The pathway between the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) is emerging as a critical mediator of stress-related affective processes. Evidence also indicates that exposure to drugs of abuse, like opioids, is associated with NMDA-type glutamate receptor-dependent plasticity in the CeA and BNST. However, there is little evidence that NMDA receptors are expressed in CeA neurons projecting to the BNST, or are required for opioid-induced BNST neural activation. Immunoelectron microscopy, tract tracing, and conditional gene deletion technology were used to investigate the synaptic organization of the NMDA receptor and the mu-opioid receptor (μOR) in the CeA-BNST pathway. By dual labeling electron microscopy, numerous CeA-BNST projection neurons expressed the NMDA-NR1 receptor subunit (NR1) or μOR. By triple labeling, it was also found that NR1 and μOR were co-expressed in some CeA-BNST projection neurons. Despite being colocalized in somato-dendritic compartments of CeA neurons, NR1 and μOR were rarely expressed in their axonal terminations in the BNST. Deleting the NR1 gene in CeA neurons resulted in a reduction of morphine-induced Fos protein labeling in the ventral BNST. In summary, NR1 and μOR are coexpressed in somatodendritic sites of CeA neurons, including those projecting to the BNST. In addition, expression of the NR1 gene in CeA neurons is required for morphine-induced BNST neural activation. Thus, postsynaptic NMDA receptors and μORs are positioned for the co-modulation of CeA projection neurons to the BNST, which may provide a synaptic substrate for stress-induced emotional processes critically involved in opioid addictive behaviors.

Corticotropin-releasing factor (CRF) and glutamate are critical signaling molecules in the central nucleus of the amygdala (CeA). Central amygdala CRF, acting via the CRF type 1 receptor (CRF-R1), plays an integral role in stress responses and emotional learning, processes that are generally known to involve functional NMDA-type glutamate receptors. There is also evidence that CRF expressing CeA projection neurons to the bed nucleus of the stria terminalis (BNST) play an important role in stress related behaviors. Despite the potentially significant interactions between CRF and NMDA receptors in the CeA, the synaptic organization of these systems is largely unknown. Using dual labeling high resolution immunocytochemical electron microscopy, it was found that individual somata and dendrites displayed immunoreactivity for CRF and the NMDA-NR1 (NR1) subunit in the mouse CeA. In addition, CRF-containing axon terminals contacted postsynaptic targets in the CeA, some of which also expressed NR1. Neuronal profiles expressing the CRF type 1 receptor (CRF-R1), identified by the expression of green fluorescent protein (GFP) in bacterial artificial chromosome (BAC) transgenic mice, also contained NR1, and GFP immunoreactive terminals formed synapses with NR1 containing dendrites. Although CRF and GFP were only occasionally co-expressed in individual somata and dendritic profiles, contacts between labeled axon terminals and dendrites were frequently observed. A combination of tract tracing and immunocytochemistry revealed that a population of CeA CRF neurons projected to the BNST. It was also found that CRF, or GFP expressing terminals directly contacted CeA-BNST projection neurons. These results indicate that the NMDA receptor is positioned for the postsynaptic regulation of CRF expressing CeA neurons and the modulation of signals conveyed by CRF inputs. Interactions between CRF and NMDA receptor mediated signaling in CeA neurons, including those projecting to the BNST, may provide the synaptic basis for integrating the experience of stress and relevant environmental stimuli with behaviors that may be of particular relevance to stress-related learning and the emergence of psychiatric disorders, including drug addiction.

Activation of GluR2 expressing non-calcium permeable AMPA-type glutamate receptors in the central nucleus of the amygdala (CeA) may play an important role in integrating emotion and memory with goal directed behaviors involved in opioid addiction. The location of non-calcium permeable AMPA receptors within distinct neuronal compartments (i.e. soma, dendrite, or axon) is an important functional feature of these proteins, however, their ultrastructural location and subcellular relationship with mu-opioid receptors (μOR) in the CeA are unknown. Immunocytochemical electron microscopy was used to characterize the ultrastructural distribution of GluR2 and its association with μOR in the mouse CeA. A single labeling analysis of GluR2 distribution employing immunoperoxidase or immunogold markers revealed that this protein was frequently affiliated with intracellular vesicular organelles, as well as the plasma membrane of CeA neuronal profiles. Among all GluR2 labeled neuronal structures, over 85% were dendrites or somata. Unlabeled axon terminals frequently formed asymmetric excitatory-type synaptic junctions with GluR2 labeled dendritic profiles. Dual labeling immunocytochemical analysis showed that GluR2 and μOR were co-localized in neuronal compartments. Among all dual labeled structures, approximately 80% were dendritic. Synaptic inputs to these dual labeled dendrites were frequently from unlabeled axon terminals forming asymmetric excitatory-type synapses. The presence of GluR2 in dendritic profiles receiving asymmetric synapses suggests that activation of the non-calcium permeable AMPA receptor plays a role in the postsynaptic modulation of excitatory signaling involving CeA neuronal circuits that coordinate sensory, affective, and behavioral processes involved in drug addiction. Given the critical role of non-calcium permeable AMPA receptor function in neural and behavioral adaptability, their dendritic association with μOR in CeA dendrites provides a neuronal substrate for opioid-mediated plasticity.

Within the amygdala, AMPA receptors expressing the AMPA-GluR1 (GluR1) subunit play an important role in basal glutamate signaling as well as behaviors associated with exposure to drugs of abuse like opiates. Although the ultrastructural location of GluR1 is an important functional feature of this protein, the basal distribution of GluR1, as well as its sensitivity to acute morphine, has never been characterized in the mouse central nucleus of the amygdala (CeA). Electron microscopic immunocytochemistry employing visually distinct gold and peroxidase markers was used to explore the distribution of GluR1 and its relationship with the mu-opioid receptor (μOR) in the mouse CeA under basal conditions and after morphine. We also looked at the effect of morphine on other glutamate receptor subunits, including AMPA-GluR2 (GluR2) and NMDA-NR1 (NR1). In opiate naive animals, GluR1 and μOR were present in diverse populations of neuronal profiles, but mainly in somatodendritic structures that expressed exclusive labeling for either antigen, as well as those co-expressing both proteins. Compared to saline treated animals, mice given morphine showed significant differences in the subcellular location of GluR1 in dendrites without co-expression of μOR. Although GluR2 also showed similar changes in non-μOR expressing dendrites, contrasting effects were seen in GluR2 and μOR co-expressing profiles. These results provide the ultrastructural basis for basal interactions involving the modulation of GluR1 or μOR activity in the mouse CeA. Further, they indicate that the subcellular distribution of GluR1 is modified by acute opiates in a manner that compares, as well as contrasts, with GluR2.