Isolation of Projection-Specific and Behavior-Relevant Amygdala Circuit for RNA Sequencing.

One of the most sought-after topics in neuroscience is to understand how the environment regulates the activity and function of neural circuitry and subsequently influences relevant behaviors. In response to alterations in the environment, the neural circuits undergo adaptive changes ranging from gene expression changes to altered cellular function. Performing sequencing of the transcriptome involved in these behavior-related circuits will provide clues to accurately dissect the detailed mechanisms of related behavior. Here, we describe methods for marking and collecting the ventral hippocampus-projecting basolateral amygdala neurons, which have been repeatedly implicated in regulation of anxiety-like behavior, and subsequently constructing a library ready for sequencing. Specifically, the reported approaches include adeno-associated virus injection, acute brain slice isolation, cell suspension preparation, cell extraction, and cDNA library construction. By utilizing the techniques described here, researchers can comprehensively investigate the transcriptional levels of neural clusters embedded in particular circuits and discover potential pathogenic and therapeutic targets for behavior-relevant disorders. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Tagging of behavior-related neural circuits Basic Protocol 2: Isolation and capture of fluorescent-positive cells Basic Protocol 3: Foundation of sequencing library.

GABAA(δ) receptor hypofunction in the amygdala-hippocampal circuit underlies stress-induced anxiety.

Dysregulated GABAergic inhibition in the amygdala has long been implicated in stress-related neuropsychiatric disorders. However, the molecular and circuit mechanisms underlying the dysregulation remain elusive. Here, by using a mouse model of chronic social defeat stress (CSDS), we observed that the dysregulation varied drastically across individual projection neurons (PNs) in the basolateral amygdala (BLA), one of the kernel amygdala subregions critical for stress coping. While persistently reducing the extrasynaptic GABAA receptor (GABAAR)-mediated tonic current in the BLA PNs projecting to the ventral hippocampus (BLA â†’ vHPC PNs), CSDS increased the current in those projecting to the anterodorsal bed nucleus of stria terminalis (BLA â†’ adBNST PNs), suggesting projection-based dysregulation of tonic inhibition in BLA PNs by CSDS. Transcriptional and electrophysiological analysis revealed that the opposite CSDS influences were mediated by loss- and gain-of-function of δ-containing GABAARs (GABAA(δ)Rs) in BLA â†’ vHPC and BLA â†’ adBNST PNs, respectively. Importantly, it was the lost inhibition in the former population but not the augmentation in the latter population that correlated with the increased anxiety-like behavior in CSDS mice. Virally mediated maintenance of GABAA(δ)R currents in BLA â†’ vHPC PNs occluded CSDS-induced anxiety-like behavior. These findings clarify the molecular substrate for the dysregulated GABAergic inhibition in amygdala circuits for stress-associated psychopathology.

Early life stress induces anxiety-like behavior during adulthood through dysregulation of neuronal plasticity in the basolateral amygdala.

AIMS: Early life stress (ELS) increases the risk of psychiatric diseases such as anxiety disorders and depression in later life. Hyperactivation of the basolateral amygdala (BLA) neurons plays a pivotal role in the pathogenesis of stress-related diseases. However, the functional roles of BLA neurons in ELS-induced anxiety disorders are not completely understood. MAIN METHODS: Mice were subjected to maternal separation (MS) during postnatal days 3 to 21 to mimic ELS. Anxiety-like behavior was tested by open field test (OFT), elevated plus maze (EPM), and novelty suppressed feeding (NSF). Then, c-fos expression, a proxy for neuronal activity, was evaluated by immunofluorescence. Finally, synaptic transmission and intrinsic excitability were measured by whole-cell patch-clamp recordings. KEY FINDINGS: MS significantly increased anxiety-like behavior in adulthood, as indicated by less time spent in the center area of the OFT, less time spent in and fewer entries to the open arms of the EPM, and increased latency to feed in NSF. Mechanistically, MS increased the expression of c-fos in BLA. MS enhanced the excitatory, but not inhibitory, synaptic transmission onto BLA projection neurons (PNs), which was caused by enhanced presynaptic glutamate release. Moreover, MS also markedly increased the intrinsic neuronal excitability of BLA PNs, probably due to the reduced medium afterhyperpolarization (mAHP) in BLA PNs. SIGNIFICANCE: Our results suggest that the changes of neuronal activity and synaptic transmission in the BLA PNs may play a crucial role in ELS-induced anxiety-like behavior, and these findings provide new insights into the pathological mechanisms of stress-related anxiety disorders.

[Analysis of Pollution Characteristics and Sources of Atmospheric VOCs in Ezhou City].

From March 2018 to February 2019, quantitative detection was made of 102 kinds of atmospheric volatile organic compounds (VOCs) using online gas chromatography in Ezhou City. We compared and analyzed the composition, seasonal variation, and diurnal variation of VOCs. Using maximum incremental reactivity (MIR), we estimated the ozone generation potential (OFP) of VOCs. The results show that the annual average volume fraction of atmospheric VOCs in Ezhou is (30.78±15.89)×10-9, and is overall higher in winter than summer, represented by alkane > oxygen > halogenated hydrocarbon > olefin > aromatic hydrocarbon > alkyne. The night volume fraction is higher than in the daytime, and overall the distribution is "double peak". The aromatic hydrocarbons, halogenated hydrocarbons, and OVOCs appear as a "third peak" at 00:00-02:00. Aromatic hydrocarbons and olefins contribute more to the OFP potential of VOCs, with contribution rates of 35.45% and 29.5%, respectively. The highest contribution rate to OFP is ethylene, reaching 24.217%. Analysis of VOC characteristic species found that vehicle exhaust fumes and solvent volatilization are the main sources of VOCs in Ezhou. Of these, motor vehicle emissions are the most important source. Controlling Ezhou's motor vehicle emissions helps to reduce the composition of atmospheric VOCs, thereby reducing ozone production.

Chronic Stress Oppositely Regulates Tonic Inhibition in Thy1-Expressing and Non-expressing Neurons in Amygdala.

Chronic or prolonged exposure to stress ranks among the most important socioenvironmental factors contributing to the development of neuropsychiatric diseases, a process generally associated with loss of inhibitory tone in amygdala. Recent studies have identified distinct neuronal circuits within the basolateral amygdala (BLA) engaged in different emotional processes. However, the potential circuit involved in stress-induced dysregulation of inhibitory tones in BLA remains elusive. Here, a transgenic mouse model expressing yellow fluorescent protein under control of the Thy1 promoter was used to differentiate subpopulations of projection neurons (PNs) within the BLA. We observed that the tonic inhibition in amygdala neurons expressing and not expressing Thy1 (Thy1+/-) was oppositely regulated by chronic social defeat stress (CSDS). In unstressed control mice, the tonic inhibitory currents were significantly stronger in Thy1- PNs than their Thy1+ counterparts. CSDS markedly reduced the currents in Thy1- projection neurons (PNs), but increased that in Thy1+ ones. By contrast, CSDS failed to affect both the phasic A-type γ-aminobutyric acid receptor (GABAAR) currents and GABABR currents in these two PN populations. Moreover, chronic corticosterone administration was sufficient to mimic the effect of CSDS on the tonic inhibition of Thy1+ and Thy1- PNs. As a consequence, the suppression of tonic GABAAR currents on the excitability of Thy1- PNs was weakened by CSDS, but enhanced in Thy1+ PNs. The differential regulation of chronic stress on the tonic inhibition in Thy1+ and Thy1- neurons may orchestrate cell-specific adaptation of amygdala neurons to chronic stress.

Identification of a prefrontal cortex-to-amygdala pathway for chronic stress-induced anxiety.

Dysregulated prefrontal control over amygdala is engaged in the pathogenesis of psychiatric diseases including depression and anxiety disorders. Here we show that, in a rodent anxiety model induced by chronic restraint stress (CRS), the dysregulation occurs in basolateral amygdala projection neurons receiving mono-directional inputs from dorsomedial prefrontal cortex (dmPFC→BLA PNs) rather than those reciprocally connected with dmPFC (dmPFC↔BLA PNs). Specifically, CRS shifts the dmPFC-driven excitatory-inhibitory balance towards excitation in the former, but not latter population. Such specificity is preferential to connections made by dmPFC, caused by enhanced presynaptic glutamate release, and highly correlated with the increased anxiety-like behavior in stressed mice. Importantly, low-frequency optogenetic stimulation of dmPFC afferents in BLA normalizes the enhanced prefrontal glutamate release onto dmPFC→BLA PNs and lastingly attenuates CRS-induced increase of anxiety-like behavior. Our findings thus reveal a target cell-based dysregulation of mPFC-to-amygdala transmission for stress-induced anxiety.

Moderate maternal separation mitigates the altered synaptic transmission and neuronal activation in amygdala by chronic stress in adult mice.

Exposure to moderate level of stress during the perinatal period helps the organisms to cope well with stressful events in their later life, an effect known as stress inoculation. Amygdala is one of the kernel brain regions mediating stress-coping in the brain. However, little is known about whether early life stress may affect amygdala to have its inoculative effect. Here, we observed that moderate maternal separation (MS) from postnatal day 3 to day 21 (D3-21, 1 h per day) significantly alleviated the increased anxiety-like behavior induced by chronic social defeat stress (CSDS) in adulthood, suggesting an obvious inoculative effect of moderate MS. Further studies revealed that MS prevented CSDS-evoked augmentation of glutamatergic transmission onto principal neurons (PNs) in the basolateral amygdala (BLA) by inhibiting presynaptic glutamate release. By contrast, it did not affect GABAergic transmission in BLA PNs, as indicated by unaltered frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Moreover, the CSDS-induced increase of neuronal excitability was also mitigated by MS in BLA PNs. In conclusion, our results suggest that MS may have its inoculative effect through alleviating the influences of later life stress on the glutamatergic transmission and neuronal activity in amygdala neurons.