The roles of gluk4 in amygdala and associated behaviours

Abstract

ABSTRACT Kainate receptors (KARs) are a class of ionotropic glutamate receptors ubiquitously present throughout the central nervous system. Mounting evidence indicates the presence of these receptors at both pre- and post-synaptic sides of the synapses. Presynaptically KARs are involved in controlling both inhibitory and excitatory synaptic transmission via the modulation of the release of neurotransmitters like GABA and Glutamate. Postsynaptically, KARs cause membrane depolarisation and mediate postsynaptic responses. KARs have a small contribution to the postsynaptic currents, but they do impart synapses with certain integrative properties and have capabilities to regulate synaptic transmission beyond their role as ion channel forming receptor. GluK1 subunit containing KARs have been found in presynaptic terminals of interneurons and postsynaptically in interneurons and pyramidal cells of the basolateral amygdala (BLA). In this structure, they mediate a fraction of the postsynaptic depolarization, while presynaptically, GluK1 containing KARs in interneurons controls GABA release. Altered glutamatergic neurotransmission is considered to be one of the primary factors contributing to mental diseases such as autism spectral disorders (ASD). The amygdala is involved in emotional behaviours and its relation with some psychiatric illnesses is subject of active research. Alterations in copy number of genes coding for KAR subunits have been linked to neuropsychiatric syndromes such as ASD and bipolar disorders. This is the case of Grik4, a gene coding for a high affinity subunit, GluK4, as de novo duplications of this gene have been described in cases of ASD and schizophrenia. To start delineating the role played by GluK4 containing KARs in brain circuits underlying emotionally relevant behaviours, we followed a gain of function strategy and generated the transgenic mouse C57BL/6J-Tg(Camk2a-grik4), which overexpresses Grik4 in the forebrain under the control of the CaMKII promoter and that was nicknamed GluK4Over. These mice displayed anhedonia, enhanced anxiety and depressive states and impaired social interaction, common endophenotypes associated to ASD. To start looking for functional neural correlates of these abnormal behaviours in amygdala circuits, we studied the effect of Grik4 overexpression on the presynaptic and postsynaptic excitatory activity in identified cells of BLA and Centrolateral amygdala (CeLA). Overexpression of Grik4 led to enhanced spontaneous and external capsule-evoked glutamate excitatory activity in the BLA and consequently increased excitatory inputs to the regular firing cells (RFCs) of CeLA. Overexpressed GluK4 subunits were present both presynaptically and postsynaptically leading to higher release probability of glutamate in cortico-amygdala synapses. In addition to the enhanced probability of release, amplitude was also enhanced in evoked responses mediated by AMPARs in BLA pyramidal cells. Decreased rectification index in these evoked responses mediated by AMPARs in BLA neurons indicated a change in subunit composition of AMPARs, which imposed an increased conductance of postsynaptic AMPARs in BLA pyramidal cells, leading to increased postsynaptic response. In synaptic connections between BLA pyramidal cells and RFCs of CeLA, the probability of release was also enhanced. On the other hand, the probability of release of glutamate and thus excitatory input in synaptic connections between BLA pyramidal cells and late firing cells (LFCs) of CeLA was decreased in GluK4Over mice. A large population of cells in CeLA consists of these LFCs and their activation is known to take part of an anxiolytic circuit. Accordingly, depression of excitatory input to LFCs in GluK4Over mice led to an anxiogenic phenotype in GluK4Over mice. Moreover, in-vivo c-Fos expression studies indicated enhanced resting activity in the BLA and Centromedial amygdala (CeMA) in the GluK4Over mice. Thus overall, depression of activity of late firing cells in the CeLA forming part of the anxiolytic circuit leads to disinhibition of CeMA neurons, which further inhibit neurons of downstream targets like Bed Nucleus of the Stria Terminalis (BNST) and Periaqueductal Gray (PAG) and this increased inhibition is known to increase anxiety and fear related behaviour. Finally, we showed that restoring levels of GluK4 protein in GluK4Over mice to normal levels rescue synaptic and behaviour alterations thus indicating excess of GluK4 as the major cause behind increased synaptic gain in anxiogenic circuits provoking aberrant behaviours that are evident in neuropsychiatric diseases like autism and schizophrenia. Taken together, these observations suggest that GluK4 subunit containing high affinity KARs have a significant role in regulating excitatory transmission in the BLA and from BLA to CeLA networks and modest increases in GluK4 protein are associated with increased synaptic gain at selected synapses in the amygdala producing an unbalanced circuit output, which may account for the behavioural abnormalities that concur with diseases such as autism and schizophrenia. Grik4 duplication may be relevant to such disease behaviours in humans given that amygdala is structurally and functionally conserved across species.