Adolescent social isolation enhances the plasmalemmal density of NMDA NR1 subunits in dendritic spines of principal neurons in the basolateral amygdala of adult mice

Highlights

• Adolescent social isolation (ASI) produces maladaptive adult behaviors.

• The affected behaviors are principally those that require emotional processing.

• ASI produces an increase in NMDA receptors in the spines of amygdala output neurons.

• Potentiation of these output neurons may contribute to ASI behavioral abnormality.

Abstract

Social isolation during the vulnerable period of adolescence produces emotional dysregulation manifested by abnormalities in adult behaviors that require emotional processing. The affected brain regions may include the basolateral amygdala (BLA), where plasticity of glutamatergic synapses in principal neurons plays a role in conditioned emotional responses. This plasticity is dependent on NMDA receptor trafficking denoted by intracellular mobilization of the obligatory NR1 NMDA subunit. We tested the hypothesis that the psychosocial stress of adolescent social isolation (ASI) produces a lasting change in NMDA receptor distribution in principal neurons in the BLA of adults that express maladaptive emotional responses to sensory cues. For this, we used behavioral testing and dual electron microscopic immunolabeling of NR1 and calcium calmodulin-dependent protein kinase II (CaMKII), a protein predominantly expressed in principal neurons of the BLA in adult C57Bl/6 mice housed in isolation or in social groups from post-weaning day 22 until adulthood (∼3 months of age). The isolates showed persistent deficits in sensorimotor gating evidenced by altered prepulse inhibition (PPI) of acoustic startle and hyperlocomotor activity in a novel environment. Immunogold-silver labeling for NR1 alone or together with CaMKII was seen in many somatodendritic profiles in the BLA of all mice irrespective of rearing conditions. However, isolates compared with group-reared mice had a significantly lower cytoplasmic (4.72 ± 0.517 vs 6.31 ± 0.517) and higher plasmalemmal (0.397 ± 0.0779 vs 0.216 ± 0.026) density of NR1 immunogold particles in CaMKII-containing dendritic spines. There was no rearing-dependent difference in the size or number of these spines or those of other dendritic profiles within the neuropil, which also failed to show an impact of ASI on NR1 immunogold labeling. These results provide the first evidence that ASI enhances the surface trafficking of NMDA receptors in dendritic spines of principal neurons in the BLA of adult mice showing maladaptive behaviors that are consistent with emotional dysregulation.

Introduction

Animal models and human imaging studies suggest that disturbances in the basolateral amygdala (BLA) play a role in the emotional dysregulation characteristic of many mental health disorders including schizophrenia and bipolar disorder (e.g. dysregulation categorized in the negative valence systems and systems for social process in the Research Domain Criteria classification (Insel et al., 2010). Such dysregulation, loosely defined as an inability to properly process emotionally-charged stimuli, often first manifests during the critical neurodevelopmental period of adolescence, when the prefrontal cortex and other brain regions providing glutamatergic inputs to the BLA continue to develop and mature (Cunningham et al., 2002). The critical involvement of the lateral amygdaloid complex in emotional processing (LeDoux, 2000) suggests that improper development of these glutamatergic inputs and ensuing adaptations in BLA output neurons during adolescence may thus contribute to emotional dysregulation seen in mental health disorders (Einon and Morgan, 1977, Lukkes et al., 2012).

BLA projection neurons are pyramidal-like glutamatergic principal neurons that comprise 85% of the neuronal population in this region, distinguishable from interneurons by their content of calcium calmodulin-dependent protein kinase II (CaMKII; McDonald, 1992). Ontogenetic studies specifically implicate activity of BLA principal neurons in the mediation of anxiety-related behaviors (Tye et al., 2011). This is consistent with evidence that severe ablations of the BLA lead to the loss of fear response in both animal models and schizophrenics (Bechara et al., 1999). However, more subtly, increased neuronal activity in the BLA can result in decreased social interaction and altered responses to novel stimuli (Benes, 2009, Stuber et al., 2011). Moreover, pharmacological manipulation of glutamate and dopamine in the BLA alters prepulse inhibition (PPI) to acoustic startle response (Bakshi and Geyer, 1998, Stevenson and Gratton, 2004). PPI, a phenomenon in which involuntary reactions to a startling stimulus is decreased by a preceding non-startling stimulus, is a measure of sensorimotor gating. Deficits in PPI are thought to indicate the inability of schizophrenics to filter environmental stimuli, leading to cognitive overload and emotional dysregulation (Geyer and Moghaddam, 2002). Sensorimotor gating deficits also manifest in rodents as sustained hyperlocomotion in a novel environment due to an inability to habituate to novel stimuli (Levine et al., 2007). Stress during adolescence, such as post-weaning adolescent social isolation (ASI), in which rodents are housed individually from weaning until adulthood, alters animal behavior similar to direct BLA disruption (Geyer and Moghaddam, 2002, Lukkes, 2009). As a result of this stressful early-life experience, rodents display irreversible abnormalities in their responses to novel environments and have deficits in PPI (Braff and Geyer, 1990).

The processing of emotional information in these complex behaviors may be susceptible to disruption through improper ontogenetic development of NMDA receptor populations on principal neurons in the BLA (Benes, 2009, Fendt, 2001). NMDA receptors are often required for activity-dependent plasticity of glutamatergic synapses (Wenthold et al., 2003). These receptors, which are comprised of NR1 subunits or a combination of an obligatory NR1 and various NR2 subunits, undergo ontogenetic changes in subunit composition making them more activity-dependent (Barria and Malinow, 2002, Barria and Malinow, 2005). The NR2B expression may make these receptors more susceptible to isolation rearing-induced diminution in the sensory signaling that is essential for social recognition memory (Jacobs and Tsien, 2012). Abnormal NMDA signaling and plasticity are highly implicated in the neuropathology of schizophrenia (Belforte et al., 2009, Adell et al., 2012;). Specifically, postmortem analysis has shown abnormal trafficking of selective isoforms of NR1- and NR1/NR2-containing NMDA receptors in the prefrontal cortex of schizophrenics (Perez-Otano and Ehlers, 2005, Kristiansen et al., 2010, Rubio et al., 2013). Impaired receptor trafficking can affect neuronal activity by changing receptor availability, possibly contributing to the underlying neuropathology (Radley et al., 2007). It is unknown, however, whether the same trafficking abnormalities are present in the BLA, or whether impairments are caused by environmental/developmental factors.

In this study, we sought to determine whether adolescent isolation resulting in emotional dysregulation produces abnormalities in the surface/synaptic distribution of the requisite NMDA NR1 subunit in CaMKII-containing principal neurons of the adult BLA. For this we used behavioral testing and electron microscopic immunolabeling of the BLA in C57Bl/6 mice reared in isolation or in small groups from weaning to adulthood.