Zhang, Liang, Kenneth T. Sykes, Amber V. Buhler, and Donna L. Hammond. Electrophysiological heterogeneity of spinally projecting serotonergic and nonserotonergic neurons in the rostral ventromedial medulla. J Neurophysiol 95: 1853-1863, 2006. First published December 7, 2005 doi:10.1152/jn.00883.2005. This study examined the passive membrane and action potential properties of serotonergic and nonserotonergic neurons in the rostral ventromedial medulla (RVM) of the rat using whole cell patch-clamp recording techniques in the slice. Serotonergic neurons were identified by immunoreactivity for tryptophan hydroxylase (TrpH). Spinally projecting neurons were retrogradely labeled with 1Ј-dioactadecyl-3,3,3Ј,3Ј-tetramethylindocarbodyanine perchlorate (DiI). Three types of neurons were identified within both spinally projecting serotonergic and nonserotonergic populations. Type 1 neurons exhibited irregular or sporadic spontaneous activity interspersed with periods of quiescence. Type 2 neurons were not spontaneously active and were additionally discriminated by a more negative resting membrane potential and a largeramplitude action potential. Type 3 neurons fired repetitively without pause. Serotonergic neurons had a higher membrane resistance and greater action potential half-width than their nonserotonergic counterparts and rarely exhibited a fast afterhyperpolarization. Serotonergic type 3 neurons also fired more slowly and regularly than nonserotonergic type 3 neurons. Comparison of electrophysiological and immunohistochemical characteristics suggested that the smallest type 3 serotonergic neurons had an increased risk of immunohistochemical "misclassification" due to failure to detect TrpH, possibly due to more complete dialysis of intracellular contents during lengthy recordings. This risk was minimal for type 1 or 2 serotonergic neurons. The three different types of spinally projecting serotonergic neurons also differed markedly in their responsiveness to the mu opioid receptor agonist D-Ala 2 , NMePhe 4 , Gly 5 -ol]enkephalin. These results provide important new electrophysiological and pharmacological evidence for a significant heterogeneity among spinally projecting serotonergic RVM neurons. They may also provide a basis for resolving the controversy concerning the role of serotonergic RVM neurons in opioid analgesia.

Loss of GABA-mediated inhibition in the spinal cord is thought to mediate allodynia and spontaneous pain after nerve injury. Despite extensive investigation of GABA itself, relatively little is known about how nerve injury alters the receptors at which GABA acts. This study examined levels of GABA(B) receptor protein in the spinal cord dorsal horn, and in the L4 and L5 (lumbar designations) dorsal root ganglia one to 18 weeks after L5 spinal nerve ligation. Mechanical allodynia was maximal by 1 week and persisted at blunted levels for at least 18 weeks after injury. Spontaneous pain behaviors were evident for 6 weeks. Western blotting of dorsal horn detected two isoforms of the GABA(B(1)) subunit and a single GABA(B(2)) subunit. High levels of GABA(B(1a)) and low levels of GABA(B(1b)) protein were present in the dorsal root ganglia. However, GABA(B(2)) protein was not detected in the dorsal root ganglia, consistent with the proposed existence of an atypical receptor composed of GABA(B(1)) homodimers. The levels of GABA(B(1a)), GABA(B(1b)), and GABA(B(2)) protein in the ipsilateral dorsal horn were unchanged at any time after injury. Immunohistochemical staining also did not detect a change in GABA(B(1)) or GABA(B(2)) subunits in dorsal horn segments having a robust loss of isolectin B4 staining. The levels of GABA(B(1a)) protein were also unchanged in the L4 or L5 dorsal root ganglia at any time after spinal nerve ligation. Levels of GABA(B(2)) remained undetectable. Finally, baclofen-stimulated binding of guanosine-5'-(gamma-O-thio)triphosphate in dorsal horn did not differ between sham and ligated rats. Collectively, these results argue that a loss of GABA(B) receptor-mediated inhibition, particularly of central terminals of primary afferents, is unlikely to mediate the development or maintenance of allodynia or spontaneous pain behaviors after spinal nerve injury.

Peripheral inflammatory injury enhances the antinociceptive or antihyperalgesic effects of -opioid receptor (MOR) and ␦-opioid receptor (DOR) agonists after systemic administration, local injection into the hindpaw, or intrathecal administration in male, but not female rats (for review, see Stein, 1995;Hammond, 2004;Wang et al., 2006). This enhancement is attributed to increases in opioid receptor number, enhanced coupling of the receptor to G proteins, and also to increased trafficking of MOR or DOR to the plasma membrane. For example, intraplantar injection of complete Freund's adjuvant (CFA) or carrageenan increases both the number of MOR and efficiency with which MOR couples with