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The synthesis enzyme glutamic acid decarboxylase (GAD65 or GAD67) identifies neurons as GABAergic. Recent studies have characterized the physiological properties of spinal cord GABAergic interneurons using lines of GAD67-green fluorescent protein (GFP) transgenic mice. A more complete characterization of their phenotype is required to better understand the role of this population of inhibitory neurons in spinal cord function. Here, we characterize the distribution of lumbar spinal cord GAD67-GFP neurons at postnatal days (P) 0, 7, and 14, and adult based on their co-expression with GABA and determine the molecular phenotype of GAD67-GFP neurons at P14 based on the expression of various neuropeptides, calcium binding proteins, and other markers. At all ages >67% of GFP(+) neurons were also GABA(+). With increasing age; (i) GFP(+) and GABA(+) cell numbers declined, (ii) ventral horn GFP(+) and GABA(+) neurons vanished, and (iii) somatic labeling was reduced while terminal labeling increased. At P14, vasoactive intestinal peptide and bombesin were expressed in approximately 63% and approximately 35% of GFP(+) cells, respectively. Somatostatin was found in a small number of neurons, whereas calcitonin gene-related peptide never co-localized with GFP. Moderate co-expression was found for all the Ca(2+) binding proteins examined. Notably, most laminae I-II parvalbumin(+) neurons were also GFP(+). Neurogranin, a protein kinase C substrate, was found in approximately 1/2 of GFP(+) cells. Lastly, while only 7% of GFP(+) cells contain nitric oxide synthase (NOS), these cells represent a large fraction of all NOS(+) cells. We conclude that GAD67-GFP neurons represent the majority of spinal GABAergic neurons and that mouse dorsal horn GAD67-GFP(+) neurons comprise a phenotypically diverse population.

Dopamine is a catecholaminergic neuromodulatory transmitter that acts through five molecularly-distinct G protein–coupled receptor subtypes (D1–D5). In the mammalian spinal cord, dopaminergic axon collaterals arise predominantly from the A11 region of the dorsoposterior hypothalamus and project diffusely throughout the spinal neuraxis. Dopaminergic modulatory actions are implicated in sensory, motor and autonomic functions in the spinal cord but the expression properties of the different dopamine receptors in the spinal cord remain incomplete. Here we determined the presence and the regional distribution of all dopamine receptor subtypes in mouse spinal cord cells by means of quantitative real time polymerase chain reaction (PCR) and digoxigenin-label in situ hybridization. Real-time PCR demonstrated that all dopamine receptors are expressed in the spinal cord with strongly dominant D2 receptor expression, including in motoneurons and in the sensory encoding superficial dorsal horn (SDH). Laser capture microdissection (LCM) corroborated the predominance of D2 receptor expression in SDH and motoneurons. In situ hybridization of lumbar cord revealed that expression for all dopamine receptors was largely in the gray matter, including motoneurons, and distributed diffusely in labeled cell subpopulations in most or all laminae. The highest incidence of cellular labeling was observed for D2 and D5 receptors, while the incidence of D1 and D3 receptor expression was least. We conclude that the expression and extensive postsynaptic distribution of all known dopamine receptors in spinal cord correspond well with the broad descending dopaminergic projection territory supporting a widespread dopaminergic control over spinal neuronal systems. The dominant expression of D2 receptors suggests a leading role for these receptors in dopaminergic actions on postsynaptic spinal neurons.

Neuronal dysfunction and cell death in patients with human immunodeficiency virus type-1 (HIV-1) infection may be mediated by HIV-1 proteins and products released from infected cells. Two HIV-1 proteins, the envelope glycoprotein gp120 and nonstructural protein Tat, are neurotoxic. We have determined the neuroexcitatory properties of HIV-1 tat protein using patch-clamp recording techniques. When fmoles of Tat were applied extracellularly, it elicited dose-dependent depolarizations of human fetal neurons in culture and rat CA1 neurons in slices, both in the absence and presence of tetrodotoxin. These responses were voltage-dependent, reversed at approximately 0 mV, and were significantly increased by repetitive applications with no evidence of desensitization. That these responses to Tat were due to direct actions on neurons was supported by observations that Tat dose-dependently depolarized outside-out patches excised from cultured human neurons. Removal of extracellular Ca2+ decreased the responses both in neurons and membrane patches. This is the first demonstration that an HIV-1 protein can, in the absence of accessory cells, directly excite neurons and leads us to speculate that Tat may be a causative agent in HIV-1 neurotoxicity.