Proliferative effects of different hormone regimens on mammary glands in ovariectomized rats.

OBJECTIVE: To compare the proliferative effect of different hormone regimens and estrogen receptor modulation on mammary glands in a rat model of surgical menopause. DESIGN: Experimental animal study. SETTING: University Hospital. INTERVENTION: In a rat model of surgical menopause, 78 adult Sprague Dawley female rats were ovariectomized and treated with estrogen, estrogen combined with continuous or intermittent progesterone or the estrogen receptor modulator raloxifene and their respective vehicle controls. Following intraperitoneal drug administration for 20 days, rats were perfused, mammary glands were removed, tissues were processed for immunohistochemical (Ki-67) and hematoxylin-eosin staining, and investigated under light microscope. MAIN OUTCOME MEASURE: Histopathological examination of mammary glands and Ki-67 positive cells (proliferation index). RESULTS: Histological examination showed dilatation in the duct cysts and vacuolization in the epithelial cells in groups receiving progestin, either intermittent or continuous. Histological findings in the raloxifene group were no different from the control group, and the atrophic terminal ductal lobular unit in adipose tissue rich stroma was similar to postmenopausal breast. In animals with a proliferative response, increased proliferation started and dominated in the terminal ductal lobular unit epithelium. Comparison of Ki-67 proliferation indices between groups revealed that estrogen alone or combined with intermittent progesterone yielded significantly higher Ki-67 indices compared to controls; estrogen combined with continuous progesterone also resulted in increasing the probability of proliferation, but the effect was not as pronounced as the other two groups. Raloxifene treatment, on the other hand, did not cause proliferation. CONCLUSION: Estrogen alone or combined with progesterone may increase the risk of breast cancer by enhancing proliferation in the TDLU; raloxifen does not induce proliferation and may be a safe estrogen receptor modulator regarding its effects on mammary glands during menopause.

Effects of laterality and sex on cognitive strategy in a water maze place learning task and modification by nicotine and nitric oxide synthase inhibition in rats.

The aim of the present study was to investigate sex differences in learning strategies and to elucidate the mechanisms, which may underlie these differences. In two separate experiments, rats were presented with different strategies that could be employed to learn the position of a platform in a water maze (WM); furthermore, rats received treatments that could influence these strategies. In the first experiment, we demonstrated that the response-learning paradigm can be applied to the WM and can be compared with visually cued learning and reversal learning. Naïve rats of either sex could acquire this protocol relatively easily. On the probe trial, where the rats are presented with a choice between using response versus visually cued learning, initially response learning was preferred, however, during these experiments, laterality emerged as a significant factor and rats trained to turn right had difficulty in reversing the learned pattern to find the platform. The second part of our study evaluated the effects of nicotine and nitric oxide synthase (NOS) inhibition on the aforementioned parameters. Drug treatments impaired acquisition compared to saline treatments and the effect was more pronounced with NOS inhibition. During the probe trial, while NOS inhibition enhanced the right-side bias in both sexes, nicotine treatment had the same effect only in males. In conclusion, naïve rats can acquire place learning using visible cues or response learning; however, there is a right side bias in both sexes and the laterality effect is more pronounced in male rats. In drug-treated animals, while NOS inhibition enhances laterality (right bias) in both sexes similarly, nicotine modifies the cognitive strategy in a sexually dimorphic manner by augmenting the right bias only in male rats.

The effect of octreotide on kainate-induced wet dog shakes and seizure activity in male and female rats.

Systemic kainic acid (KA) administration to rats triggers wet dog shakes (WDS) followed by epileptic seizures. Although WDS are often associated with the occurrence of seizures, we have recently shown that following nitric oxide (NO) synthesis inhibition, the number of WDS decreased; subsequently the onset of seizure activity was shortened, and the number of convulsions was increased. Somatostatin (SS), whose release appears to be controlled by NO, inhibits seizure activity. There are sex differences in seizure susceptibility as well as in SS and NO activities in brain. The present study was undertaken to assess the effect of octreotide (OC), a stable SS analogue, on KA-induced WDS and seizures in rats, with emphasis on possible sex differences. WDS and seizures were induced by KA in male and female (proestrus) Sprague Dawley rats; OC or saline was injected 30 min before KA and the behavior was monitored for 120 min after KA. Octreotide increased the number of WDS and decreased the number of convulsions; this effect was more pronounced in males. Onset of KA-induced seizure activity was earlier in females than males; however, there was no effect of OC on seizure latency. Seizure activity started after the termination of WDS. These results show OC has opposite effects on WDS and convulsions, in that it stimulates the former and inhibits the latter. These results support our previous findings that WDS and seizure activity involve separate mechanisms and suggest that WDS may have an inhibitory effect on limbic seizures.

Serum nitric oxide metabolites in patients with multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system characterized by myelin breakdown. The free radical nitric oxide (NO), which is considered to be a major metabolite in immune function and in autoimmune disorders, is among the possible mediators causing the inflammatory reactions in MS. Consequently, NO has been implicated in the pathogenesis of MS and its animal model experimental allergic encephalomyelitis (EAE). In this study, stable metabolites of NO (NO(2-)+NO(3-)) levels were determined in sera of MS patients (n=23) and control subjects (n=16). NO(2-)+NO(3-) levels were higher in MS patients when compared to control subjects. However, there was not any correlation with serum NO(2-)+NO(3-) values and clinical features of the disease such as duration of sickness, the time elapsed from the last attack and EDSS values. Our results imply that nitric oxide may be involved in the pathogenesis of MS although further studies are required to elucidate underlying mechanisms.

The effect of adrenalectomy on cocaine and amphetamine-regulated transcript (CART) expression in the hypothalamic nuclei of the rat.

CART peptide is a neurotransmitter involved in various physiological processes including feeding, sensory processing, development, addiction, and stress. Substantial amounts of CART mRNA and CART peptide expression have been demonstrated in the hypothalamic periventricular area, the paraventricular nucleus of hypothalamus, the anterior lobe of the pituitary gland and the adrenal gland in addition to many other brain areas. This localization defines the HPA axis, responsible for the stress response. The aim of the present study was to assess the possible mediation of the CART peptides in the stress response by testing for changes in CART in adrenalectomized animals. Three groups of male Sprague-Dawley rats were used for the study: sham operated, adrenalectomized (ADX), and ADX+hormone replacement (corticosterone, 30 microg/ml in drinking water/5 days). All rats were perfused 7 days after the surgery, brains were removed and serial coronal sections were prepared. Immunohistochemistry was used to assess CART peptide expression in paraventricular and supraoptic cells. ADX lowered both the number and percentage of CART-positive cells compared to the sham-operated group, and hormone replacement partially restored the decrease in the CART cell numbers in ADX animals. There were no significant changes in the supraoptic nucleus. Our results suggest a role for CART peptides in the stress response.

Are attractors 'strange', or is life more complicated than the simple laws of physics?

Interesting and intriguing questions involve complex systems whose properties cannot be explained fully by reductionist approaches. Last century was dominated by physics, and applying the simple laws of physics to biology appeared to be a practical solution to understand living organisms. However, although some attributes of living organisms involve physico-chemical properties, the genetic program and evolutionary history of complex biological systems make them unique and unpredictable. Furthermore, there are and will be 'unobservable' phenomena in biology which have to be accounted for.

Sex differences in brain and behavior: emphasis on nicotine, nitric oxide and place learning.

Although males and females are unmistakably different, the recognition of sex as a key variable in science and medicine is considered a revolution in some circles. Sex differences transcend reproductive functions, are evident in the structural and functional organization of the brain, and are reflected in group differences in cognitive abilities and behavior. Males and females have different neural organizational patterns for information processing and different strategies in problem solving. Research on sex differences not only provides descriptive data, but also allows us to elucidate mechanisms that underlie our behavior. In this review, sex differences in the central actions of nicotine (an addictive substance) and nitric oxide, and performance on active avoidance and place learning tasks are discussed as examples, and biobehavioral approaches relating to these topics are presented.

Nicotine modulates nitric oxide in rat brain.

Nicotine exerts its central actions by regulating cationic fluxes through nicotinic acetylcholine receptors (nAChRs). By this effect, the drug likely also modifies events occurring beyond the nAChR, including the regulation of nitric oxide (NO) synthesis. The present study was undertaken to assess the effects of acute and chronic nicotine administration (0.4 mg/kg, s.c.) on levels of NO(-)(2)+NO(-)(3), stable metabolites of NO, in brain regions of male and female rats. Nicotine increased levels of the metabolites, and therefore presumably of NO, with sex differences in the degree of stimulation, the brain regions affected, and the variance between the effects of acute and chronic administration. Prior inhibition of NO synthase eliminated the effect of nicotine in all regions studied. While nicotine appeared to increase NO indirectly via glutamate receptors in the cortex and hippocampus, this was not true of the corpus striatum, where blocking NMDA-type glutamate receptors with MK-801 had no effect. The findings support the view that NO is likely involved in some of the central effects of nicotine.

Nitric oxide synthetase inhibition hinders facilitation of active avoidance learning by nicotine in rats.

Nicotine produces dose-dependent enhancement of performance in an active avoidance test, and also increases the levels of NO2- and NO3-, which are stable metabolites of nitric oxide (NO), in various brain regions of rats. On the basis of these two observations, we hypothesized that the beneficial effect of nicotine on learning could result from increased NO in relevant brain regions. We therefore tested active avoidance performance in rats given L-Nomega-nitroarginine (L-NA) to inhibit NO synthetase (NOS) prior to nicotine administration. Male Sprague-Dawley rats received L-NA (30 or 50 mg/kg), nicotine (0.4 mg/kg), saline or combinations of these treatments before learning trials. Rats were also tested on the inclined plane, to assess the possible effects due to impairment of motor function by drug treatments on active avoidance learning. L-NA treatment impaired the acquisition of active avoidance learning, and this defect was partially overcome by the co-administration of nicotine. Nicotine facilitated learning and significantly increased the number of correct responses. The threshold for the effect of NOS inhibition on performance exceeded 30 mg/kg L-NA, whereas 50 mg/kg impaired learning and also eliminated the nicotine-induced enhancement of learning. On the fifth day of learning trials, no facilitation of learning by nicotine was observed in rats receiving either dose of L-NA. Our results suggest that NO is involved in the facilitation of active avoidance learning by nicotine.

Co-localization of cart peptide immunoreactivity and nitric oxide synthase activity in rat hypothalamus.

Because of the reported presence of both CART peptide and NOS activity in the same hypothalamic nuclei, their colocalization was examined. Eighteen percent of the neurons in the supraoptic nuclei, and 16% of the neurons in the paraventricular nucleus contained both CART immunoreactivity and NOS activity. Many other neurons in these regions stained for only one marker although they were often close by. Thus, CART peptides and NO may interact in these regions.

Sexually dimorphic cognitive style in rats emerges after puberty.

In a water maze (WM), rats employ different and sexually dimorphic behavioral strategies to solve a place-learning task, a test of cognitive/propositional ability. Puberty is the last step in brain development and marks an important phase with regard to sexually dimorphic cognitive performance and behavior. The present study assessed possible sex differences in cognitive style before and after puberty in a WM place-learning task. Since nitric oxide (NO) is implicated in spatial learning and hippocampal function, and since brain NO(-)(2) + NO(-)(3) levels (stable metabolites of NO) display region-specific sex differences in rat brain, NO(-)(2) + NO(-)(3) levels were determined after behavioral testing. The sex-related style difference emerged very clearly but only in the adult rats, which suggests that the female behavioral strategy in the WM place-learning task requires the presence of female sex hormones at puberty. Although NO(-)(2) + NO(-)(3) levels were higher in the adult rats and males compared to prepubertal and female rats, respectively, no significant correlations emerged between brain NO and behavior. The fact that the behavioral sexually dimorphic cognitive-style effect observed here and in previous studies appears to emerge only after puberty suggests that awareness of such postpubertal sex differences may also be important in human educational and therapeutic contexts.

Increased cerebrospinal fluid and serum nitrite and nitrate levels in amyotrophic lateral sclerosis.

Abnormal glutamate metabolism is implied in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and cerebrospinal fluid (CSF) glutamate levels appear to be elevated. Since nitric oxide (NO) inhibits glutamate transport, excessive amounts of nitric oxide could underlie the glutamate induced neurotoxicity in ALS. Stable metabolites of NO (NO2- + NO3-) levels were determined in serum and CSF of sporadic ALS patients and control subjects. NO2- + NO3- levels were higher in ALS, in males and in serum samples compared to controls, females and CSF, respectively. Furthermore, while the difference between serum and CSF NO2- + NO3- levels was significant in males (higher in serum) no such difference was observed in females. Our results suggest that nitric oxide may be involved in the pathogenesis of ALS directly or indirectly and in a sexually dimorphic manner.

Sexually dimorphic cognitive style, female sex hormones, and cortical nitric oxide.

Recent studies using the water maze (WM) found marked sex differences in behavioral strategy employed in place learning tasks in adult rats. When a change in the platform position is introduced following learning the place of a platform (visible or hidden) in a different position, female rats escape to the newly positioned visible platform faster than males. Nitric oxide (NO) is implicated in place learning, and there are regional sex differences in its stable metabolites, NO(2)(-)+NO(3)(-), in rat brain. Furthermore, NO(2)(-)+NO(3)(-) levels are sensitive to ovariectomy in female rats. The effect of sex hormones on brain development and function is well documented. The present study was undertaken to study the effects of ovariectomy and hormonal manipulations on cognitive performance in a WM task designed to test differences in behavioral strategy in Sprague-Dawley rats (n=48) of both sexes. Some of the females rats were ovariectomised and received either hormone replacement (estrogen or progesterone alone or in combination) or the vehicle. Cortical and hippocampal NO(2)(-)+NO(3)(-) levels were determined after behavioral testing. There were no group differences in cognitive ability or non-cognitive factors such as motivation or swim speed. Males and intact females differed in their cognitive style, but hormonal manipulations in female rats did not affect this relative use of behavioral strategy. There was a correlation between performance on the trial where sex differences were most prominent and NO(2)(-)+NO(3)(-) levels in the cortex. Our results suggest that the activational effects of circulating gonadal hormones do not play a major role in sexually dimorphic cognitive styles.

Influence of sex and female hormones on nicotine-induced changes in locomotor activity in rats.

The acute and chronic effects of nicotine (0.4 mg/kg s.c.) on locomotor activity in photocell cages have been compared in male, female, and ovariectomized hooded rats. In Experiment 1, female rats displayed higher locomotion than males (n = 12); acutely, nicotine-reduced locomotion, and this effect was slightly larger in females than males. Daily administration of nicotine for 21 days produced a similar, gradual increase in activity in both sexes. Tests then confirmed greater activity in females than males and as a function of previous chronic exposure to nicotine (n = 6); there was an activating effect of nicotine challenge but no interaction of nicotine effects with sex. In Experiment 2, ovariectomized rats were primed with 17-beta-estradiol (50 microg/kg s.c.) and progesterone (2.5 mg/kg s.c.) or vehicle only. Acute administration of nicotine reduced activity in both groups similarly (n = 12). After nicotine daily for 21 days, there was increased activity as a function of both chronic nicotine and hormonal priming, and challenge with nicotine increased activity (n = 6). The effects of these challenges with nicotine were also slightly greater, as a function of previous nicotine exposure and priming. As a whole, these experiments showed robust effects of acute and chronic nicotine administration, sex, and hormonal priming; neither sex nor gonadal hormones had marked influences on changes in locomotor activity produced by nicotine.

Sexually dimorphic effect of an acute smoking manipulation on skin resistance but not on heart-rate during a cognitive verbal task.

In a two-day, two-session experiment where smokers male and female college-student subjects worked on a cognitive verbal task during either the first or second day, and on a cognitive spatial task on the second or first day, smoking was manipulated as an acute independent variable by requiring 10+ hours of pre-experimental abstention, and providing a cigarette during the 15-minute rest period between the two sessions. Non-smoker female and male subjects underwent the same experiment, and hence served as controls for the effects of this acute-smoking manipulation. Overall adaptation (decreased arousal) to the experiment was manifested in a significant increase in skin resistance level (SRL) in all subjects, but when this adaptation effect was statistically controlled, there was a significant smokers by sex interaction during the verbal task only, such that SRL was increased by the cigarette in males, but decreased in females. In contrast, the same analysis indicated only a marked increase in heart-rate (HR) due to smoking, which was unaffected either by sex or by whether the task was the verbal or the (easier) spatial one. We interpret the SRL results as reflecting a sex difference in the direction of transient psychological arousal, and discuss it in relation to evidence in the literature based on self reports, and to evidence (based on HR in this study and on blood pressure in other studies) on physiological (cardiovascular) arousal. Key Words: Electrodermal activity, heart-rate, psychological vs. physiological, verbal and spatial cognitive tasks.

Nicotine interacts with sex in affecting rat choice between "look-out" and "navigational" cognitive styles in the Morris water maze place learning task.

The effect of sex and nicotine on cognitive style was examined in rats using a water maze task that allows differentiation between cognitive ability and style. During the 12-day acquisition period with the platform in the same location (either visible or hidden) there were no effects or interactions attributable to nicotine and sex, either in terms of learning rate or asymptotic latency. On the final test day the platform was visible and shifted in its location, and on the first trial the new location was proximal to the rats starting position, in contrast to the more distal location of the platform during the previous acquisition days. This platform relocation presented the rats with a choice between two competing cognitive styles: using local visual (look-out) cues vs. navigational cues. Performance on the test day yielded a nicotine x sex interaction, such that only saline-treated female rats showed a clear preference for the perceptual-proximal look-out cognitive style by swimming straight to the newly-relocated visible platform with mean escape latency that approximated the limits of swimming speed. The other three groups did not differ from each other, and preferred navigational cues. The results show that male and female rats use different strategies in problem solving, and that nicotine shifts the female pattern to that of the male.

Intersession interval affects performance in the Morris Water Maze.

We have recently reported an effect that shows a sexually dimorphic difference in cognitive style rather than ability. The preparation for potentially producing this proximal perceptual style effect is one where rats are first given 4-trial daily acquisition sessions for 12 days with the platform always in the same position, but sometimes visible (perceptual, "look-out" condition) and sometimes hidden (conceptual, "navigational" condition). On the first, probe trial of the 13th day, the platform's position is shifted to a point very close (proximal) to the rat's starting position, and made visible. The proximal perceptual style (PPS) effect has emerged sexually dimorphically in that only females swam straight to the newly positioned proximal platform. Other studies have shown that the PPS effect is eliminated (with females behaving like males) by nicotine and prepubertal ovariectomy, and does not occur in prepubertal females. Also, as no sex-related effects emerged during acquisition during these studies, the PPS effect appears to be a function of cognitive style rather than ability. The present study varied age, and, in an effort to economize on time, shortened acquisition to 6 days by having morning and afternoon sessions each day. To our surprise, this relatively subtle psychological manipulation eliminated the PPS effect, and also yielded some sex- and age-related effects during acquisition: A male advantage was observed and prepubertal rats had longer escape latencies; there was no significant interaction between sex and age.

Effects of nicotine on active avoidance learning in rats: sex differences.

Nicotine, a cognition enhancer and a drug of abuse, exerts its central actions through neuronal nicotinic acetylcholine receptors which show sexual dimorphism. This study tested the effects of nicotine on active avoidance learning in male and female rats. Three month-old Sprague-Dawley rats received varying doses of nicotine (0.2, 0.4 and 0.6 mg/kg) or saline s.c. before active avoidance learning trials (15 trials/day) for a period of 5 days. Behavioural testing was continued after the termination of nicotine treatment once a week for four weeks. Nicotine improved acquisition of active avoidance learning in a dose-dependent manner. In male rats nicotine was active at all doses tested whereas, in females, learning performance deteriorated at a dose of 0.6 mg/kg. In conclusion, nicotine pre-treatment affects active avoidance in a sexually dimorphic and dose-dependent manner.

Sex differences in nitrite/nitrate levels and antioxidant defense in rat brain.

This study assessed sex differences in stable metabolites of nitric oxide and major enzymes involved in antioxidant defense in various regions of rat brain. Nitrite/nitrate levels and activities of superoxide dismutase and catalase were determined in cortex, hippocampus, corpus striatum, midbrain and cerebellum of adult male and female Sprague-Dawley rats. Nitrite/nitrate levels were significantly higher in the cortex and the hippocampus of male than female rats, while catalase activity was higher in the cortex of females than in males. These sex differences may have significant effects on brain function in health and disease.

Sex difference in up-regulation of nicotinic acetylcholine receptors in rat brain.

This study tested for sex differences in the effects of chronic nicotine administration and withdrawal on nicotinic acetylcholine receptor binding in brain. Rats received nicotine (0.6 mg/kg, s.c.) or saline once daily for 15 days, and were sacrificed 1 or 20 days after termination of treatment. Saturation studies of nAChR binding were performed using [3H]cytisine as the radioligand in whole brain minus cerebellum taken from animals in the chronic treatment groups and from naive rats. Male but not female rats that received chronic nicotine had higher receptor densities than corresponding control groups; up-regulation of nAChR was not seen 20 days after withdrawal. Furthermore, in groups that showed no up-regulation (controls and rats withdrawn for 20 days), nAChR densities were higher in female rats than males. The findings underscore the importance of sex differences in pharmacological responses as well as in basal neurochemical parameters.