Probes for VGLUT2

IRSp53 (or BAIAP2) is an abundant excitatory postsynaptic scaffolding/adaptor protein that is involved in actin regulation and has been implicated in autism spectrum disorders, schizophrenia, and attention-deficit/hyperactivity disorder. IRSp53 deletion in mice leads to enhanced NMDA receptor (NMDAR) function and social deficits that are responsive to NMDAR inhibition. However, it remains unclear whether IRSp53 re-expression in the adult IRSp53-mutant mouse brain after the completion of brain development could reverse these synaptic and behavioral dysfunctions. Here we employed a brain-blood barrier (BBB)-penetrant adeno-associated virus (AAV) known as PHP.eB to drive adult IRSp53 re-expression in IRSp53-mutant mice. The adult IRSp53 re-expression normalized social deficits without affecting hyperactivity or anxiety-like behavior. In addition, adult IRSp53 re-expression normalized NMDAR-mediated excitatory synaptic transmission in the medial prefrontal cortex. Our results suggest that adult IRSp53 re-expression can normalize synaptic and behavioral deficits in IRSp53-mutant mice and that BBB-penetrant adult gene re-expression has therapeutic potential.

Although bradykinesia, tremor and rigidity are the hallmark motor defects in patients with Parkinson's disease (PD), patients also experience motor learning impairments and non-motor symptoms such as depression1. The neural circuit basis for these different symptoms of PD are not well understood. Although current treatments are effective for locomotion deficits in PD2,3, therapeutic strategies targeting motor learning deficits and non-motor symptoms are lacking4-6. Here we found that distinct parafascicular (PF) thalamic subpopulations project to caudate putamen (CPu), subthalamic nucleus (STN) and nucleus accumbens (NAc). Whereas PF→CPu and PF→STN circuits are critical for locomotion and motor learning, respectively, inhibition of the PF→NAc circuit induced a depression-like state. Whereas chemogenetically manipulating CPu-projecting PF neurons led to a long-term restoration of locomotion, optogenetic long-term potentiation (LTP) at PF→STN synapses restored motor learning behaviour in an acute mouse model of PD. Furthermore, activation of NAc-projecting PF neurons rescued depression-like phenotypes. Further, we identified nicotinic acetylcholine receptors capable of modulating PF circuits to rescue different PD phenotypes. Thus, targeting PF thalamic circuits may be an effective strategy for treating motor and non-motor deficits in PD.

Innate vocal sounds such as laughing, screaming or crying convey one's feelings to others. In many species, including humans, scaling the amplitude and duration of vocalizations is essential for effective social communication1-3. In mice, female scent triggers male mice to emit innate courtship ultrasonic vocalizations (USVs)4,5. However, whether mice flexibly scale their vocalizations and how neural circuits are structured to generate flexibility remain largely unknown. Here we identify mouse neurons from the lateral preoptic area (LPOA) that express oestrogen receptor 1 (LPOAESR1 neurons) and, when activated, elicit the complete repertoire of USV syllables emitted during natural courtship. Neural anatomy and functional data reveal a two-step, di-synaptic circuit motif in which primary long-range inhibitory LPOAESR1 neurons relieve a clamp of local periaqueductal grey (PAG) inhibition, enabling excitatory PAG USV-gating neurons to trigger vocalizations. We find that social context shapes a wide range of USV amplitudes and bout durations. This variability is absent when PAG neurons are stimulated directly; PAG-evoked vocalizations are time-locked to neural activity and stereotypically loud. By contrast, increasing the activity of LPOAESR1 neurons scales the amplitude of vocalizations, and delaying the recovery of the inhibition clamp prolongs USV bouts. Thus, the LPOA disinhibition motif contributes to flexible loudness and the duration and persistence of bouts, which are key aspects of effective vocal social communication.

Angiotensin-II acts at its type-1 receptor (AT1R) in the brain to regulate body fluid homeostasis, sympathetic outflow and blood pressure. However, the role of the angiotensin type-2 receptor (AT2R) in the neural control of these processes has received far less attention, largely because of limited ability to effectively localize these receptors at a cellular level in the brain. The present studies combine the use of a bacterial artificial chromosome transgenic AT2R-enhanced green fluorescent protein (eGFP) reporter mouse with recent advances in in situ hybridization (ISH) to circumvent this obstacle. Dual immunohistochemistry (IHC)/ISH studies conducted in AT2R-eGFP reporter mice found that eGFP and AT2R mRNA were highly co-localized within the brain. Qualitative analysis of eGFP immunoreactivity in the brain then revealed localization to neurons within nuclei that regulate blood pressure, metabolism, and fluid balance (e.g., NTS and median preoptic nucleus [MnPO]), as well as limbic and cortical areas known to impact stress responding and mood. Subsequently, dual IHC/ISH studies uncovered the phenotype of specific populations of AT2R-eGFP cells. For example, within the NTS, AT2R-eGFP neurons primarily express glutamic acid decarboxylase-1 (80.3 ± 2.8 %), while a smaller subset express vesicular glutamate transporter-2 (18.2 ± 2.9 %) or AT1R (8.7 ± 1.0 %). No co-localization was observed with tyrosine hydroxylase in the NTS. Although AT2R-eGFP neurons were not observed within the paraventricular nucleus (PVN) of the hypothalamus, eGFP immunoreactivity is localized to efferents terminating in the PVN and within GABAergic neurons surrounding this nucleus. These studies demonstrate that central AT2R are positioned to regulate blood pressure, metabolism, and stress responses.

The nucleus tractus solitarii (NTS) is the primary central station that integrates visceral afferent information and regulates respiratory, gastrointestinal, cardiovascular, and other physiological functions. Leptin receptor b (LepRb)-expressing neurons of the NTS (NTSLepRb neurons) are implicated in central respiration regulation, respiratory facilitation, and respiratory drive enhancement. Furthermore, LepRb dysfunction is involved in obesity, insulin resistance, and sleep-disordered breathing. However, the monosynaptic inputs and outputs of NTSLepRb neurons in whole-brain mapping remain to be elucidated. Therefore, the exploration of its whole-brain connection system may provide strong support for comprehensively understanding the physiological and pathological functions of NTSLepRb neurons. In the present study, we used a cell type-specific, modified rabies virus and adeno-associated virus with the Cre-loxp system to map monosynaptic inputs and outputs of NTSLepRb neurons in LepRb-Cre mice. The results showed that NTSLepRb neurons received inputs from 48 nuclei in the whole brain from five brain regions, including especially the medulla. We found that NTSLepRb neurons received inputs from nuclei associated with respiration, such as the pre-Bötzinger complex, ambiguus nucleus, and parabrachial nucleus. Interestingly, some brain areas related to cardiovascular regulation-i.e., the ventrolateral periaqueductal gray and locus coeruleus-also sent a small number of inputs to NTSLepRb neurons. In addition, anterograde tracing results demonstrated that NTSLepRb neurons sent efferent projections to 15 nuclei, including the dorsomedial hypothalamic nucleus and arcuate hypothalamic nucleus, which are involved in regulation of energy metabolism and feeding behaviors. Quantitative statistical analysis revealed that the inputs of the whole brain to NTSLepRb neurons were significantly greater than the outputs. Our study comprehensively revealed neuronal connections of NTSLepRb neurons in the whole brain and provided a neuroanatomical basis for further research on physiological and pathological functions of NTSLepRb neurons.

Sensorimotor gating is a fundamental pre-attentive process that is defined as the inhibition of a motor response by a sensory event. Sensorimotor gating, commonly measured using the prepulse inhibition (PPI) of the auditory startle reflex task, is impaired in patients suffering from various neurological and psychiatric disorders. PPI deficits are a hallmark of schizophrenia, and they are often associated with attention and other cognitive impairments. Although the reversal of PPI deficits in animal models is widely used in pre-clinical research for antipsychotic drug screening, the neurotransmitter systems and synaptic mechanisms underlying PPI are still not resolved, even under physiological conditions. Recent evidence ruled out the longstanding hypothesis that PPI is mediated by midbrain cholinergic inputs to the caudal pontine reticular nucleus (PnC). Instead, glutamatergic, glycinergic, and GABAergic inhibitory mechanisms are now suggested to be crucial for PPI, at the PnC level. Since amygdalar dysfunctions alter PPI and are common to pathologies displaying sensorimotor gating deficits, the present study was designed to test that direct projections to the PnC originating from the amygdala contribute to PPI.Using wild type and transgenic mice expressing eGFP under the control of the glycine transporter type 2 promoter (GlyT2-eGFP mice), we first employed tract-tracing, morphological reconstructions, and immunohistochemical analyses to demonstrate that the central nucleus of the amygdala (CeA) sends glutamatergic inputs lateroventrally to PnC neurons, including GlyT2+ cells. Then, we showed the contribution of the CeA-PnC excitatory synapses to PPI in vivo by demonstrating that optogenetic inhibition of this connection decreases PPI, and optogenetic activation induces partial PPI. Finally, in GlyT2-Cre mice, whole-cell recordings of GlyT2+ PnC neurons in vitro paired with optogenetic stimulation of CeA fibers, as well as photo-inhibition of GlyT2+ PnC neurons in vivo, allowed us to implicate GlyT2+ neurons in the PPI pathway.Our results uncover a feedforward inhibitory mechanism within the brainstem startle circuit by which amygdalar glutamatergic inputs and GlyT2+ PnC neurons contribute to PPI. We are providing new insights to the clinically relevant theoretical construct of PPI, which is disrupted in various neuropsychiatric and neurological diseases.