Anne Z. Murphy

Ovarian steroid modulation of seizure severity and hippocampal cell death after kainic acid treatment

To determine whether maintained estrogen or progesterone levels affect kainic acid (KA) seizure patterns or the susceptibility of hippocampal neurons to death from seizures, ovariectomized Sprague-Dawley rats were implanted with estrogen pellets, 0.1 or 0.5 mg, that generated serum levels of 42.4 +/- 6.6 (mean +/- SEM) and 242.4 +/- 32.6 pg/ml or one to six capsules of progesterone that generated serum levels of 11.00 +/-.72 to 48.62 +/- 9.4 ng/ml. Seven days later, the rats were administered KA (8.5mg/kg, ip) and scored for seizure activity; 96 h later, the rats were killed and their brains processed for localization of neuron nuclear antigen (NeuN), a general neuronal marker. The hippocampus was scored for spread (the number of separate regions showing cell loss), and the area within the CA fields occupied by NeuN immunoreactivity was measured (indicating surviving neurons). Administration of estrogen or progesterone (independent of dose) significantly reduced mortality from KA seizures. Progesterone reduced seizure severity in animals that received one to four implants; compared with controls, no difference in seizure severity was noted for animals with six progesterone implants. The reduced seizures in progesterone-treated animals were accompanied by a reduction in the spread of hippocampal damage (r(2) = 0.87; P < 0.05). Likewise, in progesterone-treated rats, neuron survival and reduction in seizure scores were correlated (r(2) = 0.76; P < 0.0001). Estrogen had no effect on seizure severity (P > 0.05), but reduced both the spread (P < 0.05) and degree of neuronal loss (P < 0.05). Indeed, in the estrogen-treated rats, neuronal death was significantly lower than that observed in progesterone-treated animals with equally severe seizures (P < 0.05). These data are consistent with the hypothesis that progesterone produces its effects by reducing seizures, whereas estrogen has little beneficial effect on seizure behavior but protects the hippocampus from the damage seizures produce.

Distribution of gonadal steroid receptor–containing neurons in the preoptic–periaqueductal gray–brainstem pathway: A potential circuit for the initiation of male sexual behavior

The present study used anterograde and retrograde tract tracing techniques to examine the organization of the medial preoptic—periaqueductal gray—nucleus paragigantocellularis pathway in the male rat. The location of neurons containing estrogen (alpha subtype; ERα) and androgen receptors (AR) were also examined. We report here that injection of the anterograde tracer biotinylated dextran amine (BDA) into the medial preoptic (MPO) produced dense labeling within the periaqueductal gray (PAG); anterogradely labeled fibers terminated in close juxtaposition to neurons retrogradely labeled from the nucleus paragigantocellularis (nPGi). Dual immunostaining for Fluoro‐Gold (FG) and ERα or FG and AR showed that over one‐third of MPO efferents to the PAG contain receptors for either estrogen or androgen. In addition, approximately 50% of PAG neurons retrogradely labeled from the nPGi were immunoreactive for either ERα or AR. These results are the first to establish an MPO→PAG→nPGi circuit and further indicate that gonadal steroids can influence neuronal synaptic activity within these sites. We reported previously that nPGi reticulospinal neurons terminate preferentially within the motoneuronal pools of the lumbosacral spinal cord that innervate the pelvic viscera. Together, we propose that the MPO→PAG→nPGi circuit forms the final common pathway whereby MPO neural output results in the initiation and maintenance of male copulatory reflexes. J. Comp. Neurol. 438:191–212, 2001.

Physiologic progesterone reduces mitochondrial dysfunction and hippocampal cell loss after traumatic brain injury in female rats.

Growing literature suggests important sex-based differences in outcome following traumatic brain injury (TBI) in animals and humans. Progesterone has emerged as a key hormone involved in many potential neuroprotective pathways after acute brain injury and may be responsible for some of these differences. Many studies have utilized supraphysiologic levels of post-traumatic progesterone to reverse pathologic processes after TBI, but few studies have focused on the role of endogenous physiologic levels of progesterone in neuroprotection. We hypothesized that progesterone at physiologic serum levels would be neuroprotective in female rats after TBI and that progesterone would reverse early mitochondrial dysfunction seen in this model. Female, Sprague-Dawley rats were ovariectomized and implanted with silastic capsules containing either low or high physiologic range progesterone at 7 days prior to TBI. Control rats received ovariectomy with implants containing no hormone. Rats underwent controlled cortical impact to the left parietotemporal cortex and were evaluated for evidence of early mitochondrial dysfunction (1 h) and delayed hippocampal neuronal injury and cortical tissue loss (7 days) after injury. Progesterone in the low physiologic range reversed the early postinjury alterations seen in mitochondrial respiration and reduced hippocampal neuronal loss in both the CA1 and CA3 subfields. Progesterone in the high physiologic range had a more limited pattern of hippocampal neuronal preservation in the CA3 region only. Neither progesterone dose significantly reduced cortical tissue loss. These findings have implications in understanding the sex-based differences in outcome following acute brain injury.

The organization of preoptic-medullary circuits in the male rat: evidence for interconnectivity of neural structures involved in reproductive behavior, antinociception and cardiovascular regulation.

The present studies used anatomical tract-tracing techniques to delineate the organization of pathways linking the medial preoptic area and the ventral medulla, two key regions involved in neuroendocrine, autonomic and sensory regulation. Wheatgerm agglutinin-horseradish peroxidase injections into the ventromedial medulla retrogradely labeled a large number of neurons in the medial preoptic area, including both the median and medial preoptic nuclei. The termination pattern of preoptic projections to the medulla was mapped using the anterograde tracers Phaseolus vulgaris leucoagglutinin and biotinylated dextran amine. Tracer injections into the preoptic area produced a dense plexus of labeled fibers and terminals in the ventromedial and ventrolateral pons and medulla. Within the caudal pons/rostral medulla, medial preoptic projections terminated heavily in the nucleus raphe magnus; strong anterograde labeling was also present in the pontine reticular field. At mid-medullary levels, labeled fibers focally targeted the nucleus paragigantocellularis, in addition to the heavy fiber labeling present in the midline raphe nuclei. By contrast, very little labeling was observed in the caudal third of the medulla. Experiments were also conducted to map the distribution of ventral pontine and medullary neurons that project to the medial preoptic area. Wheatgerm agglutinin-horseradish peroxidase injections in the preoptic area retrogradely labeled a significant population of neurons in the ventromedial and ventrolateral medulla. Ascending projections from the medulla to the preoptic area were organized along rostral-caudal, medial-lateral gradients. In the caudal pons/rostral medulla, retrogradely labeled cells were aggregated along the midline raphe nuclei; no retrograde labeling was present laterally at this level. By contrast, in the caudal half of the medulla, cells retrogradely labeled from the medial preoptic area were concentrated as a discrete zone dorsal to the lateral reticular nucleus; labeled cells were not present in the ventromedial medulla at this level. The present findings suggest that the medial preoptic area and ventral midline raphe nuclei share reciprocal connections that are organized in a highly symmetrical fashion. By contrast, preoptic-lateral medullary pathways are not reciprocal. These preoptic-brainstem circuits may participate in antinociceptive, autonomic and reproductive behaviors.

Androgen and Estrogen (α) Receptor Distribution in the Periaqueductal Gray of the Male Rat

The midbrain periaqueductal gray (PAG) has been strongly implicated in numerous behaviors heavily influenced by the gonadal steroids estrogen and testosterone, including reproductive behavior, autonomic regulation, and antinociception. However, the location of receptors for these steroids within the PAG has not been carefully characterized. Immunocytochemical techniques were used to map the distribution of neurons immunoreactive for the androgen (AR) and estrogen receptor (α subtype; ERα) along the rostrocaudal axis of the PAG in the male rat. The results show that the PAG contains a large population of both androgen and estrogen receptor containing neurons. Neurons immunoreactive for either receptor were concentrated within the caudal two-thirds of the PAG. At midlevels of the PAG, ERα and AR immunoreactive neurons were located primarily within the dorsomedial and lateral PAG. In the caudal third of the PAG, immunoreactive cells were distributed primarily within the dorsal half. The distributions of ERα and AR were remarkably similar, and it is likely that some PAG neurons contain receptors for both gonadal steroids, similar to what has been previously reported for the male rat hypothalamus. The results of this study suggest that the PAG may provide the anatomical substrate for steroid mediated changes in nociceptive thresholds and reproductive behavior.

Progesterone attenuates persistent inflammatory hyperalgesia in female rats: involvement of spinal NMDA receptor mechanisms.

The relationship between endogenous gonadal steroid levels and persistent or chronic pain is poorly understood. These studies used an inflammation model to examine the role of the gonadal steroid, progesterone, in the development of persistent pain and hyperalgesia in lactating ovary-intact and ovariectomized rats. The results indicate that constant high plasma levels of progesterone attenuate inflammatory hyperalgesia by a mechanism involving inhibition of N-methyl-D-aspartate receptor activation at the spinal cord level. Since the pattern of high progesterone in lactating rats mimics the progesterone component of the luteal phase of the human menstrual cycle, these findings have significance in persistent or chronic pain conditions that are most prevalent in females.

Pain: Sex/Gender Differences

The issue of how sex and gender differences influence pain continues to be a focus of intense investigation at many levels of inquiry. Epidemiological studies consistently report a higher prevalence of chronic pain disorders in females in comparison to males. Indeed, very few persistent pain conditions show a higher male prevalence. Psychophysically, women will generally rate a stimulus as being noxious at a lower intensity level than males; however, there are many exceptions to this generalization, with individual and situational variations being greater than the sex differences. On the other hand, substantial sex and gender differences do appear to exist in the mechanisms by which pain is generated and relayed centrally. The many factors that underlie sex differences in pain mechanisms interact dynamically, and develop and change progressively throughout the life span of each individual. Sex differences in the actions of the gonadal steroids estradiol, progesterone, and testosterone have also been shown to contribute to these differences, affecting wide regions of the central and peripheral nervous system. However, despite their potency, gonadal steroids represent only one of many factors that influence the experience of pain. While these differences would appear to have potent clinical implications for the development of different strategies to diagnose and treat pain in women and men, it remains the case that such strategies are most effective when they are focused on the individual, with the sex of that individual being only one of many factors being considered.

Anatomical Markers of Activity in Hypothalamic Systems

Attempts to study the activity of individual hypothalamic neurons directly have been fraught with problems. The location of some hypothalamic nuclei close to the base of the brain or ventricles makes access difficult. Moreover, with the exception of the magnocellular neurons that contain vasopressin (VP) and oxytocin (OT), the remaining hypothalamic cells are small and rarely concentrated within cytoarchitectonically defined nuclear boundaries. Even within some of the distinct nuclei of the hypothalamus, functionally diverse neuronal populations are present. For example, in the paraventricular nucleus (PVN), whereas the VP cells that regulate water balance are clustered into large magnocellular groups in the lateral subnucleus, the OT neurons of the same nucleus do not form homogeneous groups. Moreover, OT neurons that regulate posterior pituitary function are interspersed among OT neurons that do not innervate the posterior pituitary, but rather, project to the brain stem and spinal cord autonomic regions. In the more medial regions of the PVN, OT neurons intermix with parvocellular neurons (not containing OT) that participate in a number of diverse functions, such as regulation of temperature, blood pressure, and gastric motility and secretion. This heterogeneity is widespread within the hypothalamus and points to the necessity of being able to identify, a priori, which populations of neurons participate in a specific function.

Olfaction and brainstem circuits of reproductive behavior in the rat.

Publisher Summary This chapter discusses evidence for relatively direct anatomical linkages from the olfactory system to subcortical forebrain, midbrain, and brainstem structures that mediate autonomic and antinociceptive functions, as well as neuroendocrine and behavioral aspects of reproduction and defensive behaviors. Reproductive behavior includes pheromonal-motivational, neuroendocrine, and behavioral-performance components. Additionally, nociceptive thresholds and cardiovascular measures change dramatically. These autonomic and sensory adjustments are likely to be mediated via descending projections from the medulla to the spinal cord. Investigations have shown that activation of the medial preoptic area (MPO), a forebrain region intimately involved in sexual behavior, excites a substantial population of periaqueductal gray matter (PAG) output neurons, which in turn, project to the medulla. This finding establishes a functional connection between MPO ￫ PAG input columns and the PAG ￫ medullary output columns. Additional new results suggest that these MPO projections directly influence PAG and rostral ventral medulla neurons involved in reproductive behaviors, as well as autonomic, neuroendocrine, and sensory functions. These midbrain and medullary circuits are integrated with additional networks involved in antinociception and defensive/aggressive behaviors.

Fos expression in rat pontine tegmental neurons following activation of the medial preoptic area

Fos immunohistochemistry was used to map the distribution of pontine neurons excited by activation of the medial preoptic area (MPO). Although we have previously shown that Barringtons nucleus receives a very dense focal input from the MPO, electrical stimulation of the preoptic area unexpectedly induced very little Fos expression in Barringtons neurons. These results suggest that the MPO-->Barringtons projection utilizes a transmitter(s) that does not involve transduction of the Fos protein; alternatively, MPO afferents to Barringtons nucleus may be inhibitory in nature. As Barringtons nucleus plays a critical role in micturition, MPO projections to Barringtons nucleus may regulate voiding reflexes during sexual behavior. Interestingly, while the locus coeruleus (LC) proper receives only a sparse projection from the MPO, extensive Fos expression was present in LC. The finding of Fos immunoreactive LC neurons suggests that the excitatory influence of MPO may regulate LC neuronal activity and NE release during reproductive behaviors.