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

High levels of 17ß-estradiol (E2) have been found to reduce inflammatory temporomandibular joint (TMJ) pain. A search for genes effected by a high concentration of estradiol showed an increase in GABAA receptor subunit alpha 6 (Gabrα6) in the trigeminal ganglia (TG). Blockade of Gabrα6 expression in the TG increases masseter muscle nociception in male rats, but the relationship between estradiol's effect on nociception and Gabrα6 expression remains unclear in females. To address this knowledge gap we hypothesized that reducing Gabrα6 expression in the TG will increase the orofacial nociceptive response of ovariectomized female rats treated with estradiol. To administer hormone osmotic pumps were placed in rats that dispensed a low diestrus plasma concentration of 17ß-estradiol, in addition, 17ß-estradiol was injected to produce a high proestrus plasma concentration of estradiol. A ligature was then placed around the masseter tendon to induce a nociceptive response; a model for TMJ muscle pain. Gabrα6 small interfering RNA (siRNA) was later infused into the TG and the nociceptive response was measured using von Frey filaments and a meal duration assay. GABAA receptor expression was measured in the TG and trigeminal nucleus caudalis and upper cervical region (Vc-C1). Ligature significantly increased the nociceptive response but a high proestrus concentration of 17ß-estradiol attenuated this response. Gabrα6 siRNA infusion decreased Gabrα6 expression in the TG and Vc-C1 but increased the nociceptive response after 17ß-estradiol treatment. The results suggest estradiol decreased the orofacial nociceptive response, in part, by causing an increase in Gabrα6 expression.

Reduced GABAA receptor α6 expression in the trigeminal ganglion enhanced myofascial nociceptive response.

Activation of the GABAA receptor results in inhibition of neuronal activity. One subunit of this multi-subunit receptor termed alpha 6 (Gabrα6) contributed to inflammatory temporomandibular joint (TMJ) nociception but TMJ disorders often include myofascial pain. To address Gabrα6 role in myofascial pain we hypothesized that Gabrα6 has an inhibitory role in myofascial nociceptive responses similar to inflammatory TMJ arthritis. To test this hypothesis a, myofascial nociceptive response was induced by placing a ligature bilaterally on the tendon attachment of the anterior superficial part of a male rat's masseter muscle. Four days after ligature placement Gabrα6 expression was reduced by infusing the trigeminal ganglia (TG) with small interfering RNA (siRNA) having homology to either the Gabrα6 gene (Gabrα6 siRNA) or no known gene (control siRNA). After siRNA infusion nociceptive behavioral responses were measured, i.e., feeding behavior and head withdrawal after pressing upon the region above the ligature with von Frey filaments. Neuronal activity in the TG and trigeminal nucleus caudalis and upper cervical region (Vc-C1) was measured by quantitating the amount of phosphorylated extracellular signal-regulated kinase (p-ERK). Total Gabrα6 and GABAA receptor contents in the TG and Vc-C1 were determined. Gabrα6 siRNA infusion reduced Gabrα6 and GABAA receptor expression and significantly increased the nociceptive response in both nociceptive assays. Gabrα6 siRNA infusion also significantly increased TG p-ERK expression of the ligated rats. From these results we conclude GABAA receptors consisting of the Gabrα6 subunit inhibit TG nociceptive sensory afferents in the trigeminal pathway and have an important role in the regulation of myofascial nociception.

Modulation of temporomandibular joint nociception and inflammation in male rats after administering a physiological concentration of 17ß-oestradiol.

BACKGROUND: Previous studies have shown 17ß-estradiol will reduce temporomandibular joint (TMJ) inflammation and hypersensitivity in female rats. Although male rats contain significant amounts of oestradiol, it was unknown whether a physiological concentration of 17ß-estradiol would attenuate male TMJ inflammation and nociception. METHODS: Intact and castrated rats were given a physiological concentration of oestradiol to examine first, if oestradiol will affect male TMJ nociception/inflammation and, second, if administration of oestradiol would act synergistically with endogenous male hormones to attenuate TMJ nociception. The hormonally treated rats were given TMJ injections of complete Freund's adjuvant (CFA) and then nociception was measured using a validated method in which a lengthening in meal duration is directly correlated to the intensity of deep TMJ nociception. Inflammation was assayed by quantitating pro-inflammatory gene expression. RESULTS: Meal duration was significantly lengthened after TMJ CFA injection and this lengthening was significantly attenuated in the castrated but not intact males after administering a physiological concentration of oestradiol. A physiological concentration of 17ß-estradiol also significantly increased IL-6 expression in the inflamed TMJ of castrated males while 17ß-estradiol did not alter IL-1ß, CXCL2 and CCL20 expression. Castration increased pro-inflammatory mediators IL-6, IL-1ß and CXCL2 suggesting male sex hormones were anti-inflammatory. Calcitonin gene-related peptide in the trigeminal ganglia was unchanged. CONCLUSIONS: Similar to females, male rats with TMJ inflammation showed a reduced nociceptive response after treatment with a physiological concentration of oestradiol suggesting the effects of oestradiol treatment were not constrained by organizational processes in the males.

A non-invasive model for measuring nociception after tooth pulp exposure.

Temporomandibular arthritis will lengthen a rodent's meal duration. We hypothesized that meal duration would also lengthen after tooth pulp exposure, suggesting that this behavior could be used to measure tooth nociception. To test this hypothesis, we placed rats in feeding units and subjected 4 anterior mandibular molars to pulp exposure, with and without pre-treatment with the analgesic buprenorphine-HCl. In the first study, male Sprague-Dawley rats were placed in computerized sound-attenuated feeding modules, the pulp of 4 molars on the mandible were exposed, and meal duration was measured for 13 days. In a second study, rats were injected with either the analgesic buprenorphine-HCl or saline every 12 hrs; injections were started one day before pulp exposure. Meal duration was determined before and after treatment. In the first study, pulp exposure significantly increased daily meal duration for 8 days. In the second study, pulp exposure lengthened daily meal duration, but the group that was treated with buprenorphine-HCl showed no significant difference compared with control rats without pulp exposure. Evidence supports that a lengthening in meal duration is a response to tooth nociception and that this nociception can be measured for over a week.

Reduced GABA(A) receptor α6 expression in the trigeminal ganglion alters inflammatory TMJ hypersensitivity.

Trigeminal ganglia neurons express the GABA(A) 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.

Local kinetic interpretation of entropy production through reversed diffusion.

The time reversal of stochastic diffusion processes is revisited with emphasis on the physical meaning of the time-reversed drift and the noise prescription in the case of multiplicative noise. The local kinematics and mechanics of free diffusion are linked to the hydrodynamic description. These properties also provide an interpretation of the Pope-Ching formula for the steady-state probability density function along with a geometric interpretation of the fluctuation-dissipation relation. Finally, the statistics of the local entropy production rate of diffusion are discussed in the light of local diffusion properties, and a stochastic differential equation for entropy production is obtained using the Girsanov theorem for reversed diffusion. The results are illustrated for the Ornstein-Uhlenbeck process.

Intra-articular microparticles for drug delivery to the TMJ.

This study describes the in vivo biocompatibility of intra-articular poly(DL-lactic-co-glycolic acid) (PLGA) microparticle (MP) formulations in the rat temporomandibular joint (TMJ). To our knowledge, this is the first intra-articular microparticle-based drug delivery system for the TMJ. The impact of PLGA MP concentration on rat TMJ function was quantified via computerized meal pattern analysis; in this non-invasive technique, previously validated markers of TMJ pain or nociception (specifically, meal duration and food intake) were recorded by computer-monitored pellet feeders. Bilateral intra-articular injection of 15, 30, or 50 mg/mL PLGA MPs had no impact on meal duration or food intake over 6 days, compared with controls that did not receive injections. Histological analysis showed that the MPs were retained within the synovial lining. These findings indicate that the PLGA MPs described herein are biocompatible and suitable for intra-articular delivery to the rat TMJ, a finding that has significant implications for the improvement of TMJ therapeutics.

The effects of chronic nicotine on meal patterns, food intake, metabolism and body weight of male rats.

It is unclear what contribution food intake and metabolism have in causing weight loss after administering a dose of nicotine equivalent to smoking one to three packs of cigarettes per day because previous studies have been of a very short duration. To address this question, male Sprague Dawley rats were housed in computerized food intake modules and fed 45 mg pellets: Group 1 [nicotine injected with 1.4 mg/kg/day (free base), fed ad libitum]; and Group 2 [saline injected and pair-fed by computer with Group 2]; and Group 3 [saline injected (i.p.), fed ad libitum]. The rats received 4 equally spaced injections over the dark phase. Treatment consisted of: Phase 1 (nicotine or saline for 14 days), Phase 2 (all rats saline for 8 days and Phase 3 (pair-fed group "unyoked" for 6 days)). Nicotine inhibited food intake over the first 6 days. On termination of nicotine, there was no compensatory hyperphagia in either Groups 1 or 2; and their body weight was reduced starting on day 5 until day 28. In another study, rats were housed in an indirect calorimetry system. Saline or nicotine was injected for 14 days, as noted above; then all rats were injected with saline for 4 days and then no injections for 10 days to follow changes in body weight. Energy expenditure (Kcal/Kg(0.75)) was measured for 18 days. Nicotine significantly reduced food intake on 7 of 14 days of nicotine injections. The body weight of the nicotine injected rats was significantly reduced starting on day 3 until day 25. There were no differences in energy expenditures of the groups, which suggested that a decrease in food intake and not an increase in metabolism was the reason the rats lost weight after administering nicotine.

The effects of cycling levels of 17beta-estradiol and progesterone on the magnitude of temporomandibular joint-induced nociception.

A greater incidence of temporomandibular joint (TMJ) pain is reported in females, suggesting that gonadal hormones may play a role in this condition. However, the exact roles of 17beta-estradiol (E2) and progesterone (P4) in TMJ pain are not completely known. Two experiments were performed to determine the separate roles of E2 and P4 in TMJ nociception at various stages of the estrous cycle. Ovariectomized (OVX) rats were cycled with physiological concentrations of E2 or P4. The E2-cycled rats then received bilateral TMJ injections of saline (SAL) or complete Freund's adjuvant (CFA) on the morning of diestrus-2 (low E2 condition) or proestrus (high E2 condition). As a control, OVX rats (no ovarian E2 and no replacement) were injected with SAL or CFA. The TMJ nociception was measured using a validated novel method in which an increase in meal duration directly correlated to the intensity of deep TMJ nociception. In the E2 experiment, CFA injection, but not SAL, increased TMJ nociception in the OVX group, but the effect was less pronounced in diestrus-2 and even less in proestrus. In the P4 experiment, the rats receiving TMJ CFA in diestrus-2 (end of minor P4 surge) did not show an increase in TMJ nociception, whereas the rats injected in proestrus (major P4 surge), estrus (low P4), and metestrus (low P4) had similar increases in TMJ nociception. The hormones' concentration did not affect TMJ IL-1beta, IL-6, C-C motif ligand 20, or C-X-C motif ligand 2 or the trigeminal ganglia calcitonin gene-related peptide. The high physiological concentrations of E2 observed at proestrus and the low P4 concentrations observed at diestrus-2 attenuated or eliminated CFA-induced TMJ nociception. The results suggest that the cyclic estrous cycle concentrations of E2 and P4 can influence CFA-induced TMJ nociception in the rat.

17beta-Estradiol utilizes the estrogen receptor to regulate CD16 expression in monocytes.

Estrogen can significantly influence CD16 expression and alter monocytic cytokine release upon CD16 receptor activation. However, the function of the estrogen receptor (ER) alpha and beta in this response is unclear. To test whether estrogen binds ERalpha and/or ERbeta to affect CD16 expression, monocytic cells were treated with and without physiological levels of 17beta-estradiol and various doses of the ERalpha and ERbeta antagonist fulvestrant followed by measurement of CD16 transcript levels. To determine how estrogen induced changes in TNF-alpha and IL-1beta release due to CD16 activation we quantitated the amount of cytokines after treatment with estrogen, fulvestrant and antibodies that specifically bind and activate the CD16 receptor. Interaction of ERalpha and the CD16 promoter was then determined by chromatin immunoprecipitation. Furthermore, specific promoter elements utilized by estrogen to control CD16 expression were mutated and expression from a luciferase reporter quantitated after transfection. Using the luciferase reporter construct containing a wild type CD16 promoter, the role of ERalpha and ERbeta in the estrogen response was tested by treating transfected monocytes with an ERalpha specific agonist or an ERbeta specific agonist and measuring expression. Our results show that CD16 transcript levels significantly decreased in monocytic cells due to estrogen and that the observed decrease in message was blocked by the antagonist fulvestrant. Estrogen reduced CD16 expression and decreased TNF-alpha and IL-1beta release upon CD16 activation but the administration of fulvestrant blocked this decrease. ERalpha was found to interact with a region 5' of the CD16 gene in the presence of estrogen, and site-directed mutational analysis of this region indicated the necessity for an estrogen response element in modulating estrogen effects on CD16 expression. Moreover, both an ERalpha and an ERbeta agonist reduced expression of the CD16 reporter construct suggesting both receptors can play a role in CD16 regulation. In conclusion, CD16 expression can be altered by the activity of ERalpha or ERbeta and our results also show that ERalpha can associate with a region within the CD16 promoter that is important in production of transcript.

Specificity of meal pattern analysis as an animal model of determining temporomandibular joint inflammation/pain.

Analyzing feeding behavior, and in particular meal duration, can be used as a biological marker for temporomandibular joint (TMJ) inflammation/pain. The present study determined the specificity of meal duration as a measure of TMJ inflammation/pain in a rodent model. The model was also used to test the efficacy of dexamethasone (DEX) as a treatment for TMJ inflammation/pain that was induced by TMJ injection of complete Freund's adjuvant (CFA). In the first study, anesthetized male Sprague-Dawley rats housed in computerized feeding modules received bilateral intra-articular knee injections of CFA or saline. The next day, CFA-injected rats had significant knee swelling and impaired mobility. Food intake in the CFA-injected group was reduced over the next two days and this was due to reduced meal number with no change in meal size. Notably, meal duration was normal in both the CFA and saline knee-injected groups. In the second study, male rats were assigned to one of four groups: Group 1, no CFA and no DEX treatment; Group 2, no CFA and treatment with DEX (0.4 mg/kg i.m. once daily); Group 3, bilateral TMJ CFA injection and no DEX treatment; and Group 4, bilateral TMJ CFA injection and treatment with DEX. CFA significantly increased TMJ swelling and stress-induced chromodacryorrhea in Group 3, but treatment with DEX attenuated these effects in Group 4. Compared to the controls, meal duration was significantly lengthened 24 and 48 h post-CFA injection in Group 3, whereas DEX treatment attenuated TMJ swelling, chromodacryorrhea and normalized meal duration. The data demonstrate that meal pattern analysis, and in particular meal duration, can be used as a non-invasive specific measure of TMJ inflammation/pain and can be used as a marker of DEX treatment efficacy.

17 beta-estradiol regulates cytokine release through modulation of CD16 expression in monocytes and monocyte-derived macrophages.

OBJECTIVE: Macrophages release cytokines, such as tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), and IL-6, which modulate the symptoms of rheumatoid arthritis (RA). Macrophage release of these cytokines can be modulated by estrogen. Fc gamma receptor type IIIA (CD16a) is a receptor expressed on macrophages that selectively binds IgG molecules, an important rheumatoid factor in RA. Binding of CD16 by anti-CD16 monoclonal antibodies stimulates macrophage cytokine release. We undertook this study to test the hypothesis that decreased concentrations of estrogen (17 beta-estradiol) directly cause an increase in CD16 expression, resulting in increased release of proinflammatory cytokines from monocytes and/or macrophages upon receptor binding. METHODS: THP-1 cells and female human primary monocytes and monocyte-derived macrophages were treated with no 17 beta-estradiol, physiologic levels (1 x 10(-8)M) of 17 beta-estradiol, or 1 x 10(-8)M 17 beta-estradiol followed by withdrawal of 17 beta-estradiol. Surface expression of CD16 and CD16 messenger RNA was measured using fluorescence-activated cell sorting (FACS) and semiquantitative reverse transcription-polymerase chain reaction, respectively. Cytokine release from 17 beta-estradiol-treated or untreated monocytes was then quantitated by enzyme-linked immunosorbent assay and FACS after crosslinking the receptor with anti-CD16 antibodies. RESULTS: CD16 transcript significantly increased in macrophage-like THP-1 cells and in primary, peripheral blood macrophages in the absence of 17 beta-estradiol, and the observed increase in message was dependent on transcription. CD16 receptor levels on CD14+, transforming growth factor beta-treated primary monocytes also increased in cells deprived of 17 beta-estradiol. Analysis of the cytokines released showed that CD16 crosslinking stimulated significant increases in TNF alpha, IL-1 beta, and IL-6 due to the absence of estrogen. CONCLUSION: Estrogen can modulate proinflammatory cytokine release from activated monocytes and/or macrophages, in part through modulation of CD16 expression.

Mesenchymal stem cells acquire characteristics of cells in the periodontal ligament in vitro.

Mesenchymal stem cells differentiate into multiple types of cells derived from mesenchyme. Periodontal ligament cells are primarily derived from mesenchyme; thus, we expected mesenchymal stem cells to differentiate into periodontal ligament. Using a combination of immunohistochemistry and in situ hybridization on co-cultures of mesenchymal stem cells and periodontal ligament, we observed a significant increase in mesenchymal stem cells' expression of osteocalcin and osteopontin and a significant decrease in expression of bone sialoprotein, characteristics of periodontal ligament in vivo. Increased osteopontin and osteocalcin and decreased bone sialoprotein expression was detected within 7 days and maintained through 21 days of co-culture. We conclude that contact or factors from periodontal ligament induced mesenchymal stem cells to obtain periodontal-ligament-like characteristics. Importantly, analysis of the data suggests the feasibility of utilizing mesenchymal stem cells in clinical applications for repairing and/or regenerating periodontal tissue.

17-Beta-estradiol regulates expression of genes that function in macrophage activation and cholesterol homeostasis.

Macrophage activation and cholesterol processing can be affected by changes in estrogen concentrations. However, there is a paucity of information about the genes and mechanisms regulating this estrogen effect. In primary monocyte-derived macrophages we detected transcript and protein for estrogen receptor beta (ERbeta). Determination of genes regulated by estrogen was completed using cDNA arrays and semiquantitative RT-PCR on RNA isolated from macrophages cultured in serum free media containing (5-10) x 10(-9)M 17-beta-estradiol and subsequently deprived of estrogen for a 24h period. The data indicate that the transcript levels of interleukin 1 receptor antagonist (IL-1ra), beta 2-microglobulin, annexin XI and the LXR(alpha) receptor significantly increased and that Ly-GDI transcript levels significantly decreased after estrogen withdrawal; data congruent with estrogen depletion regulating macrophage inflammatory and biochemical processes. Treatment of THP-1 cells with phorbol 12-myristate-13-acetate in the presence or absence of estrogen indicate that differentiation to a macrophage-like cell type was a prerequisite for production of the estrogen response. In addition, experiments using cycloheximide treatment, that blocks nascent protein synthesis, indicated that estrogen withdrawal affected the transcript levels of LXR(alpha) and IL-1ra through dissimilar pathways.

Nasal embryonic LHRH factor (NELF) expression within the CNS and PNS of the rodent.

A novel protein (NELF) was identified screening embryonic luteinizing hormone releasing hormone (LHRH) neurons at different migrational states. Experiments in vitro revealed that NELF functions in olfactory axon outgrowth and subsequently alters LHRH neuronal migration. NELF was not restricted to LHRH neurons in the developing rodent. Multiple CNS and PNS tissues expressed this gene. To characterize the specific regions that express NELF in situ hybridization histochemistry was performed. Within the CNS, cells in the cortex, hippocampus, thalamus and olfactory regions express NELF pre- and postnatally.

Midline nasal tissue influences nestin expression in nasal-placode-derived luteinizing hormone-releasing hormone neurons during development.

Neurons differentiating into the luteinizing hormone-releasing hormone (LHRH) neuroendocrine phenotype are derived from the nasal placode. Cells within the vomeronasal organ anlage that turn on LHRH gene and peptide expression subsequently migrate into the forebrain where they influence reproductive function. The molecular and cellular cues regulating differentiation and migration of these cells are unknown. Discovery of developmental markers can indicate proteins directing or associated with differentiation. Analysis of such markers after manipulation of external cues can elucidate important extracellular differentiation signals. Embryonic LHRH neurons were examined in vivo for Mash-1 and nestin, two factors that delineate precursor populations in PNS and forebrain CNS cells. Nestin, but not Mash-1, was detected in early expressing LHRH cells in the vomeronasal organ anlage. These results were duplicated in LHRH neurons maintained in vitro in nasal explants. Such LHRH cells expressed nestin mRNA but not Mash-1 mRNA and were also negative for three other olfactory epithelial developmental transcription factors, Math4A, Math4C/neurogenin1, and NeuroD mRNA. Experimental manipulation of nasal explants revealed dual expression of nestin protein and LHRH in cells proximal to the vomeronasal organ anlage that was dependent upon midline cartilaginous/mesenchymal tissues. Prolonged nestin expression in LHRH cells after midline removal is consistent with nasal midline tissues modulating differentiation of LHRH neurons from the nasal placode.

Novel gene expressed in nasal region influences outgrowth of olfactory axons and migration of luteinizing hormone-releasing hormone (LHRH) neurons.

Although a variety of cues have been implicated in axonal targeting during embryogenesis and regeneration, the precise mechanisms guiding olfactory axons remain unclear. Appropriate olfactory axon pathfinding is essential for functional chemoreceptive and pheromone receptive systems. Olfactory axon pathfinding is also necessary for establishment of the neuroendocrine LHRH system, cells critical for reproductive function. LHRH cells exhibit neurophilic migration moving from the nasal region along olfactory axons into the brain. Factors involved in the migration of these neuroendocrine cells are as yet unresolved. We report identification of a novel factor termed nasal embryonic LHRH factor (NELF) that was discovered in a differential screen of migrating versus nonmigrating primary LHRH neurons. NELF is expressed in PNS and CNS tissues during embryonic development, including olfactory sensory cells and LHRH cells. NELF antisense experiments indicate that a reduction in NELF expression decreases olfactory axon outgrowth and the number of LHRH neurons that migrate out of the nasal tissue. These results demonstrate that NELF plays a role as a common guidance molecule for olfactory axon projections and subsequently, either directly or indirectly, in the neurophilic migration of LHRH cells.

Ectopic expression of luteinizing hormone-releasing hormone and peripherin in the respiratory epithelium of mice lacking transcription factor AP-2alpha.

The vertebrate transcription factor activator protein-2 (AP-2alpha) is involved in craniofacial morphogenesis. In the nasal placode AP-2alpha expression delineates presumptive respiratory epithelia from olfactory epithelia, with AP-2alpha expression restricted to the anterior region of the respiratory epithelium (absent from the olfactory epithelium) at later stages. To address the role AP-2alpha plays in differentiation of cell groups in the nasal placode, the spatiotemporal expression pattern of four markers normally associated with olfactory epithelial structures was analyzed in mice lacking AP-2alpha. These markers were the intermediate filament protein peripherin, the neuropeptide luteinizing hormone-releasing hormone (LHRH), the neural cell adhesion molecule (NCAM) and the olfactory transcription factor Olf-1. Development of cells expressing these markers was similar in both genotypes until embryonic day 12.5 (E12.5), indicating that the main olfactory epithelium and olfactory pit formation was normal. At E13.5 in mutant mice, ectopic LHRH neurons and peripherin axons were detected in respiratory epithelial areas, areas devoid of Olf-1 and NCAM staining. Over the next few days, an increase in total nasal LHRH neurons occurred. The increase in nasal LHRH neurons could be accounted for by LHRH neurons arising and migrating out of respiratory epithelial regions on peripherin-positive fibers. These results indicate that AP-2alpha is not essential for the separation of the olfactory and respiratory epithelium from the nasal placode and is consistent with AP-2alpha preventing recapitulation of developmental programs within the respiratory epithelium that lead to expression of LHRH and peripherin phenotypes.

Molecular cloning, expression, and characterization of rat homolog of human AP-2alpha that stimulates neuropeptide Y transcription activity in response to nerve growth factor.

Neuropeptide Y (NPY) plays an important role in the central regulation of neuronal activity, endocrine and sexual behavior, and food intake. Although transcription activity of the NPY gene in PC12 cells is regulated by a number of agents such as nerve growth factor (NGF), the mechanism responsible for the NGF-elicited increase in the transcription of the NPY gene remains to be explored. In this study, we isolated and characterized a nuclear protein that is bound to NGF-response elements (NGFRE) that lie between nucleotide -87 and -33 of the rat NPY promoter gene. This nuclear protein is identical to the rat homolog of human transcription factor AP-2alpha. We further demonstrated that rat AP-2a promotes efficient NPY transcription activity in response to NGF. Finally, we provide direct evidence that the mice lacking transcription factor AP-2alpha exhibit reduced expression of NPY mRNA compared with wild-type mice, further supporting the hypothesis that AP-2alpha is an important transcription factor in regulating NPY transcription activity.

Transcription factor activator protein-2 is required for continued luteinizing hormone-releasing hormone expression in the forebrain of developing mice.

LHRH is the neuropeptide responsible for reproductive function. Prenatally, LHRH expression begins when neurons are in the olfactory pit and continues as these cells migrate into the brain. Thus, LHRH neurons maintain neuropeptide expression through very distinct environments. The regulatory interactions that control onset and continued expression of the LHRH phenotype are unknown. To begin to address this question primary LHRH neurons were removed from nasal explants at different ages. A complementary DNA (cDNA) subtraction screen was performed comparing a 3.5-days in vitro LHRH neuron [approximately embryonic day 15 (E15) in vivo] to two 10.5-days in vitro LHRH neurons (approximately postnatal day 1 in vivo). The transcription factor activator protein-2 (AP-2alpha) was differentially expressed and was present in the developmentally younger LHRH neuron. In vivo analysis revealed that LHRH neurons expressed AP-2 as they migrated across the cribriform plate and into the forebrain beginning on E13.5, but that coexpression of LHRH and AP-2 was no longer detected in postnatal day 1 animals. This suggested a regulatory role for AP-2 in LHRH neurons. Analysis of animals lacking AP-2alpha revealed a dramatic decrease in forebrain LHRH neurons between E13.5 and E14.5, correlating with normal onset of AP-2 expression in LHRH neurons as they entered the central nervous system. Nasal cells robustly expressing LHRH were still present on E 14.5. The continued presence of forebrain LHRH cells is proposed based on a second marker, galanin, and lack of increased apoptotic/necrotic cells in this region. A decrease in LHRH messenger RNA in forebrain neurons indicates regulation of LHRH occurred at the transcriptional or posttranscriptional level in mutant animals. These results indicate a developmentally restricted involvement of the transcription factor AP-2 in LHRH expression once the LHRH neurons have migrated into the forebrain, but before establishment of an adult-like distribution.