VGLUT1 and VGLUT2 innervation in autonomic regions of intact and transected rat spinal cord.

Fast excitatory neurotransmission to sympathetic and parasympathetic preganglionic neurons (SPN and PPN) is glutamatergic. To characterize this innervation in spinal autonomic regions, we localized immunoreactivity for vesicular glutamate transporters (VGLUTs) 1 and 2 in intact cords and after upper thoracic complete transections. Preganglionic neurons were retrogradely labeled by intraperitoneal Fluoro-Gold or with cholera toxin B (CTB) from superior cervical, celiac, or major pelvic ganglia or adrenal medulla. Glutamatergic somata were localized with in situ hybridization for VGLUT mRNA. In intact cords, all autonomic areas contained abundant VGLUT2-immunoreactive axons and synapses. CTB-immunoreactive SPN and PPN received many close appositions from VGLUT2-immunoreactive axons. VGLUT2-immunoreactive synapses occurred on Fluoro-Gold-labeled SPN. Somata with VGLUT2 mRNA occurred throughout the spinal gray matter. VGLUT2 immunoreactivity was not noticeably affected caudal to a transection. In contrast, in intact cords, VGLUT1-immunoreactive axons were sparse in the intermediolateral cell column (IML) and lumbosacral parasympathetic nucleus but moderately dense above the central canal. VGLUT1-immunoreactive close appositions were rare on SPN in the IML and the central autonomic area and on PPN. Transection reduced the density of VGLUT1-immunoreactive axons in sympathetic subnuclei but increased their density in the parasympathetic nucleus. Neuronal cell bodies with VGLUT1 mRNA occurred only in Clarke's column. These data indicate that SPN and PPN are densely innervated by VGLUT2-immunoreactive axons, some of which arise from spinal neurons. In contrast, the VGLUT1-immunoreactive innervation of spinal preganglionic neurons is sparse, and some may arise from supraspinal sources. Increased VGLUT1 immunoreactivity after transection may correlate with increased glutamatergic transmission to PPN.

Immunoreactivity for cocaine- and amphetamine-regulated transcript in rat sympathetic preganglionic neurons projecting to sympathetic ganglia and the adrenal medulla.

Many sympathetic preganglionic neurons (SPN) in the intermediolateral cell column (IML) contain cocaine- and amphetamine-regulated transcript (CART), but the function of these CART-immunoreactive (IR) neurons is unknown. To test the possibility that CART might mark SPN involved in cardiovascular regulation, we first established whether all CART neurons in the spinal cord were SPN by double-immunofluorescent labelling for CART and choline acetyltransferase (ChAT). All autonomic subnuclei contained SPN immunoreactive for ChAT plus CART. Occasional ChAT-negative, CART-positive neurons occurred adjacent to the IML, indicating the existence of CART-IR interneurons. We then retrogradely labelled SPN with cholera toxin subunit B (CTB) from a variety of targets and used double immunofluorescence to detect CTB and CART. Among SPN in the IML, 43% projecting to the coeliac ganglion, 34% projecting to the major pelvic ganglion, and about 15% projecting to the superior cervical ganglion or adrenal medulla contained CART. CART also occurred in most SPN projecting to the major pelvic ganglion from either the central autonomic area (63%) or the intercalated nucleus (58%). Finally, we used drug-induced hypotension in conscious rats to evoke Fos immunoreactivity in barosensitive SPN and immunostained to reveal Fos and CART. CART immunoreactivity was present in 41% of the Fos-IR barosensitive neurons, which were concentrated in the IML of segments T5-T13. CART-positive, Fos-negative neurons also occurred in the same segments. These results indicate that CART occurs in barosensitive SPN, nonbarosensitive SPN, and interneurons. Thus, CART is not an exclusive marker for cardiovascular SPN but is likely to influence many autonomic activities.