Immunolabeling reveals cellular localization of the N-methyl-D-aspartate receptor subunit NR2B in neurosecretory cells but not astrocytes of the rat magnocellular nuclei

Abstract

Previous studies suggest that activation of N-methyl-D-aspartate (NMDA) receptors facilitates phasic firing and spike clustering displayed by magnocellular neuroendocrine cells (MNCs) of the supraoptic (SON) and paraventricular nucleus of the hypothalamus (PVN). Osmotic stimulation produces similar activity patterns which, in turn, can lead to enhanced release of vasopressin and oxytocin from MNCs. Our laboratory has shown that dehydration regulates the expression of the NMDA receptor subunits, NR1 and NR2B, in the SON and PVN, suggesting their involvement in osmoregulation. In the present study, we examined the cellular localization of NR2B, one of the glutamate-binding subunits of the NMDA receptor, with an NR2B-specific antibody. Using double-label immunohistochemistry and three different detection methods with metallic, peroxidase, and fluorescence markers, it was found that both vasopressin and oxytocin-producing MNC populations synthesize NR2B. The incidence of NR2B colocalization with vasopressin-neurophysin in the SON and lateral magnocellular PVN (PVL) was 0.95 and 0.91, respectively. For oxytocin-neurophysin, the corresponding values were 0.97 and 0.95, respectively. Furthermore, the extent of colocalization in MNCs of the SON, PVL, retrochiasmatic SON, and accessory neurosecretory nuclei was similar. Astrocytes associated with the SON, and identified with antibodies targeting glial fibrillary acidic protein (GFAP) or vimentin, were not colabeled with NR2B. Our results demonstrate that NR2B protein is expressed by almost all MNCs and that it is equally prevalent in vasopressinergic and oxytocinergic populations of various magnocellular neuroendocrine nuclei supporting a role of NMDA receptors in MNC-mediated neurosecretory processes. Although NR2B may form part of functional NMDA receptors on MNCs, it is probably not present on astrocytes associated with nearby MNCs.

Role of AMPA and NMDA receptors on vasopressin and oxytocin secretion induced by hypertonic extracellular volume expansion

“…The subunit of NMDA receptor NR1 is expressed in both AVP and OT magnocellular neurones of the SON and PVN and its expression is up‐regulated after dehydration in these areas . Besides, the NR2B modulatory subunit of the NMDA receptor is also highly expressed in both AVP and OT magnocellular neurones . On the other hand, GLUR2, 3 and 4 subunits of AMPA receptors are heterogeneously expressed in PVN and SON neurones, whereas the GLUR1 subunit is found in all portions of the PVN and SON .…”

NMDA receptor subunit expression in the supraoptic nucleus of adult rats: Dominance of NR2B and NR2D

“…NR2B protein expression in the SON has been shown to be exclusively neuronal (Currás-Collazo et al, 2000), and NR2B is thought to be the primary NR2 subunit involved in characteristic burst firing and synaptic plasticity of SON MNCs. In these cells, ifenprodil (an antagonist at NR2B-containing receptors) blocks synaptic plasticity [LTP and LTD (Panatier et al, 2006b)], and reduces the amplitude of evoked excitatory postsynaptic currents (EPSCs) by 80% when AMPA receptors were blocked (Panatier et al, 2006).…”

“…Functional N-methyl-D-aspartate receptors (NMDAR) are ubiquitously expressed on SON neurons (Al-Ghoul et al, 1997b; Currás-Collazo et al, 2000; Currás et al, 1998; Decavel and Curras, 1997), and are required for long-term potentiation (LTP) and depression (LTD) of synaptic responses in MNCs (Panatier et al, 2006), AVP release in response to osmotic stimuli (Swenson et al, 1998) and characteristic phasic burst firing of AVP MNCs, which is associated with enhanced hormone release (Hu and Bourque, 1992; Israel et al, 2010). …”

“…The full expression profile of NMDAR subunits in the SON has not been characterized, but in addition to ubiquitous NR1 expression in MNC neurons, NR2B has been shown to be expressed in the SON of embryonic, early post-natal and adult rats (Currás-Collazo and Dao, 1999; Currás and Dao, 1998) and is localized to neurons, not astrocytes (Currás-Collazo et al, 2000). Ifenprodil (an antagonist at NR2B-containing receptors) has been shown to reduce the amplitude of evoked excitatory postsynaptic currents (EPSCs) by 80% when AMPA receptors were blocked (Panatier et al, 2006).…”

Long-lasting dephosphorylation of connexin 43 in acute seizures is regulated by NMDA receptors in the rat cerebral cortex

“…It may enter the cell via voltage-sensitive channels (43) and metabotropic glutamate receptors during A-4P seizures as assessed by in vivo two-photon microscopy (44). NMDA receptors are known to be expressed in neurons (45), while astrocytes react to glutamate by metabotropic receptors and express NMDA subunits only under pathological conditions such as ischemia or anoxia (46). In contrast, recent studies have clearly demonstrated the presence of functional NMDA subunits in cortical astrocytes in the normal brain (47,48).…”