Reduced GABAA receptor α6 expression in the trigeminal ganglion alters inflammatory TMJ hypersensitivity

Abstract

Trigeminal ganglia neurons express the GABAA receptor subunit alpha 6 (Gabrα6) but the role of this particular subunit in orofacial hypersensitivity is unknown. In this report the function of Gabrα6 was tested by reducing its expression in the trigeminal ganglia and measuring the effect of this reduction on inflammatory temporomandibular joint (TMJ) hypersensitivity. Gabrα6 expression was reduced by infusing the trigeminal ganglia of male Sprague Dawley rats with small interfering RNA (siRNA) having homology to either the Gabrα6 gene (Gabrα6 siRNA) or no known gene (control siRNA). Sixty hours after siRNA infusion the rats received a bilateral TMJ injection of complete Freund’s adjuvant to induce an inflammatory response. Hypersensitivity was then quantitated by measuring meal duration, which lengthens when hypersensitivity increases. Neuronal activity in the trigeminal ganglia was also measured by quantitating the amount of phosphorylated ERK. Rats in a different group that did not have TMJ inflammation had an electrode placed in the spinal cord at the level of C1 sixty hours after siRNA infusion to record extracellular electrical activity of neurons that responded to TMJ stimulation. Our results show that Gabrα6 was expressed in both neurons and satellite glia of the trigeminal ganglia and that Gabrα6 positive neurons within the trigeminal ganglia have afferents in the TMJ. Gabrα6 siRNA infusion reduced Gabrα6 gene expression by 30% and significantly lengthened meal duration in rats with TMJ inflammation. Gabrα6 siRNA infusion also significantly increased p-ERK expression in the trigeminal ganglia of rats with TMJ inflammation and increased electrical activity in the spinal cord of rats without TMJ inflammation. These results suggest that maintaining Gabrα6 expression was necessary to inhibit primary sensory afferents in the trigeminal pathway and reduce inflammatory orofacial nociception.

Infusion of Gabrα6 siRNA into the trigeminal ganglia increased the myogenic orofacial nociceptive response of ovariectomized rats treated with 17β-estradiol

“…Animals were alert and mobile less than 5 minutes after removal from isoflurane. A 30-40% reduction in Gabrα6 expression within the TG has been verified in several studies using this exact method (Kramer and Bellinger, 2013; Puri et al, 2012). …”

“…Stereotaxic coordinates were established in a preliminary trial by injecting India ink through the cannulas followed by a postmortem histological examination and in all animals cannula placement was confirmed by x-ray (Puri et al, 2012). …”

“…The ratio of cationic PEI amines (N) to nucleic acid phosphates (P) was N/P=6. This amount of siRNA and PEI was based on a previous study where siRNA knockdown was performed in vivo (Puri et al, 2012). The control siRNA had no homology to any known gene and was identified as Silencer Negative Control #1 siRNA (5’-AGUACUGCUUACGAUACGGtt-3’, 5'-CCGUAUCGUAAGCAGUACUtt-3’) and the Gabrα6 siRNA sequence was sense (5’-GGAACGAUCCUGUACACCAtt-3’), anti-sense (3’-UGGUGUACAGGAUCGUUCCat-5’).…”

“…A subunit of the GABA A receptor, Gabrα6, was affected by 17 β-estradiol in the TG and Vc-C 1 (Puri et al, 2011). In addition, a later study found Gabrα6 positive neurons located in the TG projected to the temporomandibular joint (TMJ) (Puri et al, 2012). GABA A receptors are expressed in sensory neurons of the face and head and have been shown to have a role in modulating orofacial pain (Almond et al, 1996; Anderson et al, 2009; Cai et al, 2001; Cairns et al, 1999; Ginestal and Matute, 1993; Hayasaki et al, 2006; Kondo et al, 1995; Kramer and Bellinger, 2013; Viggiano et al, 2004; Vit et al, 2009).…”

“…This GABA can bind the GABA A receptor to induce an inhibiting chloride current (Hayasaki et al, 2006), whereas the application of a GABA A receptor antagonist to the TG reversed neuronal inhibition that results from enhancing GABA expression (Vit et al, 2009). Inhibition of Gabrα6 expression will increase cellular activation of TG neurons and increase the orofacial nociceptive response (Kramer and Bellinger, 2013; Puri et al, 2012). Increased in Gabrα6 expression (Puri et al, 2011) was shown to be concurrent with a decrease in the nociceptive response after administering a proestrus dose of estradiol (Kramer and Bellinger, 2009).…”