PAQR8 and PAQR9 expression is altered in the ventral tegmental area of aged rats infected with varicella zoster virus.

Infection with varicella zoster virus (VZV) results in chicken pox and reactivation of VZV results in herpes zoster (HZ) or what is often referred to as shingles. Patients with HZ experience decreased motivation and increased emotional distress consistent with functions of the ventral tegmental area (VTA) of the brain. In addition, activity within the ventral tegmental area is altered in patients with HZ. HZ primarily affects individuals that are older and the VTA changes with age. To begin to determine if the VTA has a role in HZ symptoms, a screen of 10,000 genes within the VTA in young and old male rats was completed after injecting the whisker pad with VZV. The two genes that had maximal change were membrane progesterone receptors PAQR8 (mPRß) and PAQR9 (mPRε). Neurons and non-neuronal cells expressed both PAQR8 and PAQR9. PAQR8 and PAQR9 protein expression was significantly reduced after VZV injection of young males. In old rats PAQR9 protein expression was significantly increased after VZV injection and PAQR9 protein expression was reduced in aged male rats versus young rats. Consistent with previous results, pain significantly increased after VZV injection of the whisker pad and aged animals showed significantly more pain than young animals. Our data suggests that PAQR8 and PAQR9 expression is altered by VZV injection and that these changes are affected by age.

Intracranial calcification in Fam20c-deficient mice recapitulates human Raine syndrome.

FAM20C (family with sequence similarity 20-member C) is a protein kinase that phosphorylates secretory proteins, including the proteins that are essential to the formation and mineralization of calcified tissues. FAM20C loss-of-function mutations cause Raine syndrome in humans, characterized by generalized osteosclerosis, distinctive craniofacial dysmorphism, along with extensive intracranial calcification. Our previous studies revealed that inactivation of Fam20c in mice led to hypophosphatemic rickets. In this study, we examined the expression of Fam20c in the mouse brain and investigated brain calcification in Fam20c-deficient mice. Reverse transcription polymerase chain reaction (RT-PCR), Western-blotting and in situ hybridization analyses demonstrated the broad expression of Fam20c in the mouse brain tissue. X-ray and histological analyses showed that the global deletion of Fam20c (mediated by Sox2-cre) resulted in brain calcification in mice after postnatal 3 months and that the calcifications were bilaterally distributed within the brain. There was mild perifocal microgliosis as well as astrogliosis around calcospherites. The calcifications were first observed in the thalamus, and later in the forebrain and hindbrain. Furthermore, brain-specific deletion (mediated by Nestin-cre) of Fam20c in mice also led to cerebral calcification at an older age (postnatal 6 months), but no obvious skeletal or dental defects. Our results suggest that the local loss of FAM20C function in the brain may directly account for intracranial calcification. We propose that FAM20C plays an essential role in maintaining normal brain homeostasis and preventing ectopic brain calcification.

A Guide to Preclinical Models of Zoster-Associated Pain and Postherpetic Neuralgia.

Reactivation of latent varicella-zoster virus (VZV) causes herpes zoster (HZ), which is commonly accompanied by acute pain and pruritus over the time course of a zosteriform rash. Although the rash and associated pain are self-limiting, a considerable fraction of HZ cases will subsequently develop debilitating chronic pain states termed postherpetic neuralgia (PHN). How VZV causes acute pain and the mechanisms underlying the transition to PHN are far from clear. The human-specific nature of VZV has made in vivo modeling of pain following reactivation difficult to study because no single animal can reproduce reactivated VZV disease as observed in the clinic. Investigations of VZV pathogenesis following primary infection have benefited greatly from human tissues harbored in immune-deficient mice, but modeling of acute and chronic pain requires an intact nervous system with the capability of transmitting ascending and descending sensory signals. Several groups have found that subcutaneous VZV inoculation of the rat induces prolonged and measurable changes in nociceptive behavior, indicating sensitivity that partially mimics the development of mechanical allodynia and thermal hyperalgesia seen in HZ and PHN patients. Although it is not a model of reactivation, the rat is beginning to inform how VZV infection can evoke a pain response and induce long-lasting alterations to nociception. In this review, we will summarize the rat pain models from a practical perspective and discuss avenues that have opened for testing of novel treatments for both zoster-associated pain and chronic PHN conditions, which remain in critical need of effective therapies.

Sex Differences in the Role of Neurexin 3α in Zoster Associated Pain.

Varicella zoster virus (VZV) induces orofacial pain and female rats show greater pain than male rats. During the proestrus phase of the estrous cycle the VZV induce pain response is attenuated in female rats. A screen of gene expression changes in diestrus and proestrus female rats indicated neurexin 3α (Nrxn3α) was elevated in the central amygdala of proestrus rats vs. diestrus rats. GABAergic neurons descend from the central amygdala to the lateral parabrachial region and Nrxn3α is important for presynaptic γ-Aminobutyric acid (GABA) release. Thus, we hypothesized that the reduced orofacial pain in male rats and proestrus female rats is the result of increased Nrxn3α within the central amygdala that increases GABA release from axon terminals within the parabrachial and inhibits ascending pain signals. To test this hypothesis Nrxn3 α expression was knocked-down by infusing shRNA constructs in the central amygdala. Then GABA release in the parabrachial was quantitated concomitant with measuring the pain response. Results revealed that knockdown of Nrxn3α expression significantly increases the pain response in both male rats and proestrus female rats vs. diestrus rats. GABA release was significantly reduced in the parabrachial of male and proestrus female rats after Nrxn3α knockdown. Neuronal activity of excitatory neurons was significantly inhibited in the parabrachial after Nrxn3α knockdown. These results are consistent with the idea that Nrxn3 within the central amygdala controls VZV associated pain by regulating GABA release in the lateral parabrachial that then modulates ascending orofacial pain signals.

Neurexin 3α in the Central Amygdala has a Role in Orofacial Varicella Zoster Pain.

Varicella zoster virus (VZV) is responsible for chronic pain. VZV injection has similarities to herpes zoster (HZ) "shingles" pain in humans. In this study orofacial pain was induced by injecting male rats with the human VZV. The amygdala and parabrachial have been implicated to control affective/motivational orofacial pain. Recently our lab reported neurexin 3α (Nrxn3α) is expressed in the central amygdala and parabrachial. GABAergic neurons descend from the central amygdala to the lateral parabrachial region and Nrxn3α is important for presynaptic (γ-Aminobutyric acid) GABA release. Thus, we hypothesized that lateral parabrachial neuronal activity and orofacial pain are controlled by Nrxn3α within the central amygdala. To test the hypothesis Nrxn3α expression was knocked down (i.e., using short hairpin RNA or shRNA) in the central amygdala and GABA release and neuronal activity were quantitated in the parabrachial concomitant with measurement of the VZV induced pain response. Results revealed that attenuating Nrxn3 expression within the amygdala reduces GABA release in the parabrachial and increases neuronal activity within the lateral parabrachial region. Attenuating Nrxn3 expression also increases VZV associated orofacial pain. Activating GABAergic neurons within the central amygdala with opsins increase GABA release in the parabrachial and reduced the pain response after Nrxn3 shRNA treatment. These results are consistent with the idea that Nrxn3 within the central amygdala controls VZV associated pain by regulating GABA release in the lateral parabrachial that then controls the activity of ascending pain neurons.

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.

Varicella-zoster virus early infection but not complete replication is required for the induction of chronic hypersensitivity in rat models of postherpetic neuralgia.

Herpes zoster, the result of varicella-zoster virus (VZV) reactivation, is frequently complicated by difficult-to-treat chronic pain states termed postherpetic neuralgia (PHN). While there are no animal models of VZV-induced pain following viral reactivation, subcutaneous VZV inoculation of the rat causes long-term nocifensive behaviors indicative of mechanical and thermal hypersensitivity. Previous studies using UV-inactivated VZV in the rat model suggest viral gene expression is required for the development of pain behaviors. However, it remains unclear if complete infection processes are needed for VZV to induce hypersensitivity in this host. To further assess how gene expression and replication contribute, we developed and characterized three replication-conditional VZV using a protein degron system to achieve drug-dependent stability of essential viral proteins. Each virus was then assessed for induction of hypersensitivity in rats under replication permissive and nonpermissive conditions. VZV with a degron fused to ORF9p, a late structural protein that is required for virion assembly, induced nocifensive behaviors under both replication permissive and nonpermissive conditions, indicating that complete VZV replication is dispensable for the induction of hypersensitivity. This conclusion was confirmed by showing that a genetic deletion recombinant VZV lacking DNA packaging protein ORF54p still induced prolonged hypersensitivities in the rat. In contrast, VZV with a degron fused to the essential IE4 or IE63 proteins, which are involved in early gene regulation of expression, induced nocifensive behaviors only under replication permissive conditions, indicating importance of early gene expression events for induction of hypersensitivity. These data establish that while early viral gene expression is required for the development of nocifensive behaviors in the rat, complete replication is dispensable. We postulate this model reflects events leading to clinical PHN, in which a population of ganglionic neurons become abortively infected with VZV during reactivation and survive, but host signaling becomes altered in order to transmit ongoing pain.

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.

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.

A Pre-Existing Myogenic Temporomandibular Disorder Increases Trigeminal Calcitonin Gene-Related Peptide and Enhances Nitroglycerin-Induced Hypersensitivity in Mice.

Migraine is commonly reported among patients with temporomandibular disorders (TMDs), especially myogenic TMD. The pathophysiologic mechanisms related to the comorbidity of the two conditions remain elusive. In the present study, we combined masseter muscle tendon ligation (MMTL)-produced myogenic TMD with systemic injection of nitroglycerin (NTG)-induced migraine-like hypersensitivity in mice. Facial mechanical allodynia, functional allodynia, and light-aversive behavior were evaluated. Sumatriptan, an FDA-approved medication for migraine, was used to validate migraine-like hypersensitivity. Additionally, we examined the protein level of calcitonin gene-related peptide (CGRP) in the spinal trigeminal nucleus caudalis using immunohistochemistry. We observed that mice with MMTL pretreatment have a prolonged NTG-induced migraine-like hypersensitivity, and MMTL also enabled a non-sensitizing dose of NTG to trigger migraine-like hypersensitivity. Systemic injection of sumatriptan inhibited the MMTL-enhanced migraine-like hypersensitivity. MMTL pretreatment significantly upregulated the protein level of CGRP in the spinal trigeminal nucleus caudalis after NTG injection. Our results indicate that a pre-existing myogenic TMD can upregulate NTG-induced trigeminal CGRP and enhance migraine-like hypersensitivity.

Progesterone and Allopregnanolone Rapidly Attenuate Estrogen-Associated Mechanical Allodynia in Rats with Persistent Temporomandibular Joint Inflammation.

Temporomandibular joint disorder (TMD) is associated with pain in the joint (temporomandibular joint, TMJ) and muscles involved in mastication. TMD pain dissipates following menopause but returns in some women undergoing estrogen replacement therapy. Progesterone has both anti-inflammatory and antinociceptive properties, while estrogen's effects on nociception are variable and highly dependent on both natural hormone fluctuations and estrogen dosage during pharmacological treatments, with high doses increasing pain. Allopregnanolone, a progesterone metabolite and positive allosteric modulator of the GABAA receptor, also has antinociceptive properties. While progesterone and allopregnanolone are antinociceptive, their effect on estrogen-exacerbated TMD pain has not been determined. We hypothesized that removing the source of endogenous ovarian hormones would reduce inflammatory allodynia in the TMJ of rats and both progesterone and allopregnanolone would attenuate the estrogen-provoked return of allodynia. Baseline mechanical sensitivity was measured in female Sprague-Dawley rats (150-175 g) using the von Frey filament method followed by a unilateral injection of complete Freund's adjuvant (CFA) into the TMJ. Mechanical allodynia was confirmed 24 h later; then rats were ovariectomized or received sham surgery. Two weeks later, allodynia was reassessed and rats received one of the following subcutaneous hormone treatments over 5 days: a daily pharmacological dose of estradiol benzoate (E2; 50 µg/kg), daily E2 and pharmacological to sub-physiological doses of progesterone (P4; 16 mg/kg, 16 µg/kg, or 16 ng/kg), E2 daily and interrupted P4 given every other day, daily P4, or daily vehicle control. A separate group of animals received allopregnanolone (0.16 mg/kg) instead of P4. Allodynia was reassessed 1 h following injections. Here, we report that CFA-evoked mechanical allodynia was attenuated following ovariectomy and daily high E2 treatment triggered the return of allodynia, which was rapidly attenuated when P4 was also administered either daily or every other day. Allopregnanolone treatment, whether daily or every other day, also attenuated estrogen-exacerbated allodynia within 1 h of treatment, but only on the first treatment day. These data indicate that when gonadal hormone levels have diminished, treatment with a lower dose of progesterone may be effective at rapidly reducing the estrogen-evoked recurrence of inflammatory mechanical allodynia in the TMJ.

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.

Effect of Pregnancy on TMJ Nociception in Rats.

OBJECTIVE: Does TMJ nociception, as measured by a behavioral assay, differ during pregnancy, potentially due to the effect of sex steroids? METHODS: Thirty - two Sprague Dawlcy rats were randomly allocated to either mid- (EH 11) or late- (EH 17) pregnancy groups. The animals within each group were further allocated to a saline or CFA treatment. At EH 11 or EH 17, the animals were injected bilaterally into TMJ with either saline (15 µL.) or 1 µg/µL CFA (15 µL). Nociception was measured with a feeding assay (pellet recording) and analyzed by comparing in-tra-meal rates using a distance-based permutation method. RESULTS: CFA injection resulted in the animals eating longer in both the mid - pregnant and late-pregnant groups. Plasma estradiol was higher in the late - pregnant group versus the mid - pregnant group. Importantly, the CFA injected late-pregnant group ate for a shorter amount of time (i. e., shorter meal rate) than the CFA injected rats at mid - pregnancy. CONCLUSION: The reduced nociceptive response (shorter meal rate) in the CFA injected late - pregnant group may be due to greater estradiol versus the CFA injected mid - pregnancy rats. Thus, one potential reason why women report fewer TMJ symptoms during pregnancy is the higher level of circulating estradiol.

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

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

Distance-based permutation of inter-meal differences as a sensitive test of temporomandibular joint nociception in rats.

PURPOSE: Compare non-parametric permutation method using intr-meal rate as endpoint to existing ANOVA method that uses average daily meal duration as an endpoint for detection of chronic pain in Sprague-Dawley rats. METHODS: Nociception following bilateral temporomandibular joint (TMJ) injection of high-dose of Complete Freunds Adjuvant (CFA, 250 µg/50 µL per side) could be detected in young adult male Sprague-Dawley rats using average daily meal durations as a measure of nociception for up to 19 days (Kramer, Kerins, Schneiderman, & Bellinger, 2010) using ANOVA and multiple comparisons range tests. In this study, we reanalyzed the data using a non-parametric permutation procedure based on absolute differences between intra-meal feeding rate curves. In addition, to that experiment, we injected bilaterally the TMJ of naive rats with either a low-dose CFA (15 µg/50 µL per side, n=6) or saline (50 µL of 0.9%, n=4) and monitored the animals for 7 days. RESULTS: The permutation test of the intra-meal feeding rate detected the presence of nociception in the high-dose CFA treatment group for up to 40 days or twice as long as when using ANOVA on average daily meal durations. The permutation method also detected the low-dose CFA induced nociception with ten-times lower p-values and for several days longer than ANOVA of changes in meal durations. CFA-induced injury resulted in even reduction of intra-meal feeding rate and lengthening of the meals in both high- and low-dose CFA-injected animals. The rate analysis also showed when the rats first started a meal they were experiencing the same level of nociception as at the end of the meal. This demonstrated that intra-meal chewing itself did not alter the level of nociception. CONCLUSIONS: These results suggest that permutation tests based on differences in intra-meal feeding rates can be used as a sensitive test to determine and study the temporal patterns of TMJ nociception.

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.