Spinal nerve ligation decreases γ‐aminobutyric acidB receptors on specific populations of immunohistochemically identified neurons in L5 dorsal root ganglion of the rat

Loss of γ-aminobutyric acid (GABA)-mediated inhibition in the dorsal horn of the spinal cord has long been proposed to contribute to the development or maintenance of spontaneous pain behaviors, mechanical allodynia, and thermal hyperalgesia after peripheral nerve injury (reviewed by Hammond, 1997; Sandkuhler, 2009; Todd, 2010). Studies of the disposition and release of GABA in the dorsal horn have yielded contradictory neuroanatomical (Ibuki et al., 1997; Eaton et al., 1998; Polgar et al., 2003; Scholz et al., 2005) and neurochemical findings (Stiller et al., 1996; Somers and Clemente, 2002; Lever et al., 2003). However, loss of GABA-mediated inhibition can occur independently of changes in neurotransmitter release, e.g., by a decrease in the affinity, number, or function of the receptors at which GABA acts. Of these, the heterodimeric GABAB receptor (Bettler and Tiao, 2006; Padgett and Slesinger, 2010; Pinard et al., 2010) is particularly well situated to modulate synaptic transmission in the dorsal horn. Activation of postsynaptic GABAB receptors hyperpolarizes dorsal horn neurons (Kangrga et al., 1991; Yang et al., 2001) and inhibits their responses to noxious stimuli (Willcockson et al., 1984), while activation of presynaptic GABAB receptors (Price et al., 1987; Yang et al., 2002) inhibits the release of peptide and excitatory amino acid neurotransmitters from the central terminals of primary afferents in the dorsal horn (Go and Yaksh, 1987; Kangrga et al., 1991; Malcangio and Bowery, 1993; Ataka et al., 2000; Iyadomi et al., 2000; Wang et al., 2007). Several observations support the proposal that a decrease in the number, affinity, or coupling of the GABAB receptor in the dorsal horn may underlie the allodynia and hyperalgesia that occur after peripheral nerve injury. Pharmacological antagonism of spinal GABAB receptors (Hao et al., 1994) produces mechanical allodynia. Deletion of the gene for either subunit of the GABAB receptor (Schuler et al., 2001; Gassmann et al., 2004) produces hyperalgesia. Conditional deletion of the GABAB(1) subunit in C and Aδ primary afferent fibers results in exacerbated responses of Aδ fibers to mechanical stimuli, although this finding does not translate to enhanced mechanical allodynia or hyperalgesia after nerve injury (Gangadharan et al., 2009). Finally, streptozocin-induced diabetic neuropathy is associated with a loss of mRNA and protein for the GABAB(1) subunit in the dorsal horn (Wang et al., 2011), and a diminution of the presynaptic inhibitory actions of baclofen on glutamatergic, but not GABAergic or glycinergic afferents (Wang et al., 2007). The present study therefore examined whether the number of primary afferent neurons that express the GABAB receptor is decreased after L5 spinal nerve ligation (SNL), a rodent model of nerve injury that results in our hands in mechanical allodynia and spontaneous pain behaviors (Engle et al., 2006). Indirect immunofluorescence methods were used to examine the distribution of GABAB(1) subunit immunoreactivity among three different classes of primary afferent neuron in the dorsal root ganglion (DRG) of naive rats and rats that underwent sham surgery or L5 SNL. Myelinated afferents were identified by their immunoreactivity for neurofilament 200 using the N52 monoclonal antibody (Robertson et al., 1991), while unmyelinated afferents were identified by their labeling with isolectin B4 (IB4) from Griffonia simplicifonia (Kitchener et al., 1993; Wang et al., 1998) and peptidergic afferents were identified by their immunoreactivity for calcitonin gene-related peptide (CGRP). The optical fractionator method was used to obtain an unbiased estimate of the number of GABAB(1)-immunoreactive (IR) neurons, while area measurements of GABAB(1)-IR profiles identified possible changes in the distribution of cell size among treatment groups. The immunohistochemical analysis was restricted to the GABAB(1) subunit because levels of GABAB(2) protein were below detectable limits in the DRG (McCarson and Enna, 1999; Engle et al., 2006). Finally, quantitative real-time polymerase chain reaction (RT-PCR) was used to determine levels of GABAB(1) and GABAB(2) subunit mRNA in the L4 and L5 DRG of naive, sham-operated and ligated rats.