Assessment of Nerve Injury-induced Mechanical Hypersensitivity in Rats using an Orofacial Operant Pain Assay.

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results by addressing the cognitive and motivational aspects of pain in rodents. This paper presents a protocol for assessing mechanical hypersensitivity following chronic constriction injury of the infraorbital nerves (CCI-ION) in rats using an orofacial operant pain system. Before CCI-ION surgery, rats were trained in an orofacial pain assessment device (OPAD) to drink sweetened condensed milk while making facial contact with the metal spiked bars and lick-tube. In this assay, rats can choose between receiving milk as a positive reinforcer or escaping an aversive mechanical stimulus that is produced by a vertical row of small pyramid-shaped spikes on each side of the reward access hole. Following 2 weeks of training in the OPAD and before the CCI-ION surgery, baseline mechanical sensitivity data were recorded for 5 days for each rat during a 10 min testing session. During a session, the operant system automatically records the number of reward bottle activations (licks) and facial contacts, contact duration, and latency to the first lick, among other measures. Following baseline measurements, rats underwent either CCI-ION or sham surgery. In this protocol, mechanical hypersensitivity was quantified by measuring the number of licks, latency to the first lick, the number of contacts, and the ratio of licks to facial contacts (L/F). The data showed that CCI-ION resulted in a significant decrease in the number of licks and the L/F ratio and an increase in the latency to the first lick, indicating mechanical hypersensitivity. These data support the use of operant-based pain assays to assess mechanical pain sensitivity in preclinical pain research.

Operant assays for assessing pain in preclinical rodent models: highlights from an orofacial assay.

Despite an immense investment of resources, pain remains at epidemic proportions. Given this, there has been an increased effort toward appraising the process by which new painkillers are developed, focusing specifically on why so few analgesics make it from the benchside to the bedside. The use of behavioral assays and animal modeling for the preclinical stages of analgesic development is being reexamined to determine whether they are truly relevant, meaningful, and predictive. Consequently, there is a strengthening consensus that the traditional reflex-based assays upon which several decades of preclinical pain research has been based are inadequate. Thus, investigators have recently turned to the development of new preclinical assays with improved face, content, and predictive validity. In this regard, operant pain assays show considerable promise, as they are more sensitive, present better validity, and, importantly, better encompass the psychological and affective dimensions of pain that trouble human pain sufferers. Here, we briefly compare and contrast reflex assays with operant assays, and we introduce a particular operant orofacial pain assay used in a variety of experiments to emphasize how operant pain assays can be applied to preclinical studies of pain.

Medial preoptic area delta-opioid receptors inhibit lordosis.

Endogenous opioid peptides that activate the delta-opioid receptor (DOR) are thought to facilitate female receptive behavior. This facilitation of lordosis has been demonstrated by intracerebroventricular infusions and injection of DOR-active ligands into the ventromedial hypothalamic nucleus, an area with robust DOR binding. However, DOR binding is distributed throughout the hypothalamus, and the role of DOR in other areas of the hypothalamus has not been examined. In the current study, we demonstrated DOR immunoreactivity in the medial preoptic area (MPO), in particular medial preoptic nucleus (MPN) of the preoptic area. DOR immunoreactive processes were sparsely distributed in the medial and lateral parts of the MPN. Larger DOR immunoreactive fibers were localized in the ventrolateral aspect of the lateral MPN. The MPN is involved in the modulation of female sexual receptivity and the distribution of DOR in this area suggested to us that DOR may regulate lordosis. Ovariectomized rats with unilateral cannulae aimed at the MPN were given 5microg 17beta-estradiol benzoate (EB), once every 4 days and tested for lordosis. [D-Pen(2), D-Pen(5)]-enkephalin (DPDPE), a DOR agonist, microinfused into the MPO, 52-54h after EB-priming, inhibited lordosis when compared with the aCSF (vehicle) control (P <== 0.05). The inhibitory effects of DPDPE were reversed by microinjection of naltrindole, a DOR antagonist (P <== 0.05). Interestingly, the DOR inhibition of lordosis is similar to the micro-opioid receptor inhibition of lordosis in the MPN. These results indicate that DOR in the MPO, particularly in the MPNm, plays an important role in the regulation of lordosis.

Estrogen-induced mu-opioid receptor internalization in the medial preoptic nucleus is mediated via neuropeptide Y-Y1 receptor activation in the arcuate nucleus of female rats.

The endogenous peptides beta-endorphin (beta-END) and neuropeptide Y (NPY) have been implicated in regulating sexual receptivity. Both beta-END and NPY systems are activated by estrogen and inhibit female sexual receptivity. The initial estrogen-induced sexual nonreceptivity is correlated with the activation and internalization of mu-opioid receptors (MORs), in the medial preoptic nucleus (MPN). Progesterone reverses the estrogen-induced activation/internalization of MOR and induces the sexual receptive behavior lordosis. To determine whether NPY and endogenous opioids interact, we tested the hypothesis that estrogen-induced MOR activation is mediated through NPY-Y1 receptor (Y1R) activation. Retrograde tract tracing demonstrated Y1Ron beta-END neurons that projected to the MPN. Sex steroid modulation of MOR in the MPN acts through NPY and the Y1R. Estradiol administration or intracerebroventricular injection of NPY activated/internalized Y1R in the arcuate nucleus and MOR in the MPN of ovariectomized (OVX) rats. Moreover, the selective Y1R agonist [Leu31, Pro34]-Neuropeptide Y (LPNY) internalized MOR in the MPN of OVX rats. The Y1R antagonist (Cys31, Nva34)-Neuropeptide Y (27-36)2 prevented estrogen-induced Y1R and MOR activation/internalization. NPY reversed the progesterone blockade of estradiol-induced Y1R and MOR internalization in the arcuate nucleus and MPN, respectively. Behaviorally, LPNY inhibited estrogen plus progesterone-induced lordosis, and the MOR-selective antagonist D-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr amide reversed LPNY-induced inhibition of lordosis. These results suggest that a sequential sex steroid activation of NPY and MOR circuits regulates sexual receptivity.

Estrogen induces de novo progesterone synthesis in astrocytes.

The brain is an established target for peripheral steroids, but also expresses steroidogenic enzymes and is capable of de novo 'sex' steroid synthesis (neurosteroidogenesis) independent of peripheral steroidogenic organs. In adrenalectomized and ovariectomized rats that do not have peripheral sources of steroids, estrogen treatment increased progesterone levels specifically in the hypothalamus, indicating that estrogen stimulates progesterone neurosteroidogenesis. Recent studies have demonstrated that specific cell types preferentially secrete specific steroids, and that astrocytes are the primary progesterone synthesizing cells in the nervous system. We hypothesized that estrogen could directly induce de novo synthesis of progesterone in astrocytes. To determine whether estrogen stimulates progesterone synthesis in astrocytes, astrocyte-enriched cultures were grown to confluence, then grown for an additional 48 h in an estrogen- and phenol-free Dulbecco's Modified Eagle Medium (DMEM) and then treated with either 17beta-estradiol or steroid-free media. After culturing for 48 h in steroid-free, phenol red-free DMEM, low levels of progesterone were detected in the media, whereas progesterone levels were significantly increased in the media of astrocytes cultured in DMEM with 17beta-estradiol (10(-7)-10(-4)M). To determine whether estrogen regulated the mRNA expression of progesterone synthetic enzymes, P-450 side-chain cleavage and 3beta-hydroxysteroid dehydrogenase, control and 17beta-estradiol-treated astrocytes were harvested and prepared for Northern and slot blot analysis. Expression levels of enzyme mRNAs were very low and 17beta-estradiol did not significantly increase mRNA levels of either steroidogenic enzyme. These results suggest that estrogen directly stimulated the de novo synthesis of neuroprogesterone in astrocytes, and demonstrate the potential for estrogen to regulate reproductive physiology and behavior through the paracrine actions of astrocyte-derived progesterone.

The luteinizing hormone surge is preceded by an estrogen-induced increase of hypothalamic progesterone in ovariectomized and adrenalectomized rats.

As circulating estrogen levels rise on the afternoon of proestrus, they stimulate the hypothalamo-pituitary axis. This estrogen positive feedback is pivotal to stimulate the luteinizing hormone (LH) surge required for ovulation and luteinization of ovarian follicles. In addition to estrogen, pre-LH surge progesterone is critical for an LH surge as was demonstrated by blocking progesterone synthesis. In ovariectomized (OVX) rats treated with trilostane, a blocker of the enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD) that catalyzes the conversion of pregnenolone to progesterone, estrogen did not induce an LH surge. Further, estrogen induced an LH surge in OVX and adrenalectomized (ADX) rats, indicating that the source of progesterone was neither the ovary nor adrenal gland. This estrogen-only LH surge was inhibited by pretreatment with trilostane, indicating that although the adrenal gland and ovary were not necessary for positive feedback, progesterone synthesis was critical for estrogen-induced positive feedback in an OVX/ADX rat. This suggested that the LH surge is dependent on the pre-LH surge synthesis of progesterone. Estrogen-induced progesterone receptors in the hypothalamus are vital for the LH surge, so a potential location for progesterone synthesis is the hypothalamus. OVX/ADX female rats were treated with 17beta-estradiol (50 microg) and progesterone levels were assayed by RIA. Progesterone levels were elevated in hypothalamic tissue following estrogen treatment. No increases in tissue progesterone levels were found in parietal cortex, cerebellum, medulla, pituitary or plasma. Additionally, male rats that do not have an estrogen positive feedback-induced LH surge were examined. Castrated/ADX male rats had no increase in hypothalamic progesterone levels after estrogen treatment. Together, these data strongly suggest that estrogen enhances neuroprogesterone synthesis in the hypothalamus that is involved in the positive feedback regulating the LH surge.

Site-specific decrease of progesterone receptor mRNA expression in the hypothalamus of middle-aged persistently estrus rats.

Middle-aged females gradually become acyclic and spontaneously develop a persistently estrus (PE) state. PE rats, acyclic for 30 days (early PE), are unresponsive to the positive feedback action of estrogen, but respond to a progesterone challenge with a luteinizing hormone (LH) surge and ovulation; unlike long-term PE rats, acyclic for 90 days, neither estrogen nor estrogen plus progesterone will elicit an LH surge [10th International Congress of Endocrinology, San Francisco, P3 (1996) 1061]. We hypothesize that the PE state may develop due to a diminished level of estrogen-induced progesterone receptor (PR) expression in the hypothalamus that prevents progesterone from stimulating LH regulating circuits. To test this hypothesis, PR mRNA levels were measured in hypothalamic regions of young, proestrus (2-3 months of age), early PE (10-12 months) and long-term PE (13-15 months) rats. The anteroventral periventricular nucleus (AVPV), an important regulatory site of the LH surge, had decreased PR mRNA levels in early and long-term PE rats compared with proestrus rats. However, PR mRNA levels were reduced only in long-term PE rats in the ventromedial nucleus (VMH) and arcuate nucleus (ARH). In the medial preoptic nucleus (MPN), levels of PR mRNA did not change. A previous report showed that exogenous progesterone stimulates an LH surge in young and early PE animals, indicating that the expression of PR mRNA demonstrated in this study is sufficient to mediate progesterone facilitation of the LH surge in early PE rats. In acyclic, long-term PE rats, diminished estrogen-induced expression of progesterone receptors is correlated with a previously shown inability to respond to exogenous progesterone.