Catherine Ulibarri

Gonadal steroid hormone modulation of nociception, morphine antinociception and reproductive indices in male and female rats.

&NA; The purpose of this study was to examine how gonadal steroid hormones modulate basal nociception and morphine antinociception relative to regulating reproduction in the adult rat. Male and female Sprague–Dawley rats were either gonadectomized (GDX) or sham‐gonadectomized (sham); GDX males were implanted subcutaneously with capsules containing testosterone (T), estradiol (E2), dihydrotestosterone (DHT), E2 and DHT, or nothing (0). GDX females received E2, T, or empty (0) capsules immediately after surgery, and vehicle or progesterone (P4) injections at 4‐day intervals. Basal nociception and morphine antinociception were tested 28 days after surgery on 50°C and 54°C hotplate tests, and reproductive behavior and physiology were assessed shortly thereafter. There were no significant differences in baseline hotplate latencies among the male treatment groups, but morphine was significantly more potent in sham and GDX+T males than in GDX+0 males. The ability of T to increase morphines potency was approximated by its major metabolites E2 and DHT, given together but not alone. Baseline hotplate latencies were higher in sham females tested during diestrus than in those tested during estrus. Morphine was significantly more potent in sham females tested during proestrus and diestrus than in those tested during estrus. Baseline hotplate latencies were significantly higher, and morphine was significantly less potent in GDX+E2, GDX+E2/P4 and GDX+T females than in GDX+0 females. All group differences in basal nociception and morphine antinociception observed on the 50°C hotplate test were smaller and generally non‐significant on the 54°C hotplate test. Steroid manipulations produced the expected changes in reproductive behaviors and steroid‐sensitive organs. These results demonstrate that in adult rats, gonadal steroid manipulations, that are physiologically relevant, modulate (1) basal nociception in females but not males, and (2) morphines antinociceptive potency in both males and females.

Sensitization to Stress and Psychostimulants

Rats exposed to chronic stress demonstrate an enhanced motor response to psychostimulants, such as cocaine. This behavioral cross-sensitization between stress (repeated mild footshock) and cocaine (15 mg/kg, ip) is associated with an increase in extracellular dopamine in the nucleus accumbens, but a decrease in the prefrontal cortex. To determine a role for the hypothalamic-pituitary-adrenal axis in sensitization, rats were adrenalectomized prior to administering repeated cocaine injections. One and twelve days after discontinuing repeated cocaine, rats were challenged with acute cocaine. Adrenalectomy blocked the cocaine-induced sensitization observed in sham animals, but both the sham and adrenalectomized rats demonstrated behavioral sensitization to cocaine after twelve days of withdrawal. These data argue that while long-term alterations in dopamine transmission may be an important neurochemical substrate of stress and psychostimulant-induced sensitization, the hypothalamic-pituitary-adrenal axis may not have a necessary role.

Stress-induced cross-sensitization to cocaine: effect of adrenalectomy and corticosterone after short- and long-term withdrawal

Abstract We have recently shown that adrenalectomy (ADX) in rats blocks the appearance of cocaine-induced sensitization when this behavioral response is tested at early withdrawal times (1–2 days), but not after later withdrawal from cocaine (12 days). To determine if a similar phenomenon occurred with stress-induced sensitization, male Sprague-Dawley rats were given a sham ADX, ADX surgery, or ADX plus SC implanted corticosterone (CORT) pellets (CORT 12.5% pellets or CORT 50% pellets). A fifth group was given ADX surgery, but CORT 50% pellets were implanted after repeated stress treatment. One week after surgery, each group was divided into two additional groups, naive and stress. Naive animals remained unhandled, while stress rats were given a variety of daily stressors administered twice per day for 6 consecutive days. One day after the last stress, rats were given a saline injection followed by a cocaine injection (15 mg/kg, IP) the next day, and locomotor activity was monitored (early withdrawal). Two weeks after the last stress, the locomotor responses to an additional saline and cocaine injection were monitored (late withdrawal). At early withdrawal, no significant sensitization occurred for horizontal activity, but cross-sensitization was demonstrated for vertical activity. At late withdrawal, sham controls showed a stress-induced elevation in horizontal activity, with only a trend toward increased vertical activity. Animals given ADX surgery or ADX and CORT 12.5% pellets did not demonstrate sensitization to repeated stress, while CORT 50% pellets in ADX rats restored the sensitized horizontal response to cocaine challenge at late withdrawal. In contrast, stress-pretreated rats which were given CORT 50% pellets during the 2-week withdrawal period after the stress showed a marked decrease in horizontal activity in response to cocaine challenge at late withdrawal. The results provide evidence for a necessary role for adrenal hormones in long term, but not short-term, stress-induced cross-sensitization. Together with our previous study on the role of CORT in cocaine-induced sensitization, the results indicate that CORT is not the common factor mediating the long-term sensitization to cocaine and stress.

Effect of adrenalectomy on the initiation and expression of cocaine-induced sensitization

The role of corticosterone in the initiation and expression phases of sensitization to cocaine was examined. To determine the effect of corticosterone on the initiation of sensitization, male Sprague-Dawley rats were given a sham adrenalectomy (ADX), or ADX surgery. Approximately 1 week later, rats were given a cocaine (15 mg/kg, IP) injection on day 1. On days 2–6, rats were given cocaine (30 mg/kg, IP), and the next day, a cocaine challenge (15 mg/kg) was administered (= early withdrawal). Four days later, 50% of the ADX rats were given corticosterone pellets and corticosterone in the evening drinking water to mimic the circadian variation in corticosterone levels. After 1 week, rats were given a final saline challenge followed by a cocaine challenge (15 mg/kg) the next day (= late withdrawal). Locomotor activity was monitored after cocaine treatment on day 1 and after challenge at early and late withdrawal. Sham controls demonstrated a sensitized locomotor response to the cocaine challenge at early withdrawal, with a slight increase in this behavioral sensitization at the late withdrawal time. In contrast, sensitization was not observed in ADX rats after early withdrawal from cocaine, but this attenuation was not permanent, since ADX animals demonstrated control levels of sensitization by the late withdrawal time 12 days later. Animals given corticosterone replacement 1 week prior to the late cocaine challenge also demonstrated a sensitized response similar to control levels. The effect of corticosterone on the expression of sensitization was examined by administering daily cocaine as before followed by surgery a few days later. The treatment groups were sham, ADX and ADX+corticosterone replacement as described. Fourteen days later, a saline injection was given followed by a cocaine challenge the next day. Behavioral sensitization to a cocaine challenge was found in all three treatment groups. These data suggest that adrenal hormones are necessary during the initiation phase of sensitization when observed after early withdrawal (1 day), but not when sensitization is examined at a later withdrawal time (12 days). In addition, corticosterone levels do not significantly affect the expression phase of behavioral sensitization to cocaine.

Proposed animal neurosensitization model for multiple chemical sensitivity in studies with formalin

A potentially promising line of animal research relevant to multiple chemical sensitivity (MCS) is that of sensitization in the central nervous system (CNS), particularly limbic pathways in the brain. Sensitization is the progressive and enduring enhancement in behavioral and neurochemical responses that occurs after repeated exposure to psychostimulants or environmental stressors. Since the onset and progression of sensitization has many parallels with that of MCS, it has been proposed that MCS may be initiated through a mechanism similar to the sensitization of CNS components occurring in the rodent. To test this hypothesis, female Sprague-Dawley rats were exposed to formalin vapors (FORM, 11 ppm) or water vapor (control) 1 h/day for 7 days. The next day, a saline injection was given followed by a cocaine injection (15 mg/kg, i.p.) 24 h later, and locomotor activity was monitored. Animals pretreated with repeated FORM inhalation demonstrated a significantly enhanced locomotor response to cocaine compared to controls, an indicator that specific limbic pathways may have been sensitized. At 4 weeks of withdrawal from FORM exposure, a subset of animals remained sensitized to a cocaine challenge. No differences were found between groups after a saline injection. In a second experiment, animals were screened prior to FORM or water exposure for their response to a novel situation, a measure believed to reflect an animals general responsiveness to stimuli. Rats were divided into high responders (HR) or low responders (LR), based on their locomotion in a novel cage. Results from three behavioral tests demonstrated that HR and LR were differentially affected by exposure to FORM. In a passive avoidance test, HR and LR appeared to be different in their distribution of responses, while HR and LR responses in the FORM group were nearly identical. On the elevated plus maze test of anxiety, HR spent more time on the open arms than LR in both treatment groups, with significant differences between HR and LR in the FORM, but not water, treated group. On a hot plate test to measure nociceptive levels, no differences occurred between HR and LR in the control group, whereas nociception of LR tended toward an increase compared to HR in the FORM-exposed group. Results from the second experiment suggest that the effects of FORM exposure may be obscured by examining behavior in a heterogeneous population (HR and LR). This approach using animal models may help define neural substrates that mediate the amplification of responses of a subpopulation of individuals to chemicals in the environment.

Dose- and time-dependent estradiol modulation of morphine antinociception in adult female rats.

To clarify the activational role of ovarian hormones on pain and analgesia, the present study determined whether estradiol (E2) modulation of nociception and morphine antinociception in adult female rats depends on (1) the dose of E2 and (2) the interval between E2 treatment and nociceptive testing. Female rats were ovariectomized (OvX) and either oil vehicle (0), or E2 (0.25, 2.5 or 25μg/0.1ml vehicle) was injected s.c. two consecutive days of every four days for five cycles before testing. Either 4, 24, 48 or 96h after the last injection, nociception was evaluated on the 50°C hotplate and warm water tail withdrawal tests before and after escalating doses of s.c. morphine. Lordosis behavior and uterine weight were assessed in other rats at the same E2 doses and time points. E2 significantly lengthened latency to respond on the hotplate test at 24h after the last injection, but had no significant effect on tail withdrawal latencies. The lower doses of E2 significantly increased morphine antinociceptive potency at 4–24h on one or both tests, but the intermediate E2 dose significantly decreased morphine potency at 48h on the hotplate test. Thus, E2 modulation of morphine antinociception in the adult female rat is bidirectional, and occurs at E2 doses producing cyclic changes in sexual behavior, uterine weight and vaginal cytology that are similar to those observed in gonadally intact, cycling females.

Long-term castration effects motoneuron size but not number in the spinal nucleus of the bulbocavernosus in the adult male Mongolian gerbil

The spinal nucleus of the bulbocavernosus (SNB) is a sexually dimorphic group of motoneurons in Rexeds Lamina X of the lumbosacral spinal cord of the Mongolian gerbil. The SNB innervates the perineal musculature, the bulbocavernosus (BC), levator ani (LA), and external anal sphincter (EAS). Recent studies demonstrated a peripubertal component to the masculinization of the gerbil SNB with an apparent increase in both motoneuron size and number after puberty as measured with a Nissl stain. However, these studies could not determine if the apparent change in motoneuron number were due to the long-term castration involved in the methodology or due to a loss of motoneuron size beyond the point of being recognizable as motoneurons. Therefore, the current study was undertaken to examine this possibility by repeating the experimental protocol from previous peripubertal studies, on adult male gerbils with the addition of retrograde tract-tracing. Adult male gerbils were castrated at postnatal day (PND) 150 and given subcutaneous implants of testosterone proprionate (TP), dihydrotestosterone (DHT), estradiol benzoate (EB), EB and DHT, no steroid, or left intact. At PND215, the animals were injected with fluorogold (FG), a retrograde tract-tracer. At PND220, the animals were aldehyde perfused transcardially. The spinal cords were sectioned and alternate sections processed for either thionin-stain or FG visualization. Results indicated that long-term castration had no effect on SNB motoneuron number but did decrease SNB motoneuron size. TP, and to a lesser extent DHT, treatment could prevent the reduction in motoneuron size, however, EB could not. This study indicates that the maintenance of the adult male gerbil SNB-BC system is androgen, not estrogen, dependent and that long-term castration does not reduce motoneuron size to the point where they can no longer be distinguished.

Sexual dimorphism in the number and size of SNB motoneurons: delayed development during normal ontogeny

The spinal nucleus of the bulbocavernosus (SNB) is a sexually dimorphic pool of motoneurons that innervates the perineal musculature. In the Mongolian gerbil, the SNB lies dorsolateral to the central canal within the lumbosacral spinal cord. Previously, no information was available on the normal development of the sexual dimorphism of this structure in the Mongolian gerbil, although evidence exists for a peripubertal development of the SNB in the gerbil. At each age from postnatal day 1 (PND1) through PND15 and at PND25, male and female gerbils were aldehyde perfused. Spinal cords were gelatin-embedded, cryoprotected, frozen and sectioned coronally through the lumbosacral transition zone and stained for Nissl substance with thionin. Examination by light microscopy revealed that the number of visible male and female SNB motoneurons significantly increased from PND1 through PND25. The size of the motoneurons also significantly increased in both sexes, however, until PND15 male gerbil SNB showed two significantly different sized populations of motoneurons. These data suggest the development of the SNB in the Mongolian gerbil is delayed, compared to the rat, and may continue well beyond the perinatal time period.

Early postnatal response of the spinal nucleus of the bulbocavernosus and target muscles to testosterone in male gerbils.

This study examined the response of the spinal nucleus of the bulbocavernosus (SNB) and the bulbocavernosus (BC) muscle, to testosterone in male Mongolian gerbils (Meriones unguiculatus) during the early postnatal period. Male gerbil pups were given testosterone propionate (TP) or vehicle for 2 days, then perfused on postnatal day (PND) 3, 5, 10 or 15. The BC and levator ani (LA) muscles were removed, weighed, and sectioned. Cross-sections of BC muscle fibers were measured and muscle fiber morphology examined. Spinal cords were removed and coronally sectioned in order to count and measure the SNB motoneurons. Following TP treatment, male pups of all ages had significantly heavier BC-LA muscles and larger fibers in the BC muscle compared to age-matched controls. The increase in muscle weight following TP treatment was greatest at PND10, while fiber size increased to a similar degree at all ages suggesting that hyperplasia as well as hypertrophy was responsible for the increase in muscle mass at this time. SNB motoneurons increased significantly in number and size with age and TP treatment. We hypothesize that the increase in SNB motoneuron number during normal ontogeny that can be augmented by TP treatment and represents an unusual means of establishing sexual dimorphism in the nervous system of a mammal through cell recruitment to the motor pool of a postnatal animal.

Transiently catecholaminergic cells in the fetal rat express mRNA for the glutamate NMDAR1 receptor

The N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor has been shown to be vital to the development of the central nervous system. The purpose of this study was to determine if the neural crost-derived precursors which migrate to the primitive gut contain mRNA encoding for the NMDA receptor. Many of these enteric precursors briefly elaborate tyrosine hydroxylase (TH) and have been termed transiently catecholaminergic (TC) cells. TH-like immunoreactivity (TH-ir) serves as a marker for them. Immunocytochemistry combined with NMDAR1 in situ hybridization revealed that TH-ir cells in Day 14 rat embryos do express mRNA coding for the NMDAR1 receptor. However, the TC cells did not contain detectable levels of immunoreactivity for the NMDAR1 receptor peptide. The absence of detectable NMDAR1-like immunoreactivity might reflect some form of transcriptional or translational regulation, such that the onset of functional receptor activity is delayed until differentiation and/or synaptogenesis commence. Whether TC cell migration is glutamate-mediated remains unclear, since some of them successfully reached the gut without expressing NMDAR1 message. Characterizing TC cell NMDA receptor activity and determining exactly when it ensues will be of paramount importance to defining the role(s) of this receptor in ENS development. In conclusion, the expression of NMDAR1 mRNA by TH-ir cells suggests a possible developmental role for this receptor.