The Role Of Erbin In Modulation Of Anxiety And Depression-like Behaviors And The Underlying Mechanisms

Psychiatric disorders are devastating neuropsychiatric syndrome,Major depressive disorder (MDD) affects ～17% of people in the world within their lifetime, and anxiety disorders’ prevalence is ～28% of the population worldwide. Worldwide, psychiatric disorders and their comorbidities are the leading causes of disability and economic burden. Depression highly comorbids with anxiety, with 90% anxiety disorder patients showing a history of treatment for depression, and 50-60% of depressed patients reporting a lifetime history of anxiety disorders. Also antidepressant drug (AD) treatments are currently in use for both anxiety and depressive disorders. These suggest us anxiety and depression have a common pathological mechanism, but the common mechanism of anxiety and depression is primarily unknown. Therefore, to reveal common pathological mechanisms of anxiety and depression is one of the key scientific issues in anxiety and depression’s prevention and treatment.GABAergic system is the major inhibitory nervous system of central nervous system, and GABAA receptor is a major neurotransmitter GABA receptor in the central nervous system, which is mainly mediated by the GABA neurotransmitter rapid conduction. GABAA receptors are different pentamers, there are 19 kinds of classic GABAA subunits consisting GABAA receptors, normally contain two a subunits, two β subunits and one y subunit, in CNS more than 90% GABAA receptors containing γ2 subunit. Recently research through genetic manipulation and other means indicated that depression and anxiety disorders significantly associated with γ2 subunit function in the animal. Gad67 and Gad65 (GABA synthesis key enzyme) studies have shown that GABA synthesis and transfer are involved in anxiety regulation. BZ (Benzodiazepines Benzodiazepines drugs) a GABA agonist, is also a kind of classic anti-anxiety drugs, which could further support the GABA system abnormalities involved in the pathogenesis of anxiety disorders. Numerous clinical data indicate that GABAA receptor and GABA levels are significantly changed in patients with severe depression; stress is an important cause of depression and anxiety, GABA energy system is an important factor in controlling the stress response, while chronic stress caused change in GABA system; some classic antidepressants can promote the release of GABA and the upregulation of Gad67. These findings suggest that GABA system plays an important regulatory role in depression and anxiety,and GABA system abnormalities are involved in the pathogenesis of depression and anxiety.Erbin also known as Erbb2 interacting protein (ERBB2IP), is a 200kDa protein containing a PDZ domain. In humans Erbin is encoded by the ERBB2IP gene,which is a member of the leucine-rich repeat and PDZ domain (LAP) family. The encoded protein contains 17 leucine-rich repeats and one PDZ domain. It interacts with various proteins including PSD-95(a postsynaptic scaffold protein of excitatory synapses) and MAPK,Ras and TGFβ. Erbin is specifically expressed in GABAergic interneurons in the neocortex and hippocampus and localized in excitatory synapses of interneurons. Erbin is necessary for surface expression of AMPARs in interneurons via controlling TARP γ-2 levels. As we know GABAergic system is involved in anxiety and depression.The amygdala is an important component of the limbic system,including five major nuclear groups, coordinateds management of multiple brain regions’ emotional behavior (such as fear and anxiety) in different ways. The BLA (basolateral amygdala) plays an important role in the regulation of anxiety behavior, which is a kind of signal input amygdala nucleus. Numerous studies have indicated that enhancing or reducing BLA neurons’ excitement is closely related to anxiety behavior. And clinical trials have shown that contacts inside the amygdala,and between amygdala and other brain regions in patients with anxiety disorders are obvious exceptions. Before the middle prefrontal cortex (medial prefrontal cortex mPFC) is a major regulator of depression brain regions, the first onset of depression and mPFC neuronal activity is closely related; and brain imaging experiments show that there is a clear mPFC differences in depressed patients and normal people; stress is an important cause of depression, middle prefrontal cortex’ excitability, neuronal morphology and some proteins transcripted state has significant changed after stress. This above evidence indicate that the amygdala and medial prefrontal cortex are the major brain regions which regulate anxiety and depression behavior separately.Therefore, we propose our hypothesis:Erbin can be involved in the regulation of anxiety and depression-like behavior of animals by GABA system; because Erbin specifically distributed in PV neurons, thus, we propose PV neurons are involved in the regulation of anxiety and depression, which is an important mechanism for anxiety and depression complications.The role of Erbin in modulation of anxiety -like behaviors and the underlying mechanismsFirst, to determine whether Erbin plays a role in the expression of anxiety Erbin Δc/Δc mice and wild-type littermate mice were tested by the elevated plus maze (EPM), a widely used model for anxiety-like behavior. We found that ErbinΔc/Δc mice spent significantly less time in the open arms of the EPM, as compared to wild-type animals. Meanwhile ErbinΔc/Δc mice spent significantly more time in the closed arms of the EPM compared to wild-type animals. However there was no difference in the total distance between genotypes. Those suggest ErbinΔc/Δc mice have increased anxious behavior in the EPM.In order to confirm that ErbinΔc/Δc mice exhibit enhanced anxiety levels, we conducted a second behavioral test for anxiety, the open field test. Our results showed that ErbinΔc/Δc mice spent significantly less time exploring the center of the field compared to wild-type mice. To test for changes in mobility, we found no significant differences between wild-type and ErbinΔc/Δc mice in the total distance traveled within the open field for 5 min. At last, we also conducted Novelty-suppressed feeding test (NSFT), we found that ErbinΔc/Δc mice spent significantly more time before eating the food compared to wild-type animals. But there was no difference in food intake within 5min. Taken together, these all three experiment results provide strong evidence for an increase in anxiety-like behavior in ErbinΔc/Δc mice.Then, previous studies have showed that activation of the amygdala elicits anxiety whereas lesion of the amygdala impairs the perception of fear in both humans and animals. Anatomically, projecting pathways from the thalamus and cerebral cortex terminate at the lateral amygdala and basolateral amygdala (BLA). Since the amygdala is an essential component of the circuitry underlying anxious behavior, we focus on the role of Erbin in the amygdala, with particular emphasis on the BLA. However, Erbin is specifically expressed in GABAergic interneurons especially in PV-positive interneurons. Therefore, we examined the miniature excitatory postsynaptic currents (mEPSCs) in the BLA PV neurons. In order to record PV neurons, first we made the filial generation from ErbinΔc/Δc mice and Gad-67-GFP mice, then we chose Erbin homozygous mutant and wild -type mice both GFP finally, we record mEPSCs of the green fluorescent cells which are interneurons. And we also examined the action potential of recorded neurons, because a majority of PV neurons are fast spiking neurons. Experimental results showed that ErbinΔc/Δc mice had decreased amplitude of mEPSCs, while the frequency did not change significantly and the excitatory postsynaptic current (EPSC) in the pair pulse ration (PPR) has no significant change, indicating a role for Erbin in regulating AMPAR surface levels, which suggested that ErbinΔc/Δc mice had impaired AMPAR surface expression.Next, we found the excitability of pyramidal neurons in ErbinΔc/Δc mice were increased. What is the possible mechanism of Erbin contribution to behavioral anxiety? Electrophysiological recordings in a brain slice preparation were performed to answer this question. We used the whole-cell patch clamp recording technique to measure currents in pyramidal in the BLA of ErbinΔc/Δc mice and wild type mice. Interneurons and pyramidal neurons were identified by the GFP in the filial generation from GAD-67-GFP mice and ErbinΔc/Δc mice. Our results showed that the pyramidal neuron’s action potential of ErbinΔc/Δc mice increased significantly in the same current injection, compared to wild-type mice. Also when the first action potential arose the inject current was significant lower in ErbinΔc/Δc mice than in wild-type mice. Those suggested the pyramidal neurons’ excitability of ErbinΔc/Δc mice significantly increased compared to wild-type mice. Some studies have shown that Erbin regulates synaptic currents, so the effect of Erbin on pyramidal neurons’ excitability could be caused by (i) the excitatory input onto pyramidal neurons or (ii) the inhibitory input onto pyramidal neurons or (iii) direct modulation of the intrinsic excitability of pyramidal neurons. To discriminate these possibilities, we furter analyed the resting member potential and the action potential threshold of pyramidal neurons both in ErbinΔc/Δc mice and wild type mice, and no significant change was found, suggesting that the intrinsic excitability of pyramidal neuron in both two genotypes was the same.In order to discriminate these possibilities of which caused the increased excitability of pyramidal neuron in ErbinΔc/Δc mice. We examined the excitatory input of pyramid neurons in BLA, we found that spontaneous excitatory postsynaptic currents (sEPSCs) was the same both in ErbinΔc/Δc mice and wild type. Those suggested excitatory input of pyramidal neurons in both genotypes was the same. Taken together, these results provided strong evidence for the impaired inhibitory input onto pyramidal neurons in ErbinΔc/Δc mice.In order to confirm whether the inhibitory input onto pyramidal neurons was impaired in ErbinΔc/Δc mice, we examined the inhibitory input of pyramid neurons in BLA. First we examined the spontaneous inhibitory postsynaptic currents (sIPSCs), we found that the frequency of sIPSCs reduced significantly, but no significant change of the amplitude. And no significant change was found both in frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs), suggesting that the decrease of the action potential-dependent GAB A release.Taken together, these results provided strong evidence for the impaired inhibitory input onto pyramidal neurons in ErbinΔc/Δc mice. Therefore, we also checked whether this effect could be abolished by BMI (20μM), a GABAA receptor antagonist. We found the increased excitability can be abolished by BMI (20μM), suggesting that the effect of Erbin on pyramidal neurons is mediated by GABAergic transmission. All those results indicated that Erbin throught impairing action potential-dependent GABA release increased the excitability of pyramid neurons. In this part, we found ErbinΔc/Δc mice showed anxiety-like behaviors, and Erbin through impairing PV neurons’ AMPAR surface expression increased the excitability of the pyramid neurons.Secondly, all of the above experimental results were in ErbinΔc/Δc mice, as our ErbinΔc/Δc mice are not a general way of expressing the mutation induction. To prove that Erbin might involve in the pathogenesis of anxiety disorders, and further to know what type of interneurons involve in anxiety disorders, we established a PV-specific expression virus. First we constructed Erbin shRNA interference plasmids, then we used PV-Cre mice and loxp-stop virus to limite Erbin-shRNA expressed in PV-positive neurons only. Next we stereotaxicly injected Erbin-shRNA virus into the BLA region of PV-Cre mice, after viral expression and the mice recovery for three weeks, we examined the anxiety-like behavior of those mice. We found Erbin-shRNA (BLA area PV -positive neurons knockout Erbin) mice had obvious anxiety-like behavior. Also in the elevated plus maze test, Erbin-shRNA mice spent significantly less time in the open arms compared to the control mice, but no significant differences in their movement distance, which suggested no significant differences in mobility between two groups of mice. While in the open field test, the central exploration time of Erbin-shRNA mice was significantly shorter than that of control mice, but no significant difference in movement distance. Taken together, these results provided strong evidence for anxiety-like behavior increasement in Erbin-shRNA mice.Next, we examined the miniature excitatory postsynaptic currents (mEPSCs) in the BLA PV neurons of Erbin-shRNA mice. Experimental results showed that mEPSCs amplitude decreased in Erbin-shRNA mice, while the frequency did not change significantly, indicating a role for Erbin in regulating AMPAR surface levels, suggesting that there was impaired AMPAR surface expression in the ErbinΔc/Δc mice.Then, we used the whole-cell patch clamp recording technique to measure currents in pyramidal in the BLA of Erbin-shRNA mice and control mice. Our results showed that the pyramidal neuron’s action potential of Erbin-shRNA mice were significantly increased in the same current injection, compared to wild-type mice. Also when the first action potential arose the inject current was significantly lower in Erbin-shRNA mice compared to wild-type mice. Those suggested the pyramidal neuron’s excitability of Erbin-shRNA mice increased significantly compared to control mice. In this part, we knew that Erbin knocked down by Erbin-shRNA also had effect on anxiety-like behaviors.Finally, in the animal models of anxiety, to further define whether Erbin regulated of anxiety-like behaviors and its mechanisms. Six- to eight-week-old C57BL/6 male mice were used. All animals were housed under a 12 h dark-12 h light cycle with free food and water. Acute stress was induced by restraining the mice in well-ventilated Perspex restraining tubes for 2 h, during which the animals were not physically compressed or experience pain. And the mice were exposed to one or three 2 h of restraints on each of one or three consecutive days. After 24h later mice wre removed brain and BLA and hippocampus were removed from brain slices. From the result of western blotting we found that acute stress mice showed significantly reduced Erbin expression levels in the BLA area, but no significant difference was found in hippocampal tissue. Those results suggested Erbin involved in the pathological process of anxiety.Regulating effect and mechanism of depression-like behavior ErbinFirstly, to determine whether Erbin plays a role in depression ErbinΔc/Δc mice and wild-type littermate mice were tested on the Sucrose preference test, a widely used model for depression-like behavior. ErbinΔc/Δc mice showed significantly reduced interaction time in the social interaction test compared to control mice. Also ErbinΔc/Δc mice had more immobility time in the tail suspension test ompared to control mice. Taken together, all these three experiment results provided strong evidence for depression-like behavior increasement in ErbinΔc/Δc mice.Secondly, based on previous studies that the prefrontal cortex is closely related to the onset of depression, and middle prefrontal cortex mPFC region is an important area of regulating depression. According to the previous studies on depression, we also checked pyramidal neurons’excitability in mPFC. Through the action potential analysis, we found that pyramidal neurons’ excitatory was significantly increased in ErbinΔc/Δc mice, as demonstrated by significantly increasing the number of action potentials in the same electrical stimulation, and also when the first action potential arose the inject current was significant lower in ErbinΔc/Δc mice compared with wild-type mice.Then, as we had found that significantly increased pyramidal neurons’ excitability in mPFC region of ErbinΔc/Δc mice was not due to their own intrinsic excitability of pyramidal neurons, so inhibitory inputs onto pyramidal neurons might cause its excitability increasement, GABA in the central nervous system is the most important part of the inhibitory system. And GABA system plays a strong regulatory role in pyramidal neurons’ excitatory, indicating that Erbin might regulate the pyramidal neurons’excitatory by GABA system. As previous studies have shown that GABA systems play an important role in the regulation of depression, here we detected whether Erbin inhibited GABA system on mPFC. By checking the GABA system on pyramidal neurons of mPFC, we found that the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) was significantly reduced in pyramidal neurons of ErbinΔc/Δc mice, while no significant change was detected in amplitude. Those suggested due to reduced GABA system function led to increased mPFC pyramidal neurons’ excitatory, and reduced PV interneurons’ input increased excitability of pyramidal neurons, which might also be an important factor in the pathogenesis of depression.