If we are to stand any chance of understanding the circuitry of the superficial dorsal horn, it is imperative that we can identify which classes of interneuron are excitatory and which are inhibitory. Our aim was to test the hypothesis that there is a correlation between the morphology of an interneuron and its postsynaptic action. We used in vitro slice preparations of the rat spinal cord to characterize and label interneurons in laminae I-III with Neurobiotin. Labelled cells (n = 19) were reconstructed in 3D with Neurolucida and classified according to the scheme proposed by Grudt & Perl (2002). We determined if cells were inhibitory or excitatory by reacting their axon terminals with antibodies to reveal glutamate decrboxylase (for GABAergic cells) or the vesicular glutamate transporter 2 (for glutamatergic cells). All five islet cells retrieved were inhibitory. Of the six vertical (stalked) cells analysed, four were excitatory and, surprisingly, two were inhibitory. It was noted that these inhibitory cells had axonal projections confined to lamina II whereas excitatory vertical cells projected to lamina I and II. Of the remaining neurons, three were radial cells (2 inhibitory, 1 excitatory), two were antennae cells (1 inhibitory, 1 excitatory), one was an inhibitory central cell and the remaining two were unclassifiable excitatory cells. Our hypothesis appears to be correct only for islet cells. Other classes of cells have mixed actions, and in the case of vertical cells, the axonal projection appears to be a more important determinant of postsynaptic action.
Following systemic injection, several different dyes and markers are found to accumulate rapidly in cells in the arcuate nucleus and median eminence, and the capillaries in this region appear specialised for exchange of molecules. The present study used hydroxystilbamidine (FluoroGold equivalent) to identify cells that take up molecules from the circulation in these regions; 2-6 h following injection, uptake was seen in the external and intermediate zones of the median eminence and the adjacent ventral part of the arcuate nucleus, but not in other regions of the hypothalamus. The labelled cells were small; double-labelling experiments revealed that they expressed glial fibrillary acid protein (GFAP), but not NeuN, Agouti-related protein (AgRP) or beta-endorphin. They had the morphology of astrocytes and were readily distinguished from tanycytes by staining for vimentin. Many of these labelled astrocytes also expressed leptin receptors and neuropeptide Y Y1 receptors. The surrounding neurons that expressed these receptors did not take up this dye. This demonstrates that astrocytes take up molecules from the circulation in the median eminence and adjacent arcuate nucleus, and may have a significant signalling role in regulation of food intake.
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