N-methyl-d-aspartic acid receptors are altered by stress and alcohol in Wistar-Kyoto rat brain.

Previous studies have shown that the Wistar-Kyoto (WKY) rat strain is more sensitive to stressors and consumes significant quantities of alcohol under basal as well as stressful conditions when compared to other strains. Given that the glutamate neurotransmitter system has been implicated in depression and addiction, the goals of the present study were to investigate the effects of stress and stress-alcohol interactions on N-methyl-d-aspartate (NMDA) receptors in the rat brain. Thus this study measured the binding of [(3)H] MK-801 to NMDA receptors in the prefrontal cortex (PFC), caudate putamen (CPu), nucleus accumbens (NAc), hippocampus (HIP) and basolateral amygdala (BLA) in WKY rats in comparison to the Wistar (WIS) rat strain. Our results suggested that while voluntary alcohol consumption did not alter NMDA receptors in the PFC, CPu or NAc in either rat strain, it increased NMDA receptors in the HIP and BLA in both strains. In contrast, chronic stress increased NMDA receptors in the PFC, CPu, NAc in WKY rats but not in WIS rats. Chronic stress also decreased NMDA receptors in the HIP and increased NMDA receptors in the BLA in both strains. Alcohol co-treatment with stress increased NMDA receptors in the PFC, CPu and NAc in WKY rats but not in WIS rats. Interestingly, while alcohol co-treatment did not reverse stress induced decreases in NMDA receptors in the HIP, it reduced the binding of NMDA receptors in the BLA to control levels in both strains. Thus it appears that NMDA receptors in the PFC, CPu and NAc may be more sensitive to the effects of stress and could be implicated in the stress-induced alcohol consumption behavior seen in WKY rats. In contrast, NMDA receptors in the HIP and BLA may reflect an adaptive response and may not be responsible for the stress susceptible phenotype of the WKY rat strain.

Alcohol consumption alters dopamine transporter sites in Wistar-Kyoto rat brain.

Even though animal and human studies show alterations in dopamine transporter (DAT) sites after alcohol withdrawal, the role of DAT in influencing either alcoholic or depressive behavior has not been examined extensively. Given that the Wistar-Kyoto (WKY) rat is a putative animal model of depressive behavior, the present study examined the effects of chronic alcohol consumption on DAT sites in WKY versus Wistar (WIS) rats. Brains from both strains were sectioned for autoradiographic analysis of [3H]-GBR12935 binding to DAT sites after 24 days of alcohol exposure. The results indicated that WKY rats consumed a greater amount of alcohol (P < 0.001) than WIS rats did throughout the experiment. Autoradiographic analyses of discrete brain regions indicated that alcohol consumption increased DAT sites in a greater number of brain areas in WKY compared to WIS rats. In WKY rats, the binding of [3H]-GBR12935 to DAT sites was increased in the basolateral, central and lateral nuclei of the amygdala, lateral nucleus of the hypothalamus, olfactory tubercle, caudate-putamen, nucleus accumbens and substantia nigra (P < 0.05) and decreased in the ventromedial nucleus of the hypothalamus and the CA1 region of the hippocampus. In WIS rats, alcohol consumption increased DAT sites in the CA1 region of the hippocampus, basolateral nucleus of the amygdala, ventral tegmental area and substantia nigra, and decreased DAT sites in the lateral and ventromedial hypothalamus and dentate gyrus. These results indicate a strain dependent alteration in DAT sites which may be related to altered dopamine neurotransmission in select brain regions following alcohol consumption.

Differential expression of thyroid hormone receptor isoforms by thyroid hormone and lithium in rat GH3 and B103 cells.

BACKGROUND: The interaction between lithium, a mood stabilizer, and the thyroid axis has been extensively studied; however, the regulation of thyroid hormone receptors by lithium is yet to be investigated. METHODS: To test whether lithium affects thyroid hormones at the receptor level, we examined the effects of lithium in combination with triiodothyronine (T3) on gene expression of thyroid hormone receptor isoforms in GH3 and B103 cells. RESULTS: The pattern of expression as well as the magnitude of regulation of the different thyroid hormone receptor isoforms appeared to be cell line specific. Whereas T3 regulated all four isoforms in GH3 cells at both time points, T3 did not alter thyroid hormone receptor TR alpha 1 and TR alpha 2 mRNA in B103 cells. Addition of lithium to thyroid hormone-deficient GH3 cells decreased TR alpha 1, alpha 2, and beta 2 expression without affecting TR beta 1 expression at 2 but not 5 days. Addition of lithium to T3-treated GH3 cells did not further modulate gene expression of TR alpha 1, alpha 2, beta 1, or beta 2 when compared to cells treated with T3 alone. The effects of lithium in B103 cells appeared to be isoform specific as well as time dependent, since TR alpha 1 expression was selectively decreased in B103 cells, when treated with T3 in the presence of lithium. CONCLUSIONS: The present study provides direct evidence that T3 and/or lithium regulate TR gene expression in vitro in a both time-dependent and cell line-specific manner.

Lithium administration affects gene expression of thyroid hormone receptors in rat brain.

Even though lithium has received wide attention in the treatment of manic depressive illness, the mechanisms underlying its mood stabilizing effects are not understood. Lithium is known to interact with the thyroid axis and causes hypothyroidism in a subgroup of patients, which compromises its mood stabilizing effects. Since lithium was recently reported to alter thyroid hormone metabolism in the rat brain, the present study investigated whether these effects were mediated through regulation of thyroid hormone receptor (THR) gene expression. Adult male euthyroid rats were either given a diet containing 0.25% lithium or one without lithium for 14 days. Rats were sacrificed in the evening and RNA was isolated from different brain regions to quantitate the isoform specific mRNAs of THRs. Following 14 days of lithium treatment, THR alpha1 mRNA levels were increased in the cortex and decreased in hypothalamus; THR alpha2 mRNA levels were increased in the cortex and THR beta mRNA levels were decreased in the hypothalamus. No significant difference in the expression of these THR isoforms was observed in the hippocampus or cerebellum. Thus, chronic lithium treatment appeared to regulate THR gene expression in a subtype and region specific manner in the rat brain. It remains to be determined whether the observed effects of lithium on THR gene expression are related to its therapeutic efficacy in the treatment of bipolar disorder.

Reserpine modulates serotonin transporter mRNA levels in the rat brain.

Despite recent advances in determining central serotonin (5-HT) function, the basic aspects by which serotonin neurotransmission is controlled and regulated are still not understood. Since the serotonin transporter (5-HTT) is involved in terminating the action of 5-HT that is released from the presynaptic nerve terminal, the regulation of 5-HTT may be an important step in controlling 5-HT neurotransmission at the synaptic cleft. The present study investigated the effects of reserpine administration on 5-HTT gene expression as well as on tryptophan hydroxylase (TPH) expression in the rat brain. Male Sprague-Dawley rats were injected with reserpine (10 mg/kg, i.p.) and sacrificed at 8 h, 3 days, 7 days or 21 days after the injection. Control rats were injected with saline and sacrificed either at 8 h or 21 days after the injection. The midbrain region was dissected, RNA was isolated and probed for 5-HTT expression using Northern Blotting. Data were analyzed using Super-Anova followed by post-hoc Dunnett's test. While mRNA levels for 5-HTT were unchanged at 8 h after reserpine, a significant decrease was noted at 3 days and 7 days (F=10; p<0.0001). 5-HTT mRNA levels returned to control levels by 21 days. In contrast, TPH expression was unaltered at all time points examined. The results of this study provide useful information regarding the role that the 5-HTT may be playing in the homeostatic control of 5-HT neurotransmission at the synapse.

Effect of acute or repeated stress on behavior and brain norepinephrine system in Wistar-Kyoto (WKY) rats.

WKY rats develop more restraint-induced gastric ulcers and exhibit more depressive behavior compared to other rat strains. Exposure to novel stressors for 21 days exacerbates depressive behavior in WKY rats and alters beta-adrenoceptors (beta-ARs) and norepinephrine transporter (NET) sites in several limbic brain regions when compared to Sprague-Dawley rats. The present study examined whether these effects would be elaborated following an acute stressor and whether WKY rats would demonstrate adaptation after repeated stress. Rats were subjected to a 2-h supine restraint stress for either one or eight consecutive daily sessions. Open-field behavioral data were collected immediately after the daily stress sessions. Brains were sectioned for autoradiographic analysis of 125I-pindolol binding to beta-ARs and 3H-nisoxetine binding to NET sites in discrete brain regions. Acute 1-day stress resulted in a significant drop in body weight and an inhibition of behaviors in the open field. These effects were also sustained following 7 days of chronic restraint stress. In contrast, while acute stress had no effect on NET binding sites or beta-ARs, repeated stress decreased NET sites in the amygdala, hypothalamus, and locus coeruleus with little effect on beta-ARs in the brain regions examined.

Effect of stress on the behavior and 5-HT system in Sprague-Dawley and Wistar Kyoto rat strains.

The effects of chronic novel stressors, for 21 days, on the behavior and the serotoninergic (5-HT) system in Sprague-Dawley (SD) and Wistar Kyoto (WKY) rats were studied. Open-field and forced-swim tests revealed a significantly greater behavioral depression in the WKY strain. SD rats showed a decrease in 3H-DPAT binding to 5-HT1A receptors in the hippocampus, whereas WKY rats revealed an increase in 3H-DPAT binding in the hippocampus and hypothalamus. Stress did not appear to alter the binding of 3H-DPAT to 5-HT1A sites in the dorsal raphe or median raphe in either strains. SD rats revealed a modest increase in 5-HT transporter (5-HTT) sites in the cortex; WKY rats revealed a decrease in 5-HTT sites in the cortex and the hippocampus. Stress caused an increase in 3H-CNIMI binding to 5-HTT sites in the dorsal and median raphe nuclei in both strains. The results suggest that the greater susceptibility to behavioral depression in WKY rats may account for the differential effects on 5HT1A sites as well as 5-HTT sites in limbic regions and cell body area as compared to SD rats.

Elicited PGO waves in rats: lack of 5-HT1A inhibition in putative pontine generator region.

Ponto-geniculo-occipital (PGO) waves and an elicited analogue (PGOE) may be recorded in the pons of rats. Cholinergic cells in the pedunculopontine tegmental (PPT) and laterodorsal tegmental (LDT) nuclei are implicated in the generation of PGO waves. Serotonin (5-HT) may inhibit the generation of PGO waves, and possibly PGOE. We examined the role of 5-HT1A receptor mechanisms in the generation of auditory-elicited PGOE in rats. Administration of 8-OH-DPAT [8-hydroxy-2-(n-dipropylamino)tetralin] into PPT did not significantly affect PGOE amplitude or response frequency. Binding of [3H]CN-IMI ([3H]cyanoimipramine) to 5-HT uptake sites located presynaptically was used as a measure of 5-HT innervation. Quantitative autoradiographic analysis of [3H]CN-IMI binding indicated a moderate to low degree of 5-HT innervation of PPT and a moderately high innervation of LDT compared to the dorsal raphe nucleus (DRN). Binding of [3H]8-OH-DPAT to 5-HT1A receptors revealed few receptor sites in PPT, and a low to moderate number of receptors in LDT compared to binding in DRN. The results suggest that inhibitory serotonergic modulation of PGOE is probably not mediated through a 5-HT1A receptor mechanism in PPT.

Amygdaloid control of alerting and behavioral arousal in rats: involvement of serotonergic mechanisms.

The role of 5-HT mechanisms in the amygdala in the modulation of sleep and arousal states and PGO waves was examined. Studies of the amygdala suggest that it provides a neural mechanism by which emotionally-relevant or significant stimuli may influence behavioral state and alerting mechanisms. The amygdala projects massively (via the central nucleus) into brainstem regions involved in alerting and in the generation of REM and PGO waves. Serotonergic innervation of the amygdala comes from DRN and to a lesser degree MRN. Microinjections of 5-HT into the amygdala produced short-latency changes of state from NREM and REM with the effect being relatively greater in REM. Microinjections of the 5-HT antagonist, methysergide, increased sleep efficiency and increased PGO wave frequency in waking and NREM. These results demonstrate an important role for the amygdala in the control of behavioral state and alerting mechanisms and suggest that 5-HT exerts some of its regulatory effects via an influence on forebrain regions.

Acute depletion of serotonin down-regulates serotonin transporter mRNA in raphe neurons.

Serotonin transporter (5-HTT) mRNA and 5-HTT sites were measured 3 days after treatment with p-chlorophenylalanine, a tryptophan hydroxylase inhibitor. While 5-HTT mRNA levels decreased (P < 0.001) in the dorsal raphe nucleus, 5-HTT binding sites remained unchanged, suggesting that an acute depletion of 5-HT may induce an increase in the turnover of 5-HTT mRNA without affecting protein levels at 3 days.

Acute administration of alpha-methyl-para-tyrosine alters levels of norepinephrine transporter mRNA in the rat brainstem.

This study investigated whether rat norepinephrine transporter (NET) mRNA levels would be altered by alpha-methyl-p-tyrosine (alpha-MPT), a tyrosine hydroxylase inhibitor. While NE levels decreased at 1 and 3 days but recovered at 7 days after alpha-MPT, NET mRNA levels decreased at 3 and 7 days but not at 1 day after alpha-MPT. The results indicate that acute treatment with alpha-MPT led to a delayed time response in its effects on NET mRNA and NE levels in the rat brain.

Altered serotonin transporter sites in Alzheimer's disease raphe and hippocampus.

This study measured serotonin transporter (5-HTT) sites in the dorsal raphe nucleus (DRN) and hippocampus in Alzheimer's disease (AD). 5-HTT sites were significantly decreased in the DRN in AD, with significant reductions occurring in the lateral wings of the DRN complex. A significant reduction in 5-HTT sites were also observed in the CA2 subfield of the hippocampus and in the entorhinal cortex in AD. The results indicate that the integrity of 5-HT neurons in the DRN may be compromised in AD, and that region specific alterations in 5-HTT sites may occur in the hippocampal complex in AD.

Effect of repeated novel stressors on depressive behavior and brain norepinephrine receptor system in Sprague-Dawley and Wistar Kyoto (WKY) rats.

This study compared the effects of repeated novel stressors on 'depressive behaviors', defined by the forced-swim and open-field tests, in Sprague-Dawley (S-D) and Wistar Kyoto (WKY) rats. Since stress appears to alter brain norepinephrine (NE) activity, this study also investigated the effects of the stressors on beta-adrenoceptors (beta-ARs), alpha 2-adrenoceptors (alpha 2-ARs) and NE transporter (NET) sites in S-D and WKY rats. Stress did not alter 125I-iodopindolol (125I-PIN) binding to beta-ARs, nor [3H]idazoxan ([3H]IDAZ) binding to alpha 2-ARs in S-D rats, compared to non-stressed controls. However, WKY-stressed rats showed a significant reduction in 125I-IPIN binding to beta-ARs in the cortex, hippocampus, amygdala and hypothalamus, and a reduction in [3H]IDAZ binding to alpha 2-ARs in the amygdala. [3H]nisoxetine ([3H]NIS) binding to NET sites in WKY-stressed rats was also reduced in the cortex, hippocampus and amygdala. When both strains were compared, the most surprising finding was a significantly higher density of NET sites in the hippocampus and amygdala in WKY rats compared to S-D rats. The results of this study indicate that stress, not only exacerbates depressive behavior in WKY rats, but also selectively alters beta-ARs, alpha 2-ARs and NET sites in limbic brain regions. Thus, the WKY strain may serve as a useful animal model for depressive behavior and for the investigation of novel antidepressant drugs.

A time course of altered thyroid states on the noradrenergic system in rat brain by quantitative autoradiography.

The present study was designed to investigate the time course of alterations in beta 1- and beta 2-adrenoceptor subtypes, alpha 2-adrenoceptors and uptake sites for norepinephrine in the rat brain following thyroidectomy (TXT) and thyroxine replacement. The results indicate that alterations in the thyroid state lead to changes in the pre- and postsynaptic noradrenergic system that are both region- and receptor-specific. TXT caused the binding of 125-iodopindolol to beta 1-adrenoceptors to decrease in the cortex and hippocampus and caused its binding to beta 2-adrenoceptors to decrease in the hypothalamus. TXT caused 3H-idazoxan binding to alpha 2-adrenoceptors to be reduced specifically in the amygdala. Following TXT, the binding of 3H-nisoxetine to norepinephrine uptake sites was found to be increased in the hippocampus and decreased in the hypothalamus. In most brain regions, thyroxine replacement for 7 or 28 days caused radioligand binding to recover to control levels. Thus it appears that a neuromodulatory link between thyroid hormone and the noradrenergic system exists in many regions of the rat brain.

Time course of the effects of adrenalectomy and corticosterone replacement on 5-HT1A receptors and 5-HT uptake sites in the hippocampus and dorsal raphe nucleus of the rat brain: an autoradiographic analysis.

Previous studies have shown that adrenalectomy (ADX) increases the binding of 3H-DPAT to 5-HT1A receptors in the hippocampus (HIP) and this effect is partially overcome by corticosterone (CORT) replacement. The present study investigated the time course of the effects of ADX with or without CORT replacement on serotonin (5-HT) pre- and postsynaptic systems in the HIP and dorsal raphe nucleus (DR) by quantitative autoradiography. In the HIP, ADX for 7, 10 or 14 days caused a significant increase in 3H-DPAT binding in the CA1 region (pyramidal layer), CA2,3 region (molecular and pyramidal layers) and in the dentate gyrus (molecular and granular layers) which returned to control levels when measurements were made 35 days post-ADX. A decrease in 3H-DPAT binding was observed 14 days after ADX in the DR but not in the median raphe nucleus (MR). Although replacement with CORT did not lead to a reversal in 3H-DPAT binding early time points, binding was restored to control levels 7-28 days after CORT replacement in all regions of the HIP. In the DR, CORT did not cause a reversal in 3H-DPAT binding at any of the time points examined. In contrast to the effects seen on the 5-HT1A receptor subtype, no significant change was noted on the binding of 3H-CN-IMI to uptake sites for 5-HT in the HIP or DR after ADX or CORT replacement. The results of this study indicate that long-term alterations in the HPA axis lead to changes in the 5-HT1A receptor system that are both region-specific and time-dependent.

Norepinephrine transporter sites are decreased in the locus coeruleus in Alzheimer's disease.

The present study determined whether [3H]nisoxetine binding to norepinephrine transporter sites would be altered uniformly throughout the locus coeruleus in Alzheimer's disease. A significant decrease (P < 0.01) in [3H]nisoxetine binding was seen in the mid and caudal regions but not in the rostral region of the locus coeruleus. The loss of [3H]nisoxetine binding to norepinephrine transporter sites may be attributed to a loss of norepinephrine transporter sites located on terminals of noradrenergic neurons in the locus coeruleus in Alzheimer's disease.

Time course of altered thyroid states on 5-HT1A receptors and 5-HT uptake sites in rat brain: an autoradiographic analysis.

Although a link between the hypothalamic-pituitary-thyroid (HPT) axis and affective disorder has been established, the mechanism underlying this relationship remains unclear. Since the serotonin (5-HT) system appears to be involved in the pathophysiology of mood disorders, the time course of the effects of thyroidectomy (TXT) with or without thyroxine (T4) replacement on 5-HT1A receptors and 5-HT uptake sites was examined. TXT caused a significant increase in 3H-8-hydroxy-2-(di-n-propylamino)-tetralin (3H-DPAT) binding to 5-HT1A receptors in the cortex and hippocampus at 7 days and this increase was also evident at 35 days following TXT. By contrast, TXT did not have a significant effect on 3H-DPAT binding in the hypothalamus or in the dorsal raphe nucleus. TXT did not affect the binding of 3H-cyanoimipramine (3H-CN-IMI) to 5-HT uptake sites in any of the brain regions analyzed, or at any of the time points studied. Administration of high-dose T4 for 28 days caused the binding of 3H-DPAT to recover to sham levels in the cortex, to increase in the hippocampus and hypothalamus, and had no effect in the dorsal raphe nucleus. Replacement with high-dose T4 had no effect on 3H-CN-IMI binding to 5-HT uptake sites when compared to sham-operated animals at all time points examined. These results suggest that a neuromodulatory link may exist between the HPT axis and 5-HT1A receptors in the limbic regions of the rat brain. Depending on the brain region examined, a differential response to circulating levels of thyroid hormone was observed.

Effects of high-dose methamphetamine on monoamine uptake sites in rat brain measured by quantitative autoradiography.

The neurotoxicity of methamphetamine to monoaminergic neurons was examined. Neurotoxicity was assessed by quantitative autoradiography using radioligands specific for binding to norepinephrine, dopamine, and serotonin uptake sites. High-dose administration of methamphetamine led to decreases in binding to uptake sites for the three monoamines. Norepinephrine binding sites were decreased in certain amygdaloid nuclei and in the dorsomedial hypothalamic nucleus. Serotonin binding sites were reduced in widespread brain areas, while dopamine binding sites were reduced in the caudate putamen, olfactory tubercle, and nucleus accumbens. The decreases in binding site density for the three monoamines are limited to terminal field areas; cell body areas are not affected. Our results indicate that methamphetamine is neurotoxic to serotonin, dopamine, and norepinephrine neurons. The neurotoxicity to norepinephrine neurons is in selected brain areas.

Effects of repeated administration of desipramine or electroconvulsive shock on norepinephrine uptake sites measured by [3H]nisoxetine autoradiography.

To determine if repeated administration of desipramine (DMI) or electroconvulsive shock (ECS) regulate uptake sites for norepinephrine (NE) in rat brain, the binding of [3H]nisoxetine ([3H]NIS) was measured using quantitative autoradiography. Groups of animals were given DMI intraperitoneally, either a single injection or repeated doses of 10 mg/kg once daily for 21 days and were killed 48 h after the last injection. Another group of rats received ECS daily for 12 days (150 mA, 300 ms, 60 Hz) and was killed 24 h after the last shock. Repeated administration of DMI caused statistically significant decreases (20-40%) in the binding of [3H]NIS in 8 out of 17 brain regions measured; these areas included the hippocampus, thalamus and the amygdala. Acute treatment with DMI had no effect on the binding of [3H]NIS in any of the regions analyzed except the centrolateral nucleus of the amygdala. By contrast, except for the paraventricular nucleus of the thalamus where ECS caused a modest (20%) increase in binding, no other brain region was affected by ECS. Thus it appears that repeated administration of DMI and chronic ECS treatment have different effects on the binding of [3H]NIS to uptake sites for NE in rat brain.

Effect of age on [3H]nisoxetine binding to uptake sites for norepinephrine in the locus coeruleus of humans.

[3H]Nisoxetine ([3H]NIS) was used to measure uptake sites for norepinephrine (NE) in the locus coeruleus (LC) of humans. Regression analysis demonstrated an inverse correlation between age and the binding of [3H]NIS to NE uptake sites in the LC (r = -0.6; P less than 0.01). Since considerable cell loss has been reported to occur with age in the human LC, the decreased binding of [3H]NIS probably reflects loss of LC cells rather than a down-regulation of these sites.