| Fear refers to the protective reactions of the organisms in face of the actual or expected threat in the external world.These responses are developed gradually in the evolutionary process and include behavior(such as fleezing,fight or flight),endocrine(secretion of a number of stress hormones such as norepinephrine)and autonomic(decreased respiratory rate,heart rate increase)response.Appropriate fear response is essential for the organism’s survival.On the contrary,excessive and inappropriate expression of fear or anxiety is the main symptoms of a series of mental disorders such as post-traumatic stress syndrome,genernalized anxiety disorder and phobia.There is evidence that fear and anxiety disorders are seen in almost half of patients with mental illness.The occurrence of abnormal fear and anxiety can significantly exacerbate the development of mental illness.The research on the brain mechanisms underlying fear has lasted for a long period.In recent decades,accumulating experimental and clinical evidence suggests that the amygdala has a very crucial role in the occurrence of fear.Earlier experiments showed that electrical and chemical damage of amygdala resulted in the decreased or even diminished fear.Intracranial injection of chemicals such as muscimol into amygdala also blocked the ability of animals to learn fear.With the aid of functional magnetic resonance imaging technology,the clinical researchers have found the amygdala activity was substantially increased in face of risky factor in the outside world.On the other hand,amygdala activity was significantly higher in patients with anxiety disorders.Thus,the above evidence strongly suggests that amygdala serve as the key mediator of fear in the brain.The amygdala is a brain region located deep inside in the cerebral temporal lobe and is composed of more than ten nuclei including the lateral nucleus,basal ganglia and the medial nucleus.The lateral nucleus of the amygdala serves as the gatekeeper of most sensory inputs,the central nucleus is the major output station of amygdala.Once integrated inside amygdala,the information will be transferred through central amygdala to the brainstem and other areas.Compared with the neighboring brain regions,the activity of amygdala in the resting state was significantly lower,hinting a very high inhibitory tone.A large number of studies have shown that this highly inhibitory tone is essential for maintaining normal physiological functions of the amygdala.At the same time,electrophysiological and anatomical evidence suggests that the maintenance of amygdala inhibitory state is mainly due to its unique inhibitory network.Inhibitory neurons in the amygdala,include the local neurons in the nucleus and the paracapsular intercalated cells(ITCs)located inside the external capsule between the basolateral and central amygdala.ITCs is densely scattered in amygdala with high input impedance,rendering these neurons with high excitability.Thus,ITCs have the capability to generate strong inhibition over the PNs in amygdala.The disinhibition of the amygdala will lead to the occurrence of a series of mental diseases.Some clinical studies have reported increased levels of amygdala activity are closely associated with the incidence of anxiety disorders.As with the other regions of the brain,the amygdala inhibitory state is mediated by the release of y-aminobutyric acid(GABA)form the INs.Once released,GABA will activate ionotropic GABAA receptors and metabolic GABABR.It is mostly clear that GABAARs activativation mediates two forms of inhibition:phasic inhibition mediated by postsynaptic receptor and tonic inhibition mediated by para-or extrasynaptic receptors.Both types of inhibition differ not only in the place where they are generated,but also in subunit composition,sensitivity to the neurotransmitter GABA and the duration of the inhibition.It is generally believed that the phasic inhibition results from asynchronous release of GABA from the presynaptic terminals with subsequent activation of postsynaptic GABAARs in a short period.Therefore,the phasic inhibition can transfer the presynaptic activity into the postsynaptic signal in a quick and accurate manner.High-resolution single cell voltage recording showed that the duration of phasic inhibition is about hundreds of milliseconds.In contrast,tonic inhibition is mediated by the low concentration of GABA surrounding the cell currently,these two types of inhibition has been found in cerebellar granule cells,hippocampal dentate gyrus and cerebral cortex.Such clear distinctions between them suggest that they may have fundamental differences in the regulation of neural network activity.The basic function of the phasic inhibition in the mature central nervous system is to decrease the excitatory state of the target neurons,thus avoiding the overexcitement of single neurons and neural networks.In addition,phasic inhibition has been shown to be critically engaged in the regulation of the rhythmic activity of neural networks.Through synchronizing a large number of PNs in a time-locked manner,the phasic inhibition contributes substantially to the generation and maintenance of the neural THETA and GAMMA oscillation.In relative to the short term effect of phasic inhibition,tonic inhibition can continuously add the chloride conductance to the neurons.Although many studies have shown that extrasynaptic tonic inhibition is involved in a range of cognitive,emotional,and spiritual illness,understanding the role of tonic inhibition in CNS remains in its infancy.Currently,the research in the molecular composition and its physiological significance on the nervous system has aroused wide interest.So far,people have known that under physiological conditions,the tonic inhibition is primarily mediated by GABAARs containing α5 or δ subunit.For instance,in the hippocampal dentate gyrus,α5 subunit containing GABAARs underlies tonic inhibition.In contrast,in cerebellar granule cells,tonic inhibition is mediated by GABAARs containing the δ subunit and α6 subunit.To certify whether there is tonic inhibition in amygdala,we employed patch clamp technique to investigate the electrophysiological properties in amygdala slice from GAD67-GFP knockin mice.Data was analysed with SPSS 13.0 statistical sofware and was presented by mean ± SEM.Two-sample t-test for independent sample Statistical method was used between the two groups.First,we used high chloride intracellular fluid to perform the whole-cell patch-clamp recording.Cells were clamped at-70mV and the sIPSCs of the amygdala PNs and INs were recorded with the Gap-free mode.The effects of BMI on the inhibition of PNs and INs were compared.The results showed the tonic inhibition(pA/pF)inPNs was 0.015±0.008 and the INs was 0.114±0.039,significant differences(P<0.05).The results showed the sIPSCs in both PNs and INs can be completely blocked by 10μM BMI.At the same time,we found that,in INs,BMI not only blocked the sIPSCs,it also shifted up the recording baseline,which indicated that there was clear tonic inhibition in INs.In contrast,in PNs,BMI only blocked the sIPSCs,but had no significant effect on the clamp current.To further confirm the above experimental results,we clamped the neurons at OmV and replace the recording fluid with the low chloride solution.In this set of experiment,we also found that BMI blocked the sIPSCs and shifted the recording baseline in INs.But for PNs,BMI only blocked sIPSCs without any significant effect on baseline.These results showed the tonic inhibition(pA/pF)in PNs was-0.024±0.006 and the INs was-0.297 ± 0.066,significant differences(P<0.001)Thus,the above results consistently suggested that tonic inhibition is mainly present in INs but not PNs.The cell type specific expression of tonic inhibition may originate from different levels of extracellular GABA concentrations or different expression of the receptors mediating the tonic inhibition.We tested the first possibility by adding GABA(20μM)in the perfusion solution and the suppression of tonic inhibition by BMI in PNs and INs were compared.We observed that BMI selectively shifted up the recording baseline in INs but not PNs with addition of GABA(20μM).The tonic inhibition suppressed by BMI in PNs was 0.036±0.019 pA/pF,and in INs was 0.201±0.048 pA/pF(P<0.05).The result indicated that the cell type specificity of tonic inhibition current does not arise from the difference of extracellular GABA concentration in between.Since there are different subtypes of GABAARs mediating tonic inhibition,we need to identify the specific subtypes of GABAARs underlying the tonic inhibition in INs in LA.We first used THIP,which was shown to specifically activate 8 subunit containing GABAARs at a relatively low concentration and try to determine whether this type of GABAARs is involved in the generation of tonic inhibition in INs.INs were clamped and a low concentration of THIP(1-2μM)and BMI was added in turn into the perfusion solution.We observed that THIP selectively shifted down the recording baseline in INs but not PNs.THIP activated tonic inhibition(pA/pF)in PNs was-0.027 ± 0.012,and in INs was-0.168 ± 0.045,significant differences(P<0.05),which can be completely reversed by additional application of BMI(PNs(-0.005 ± 0.009pA/pF),INs(0.054±0.027pA/pF)).Thus,our findings indicated that 8 subunit containing GABAARs is preferentially expressed in INs and may underline tonic inhibition in INs.To further validate the above experimental findings,we repeated the experiments when holding the cell at OmV,similar results were observed.The results showed the THIP activated tonic inhibition(pA/pF)in PNs was 0.105 ± 0.019 and in INs was 0.609±0.126,significant differences(P<0.001).In order to further clarify the potential difference in the expression profiles of 8 subunit containing GABAARs in PNs and INs in LA,we used immunohistochemical methods to visualize&subunit containing GABAARs in these two types of neurons.In GAD67-GFP-negative mice,we used antibody against CaMKII to label the PNs,and the 8 subunit-specific antibody to label extrasynaptic GABAARs and tried to see whether the two antibodies co-exist in PNs.In GAD67-GFP positive mice,we labeled the extrasynaptic GABAARs with antibody against δ subunit and observe the potential coexsitence of δ subunit and GFP.The immunofluorescent results showed that in very few 8 subunits containing GABAARs are expressed in PNs.On the contrary,in most of the GABAergic INs,we can see the expression of extrasynaptic GABAARs containing δ subunits.Further,we found that the expression of this subtype of GABAARs is particularly abundant in parvabumin positive INs.Subsequently,we made effort to investigate the possible role of δ subunit containing GABAARs in amgydala function.First,we investigated the regulation of these receptors on the excitability of the amygdala PNs and INs.In current clamp mode,we injected depolarizing current to the clamped PNs or INs.Once reached to a certain threshold,the current can produce a certain number of action potentials in PNs and INs.The number of action potentials will gradually increase in accordance with increase of the intensity of injected current.Afterwards,we added BMI to block the δ subunit-containing extrasynaptic GABAARs and tested whether,and if yes,how BMI affected the excitability of PNs as well as INs.The results showed that after application of BMI,the number of action potential was increased in INs but not in PNs.Such effect might not result from the blockage of phasic inhibition by BMI.As generally known,the phasic inhibition is much stronger in PNs than that in INs.If blockage of phasic inhibition led to the increased number of action potential,one would expect that blockage of stronger phasic inhibition in PNs should also increase the firing of action potential.However,it was not the case.Thus,the BMI-induced enhancement of interneuronal excitability should be due to the blockage of tonic inhibition in INs.Our above results suggest that tonic inhibition is mainly expressed in the INs in the LA,however,since the activity of PNs in amygdala is also tightly controlled by INs,we consider that it is also possible that tonic inhibition may affect the activity of PNs through modulating the interaction between INs and PNs.We next tested whether,and if yes how δ subunit-containing extrasynaptic GABAARs would modulate the activity of PNs.We placed the stimulation electrode in either external capsule or internal capsule to activate cortical or thalamic afferents respectively.In the voltage-clamp mode,we held the PNs at-40mV and gradually increased the stimulation intensity until we can readily record the disynaptic EPSC/IPSC.Afterwards,we added THIP into the bath solution to active tonic inhibition in INs and examined how THIP will affect the EPSC/IPSC in PNs.We found that,regardless of the placement of stimulation electrode in cortical or thalamic afferents,THIP perfusion selectively inhibited the amplitude of the IPSC,but had no significant effect on the EPSC.Since PNs did not express δ subunit-containing GABAARs,we propose the action of THIP should be through the modulation of interneuronal activity.THIP perfusion reduced the interneuronal firing,thus weakening the inhibitory gating of PNs by INs,as reflected by a compromised IPSC with no alteration in EPSC.Therefore,the above experiment suggested,the δsubunits containing extrasynaptic GABAARs play an important role in the regulation of normal physiological function of the amygdala.In summary,our current study found that in LA,the expression pattern of the tonic inhibition markedly differed in PNs and INs.The tonic inhibition is much stronger in INs while that in PNs is rather weak.And the tonic inhibition discrepancy between PNs and INs has nothing to do with extracellular GABA concentration discrepancy.We have also verified that the tonic inhibition is primarily mediated by the δ subunits containing GABAARs.Besides reducing the excitability of local INs in amygdala,tonic inhibition also reduced the inhibitory tone in PNs,effectively ensuring their excitatory output.Our current study has provided insight into the expression of δ subunit containing GABAARs in amygdala neurons and its potential function,which established the theoretical basis for exploring the role of related receptors in the regulation of fear and anxiety and related disorders. |
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