Cellular and subcellular localization of NMDA-R1 subunit immunoreactivity in the visual cortex of adult and neonatal rats

Abstract

NMDA receptor activation can alter synaptic strength, cause cell death, and may modulate the release of glutamate and other neurotransmitters. Using a specific and selective antiserum directed against the R1 subunit of the NMDA receptor, we examined (1) whether NMDA receptors in the adult rat visual cortex are exclusively postsynaptic or also presynaptic and (2) whether NMDA-R1 subunits are incorporated into the plasma membrane prior to, contemporaneously, or following the formation of synapses during postnatal development. By light microscopy, NMDA-R1 immunoreactivity in the adult visual cortex is easily detectable within perikarya and proximal dendrites in laminae 2-6. Many of them have the morphological features of pyramidal neurons. In addition, fine punctate labeling is evident throughout the neuropil. Electron microscopy reveals these puncta to reside at postsynaptic densities of axospinous junctions and at fine astrocytic processes and axon terminals. In the deeper laminae, the majority of labeled profiles are astrocytic. Visual cortices of animals in their first postnatal week show concentrated immunoreactivity in a few nonpyramidal neurons within laminae that have just differentiated from the cortical plate. Electron microscopy reveals diffuse labeling along the plasma membrane of dendritic shafts lacking morphologically identifiable synaptic junctions or appositions to axons. Immunoreactivity is detectable in dendritic processes by postnatal day (PND) 2, in axonal processes by PND 4, and in astrocytic profiles by PND 14. Immunoreactivity also is detectable along the postsynaptic membrane of presumably transient axosomatic junctions. At all ages, the prevalence of NMDA-R1-immunoreactive profiles is lamina 1 > 4/5 > 6/6B. These results provide the cellular basis for NMDA receptors' participation in (1) postsynaptic membrane excitability, (2) regulation of transmitter release, (3) and, in the deeper laminae, astrocyte responses. During development, NMDA-R1 subunits are associated with the plasma membrane prior to axons' arrival while clustering of receptors to junctions may be promoted by axonal contact. Finally, spatial segregation of axonal growth cones may be mediated by NMDA-R1 subunits on these axonal processes.

In vivo blockade of N-methyl-d-aspartate receptors induces rapid trafficking of NR2B subunits away from synapses and out of spines and terminals in adult cortex

“…Gold label occurred postsynaptically, directly over, or near the PSD complex and also within the cytoplasm of dendritic spines. These locations agree with results from past ultrastructural studies (Aoki et al, 1994;Charton et al, 1999;Conti et al, 1999;Kharazia and Weinberg, 1999;Valtschanoff and Weinberg, 2001). In addition, NR2B subunit labeling was prominent in the cytoplasm and along membranes of presynaptic terminals.…”

“…It has been shown that layer I contains more synapses with NMDA receptors than deeper layers (Aoki et al, 1994); therefore, we predicted that a great amount of synapses would be affected by the D-APV application in this layer. Earlier published work (Aoki et al, 2003) showed that, indeed, the effect of D-APV upon NR2A subunits is restricted to layer I during the first 2 h of surface application of the drug.…”

“…Asymmetric synapses were presumed to be excitatory, and thus glutamatergic (DeFelipe et al, 1988;Aoki et al, 1994;Kharazia and Weinberg, 1999). An asymmetric synapse was identified as such using the following criteria: presence of parallelaligned plasma membranes, denoting the synaptic cleft; the presence of vesicles, indicating that the juxtaposed profile was presynaptic; and the presence of electron-densities without vesicles, denoting the postsynaptic membrane.…”

Visual Deprivation Modifies Both Presynaptic Glutamate Release and the Composition of Perisynaptic/Extrasynaptic NMDA Receptors in Adult Visual Cortex

“…A possible complication to our interpretation of the data is that presynaptic NR2B-containing NMDARs are known to exist (Aoki et al, 1994) and to enhance neurotransmitter release in the visual cortex (Sjostrom et al, 2003). The deprivation-induced elevation in NR2B proteins within the lysed synaptic membrane fraction (Fig.…”

A Role for Nitric Oxide in the Development of the Ferret Retinogeniculate Projection

“…Sublamination requires postsynaptic activation of NMDA receptors, which are present and involved in synaptic potentiation in the developing LGN (Mooney et al, 1993). Whereas presynaptic NMDA receptors may be present in visual cortex (Aoki et al, 1994), there is no evidence for their presence in the LGN. Physiological evidence suggests that NMDA receptors are postsynaptic, because holding postsynaptic LGN cells at hyperpolarizing membrane potentials reduces the NMDA component of the postsynaptic current (Esguerra et al, 1992;Ramoa and McCormick, 1994).…”