Bang H. Hwang

Corticotropin-releasing factor gene expression is down-regulated in the central nucleus of the amygdala of alcohol-preferring rats which exhibit high anxiety: a comparison between rat lines selectively bred for high and low alcohol preference

The role of amygdaloid corticotropin-releasing factor (CRF) in alcoholism is not clear. Alcohol-preferring (P) rats and high alcohol-drinking (HAD) rats are selectively bred for high alcohol preference, and have been considered suitable animal models for studying alcoholism. The CRF neurons in the central nucleus of the amygdala (CeA) of P rats and HAD rats were studied in comparison with those of their respective counterparts, namely, alcohol-nonpreferring (NP) rats and low alcohol-drinking (LAD) rats. Specifically, CRF-immunoreactivity (ir) in the CeA and paraventricular hypothalamic nucleus (PVN) was assessed using radioimmunohistochemical (RIH) assay in alcohol-naive P/NP rats, and HAD/LAD rats. Furthermore, CRF mRNA was examined using in situ hybridization in the CeA of P/NP rats. Anxiety levels were also evaluated using an elevated plus maze. Results of the present study showed that CRF-ir was significantly lower in the CeA of P rats than NP rats. Moreover, CRF mRNA in the CeA was also much lower in P rats than NP rats. Such differences were not seen in the PVN. Interestingly, those P rats exhibited higher anxiety than NP rats. In contrary, there were no innate differences of CRF-ir in both the CeA and PVN between HAD and LAD rats whose anxiety levels were similar. This study is consistent with the literature showing CRF knockout (KO) induces alcohol drinking, and central administrations of CRF reduce alcohol intake. Collectively, the present study suggests that reduced CRF gene expression in the CeA of P rats is associated with their alcohol preference and anxiety.

Lower GABAA receptor binding in the amygdala and hypothalamus of spontaneously hypertensive rats

The central GABAergic system is associated with normal blood pressure regulation, but the role of GABA receptors in genetic hypertension remains unclear. This study was conducted to investigate GABAA receptor binding in several brain regions of spontaneously hypertensive (SHR) rats during development of hypertension. GABAA receptor binding was labeled with [35S]TBPS and was assessed by quantitative autoradiography with the aid of a computer-assisted image analysis system. Densities of GABAA receptor binding sites were significantly lower in all hypothalamic and amygdaloid nuclei evaluated in 4-week-old SHR rats, when compared with their age-matched normotensive Wistar-Kyoto rats. At 12 weeks of age, GABAA receptor binding remained significantly lower in the central amygdaloid nucleus and paraventricular hypothalamic nucleus of SHR rats. Collectively, the results suggest that GABAA receptors in these nuclei are likely to be involved in the initiation and maintenance of hypertension. In conclusion, this study supports a notion that downregulation of GABAA receptor binding occurs in the hypothalamus and amygdala of SHR rats and may play a role in genetic hypertension.

Downregulation of corticotropin-releasing factor mRNA, but not vasopressin mRNA, in the paraventricular hypothalamic nucleus of rats following nutritional stress

Stress can cause disturbance of homeostasis to result in illness. Stress can also induce various gene expression in different neuronal systems. For example, nutritional stress induced by acute food deprivation upregulates corticotropin-releasing factor (CRF) mRNA, whereas osmotic stress increases vasopressin (VP) mRNA. However, it is unknown if nutritional stress induced by chronic food deprivation has synergistic effects on CRF and VP mRNAs. We have used in situ hybridization in conjunction with quantitative autoradiography to demonstrate that nutritional stress induced by a 4-day food deprivation results in a body-weight loss with a significant decrease of CRF mRNAs, but not VP mRNAs in the paraventricular hypothalamic nucleus (PVN) of Sprague-Dawley rats. The present study has thus indicated that a chronic nutritional stress does not have synergistic effects on CRF and VP mRNAs. The decrease of CRF mRNAs is obviously related to the body-weight loss induced by food deprivation. This study thus supports a notion that the CRF, but not VP, neurons in the PVN play an important role in their neuroadaptation associated with body weight loss. Thus, it is conceivable that downregulated CRF neurons in the hypothalamus could be involved in pathogenesis of human eating disorder with severe weight loss, whereas upregulated CRF neurons could be associated with an opposite form of the eating disorder that causes obesity.

Reduced neuropeptide Y mRNA expression in the central nucleus of amygdala of alcohol preferring (P) rats: its potential involvement in alcohol preference and anxiety

Levels of neuropeptide Y (NPY) mRNA expression in discrete brain regions of alcohol preferring (P) rats and alcohol nonpreferring (NP) rats were examined using in situ hybridization. NPY mRNA expression was significantly lower in the central nucleus of amygdala (CeA) of P rats than NP rats, whereas no differences were found in the medial or basolateral amygdaloid nuclei. This study suggests that reduced NPY gene expression in the CeA may contribute to differences in alcohol preference and other behavioral differences observed between P and NP rats.

Galanin-containing neurons in the solitary nucleus and locus coeruleus of spontaneously hypertensive rats are associated with genetic hypertension ☆

Spontaneously hypertensive (SHR) rats contained more galanin (GAL) content and GAL mRNA in locus coeruleus (LC) at the prehypertensive, but not at the well-established hypertensive stage, than did age-matched Wistar-Kyoto (WKY) rats. However, there was also more GAL content, but not GAL mRNA, in the nucleus tractus solitarii (NTS) of SHR rats than WKY rats at both stages. This study suggests that galaninergic neurons in the LC and NTS may participate in the pathogenesis of genetic hypertension.

Calcitonin gene-related peptide (CGRP) levels and alcohol

Calcitonin gene-related peptide (CGRP) when administered into the brain exerts stress-like effects such as increased pain sensitivity, anorexia, and potentiation of fear-related behaviours. Since alcohol consumption may be related to alcohols anxiolytic properties, the present study sought to determine if brain CGRP levels were correlated with genetic differences in preference for drinking alcohol and/or affected by alcohol exposure/withdrawal. CGRP-like immunoreactivity (CGRP-LI) was measured by radioimmunoassay (RIA) in amygdala, hippocampus, frontal cortex, hypothalamus, and caudate. In the first experiment, CGRP-LI was compared in alcohol-naive rats [preferring (P) and non-preferring (NP)], lower concentrations were found in the hippocampus (U = 153.5; d.f. = 1,28; p < 0.014) and frontal cortex (U = 183.0; d.f. = 1,28; p < 0.0001) of the P rats. In a second experiment, a group of outbred Wistar rats were exposed to alcohol in vapour chambers, or control conditions. At 7 wk of alcohol exposure there were no differences in exposed rats as compared to controls. However, at 4 wk following ethanol withdrawal, higher concentrations of CGRP-LI were found in the hippocampus (U = 26.5; d.f. = 1,20 p < 0.05), hypothalamus (U = 17.5; d.f. = 1,20; p < 0.009), and caudate-putamen (U = 17.0; d.f. = 1,20; p < 0.009) of the previously exposed animals. These studies suggest that CGRP may modulate alcohol preference and additionally, that exposure/withdrawal from ethanol produces long-lasting effects on CGRP-LI.

Calcitonin gene-related peptide (CGRP) content and CGRP receptor binding sites in discrete forebrain regions of alcohol-preferring vs. -nonpreferring rats, and high alcohol-drinking vs. low alcohol-drinking rats

This study showed that alcohol-preferring (P) rats and high alcohol-drinking (HAD) rats possess fewer calcitonin gene-related peptide (CGRP) receptor binding sites than their respective controls in the central amygdaloid nucleus (CeA) which is known to be related to anxiety. Since P and HAD rats are selectively bred for high alcohol preference, and alcohol can produce anxiolytic effect, one can postulate that P and HAD rats preferentially drink alcohol in order to obtain its anxiolytic effect. This study supports a hypothesis that deficit of CGRP receptors in the CeA of P and HAD rats may contribute to alcohol preference.

c-fos gene expression is increased in the paraventricular hypothalamic nucleus of Sprague-Dawley rats with visceral pain induced by acetic acid without detectable changes of corticotrophin-releasing factor mRNA: A quantitative approach with an image analysis system

This study is the first of its kind to demonstrate that c‐Fos immunoreactivity (ir) together with c‐fos mRNA in their immediately adjacent tissue sections of a discrete brain region can be reliably measured. The c‐fos gene expression in the paraventricular hypothalamic nucleus (PVN) of Sprague‐Dawley rats for an animal model for visceral or somatovisceral pain induced by 2% acetic acid (AA) was used in this study. Specifically, c‐fos mRNA signals were measured by quantitative autoradiography after in situ hybridization using c‐fos oligodeoxynucleotide probe, and c‐Fos‐ir signals were represented by c‐Fos immunostaining, as detected using c‐Fos antibody in a regular immunohistochemistry. Signals from both c‐Fos‐ir and c‐fos mRNA in the PVN were measured from their immediately adjacent cryostat sections. For the measurement of c‐Fos‐ir, it was carried out by reading 10 rectangles (1,000 μm2/rectangle) on each PVN section with c‐Fos immunostaining. Specific signals were obtained from subtracting the nonspecific background signal from the total signals using a computer‐assisted image analysis system. Results indicated that the AA treatment induced a significant increase of both c‐Fos‐ir and c‐fos mRNA in the PVN. Interestingly, there was no increase of corticotrophin‐releasing factor (CRF) mRNA expression in the PVN and central nucleus of the amygdala of Sprague‐Dawley rats subjected to the AA treatment. In summary, this study has demonstrated that c‐Fos‐ir in the PVN with an anatomical resolution can be semiquantitatively measured after immunohistochemistry using an image analysis system, and that increased c‐fos mRNA in the PVN 1 hr after the AA treatment is associated with no changes of the CRF mRNA expression. Anat Rec, 2007.

Quantitative autoradiography on [35S]TBPS binding sites of gamma-aminobutyric acidA receptors in discrete brain regions of high-alcohol-drinking and low-alcohol-drinking rats selectively bred for high- and low-alcohol preference

It has been documented that ethanol can potentiate brain gamma-aminobutyric acid (GABA)ergic function, and there is a close link between the GABA(A) receptor complex and effects of ethanol, including reinforcement of alcohol which is a fundamental element of alcohol preference. However, it is unknown in what discrete brain regions GABA(A) receptors might be associated with alcohol preference. In the present study, [(35)S]t-butylbicyclophosphorothionate ([(35)S]TBPS) was used to localize GABA(A) receptors in high-alcohol-drinking (HAD) rats and low-alcohol-drinking (LAD) rats which were selectively bred for high and low alcohol preference, respectively. Initial qualitative observations indicated that [(35)S]TBPS binding sites were abundant in many brain areas including the cerebral cortex, hypothalamus and amygdala of HAD and LAD rats. Furthermore, the quantitative autoradiographic analysis revealed fewer [(35)S]TBPS binding sites of GABA(A) receptors in the amygdaloid complex, central medial thalamic nucleus, lateral hypothalamic nucleus and anterior hypothalamic nucleus of HAD rats than LAD rats. Collectively, this study has indicated that HAD rats selectively bred for high alcohol preference possess lower [(35)S]TBPS binding in the brain. Since lower TBPS binding has been proposed to reflect enhanced GABAergic function, as evidenced in rats with seizure or under alcohol withdrawal, the results from the present study suggest that HAD rats might have an enhanced GABAergic function. It is thus likely that enhanced GABAergic function in the brain might be related to high alcohol preference which is characteristic in HAD rats. In addition, the present result showing no difference of [(35)S]TBPS binding in the nucleus accumbens is also in agreement with a notion that [(35)S]TBPS binding may represent only a small spectrum of the GABA(A) receptor complex which is constituted of a sophisticated subunit combination whose functional compositions are still unknown. In conclusion, the present study supports the working hypothesis that GABA(A) receptors are involved in alcohol preference in HAD rats.

Angiotensin II receptor binding in the locus ceruleus of spontaneously hypertensive rats and Wistar-Kyoto rats: a quantitative autoradiographic study with references to hypertension and cardiac/testicular hypertrophy.

The locus ceruleus (LC) contains a high density of angiotensin II (AII) receptors. The role of AII receptors at the LC in genetic hypertension and organ function is unclear. Spontaneously hypertensive (SHR) rats and Wistar-Kyoto (WKY) rats were studied, and blood pressure of animals was measured using the tail-cuff method. Animals were decapitated and the heart weight (HW) and testicular weight (TW) of animals measured. AII receptor binding was carried out by incubating the LC tissue sections with 200 pM [(125)I]-AII receptor ligand, and measured using quantitative autoradiography. Results showed that the HW/BW ratio was significantly higher in SHR rats than WKY rats. However, the TW/BW ratio was higher in SHR rats than WKY rats only at two hypertensive stages, whereas AII receptor binding capacity in the LC was also statistically higher in SHR rats than WKY rats. Results indicated that cardiac and testicular hypertrophies were related to higher AII receptor binding in the LC of SHR rats, when compared with WKY rats. Interestingly, the literature shows that there is an LC-testes axis. In conclusion, this study indicated that AII receptors in the LC are associated with genetic hypertension, and testicular weight could be a reasonable index for essential hypertension.