Comparing Gene Expression in the Parabrachial and Amygdala of Diestrus and Proestrus Female Rats after Orofacial Varicella Zoster Injection.

The orofacial pain pathway projects to the parabrachial and amygdala, and sex steroids have been shown to affect neuronal activity in these regions. GABA positive cells in the amygdala are influenced by sex steroid metabolites to affect pain, and sex steroids have been shown to alter the expression of genes in the parabrachial, changing neuronal excitability. Mechanisms by which sex steroids affect amygdala and parabrachial signaling are unclear. The expression of genes in the parabrachial and amygdala in diestrus (low estradiol) and proestrus (high estradiol) female rats were evaluated in this study. First, varicella zoster virus was injected into the whisker pad of female rats to induce a pain response. Second, gene expression was quantitated using RNA-seq one week after injection. Genes that had the greatest change in expression and known to function in pain signaling were selected for the quantitation of protein content. Protein expression of four genes in the parabrachial and seven genes in the amygdala were quantitated by ELISA. In the parabrachial, neurexin 3 (Nrnx3) was elevated at proestrus. Nrnx3 has a role in AMPA receptor and GABA signaling. Neuronatin (Nnat) and protein phosphatase, Mg2+/Mn2+ dependent 1E (Ppm1e) were elevated in the parabrachial of diestrus animals both genes having a role in pain signaling. Epoxide hydroxylase (Ephx2) was elevated in the parabrachial at proestrus and the vitamin D receptor (Vdr) was elevated in the amygdala. Ephx2 antagonists and vitamin D have been used to treat neuropathic pain. In conclusion, sex steroids regulate genes in the parabrachial and amygdala that might result in the greater pain response observed during diestrus.

Measuring calcium activity within individual neurons within the rat thalamus.

Detailed methods for imaging calcium activity in single cells within the ventral posteromedial thalamic nucleus of the rat was completed for the first time in these studies. These methods also detail the procedure to image calcium activity in individual GABAergic neurons within the reticular thalamic nucleus using GAD1-Cre rats. This activity was measured in freely behaving rats allowing for recording of activity from GABA neurons during behavioral testing. Key methods for imaging success are:•Calcium activity in the lateral thalamic region is inhibited by isoflurane anesthesia and GCAMP florescent cells are often not observed when mounting the baseplate.•If no cells are observed when mounting the baseplate then place the lens at 300 micrometers or focus on a blood vessel if present.•Depending on the virus, a one microliter infusion could be needed to produce a one millimeter field of GCAMP positive cells for imaging with a lens having a one millimeter diameter.

Reduced activity of GAD67 expressing cells in the reticular thalamus enhance thalamic excitatory activity and varicella zoster virus associated pain.

Within the reticular thalamic nucleus neurons express gamma aminobutyric acid (GABA) and these cells project to the ventral posteromedial thalamic nucleus. When GABA activity decreases the activity of excitatory cells in the ventral posteromedial nucleus would be expected to increase. In this study, we addressed the hypothesis that attenuating GABAergic cells in the reticular thalamic nucleus increases excitatory activity in the ventral posteromedial nucleus increasing varicella zoster virus (VZV) associated pain in the orofacial region. Adeno-associated virus (AAV) was infused in the reticular thalamic nucleus of Gad1-Cre rats. This virus transduced a G inhibitory designer receptor exclusively activated by designer drugs (DREADD) gene that was Cre dependent. A dose of estradiol that was previously shown to reduce VZV pain and increase GABAergic activity was administered to castrated and ovariectomized rats. Previous studies suggest that estradiol attenuates herpes zoster pain by increasing the activity of inhibitory neurons and decreasing the activity of excitatory cells within the lateral thalamic region. The ventral posteromedial nucleus was infused with AAV containing a GCaMP6f expression construct. A glass lens was implanted for miniscope imaging. Our results show that the activity of GABA cells within the reticular thalamic region decreased with clozapine N-oxide treatment concomitant with increased calcium activity of excitatory cells in the ventral posteromedial nucleus and an increased orofacial pain response. The results suggest that estradiol attenuates herpes zoster pain by increasing the activity of inhibitory neurons within the reticular thalamus that then inhibit excitatory activity in ventral posteromedial nucleus causing a reduction in orofacial pain.