Neural Circuits Of VBNST Regulate Response To Unpredictable Threat Signals

Fear is caused by specific and predictable severe threat stimuli,while anxiety is caused by vague and unpredictable threat stimuli.At present,the neural circuits related to fear and anxiety have been studied extensively.However,the neural circuits related to anxiety caused by unpredictable threat signals remain unclear.The first national epidemiological survey of mental disorders in China shows that anxiety disorders have the highest prevalence,with the annual prevalence of 5.0%and the lifetime prevalence of 7.6%.Therefore,it is of great significance to reveal the neural circuits underlying the defensive behavior to unpredictable threat signals which could guide the treatment of psychiatric disorders such as anxiety disorders.Previous studies had reported that the ventral bed nucleus of the stria terminalis(vBNST)was selectively activated by unpredictable threat signals.Moreover,the inactivation of vBNST could reduce the freezing behavior during CS-test,which suggests that vBNST was involved in the freezing behavior to unpredictable threat signals.There are different types of neurons in vBNST.However,it’s unclear that the cell-type specific neurons and neural circuits of vBNST underlying the response to unpredictable threat signals.To answer the question,we first explored which types of vBNST neurons were involved in regulation of the response to unpredictable threat signals by establishing the behavioural model of conditioned stimulus(CS)combined with transgenic mice and fiber photometry.We found that Vgat(vesicular GABA transporter)but not Vglut2(vesicular glutamate transporter 2)neurons were activated in CS-but not in CS+ test.To further clarify the causal role of vBNST Vgat and Vglut2 neurons in response to unpredictable threat signals,the optogenetic experiments was conducted.We found that inhibition of Vgat neurons but not Vglut2 neurons in vBNST could only reduce freezing behavior during CS-test.To explore vBNST neuronal circuitry which was involved in the regulation of the response to unpredictable threat signals,we traced the activated neurons of vBNST during CS’ test and analyzed the afferents in the whole brain level using retrograde trans-synaptic tracking.It was found that the main afferents included the cortex,amygdala and hypothalamus.Next,we wanted to investigate the mechanisms of vBNST neuronal circuitry.Using the technology of fiber photometry,we found that the IC(insular cortex)to vBNST projectors showed high activation during both CS-and CS+test.But optiogenetic inhibition of IC-vBNST projection could only reduce the freezing behavior to CS-but not CS+test.In addition to this,NE(noradrenaline)signals which were released to vBNST Vgat neurons during CS+and CS-test was monitored by GPCR activation-based NE(GRABNE)sensors.We found that the intensity of NE was increased during CS+ and CS-test.But inhibition of NE signals in vBSNT by microinjection could only reduce freezing behavior in CS-test.Our results revealed:1:GABAergic neurons of vBNST not glutaminergic neurons were involved in regulation of the responses to unpredictable threat signals;2:The main afferents of vBNST neurons which was activated during unpredictable threat signals included the cortex,amygdala,hypothalamus and so on;3:The IC-vBNST projection was involved in the regulation of the response to unpredictable threat signals;4:NE signals in vBNST were involved in regulation of the responses to unpredictable threat signals.This study provides direct evidence that GABAergic neurons,IC-vBNST projection and NE signals in vBNST were involved in regulation of the responses to unpredictable threat signals,which could deepen the understanding of the pathogenesis of anxiety related disorders and provide new insight for the treatment of anxiety disorders and other mental diseases.