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.

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.

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.

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.

Genetic Analysis of a Large Family with Migraine, Vertigo, and Motion Sickness.

BACKGROUND: Migraine is a common disorder most typically presenting as headache and often associated with vertigo and motion sickness. It is a genetically complex condition with multiple genes ultimately contributing to the predisposition and development of this episodic neurological disorder. We identified a large American family of 29 individuals of which 17 members suffered from at least one of these disorders, migraine, vertigo, or motion sickness. Many of these individuals suffered from several simultaneously. We hypothesized that vertigo and motion sickness may involve genes that are independent to those directly contributing to migraine susceptibility. METHODS: Genome-wide linkage analysis performed using 400 microsatellite repeat markers spaced at 10 cM throughout the genome. The members of this family were phenotyped for each condition, migraine, vertigo, and motion sickness and analyzed separately. Statistical analysis was performed using two-point and multipoint linkage analysis employing a number of models including autosomal recessive or dominant patterns of inheritance with high and low genetic penetrance. RESULTS: We identified a novel locus for migraine, 9q13-q22 (maximum two-point logarithm of odds [LOD] score-2.51). In addition, there are suggestive LOD scores that localize to different chromosomes for each phenotype; vertigo (chromosome 18, LOD score of 1.82) and motion sickness (chromosome 4, LOD score of 2.09). CONCLUSIONS: Our analysis supports our hypothesis that the migraine-associated vertigo and motion sickness may involve distinct susceptibility genes.

Analyse génétique d'une famille étendue dont les membres souffrent de migraines, de vertiges et du mal des transports. Contexte : La migraine est un trouble courant qui entraîne habituellement des maux de tête et qui est souvent associé à des vertiges et au mal des transports. Il s'agit aussi d'une condition génétique complexe en vertu de laquelle de nombreux gènes contribuent à terme à cette prédisposition et au développement de ce trouble neurologique périodique. À cet égard, nous avons identifié une famille étendue américaine comptant 29 membres. De ce nombre, 17 d'entre eux avaient souffert d'au moins un de ces troubles : des migraines, des vertiges ou le mal des transports. À noter que plusieurs d'entre eux avaient souffert de ces troubles en même temps. Nous avons émis l'hypothèse que les vertiges et le mal des transports pourraient impliquer des gènes qui sont indépendants de ceux contribuant directement à la propension aux migraines. Méthodes : Nous avons effectué une analyse de liaison au moyen de 400 marqueurs microsatellites répétés et espacés à tous les 10 cm au sein de l'ensemble du génome des membres de cette famille. Les membres de cette famille ont été « phénotypés ¼ pour chaque type de trouble (les migraines, les vertiges et le mal des transports) et ont été ensuite analysés de façon séparée. Nous avons effectué une analyse statistique au moyen de l'analyse de liaison multipoint et à deux points, utilisant pour ce faire un certain nombre de modèles, par exemple le modèle autosomique récessif ou des patterns dominants de transmission avec une pénétrance génétique élevée ou faible. Résultats : Nous avons été en mesure d'identifier un nouveau locus dans le cas de la migraine : 9q13-q22 (maximum 2-points ; score au logarithme des probabilités ou LOD : - 2,51). De plus, il est des scores révélateurs au logarithme des probabilités qui permettent de localiser divers chromosomes pour chaque phénotype : vertiges (chromosome 18 ; score au logarithme des probabilités ou LOD : 1,82) et mal des transports (chromosome 4 ; score au logarithme des probabilités ou LOD : 2,09). Conclusions : Notre analyse confirme ainsi notre hypothèse initiale, à savoir que les cas de migraine auxquels sont associés des vertiges et le mal des transports pourraient très bien impliquer différents gènes de susceptibilité.

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.

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.

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.

Attenuation of myogenic orofacial nociception and mechanical hypersensitivity by viral mediated enkephalin overproduction in male and female rats.

BACKGROUND: Clinical studies have tested the use of an engineered herpes virus to treat pain. We hypothesized that subcutaneous injections of an engineered herpes virus that expresses enkephalin would attenuate orofacial nociception and hypersensitivity in male and female rats by a central mechanism. METHODS: Herpes virus was injected subcutaneously around the mouth of male and female rats seventy-two hours before ligatures were placed on the masseter tendon, control treatment groups received either no virus or no ligature. Enkephalin expression was measured and von Frey filament testing and meal duration were utilized to measure mechanical hypersensitivity and the nociceptive response, respectively. Naloxone or naloxone methiodide was administered to rats injected with the enkephalin expressing virus to test if enkephalin was acting peripherally or centrally. RESULTS: Ligature significantly lengthened meal duration and reduced the threshold to von Frey filaments for 18 days. Infection with the enkephalin transgene significantly decreased this response for at least 11 days but only in male rats. Virus injection significantly increased expression of enkephalin in the mental nerve that innervates the mouth region, the trigeminal ganglia and the trigeminal nucleus caudalis but no increase was observed in the masseter nerve after virus injection. Naloxone but not naloxone methiodide reversed the response to the enkephaline expressing virus. CONCLUSIONS: The data suggests that sex should be a considered when using this virus and that viral transfection of the mental nerve with an enkephalin transgene can reduce nociception and hypersensitivity through a central mechanism.

Ketogenic diet mitigates opioid-induced hyperalgesia by restoring short-chain fatty acids-producing bacteria in the gut.

ABSTRACT: Opioids are commonly prescribed to patients with chronic pain. Chronic opioid usage comes with a slew of serious side effects, including opioid-induced hyperalgesia (OIH). The patients with long-term opioid treatment experience paradoxical increases in nociceptive hypersensitivity, namely, OIH. Currently, treatment options for OIH are extremely lacking. In this study, we show that the ketogenic diet recovers the abnormal pain behavior caused by chronic morphine treatment in male mice, and we further show that the therapeutic effect of the ketogenic diet is mediated through gut microbiome. Our 16S rRNA sequencing demonstrates that chronic morphine treatment causes changes in mouse gut microbiota, specifically a decrease in short-chain fatty acids-producing bacteria, and the sequencing data also show that the ketogenic diet rescues those bacteria in the mouse gut. More importantly, we show that supplementation with short-chain fatty acids (butyrate, propionate, and acetate) can delay the onset of OIH, indicating that short-chain fatty acids play a direct role in the development of OIH. Our findings suggest that gut microbiome could be targeted to treat OIH, and the ketogenic diet can be used as a complementary approach for pain relief in patients with chronic opioid treatment. We only used male mice in this study, and thus, our findings cannot be generalized to both sexes.

Neurexin 3 Regulates Synaptic Connections Between Central Amygdala Neurons and Excitable Cells of the Lateral Parabrachial Nucleus in Rats with Varicella Zoster Induced Orofacial Pain.

Introduction: Herpes Zoster in humans is the result of varicella zoster virus (VZV) infection. Injecting rats with varicella zoster virus produces pain similar to herpes zoster "shingles" pain in humans. . In a previous study, orofacial pain was induced by injecting the whisker pad of male rats with VZV and the pain response increased after attenuating neurexin 3 (Nrxn3) expression in the central amygdala. Neurons descend from the central amygdala to the lateral parabrachial nucleus and orofacial pain signals ascend to the lateral parabrachial nucleus. GABAergic neurons within the central amygdala regulate pain by inhibiting activity within the lateral parabrachial nucleus. Attenuating Nrxn3 expression in the central amygdala increased GABA release in the lateral parabrachial nucleus suggesting Nrxn3 controls pain by regulating GABA release. Nrxn3 can also control synaptic connections between neurons, and we hypothesized that Nrxn3 knockdown in the central amygdala would reduce the number of GABAergic synaptic connections in the lateral parabrachial nucleus and increase VZV associated pain. Methods: To test this idea, the number of synaptic connections between GABAergic cells of the central amygdala and excitatory or dynorphin positive neurons within the lateral parabrachial nucleus were quantitated after infusion of a virus expressing synaptophysin. Synaptophysin is a synaptic vesicle protein that labels neuronal synaptic connections. These connections were measured in rats with and without whisker pad injection of VZV and knockdown of Nrxn3 within the central amygdala. Orofacial pain was measured using a place escape avoidance paradigm. Results: GABAergic synaptic connections were reduced in the lateral parabrachial nucleus after Nrxn3 knockdown. Rats with a reduction in the number of connections had an increase in VZV associated orofacial pain. Immunostaining with the pain marker prodynorphin indicated that the reduction in GABAergic connections was primarily associated with prodynorphin positive neurons. Discussion: The results suggest Nrxn3 reduces VZV associated orofacial pain, in part, by enhancing synaptic connections between GABA cells of the central amygdala and pain neurons within the lateral parabrachial nucleus.

Dysregulation of Semaphorin7A/ß1-integrin signaling leads to defective GnRH-1 cell migration, abnormal gonadal development and altered fertility.

Reproduction in mammals is dependent on the function of specific neurons that secrete gonadotropin-releasing hormone-1 (GnRH-1). These neurons originate prenatally in the nasal placode and migrate into the forebrain along the olfactory-vomeronasal nerves. Alterations in this migratory process lead to defective GnRH-1 secretion, resulting in heterogeneous genetic disorders such as idiopathic hypogonadotropic hypogonadism (IHH), and other reproductive diseases characterized by the reduction or failure of sexual competence. Combining mouse genetics with in vitro models, we demonstrate that Semaphorin 7A (Sema7A) is essential for the development of the GnRH-1 neuronal system. Loss of Sema7A signaling alters the migration of GnRH-1 neurons, resulting in significantly reduced numbers of these neurons in the adult brain as well as in reduced gonadal size and subfertility. We also show that GnRH-1 cells differentially express the Sema7 receptors ß1-integrin and Plexin C1 as a function of their migratory stage, whereas the ligand is robustly expressed along developing olfactory/vomeronasal fibers. Disruption of Sema7A function in vitro inhibits ß1-integrin-mediated migration. Analysis of Plexin C1(-/-) mice did not reveal any difference in the migratory process of GnRH-1 neurons, indicating that Sema7A mainly signals through ß1-integrin to regulate GnRH-1 cell motility. In conclusion, we have identified Sema7A as a gene implicated in the normal development of the GnRH-1 system in mice and as a genetic marker for the elucidation of some forms of GnRH-1 deficiency in humans.

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.

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.

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.

Estrogen in cycling rats alters gene expression in the temporomandibular joint, trigeminal ganglia and trigeminal subnucleus caudalis/upper cervical cord junction.

Females report temporomandibular joint (TMJ) pain more than men and studies suggest estrogen modulates this pain response. Our goal in this study was to determine genes that are modulated by physiological levels of 17ß-estradiol that could have a role in TMJ pain. To complete this goal, saline or complete Freund's adjuvant was injected in the TMJ when plasma 17ß-estradiol was low or when it was at a high proestrus level. TMJ, trigeminal ganglion, and trigeminal subnucleus caudalis/upper cervical cord junction (Vc/C(1-2) ) tissues were isolated from the treated rats and expression of 184 genes was quantitated in each tissue using real-time PCR. Significant changes in the amount of specific transcripts were observed in the TMJ tissues, trigeminal ganglia, and Vc/C(1-2) region when comparing rats with high and low estrogen. GABA A receptor subunit α6 (Gabra6) and the glycine receptor α2 (Glra2) were two genes of interest because of their direct function in neuronal activity and a >29-fold increase in the trigeminal ganglia was observed in proestrus rats with TMJ inflammation. Immunohistochemical studies showed that Gabrα6 and Glrα2 neuronal and not glial expression increased when comparing rats with high and low estrogen. Estrogen receptors α and ß are present in neurons of the trigeminal ganglia, whereby 17ß-estradiol can alter expression of Gabrα6 and Glrα2. Also, estrogen receptor α (ERα) but not ERß was observed in satellite glial cells of the trigeminal ganglia. These results demonstrate that genes associated with neurogenic inflammation or neuronal excitability were altered by changes in the concentration of 17ß-estradiol.

Knockdown of Fcγ receptor III in an arthritic temporomandibular joint reduces the nociceptive response in rats.

OBJECTIVE: Fcγ receptor III (FcγRIII; CD16) is a receptor expressed on immune cells that selectively binds IgG molecules. IgG binding results in cellular activation and cytokine release. IgG is an important factor in arthritis and can be found in the arthritic temporomandibular joint (TMJ). We undertook this study to test the hypothesis that a reduction in FcγRIII expression in TMJ tissues would reduce the nociceptive and inflammatory responses in an inflamed joint. METHODS: Small interfering RNA (siRNA), either naked or complexed with linear polyethyleneimine, was injected into the superior joint space of the TMJ in rats. After administration of siRNA the joint was injected with saline or with Freund's complete adjuvant to induce arthritis. Nociceptive responses were quantitated in the rat by measuring the animal's meal duration. FcγRIII expression in the TMJ tissue was assayed by immunocytochemistry or Western blotting. Cleavage of FcγRIII transcript was then assayed by 5' rapid amplification of complementary DNA ends. Interleukin-1ß (IL-1ß) and IgG content was measured in the TMJ tissue by enzyme-linked immunosorbent assay. RESULTS: Injection of FcγRIII siRNA reduced the amount of FcγRIII in the TMJ tissues, and the transcript was cleaved in a manner consistent with an RNA interference mechanism. Moreover, injection of FcγRIII siRNA reduced the nociceptive response of rats with an arthritic TMJ and reduced the amount of the proinflammatory cytokine IL-1ß. CONCLUSION: FcγRIII contributes to the pain resulting from inflammatory arthritis of the TMJ, and siRNA has the potential to be an effective treatment for this disorder.

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.

PI3K limits TNF-alpha production in CD16-activated monocytes.

IgG complexes bind to Fc receptor family members FcgammaRI (CD64), FcgammaRII (CD32) and FcgammaRIII (CD16), activating cell MAPK and PI3K resulting in increased cytokine production from particular leukocytes. The signaling molecules involved in cytokine production after cross-linking CD16 have not been determined in monocytes. To address this question, TNF-alpha, IL-1beta and IL-6 were measured in activated monocytes after inhibiting MEK1/2, PI3K and glycogen synthase kinase-beta (GSK-3beta). The roles of GSK-3beta and NF-kappaB were then determined using reporter assays and siRNA treatment. The data suggested that an MAPK pathway stimulated TNF-alpha release but that active PI3K limited TNF-alpha, IL-1beta and IL-6 cytokine production after cross-linking CD16. PI3K was also shown to limit nuclear translocation of NF-kappaB. The limiting effect of PI3K on TNF-alpha production from activated monocytes depended on the decrease of GSK-3beta activity, which significantly reduced the transactivation of NF-kappaB. Moreover, the TNF-alpha production induced by CD16 cross-linking was reduced in monocytes after treatment with siRNA against NF-kappaB, implying that this transcription factor functioned in TNF-alpha production. The results suggest that CD16 cross-linking activated PI3K and that active PI3K limited TNF-alpha production by inhibiting GSK-3beta activity, that blocked the action of NF-kappaB.