GABAergic neurons in the central region of the spinal cord: a novel substrate for sympathetic inhibition

Abstract

Homeostatic maintenance of widespread functions is critically dependent on the activity of the sympathetic nervous system. This activity is generated by the CNS acting on the sole output cells in the spinal cord, sympathetic preganglionic neurons (SPNs). SPNs are subject to control from both supraspinal and spinal inputs that exert effects through activation of direct or indirect pathways. A high proportion of indirect control is attributable to activation of spinal interneurons in a number of locations. However, little is known about the different groups of interneurons with respect to their neurochemistry or function. In this study, we report on a novel group of GABAergic interneurons located in the spinal central autonomic area (CAA) that directly inhibit SPN activity. In situ hybridization studies demonstrated a group of neurons that contained mRNA for glutamic acid decarboxylase (GAD)65 and GAD67 within the CAA. Combining in situ hybridization with trans-synaptic labeling from the adrenal gland using pseudorabies virus identified presympathetic GABAergic neurons in the CAA. Electrical stimulation of the CAA elicited monosynaptic IPSPs in SPNs located laterally in the intermediolateral cell column. IPSPs were GABAergic, because they reversed at the chloride reversal potential and were blocked by bicuculline. Chemical activation of neurons in the CAA hyperpolarized SPNs, an effect that was also bicuculline sensitive. We conclude that the CAA contains GABAergic interneurons that impinge directly onto SPNs to inhibit their activity and suggest that these newly identified interneurons may play an essential role in the regulation of sympathetic activity and thus homeostasis.

Figure 1.

In situ hybridization reveals the presence of GAD₆₅ and GAD₆₇ mRNA in adult and P12 rat spinal cord. A-D, Sections have been hybridized with antisense DIG-labeled riboprobes for either GAD₆₅ or GAD₆₇ and visualized with alkaline phosphatase reaction product. A, Section of P12 spinal cord indicating the presence of GAD₆₅ mRNA. Ai, Larger magnification of the boxed area in A indicating GAD₆₅ mRNA highly expressed in a compact group of neurons dorsal to the central canal. B, GAD₆₇ mRNA in P12 spinal cord. Bi, Larger magnification of the boxed area in B showing labeled neurons dorsal to the central canal. C, GAD₆₅ mRNA in adult rat spinal cord. Ci, Larger magnification of the boxed area in C indicating a compact group of labeled neurons dorsal to the central canal. D, GAD₆₇ mRNA in adult rat spinal cord. Di, Larger magnification of the boxed area in D indicating a group of labeled neurons dorsal to the central canal. Aii-Dii, Sections of spinal cord devoid of signal after hybridization with sense (control) DIG-labeled riboprobe for GAD₆₅ (Aii, Cii) and GAD₆₇ (Bii, Dii).

Figure 2.

Distribution and appearance of GAD₆₇ mRNA-containing cells infected with PRV. A, C, GAD₆₇ mRNA labeled with a DIG riboprobe (bright field). B, D, PRV labeled with antibody visualized with Cy3 (fluorescence). The arrows point to double-labeled neurons. The example shown in A and B is dorsal to the central canal. The example shown in C and D is located lateral to the central canal. cc, Central canal. Scale bar: (in D) A-D, 50 μm.

Figure 3.

Stimulation of the CAA elicits monosynaptic IPSPs in SPNs. Electrical stimulation of either the CAA (top) or the lateral funiculus (bottom) elicited IPSPs in an SPN held at -10 mV. CAA-evoked IPSPs were not affected by kynurenic acid in this SPN, whereas the LF-evoked IPSP was reduced in amplitude (averages of 10 sweeps). The areas stimulated are shown on the spinal cord slice, as is the region of recorded SPNs.

Figure 4.

IPSPs are not elicited by stimulation outside of the CAA. A, Stimulation within the white matter dorsal to the CAA in the presence of kynurenic acid did not elicit an IPSP (bottom trace), although LF stimulation did still elicit IPSPs in the same SPN (top trace). B, Stimulation of the contralateral IML/LF region did not elicit IPSPs (bottom trace), although CAA stimulation did still yield a response (top trace). Averages of 10 consecutive sweeps are shown. SPNs were held at -10 mV.

Figure 5.

CAA IPSPs reverse at the equilibrium potential for chloride. A, IPSPs elicited at different holding potentials decreased in amplitude as the membrane was hyperpolarized and were positive-going at -90 mV. These recordings were made in the presence of NBQX (10 μm) and AP-5 (50 μm). B, Graph of IPSP amplitude against holding potential. The line of best fit gives a reversal potential of -86.8 mV.

Figure 6.

CAA IPSPs are GABAergic in nature. A, Stimulation within the CAA or LF elicited IPSPs in the presence of kynurenic acid (marked as control). Bicuculline (5 μm) almost abolished the CAA-evoked response, and strychnine had little additional effect. In contrast, bicuculline reduced the amplitude of the LF-evoked IPSP, but strychnine together with bicuculline then abolished the remaining IPSP. B, CAA IPSPs were not significantly reduced by strychnine alone. All traces are averages of 10 consecutive sweeps. SPNs were held at -10 mV. C, Pooled data showing that CAA-evoked IPSPs are GABAergic (blocked by bicuculline) and the more complex pharmacology of IPSPs elicited by LF stimulation.

Figure 7.

Glutamate microinjections in the CAA hyperpolarize SPNs, and these effects are antagonized by bicuculline. A, Repeated microinjections of glutamate into the CAA caused reproducible hyperpolarizations in SPNs in the IML. In this cell, spikelets were also observed that are likely to be attributable to action potentials in an electrically coupled SPN. These spikelets were also reduced during glutamate application; this effect is not solely attributable to hyperpolarizing the membrane, because manual hyperpolarization to this level was not sufficient to reduce the frequency of spikelets in this cell. This indicated that the coupled SPN was also inhibited by glutamate microinjection. B, Glutamate microinjection in the CAA hyperpolarized this SPN in the IML. Bicuculline (10 μm) antagonized this hyperpolarization.