Estradiol Acts in Lateral Thalamic Region to Attenuate Varicella Zoster Virus Associated Affective Pain.

Varicella zoster virus (VZV) results in chicken pox and herpes zoster. Female rats show a higher level of herpes zoster associated pain than males, consistent with human studies. In this study, we addressed the novel hypothesis that sex difference in herpes zoster associated pain is due, in part, to estradiol modulating activity in the thalamus. To test this hypothesis a high and low physiological dose of estradiol was administered to castrated and ovariectomized rats and the affective pain response was measured after injection of VZV into the whisker pad. Thalamic infusion of the estrogen receptor antagonist ICI 182,780 concomitant with a high dose of estradiol addressed the role of estradiol binding to its receptor to effect pain. Phosphorylated extracellular signal-regulated protein kinase (pERK) positive cells were measured in excitatory (glutaminase positive) and inhibitory (glutamate decarboxylase 67 positive) cells of the lateral thalamic region. Our results show that a high dose of estradiol significantly reduced the pain response in both males and females. pERK significantly increased in excitatory cells after treatment with a low dose of estradiol and increased in inhibitory cells after treatment with a high dose of estradiol. Administration of ICI 182,780 significantly increased the pain response, reduced expression of GABA related genes in the thalamic region and significantly reduced the number of inhibitory cells expressing pERK. The results suggest that estradiol attenuates herpes zoster pain by increasing the activity of inhibitory neurons within the thalamus and that this reduction includes an estrogen receptor dependent mechanism.

Aromatase Derived Estradiol Within the Thalamus Modulates Pain Induced by Varicella Zoster Virus.

Herpes zoster or shingles is the result of varicella zoster virus (VZV) infection and often results in chronic pain that lasts for months after visible symptoms subside. Testosterone often attenuates pain in males. Previous work demonstrates ovarian estrogen effects γ-aminobutyric acid (GABA) signaling in the thalamus, reducing pain but the role of testosterone within the thalamus is currently unknown. Because aromatase affects pain and is present in the thalamus we tested a hypothesis that testosterone converted to estrogen in the thalamus attenuates herpes zoster induced pain. To address this hypothesis, male Sprague-Dawley rats received whisker pad injection of either MeWo cells or MeWo cells containing VZV. To reduce aromatase derived estrogen in these animals we injected aromatase inhibitor letrozole systemically or infused it into the thalamus. To test if estrogen was working through the estrogen receptor (ER) agonist, 4, 4', 4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) was infused concomitant with letrozole. Motivational and affective pain was measured after letrozole and/or PPT treatment. Vesicular GABA transporter (VGAT) is important in pain signaling. Because estrogen effects VGAT expression we measured its transcript and protein levels after letrozole treatment. Virus injection and letrozole significantly increased the pain response but thalamic infusion of PPT reduced zoster pain. Letrozole increased the number of thalamic neurons staining for phosphorylated ERK (pERK) but decreased VGAT expression. The results suggest in male rats aromatase derived estradiol interacts with the ER to increase VGAT expression and increase neuronal inhibition in the thalamus to attenuate VZV induced pain.

Gi protein functions in thalamic neurons to decrease orofacial nociceptive response.

Orofacial pain includes neuronal pathways that project from the trigeminal nucleus to and through the thalamus. What role the ventroposterior thalamic complex (VP) has on orofacial pain transmission is not understood. To begin to address this question an inhibitory G protein (Gi) designer receptor exclusively activated by a designer drug (DREADD) was transfected in cells of the VP using adeno-associated virus isotype 8. Virus infected cells were identified by a fluorescent tag and immunostaining. Cells were silenced after injecting the designer drug clozapine-n-oxide, which binds the designer receptor activating Gi. Facial rubbing and local field potentials (LFP) in the VP were then recorded in awake, free moving Sprague Dawley rats after formalin injection of the masseter muscle to induce nociception. Formalin injection significantly increased LFP and the nociceptive behavioral response. Activation of DREADD Gi with clozapine-n-oxide significantly reduced LFP in the VP and reduced the orofacial nociceptive response. Because DREADD silencing can result from Gi-coupled inwardly-rectifying potassium channels (GIRK), the GIRK channel blocker tertiapin-Q was injected. Injection of GIRK blocker resulted in an increase in the nociceptive response and increased LFP activity. Immunostaining of the VP for glutamate vesicular transporter (VGLUT2) and gamma-aminobutyric acid vesicular transporter (VGAT) indicated a majority of the virally transfected cells were excitatory (VGLUT2 positive) and a minority were inhibitory (VGAT positive). We conclude first, that inhibition of the excitatory neurons within the VP reduced electrical activity and the orofacial nociceptive response and that the effect on excitatory neurons overwhelmed any change resulting from inhibitor neurons. Second, inhibition of LFP and nociception was due, in part, to GIRK activation.

Corrigendum: Aromatase Derived Estradiol Within the Thalamus Modulates Pain Induced by Varicella Zoster Virus.

[This corrects the article DOI: 10.3389/fnint.2018.00046.].

Role for the Ventral Posterior Medial/Posterior Lateral Thalamus and Anterior Cingulate Cortex in Affective/Motivation Pain Induced by Varicella Zoster Virus.

Varicella zoster virus (VZV) infects the face and can result in chronic, debilitating pain. The mechanism for this pain is unknown and current treatment is often not effective, thus investigations into the pain pathway become vital. Pain itself is multidimensional, consisting of sensory and affective experiences. One of the primary brain substrates for transmitting sensory signals in the face is the ventral posterior medial/posterior lateral thalamus (VPM/VPL). In addition, the anterior cingulate cortex (ACC) has been shown to be vital in the affective experience of pain, so investigating both of these areas in freely behaving animals was completed to address the role of the brain in VZV-induced pain. Our lab has developed a place escape avoidance paradigm (PEAP) to measure VZV-induced affective pain in the orofacial region of the rat. Using this assay as a measure of the affective pain experience a significant response was observed after VZV injection into the whisker pad and after VZV infusion into the trigeminal ganglion. Local field potentials (LFPs) are the summed electrical current from a group of neurons. LFP in both the VPM/VPL and ACC was attenuated in VZV injected rats after inhibition of neuronal activity. This inhibition of VPM/VPL neurons was accomplished using a designer receptor exclusively activated by a designer drug (DREADD). Immunostaining showed that cells within the VPM/VPL expressed thalamic glutamatergic vesicle transporter-2, NeuN and DREADD suggesting inhibition occurred primarily in excitable neurons. From these results we conclude: (1) that VZV associated pain does not involve a mechanism exclusive to the peripheral nerve terminals, and (2) can be controlled, in part, by excitatory neurons within the VPM/VPL that potentially modulate the affective experience by altering activity in the ACC.

Sex differences underlying orofacial varicella zoster associated pain in rats.

BACKGROUND: Most people are initially infected with varicella zoster virus (VZV) at a young age and this infection results in chickenpox. VZV then becomes latent and reactivates later in life resulting in herpes zoster (HZ) or "shingles". Often VZV infects neurons of the trigeminal ganglia to cause ocular problems, orofacial disease and occasionally a chronic pain condition termed post-herpetic neuralgia (PHN). To date, no model has been developed to study orofacial pain related to varicella zoster. Importantly, the incidence of zoster associated pain and PHN is known to be higher in women, although reasons for this sex difference remain unclear. Prior to this work, no animal model was available to study these sex-differences. Our goal was to develop an orofacial animal model for zoster associated pain which could be utilized to study the mechanisms contributing to this sex difference. METHODS: To develop this model VZV was injected into the whisker pad of rats resulting in IE62 protein expression in the trigeminal ganglia; IE62 is an immediate early gene in the VZV replication program. RESULTS: Similar to PHN patients, rats showed retraction of neurites after VZV infection. Treatment of rats with gabapentin, an agent often used to combat PHN, ameliorated the pain response after whisker pad injection. Aversive behavior was significantly greater for up to 7 weeks in VZV injected rats over control inoculated rats. Sex differences were also seen such that ovariectomized and intact female rats given the lower dose of VZV showed a longer affective response than male rats. The phase of the estrous cycle also affected the aversive response suggesting a role for sex steroids in modulating VZV pain. CONCLUSIONS: These results suggest that this rat model can be utilized to study the mechanisms of 1) orofacial zoster associated pain and 2) the sex differences underlying zoster associated pain.

Lateral thalamic control of nociceptive response after whisker pad injection of varicella zoster virus.

Pain is a common complication of herpes zoster (HZ) infection which results from reactivation of a latent varicella zoster virus (VZV). A third of HZ patients' progress to a chronic pain state known as post herpetic neuralgia (PHN), and about a quarter of these patients' have orofacial pain. The mechanisms controlling the pain responses are not understood. Studies suggest central pathways involving the thalamus could control pain related to HZ, and studies in our lab suggest (VGAT) in the lateral thalamus influences orofacial pain. We hypothesized that thalamic VGAT functions, in part, to reduce pain, particularly orofacial pain, associated with VZV. To address this hypothesis VZV was injected into the whisker pad. Affective and motivational aspects of pain were measured using the Place Escape/Avoidance Paradigm. Thalamic neuronal activity was modulated after injecting an adeno-associated virus (AAV) expressing an engineered acetylcholine Gi-protein-coupled receptor. This receptor inhibits neuronal firing when bound by clozapine-n-oxide (CNO). VGAT expression was attenuated in the thalamus by injecting an AAV construct that expressed a VGAT silencing shRNA. VZV-induced nociception was significantly decreased after administering CNO in male rats. Nociception significantly increased concomitant with increased thalamic c-fos expression after attenuating thalamic VGAT expression. These data establish that the lateral thalamus (posterior, ventral posteromedial, ventral posterolateral and/or reticular thalamic nucleus) controls VZV-induced nociception in the orofacial region, and that GABA in this region appears to reduce the response to VZV-induced nociception possibly by gating facial pain input.

Genes in the GABA Pathway Increase in the Lateral Thalamus of Sprague-Dawley Rats During the Proestrus/Estrus Phase.

Pain can vary over the estrous cycle as a result of changes in estradiol concentration but the mechanism causing this variation is unclear. Because the thalamus is important in pain control, gene expression in the lateral thalamus (ventral posteromedial, ventral posterolateral, reticular thalamic nuclei) was screened at different phases of the estrous cycle. Gene expression changes in Sprague-Dawley rats were further analyzed by real-time PCR and ELISA and plasma estradiol levels were measured by RIAs at different phases of the estrous cycle. Our results indicated that both the RNA and protein expression of glutamate decarboxylase 1 and 2 (GAD1, GAD2), GABA(A) receptor-associated protein like 1 (GABARAPL1), and vesicular GABA transporter (VGAT) significantly increased in the lateral thalamus when plasma estradiol levels were elevated. Estradiol levels were elevated during the proestrus and estrus phases of the estrous cycle. Estrogen receptor α (ERα) was observed to be co-localized in thalamic cells and thalamic infusion of an ERα antagonist significantly reduced GAD1 and VGAT transcript. GAD1, GAD2, GABARAPL1, and VGAT have been shown to effect neuronal responses suggesting that attenuation of pain during the estrous cycle can be dependent, in part, through estradiol induced changes in thalamic gene expression.