate cortex.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly. At the early stages of AD development, the soluble ß-amyloid (Aß) induces synaptic dysfunction, perturbs the excitation/inhibition balance of neural circuitries, and in turn alters the normal neural network activity leading to cognitive decline, but the underlying mechanisms are not well established. Here by using whole-cell recordings in acute mouse brain slices, we found that 50 nM Aß induces hyperexcitability of excitatory pyramidal cells in the cingulate cortex, one of the most vulnerable areas in AD, via depressing inhibitory synaptic transmission. Furthermore, by simultaneously recording multiple cells, we discovered that the inhibitory innervation of pyramidal cells from fast-spiking (FS) interneurons instead of non-FS interneurons is dramatically disrupted by Aß, and perturbation of the presynaptic inhibitory neurotransmitter gamma-aminobutyric acid (GABA) release underlies this inhibitory input disruption. Finally, we identified the increased dopamine action on dopamine D1 receptor of FS interneurons as a key pathological factor that contributes to GABAergic input perturbation and excitation/inhibition imbalance caused by Aß. Thus, we conclude that the dopamine receptor 1-dependent disruption of FS GABAergic inhibitory input plays a critical role in Aß-induced excitation/inhibition imbalance in anterior cingulate cortex.

Distinct Roles of PKCι/λ and PKMζ in the Initiation and Maintenance of Hippocampal Long-Term Potentiation and Memory.

PKMζ has been proposed to be essential for maintenance of long-term potentiation (LTP) and long-term memory (LTM). However, recent data from PKMζ-knockout mice has called this role into question. Instead, the other atypical isoform, protein kinase C iota/lambda (PKCι/λ), has emerged as a potential alternative player. Therefore, the nature of the "memory molecule" maintaining learned information remains uncertain. Here, we report knockdown (KD) of PKCι/λ and PKMζ in the dorsal hippocampus and find deficits in early expression and late maintenance, respectively, during both LTP and hippocampus-dependent LTM. Sequential increases in the active form of PKCι/λ and PKMζ are detected during LTP or fear conditioning. Importantly, PKMζ, but not PKCι/λ, KD disrupts previously established LTM. Thus, PKCι/λ and PKMζ have distinct functions in LTP and memory, with PKMζ playing a specific role in memory maintenance. This relaying pattern may represent a precise molecular mechanism by which atypical PKCs regulate the different stages of memory.

Vascular Influence on Ventral Telencephalic Progenitors and Neocortical Interneuron Production.

The neocortex contains glutamatergic excitatory neurons and γ-aminobutyric acid (GABA)ergic inhibitory interneurons. Extensive studies have revealed substantial insights into excitatory neuron production. However, our knowledge of the generation of GABAergic interneurons remains limited. Here we show that periventricular blood vessels selectively influence neocortical interneuron progenitor behavior and neurogenesis. Distinct from those in the dorsal telencephalon, radial glial progenitors (RGPs) in the ventral telencephalon responsible for producing neocortical interneurons progressively grow radial glial fibers anchored to periventricular vessels. This progenitor-vessel association is robust and actively maintained as RGPs undergo interkinetic nuclear migration and divide at the ventricular zone surface. Disruption of this association by selective removal of INTEGRIN ß1 in RGPs leads to a decrease in progenitor division, a loss of PARVALBUMIN and SOMATOSTATIN-expressing interneurons, and defective synaptic inhibition in the neocortex. These results highlight a prominent interaction between RGPs and periventricular vessels important for proper production and function of neocortical interneurons.

Aß induces acute depression of excitatory glutamatergic synaptic transmission through distinct phosphatase-dependent mechanisms in rat CA1 pyramidal neurons.

Beta-amyloid peptide (Aß) has a causal role in the pathophysiology of Alzheimer's disease (AD). Recent studies indicate that Aß can disrupt excitatory glutamatergic synaptic function at synaptic level. However, the underlying mechanisms remain obscure. In this study, we recorded evoked and spontaneous EPSCs in hippocampal CA1 pyramidal neurons via whole-cell voltage-clamping methods and found that 1 µM Aß can induce acute depression of basal glutamatergic synaptic transmission through both presynaptic and postsynaptic dysfunction. Moreover, we also found that Aß-induced both presynaptic and postsynaptic dysfunction can be reversed by the inhibitor of protein phosphatase 2B (PP2B), FK506, whereas only postsynaptic disruption can be ameliorated by the inhibitor of PP1/PP2A, Okadaic acid (OA). These results indicate that PP1/PP2A and PP2B have overlapping but not identical functions in Aß-induced acute depression of excitatory glutamatergic synaptic transmission of hippocampal CA1 pyramidal neurons.

PKCλ is critical in AMPA receptor phosphorylation and synaptic incorporation during LTP.

Direct phosphorylation of GluA1 by PKC controls α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor (AMPAR) incorporation into active synapses during long-term potentiation (LTP). Numerous signalling molecules that involved in AMPAR incorporation have been identified, but the specific PKC isoform(s) participating in GluA1 phosphorylation and the molecule triggering PKC activation remain largely unknown. Here, we report that the atypical isoform of PKC, PKCλ, is a critical molecule that acts downstream of phosphatidylinositol 3-kinase (PI3K) and is essential for LTP expression. PKCλ activation is required for both GluA1 phosphorylation and increased surface expression of AMPARs during LTP. Moreover, p62 interacts with both PKCλ and GluA1 during LTP and may serve as a scaffolding protein to place PKCλ in close proximity to facilitate GluA1 phosphorylation by PKCλ. Thus, we conclude that PKCλ is the critical signalling molecule responsible for GluA1-containing AMPAR phosphorylation and synaptic incorporation at activated synapses during LTP expression.

Glycine exerts dual roles in ischemic injury through distinct mechanisms.

BACKGROUND AND PURPOSE: We characterized the differential effects of glycine at different levels in the induction of postischemic long-term potentiation, as well as in the neuronal damage induced by focal ischemia. METHODS: Whole-cell patch clamp recordings were obtained from rat hippocampal slice preparations. In vitro ischemia and postischemic long-term potentiation were induced by oxygen and glucose deprivation. In vivo ischemia was induced by transient middle cerebral artery occlusion. RESULTS: In both in vitro and in vivo ischemia models, glycine at low level exerts deleterious effects in postischemic long-term potentiation and ischemic neuronal injury by modulation of the N-methyl-d-aspartate receptor coagonist site; whereas glycine at high level exerts neuroprotective effects by activation of glycine receptor and subsequent differential regulation of N-methyl-d-aspartate receptor subunit components. CONCLUSIONS: Our results provide a molecular basis for the dual roles of glycine in ischemic injury through distinct mechanisms, and they suggest that glycine receptors could be a potential target for clinical treatment of stroke.

Regulation of spike timing-dependent plasticity of olfactory inputs in mitral cells in the rat olfactory bulb.

The recent history of activity input onto granule cells (GCs) in the main olfactory bulb can affect the strength of lateral inhibition, which functions to generate contrast enhancement. However, at the plasticity level, it is unknown whether and how the prior modification of lateral inhibition modulates the subsequent induction of long-lasting changes of the excitatory olfactory nerve (ON) inputs to mitral cells (MCs). Here we found that the repetitive stimulation of two distinct excitatory inputs to the GCs induced a persistent modification of lateral inhibition in MCs in opposing directions. This bidirectional modification of inhibitory inputs differentially regulated the subsequent synaptic plasticity of the excitatory ON inputs to the MCs, which was induced by the repetitive pairing of excitatory postsynaptic potentials (EPSPs) with postsynaptic bursts. The regulation of spike timing-dependent plasticity (STDP) was achieved by the regulation of the inter-spike-interval (ISI) of the postsynaptic bursts. This novel form of inhibition-dependent regulation of plasticity may contribute to the encoding or processing of olfactory information in the olfactory bulb.

Protein kinase C promotes N-methyl-D-aspartate (NMDA) receptor trafficking by indirectly triggering calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation.

Regulation of neuronal NMDA receptor (NMDAR) is critical in synaptic transmission and plasticity. Protein kinase C (PKC) promotes NMDAR trafficking to the cell surface via interaction with NMDAR-associated proteins (NAPs). Little is known, however, about the NAPs that are critical to PKC-induced NMDAR trafficking. Here, we showed that calcium/calmodulin-dependent protein kinase II (CaMKII) could be a NAP that mediates the potentiation of NMDAR trafficking by PKC. PKC activation promoted the level of autophosphorylated CaMKII and increased association with NMDARs, accompanied by functional NMDAR insertion, at postsynaptic sites. This potentiation, along with PKC-induced long term potentiation of the AMPA receptor-mediated response, was abolished by CaMKII antagonist or by disturbing the interaction between CaMKII and NR2A or NR2B. Further mutual occlusion experiments demonstrated that PKC and CaMKII share a common signaling pathway in the potentiation of NMDAR trafficking and long-term potentiation (LTP) induction. Our results revealed that PKC promotes NMDA receptor trafficking and induces synaptic plasticity through indirectly triggering CaMKII autophosphorylation and subsequent increased association with NMDARs.

Role of glycine receptors in glycine-induced LTD in hippocampal CA1 pyramidal neurons.

Glycine in the hippocampus can exert its effect on both synaptic NMDA receptors (NMDARs) and extrasynaptic functional glycine receptors (GlyRs) via distinct binding sites. Previous studies have reported that glycine induces long-term potentiation (LTP) through the activation of synaptic NMDARs. However, little is known about the potential role of the activated GlyRs that are largely located in extrasynaptic regions. We report here that relatively high levels of glycine achieved either by exogenous glycine application or by the elevation of endogenous glycine accumulation with an antagonist of the glycine transporter induced long-term depression (LTD) of excitatory postsynaptic currents (EPSCs) in hippocampal CA1 pyramidal neurons. The co-application of glycine with the selective GlyR antagonist strychnine changed glycine-induced LTD (Gly-LTD) to LTP. Blocking the postsynaptic GlyR-gated net chloride flux by manipulating intracellular chloride concentrations failed to elicit any changes in EPSCs. These results suggest that GlyRs are involved in Gly-LTD. Furthermore, this new form of chemical LTD was accompanied by the internalization of postsynaptic AMPA receptors and required the activation of NMDARs. Therefore, our present findings reveal an important function of GlyR activation and modulation in gating the direction of synaptic plasticity.