Distribution of NMDA receptor subunit proteins NR2A, 2B, 2C and 2D in rat brain.

The regional distribution of the NMDA receptor subunits NR2A, 2B, 2C and 2D was visualized in adult rat brain using the histo-blot technique with newly developed subunit-specific antisera. NR2A immunoreactivity was found in almost all regions of the brain, whereas NR2B staining was restricted to forebrain, and NR2D immunoreactivity to diencephalic, mesencephalic and brain stem structures. NR2C staining was confined to cerebellum, thalamus and olfactory bulb. Thus, NMDA receptors containing the NR2A subunit are likely to represent a receptor subtype predominant throughout the brain, while those containing the NR2B, NR2C or NR2D subunit represent more region-specific receptor subtypes. The regionally overlapping distribution of certain NR2 subunits points to the existence of NMDA receptors containing more than one NR2 subunit variant.

Photoaffinity labeling of the NMDA receptor.

The structure of NMDA receptors in situ has been probed with the novel photoaffinity ligand 125I-CGP 55802A. By covalently linking the radioactive high-affinity photolabel to NMDA receptors in bovine brain we have identified a protein of 175 kDa associated with the binding site for NMDA receptor agonists and competitive antagonists. Based on its molecular size the photolabeled protein is likely to correspond to the NR2A and/or NR2B subunit. The photoaffinity ligand will permit the assessment of regulatory changes in NMDA receptor subunit expression.

Axonal projections and synaptogenesis by supraspinal descending neurons in the spinal cord of the chick embryo.

Following the injection of horeseradish peroxidase (HRP) into the brachial spinal cord of the chick on embryonic day (E)4.5, retrogradely labeled neurons can be found in the brainstem (Okado and Oppenheim: Journal of Comparative Neurology 232: 143-161, 1985). By contrast, following high cervical spinal transection, functional (behavioral) deficits are not observed until E10 (Oppenheim: Journal of Comparative Neurology 160: 37-50, 1975). To determine whether this temporal difference between projections and function reflects a delay in synaptogenesis, we looked for the presence of anterogradely HRP-labeled pre-synaptic terminals in brachial cord following injection of HRP into the boundary between brainstem and spinal cord at ages between E3.5 and E7. HRP-labeled fibers were observed in the branchial cord by E4.5 and were diffusely distributed in the ventral and lateral marginal zones (presumptive ventral and lateral funiculi, respectively). Although some axo-dendritic and axo-somatic synapses were observed in the brachial cord prior to E6, the presynaptic profiles were always unlabeled by HRP and thus must originate from propriospinal sources. The first HRP-labeled supraspinal synapses were found in the ventral and lateral funiculi on E6. They contained several clear spherical synaptic vesicles and were axo-dendritic in nature. The cells of origin of the postsynaptic dendrites were determined by injecting HRP into the wing-bud to label the brachial motoneurons retrogradely and the presynaptic component was identified as supraspinal by HRP injections into the brainstem/spinal cord boundary to orthogradely label the descending fibers. Several double-labeled axo-dendritic synapses were found in the ventral and lateral funiculi of E6 brachial cord. Therefore, at least some descending supraspinal fibers make synapses directly onto motoneuron dendrites. We conclude that 1) there is a delay of about 1.5 days between the arrival of supraspinal fibers and synapse formation in the brachial cord, 2) the earliest synapses are axo-dendritic in nature, 3) at least some supraspinal fibers make direct contact with motoneuron dendrites as early as E6, and 4) synaptogenesis from propriospinal sources precedes that from supraspinal descending axons. These observations provide evidence indicating that the temporal difference between the onset of projections of supraspinal descending fibers and the onset of their function may be partly owing to delayed synaptogenesis.

Pathfinding by growth cones of commissural interneurons in the chick embryo spinal cord: a light and electron microscopic study.

To investigate putative axonal guidance mechanisms used by commissural interneurons in the chick embryo spinal cord, we have examined growth cone morphology, the microenvironment through which the growth cones advance, and interactions between growth cones and their surroundings. Growth cones of both early and late developing commissural interneurons were examined. The growth cones were visualized by injection of either horseradish peroxidase (HRP) or the fluorescent dye Di-I. Unlabelled growth cones as well as HRP-labelled growth cones were also examined by electron microscopy. The early developing growth cones project circumferentially without fasciculation until they reach the region of the longitudinal pathway in the contralateral ventral funiculus (CVF). In their trajectory towards the floor plate, axons exhibited elaborate growth cones with filopodia and lamellipodia. They projected between processes of neuroepithelial cells within abundant extracellular spaces. Upon arrival at the ipsilateral ventral funiculus, growth cones did not appear to contact preexisting longitudinal axons. Within the floor plate, the growth cones were less complex and lacked long filopodia and exhibited bulbous or varicose shapes with short processes. Electron microscopic observations of the floor plate at this stage revealed that there was only a small amount of extracellular space and that the basal portion of the floor plate cells were directionally oriented (polarized) in the transverse plane. It is of particular interest that contacts between growth cones and the basement membrane in the floor plate were often observed. When the growth cones reached the contralateral ventrolateral region, they again exhibited an elaborate morphology. Close contacts between growth cones and the preexisting contralateral longitudinal axons were observed. Growth cones advancing in the contralateral longitudinal pathway exhibited various shapes and were observed to contact other axons and processes of neuroepithelial cells. Most of the later developing growth cones of commissural cells exhibited lamellipodial shapes irrespective of their location along the circumferential trajectory. Electron microscopic observations revealed that these late developing growth cones always contacted or fasciculated with preexisting axons and that the cellular environment through which they grow is oriented in such a way that the growth cones appear to be guided in specific directions. Growth cones entering the CVF exhibited more elaborated shapes with ramified lamellipodia that made multiple contacts with preexisting longitudinal axons. The present results indicate that differential axonal guidance mechanisms may be employed along the pathway followed by spinal commissural interneurons and that axons and growth cones projecting along this pathway at different developmental stages employ different mechanisms for pathfinding and guidance.(ABSTRACT TRUNCATED AT 400 WORDS)