Injections of Galanin-Like Peptide directly into the nucleus of the tractus solitarius (NTS) reduces food intake and body weight but increases metabolic rate and plasma leptin.

Galanin-Like Peptide (GALP) is a hypothalamic neuromediator of metabolism and reproduction. GALP is known to stimulate reproduction and alter food intake and body weight in multiple species. The regulation of body weight involves control of both energy intake and energy expenditure. Since GALP is known to alter food intake - possibly via the autonomic nervous system - we first hypothesized that GALP would increase metabolic rate. First, male Sprague-Dawley rats were implanted with intracerebroventricular (ICV) cannulae and abdominal radiotelemetry temperature transmitters. Following ICV injection with either 5nmol GALP or vehicle, the oxygen consumption of each rat was monitored for 8h. Food intake, core temperature, and general motor activity were monitored for 24h. GALP significantly increased oxygen consumption, an indirect estimator of metabolic rate, without having any significant effect on motor activity. Compared to controls, GALP increased core body temperature during the photophase and reduced food intake over the 24h period following injection. ICV GALP also increased plasma levels of luteinizing hormone (LH). A second group of male Sprague-Dawley rats were implanted with abdominal transmitters and given injections of GALP directly into the nucleus of the tractus solitarius (NTS). These injections resulted in a significant reduction in food intake, and a significant increase in both oxygen consumption and core body temperature compared to vehicle injections. Direct injections of GALP into the NTS compared to vehicle also resulted in a significant increase in plasma leptin levels, but not LH levels. GALP appears to increase energy expenditure in addition to decreasing energy input by actions within the NTS and thus may play an important role in the hypothalamic regulation of body weight.

Intermittent heat exposure and thirst in rats.

Adequate water intake, supporting both cardiovascular function and evaporative cooling, is a critical factor in mitigating the effects of heat waves, which are expected to increase with global warming. However, the regulation of water intake during periods of intermittent heat exposure is not well understood. In this study, the effects of access to water or no access during intermittent heat exposure were assessed using male Sprague-Dawley rats exposed to 37.5°C for 4 h/day. After 7 days of intermittent heat exposure, reductions in evaporative water loss were observed in all animals and reductions in water intake following heat exposure occurred as the days of heat exposure increased. Rats that were not allowed water during the 7 days of exposure had decreased rehydration levels, however, rats allowed access to water increased water intake during exposure and exhibited higher overall rehydration levels over the same time period. Peripheral administration of angiotensinII, mimicking activation of volemic thirst, or hypertonic saline solution, activating intracellular thirst, did not result in alteration of water intake in rats exposed to heat with access to water compared to control rats. In contrast, rats exposed to heat without access to water had reduced water intake after administration of hypertonic saline and increased water intake after administration of angiotensinIIcompared to control rats. These experiments demonstrate that thirst responses to intermittent heat exposure are altered by providing water during heat exposure and that intermittent heat exposure without access to water alters drinking responses to both intracellular and extracellular thirst challenges.

Heat acclimation and thirst in rats.

The effects of heat acclimation on water intake and urine output responses to thermal dehydration and other thirst stimuli were studied in male Sprague-Dawley rats. Rats were heat acclimated by continuous exposure to a 34°C environment for at least 6 weeks. Thermal dehydration-induced thirst was brought about by exposing the heat-acclimated rats and control rats housed at 24°C to a 37.5°C environment for 4 h without access to food or water. Heat acclimation reduced evaporative and urinary water losses and the increases in plasma sodium and osmolality during thermal dehydration, which led to a reduction in thermal dehydration-induced thirst. Heat acclimation reduced the rate of rehydration following thermal dehydration but did not alter the final rehydration level, indicating that heat acclimation does not alter the primary control of thermal dehydration-induced thirst. Heat acclimation did not alter water intake or urine output following administration of hypertonic saline, which selectively stimulates intracellular thirst, but led to greater water intake following administration of angiotensin II, which plays an important role in extracellular/volemic thirst, and following water deprivation, which activates both thirst pathways. Cardiovascular responses to angiotensin II were not altered by heat acclimation. Heat acclimation thus reduces water loss during heat exposure in rats, but does not have major effects on thermal dehydration-induced or extracellular thirst but does appear to alter volemic thirst.

New insights into the mechanism for VACM-1/cul5 expression in vascular tissue in vivo.

Vasopressin-activated calcium-mobilizing (VACM-1)/cul5 is the least conserved member of a cullin protein family involved in the formation of E3-specific ligase complexes that are responsible for delivering the ubiquitin protein to their target substrate proteins selected for ubiquitin-dependent degradation. This chapter summarizes work to date that has focused on VACM-1/cul5's tissue-specific expression in vivo and on its potential role in the control of specific cellular signaling pathways in those structures. As mammalian cells may contain hundreds of E3 ligases, identification VACM-1/cul5 as a specific subunit of the system that is expressed in the endothelium and in collecting tubules, structures known for their control of cellular permeability, may have significant implications when designing studies to elucidate the mechanism of water conservation. For example, VACM-1/cul5 expression is affected by water deprivation in some tissues and there is a potential relationship between neddylated VACM-1/cul5 and aquaporins.

Aquaporin-2 levels in vitro and in vivo are regulated by VACM-1, a cul 5 gene.

BACKGROUND: In the renal collecting duct, vasopressin regulates water permeability by a process that involves stimulation of adenylyl cyclase activity, cAMP production and subsequent translocation of water channel aquaporin-2 (AQP2) into the apical plasma membrane. We have previously shown that in cos 1 cells in vitro, both adenylyl cyclase activity and cAMP production can be regulated by VACM-1, a cul 5 gene that forms complexes involved in protein ubiquitination and subsequent degradation. METHODS: To extend these observations further, the effects of changes in hydration state on the expression of VACM-1 at the mRNA and the protein level were examined in rats deprived of water (WD) for 24 hrs. RESULTS: In the kidney of WD rats Western blot analyses of kidney tissue showed that the decrease in VACM-1 protein concentration was correlated with the increase in the AQP2 protein level. The immunostaining data suggested that VACM-1/cul5 may be decreased in renal collecting duct but increases in the vasculature of the inner medullary region in response to WD. To determine the possible consequences of the WD dependent decrease in VACM-1/cul5, we next examined the effects of VACM-1 expression on AQP2 protein in vitro. Immunocytochemistry and Western blot analyses data indicate that VACM-1/cul5 expression in MDCK line stably expressing AQP2 gene and in cos 1 cells co-transfected with the AQP2 and VACM-1/cul5 cDNAs decreased AQP2 protein concentration when compared to the vector transfected control groups. CONCLUSION: In summary, our data demonstrate that VACM-1 is involved in the regulation of AQP2 protein concentration and may play a role in regulating water balance.

VACM-1/cul5 expression in vascular tissue in vivo is induced by water deprivation and its expression in vitro regulates aquaporin-1 concentrations.

VACM-1, a cul5 gene product, when overexpressed in vitro, has an antiproliferative effect. In vivo, VACM-1/cul5 is present in tissues involved in the regulation of water balance. Neither proteins targeted for VACM-1/cul5-specific degradation nor factors that may regulate its expression in those tissues have been studied. To identify genes that may be misregulated by VACM-1 cDNA, we performed microarray analysis. Our results indicate that in cos-1 cells transfected with VACM-1 cDNA, mRNA levels for several genes, including AQP1, were decreased when compared to the control group. Our results also indicate that in cos-1 cells transfected with VACM-1 cDNA, endogenous AQP1 protein was decreased about 6-fold when compared to the controls. To test the hypothesis that VACM-1/cul5 may be regulated by conditions that compromise water homeostasis in vivo, we determined if 24 h of water deprivation affects VACM-1/cul5 levels or the effect of VACM-1/cul5 on AQP1. VACM-1 mRNA and protein levels were significantly higher in rat mesenteric arteries, skeletal muscle and the heart ventricle but not in the heart atrium from 24-h water-deprived rats when compared to the controls. Interestingly, 24 h of water deprivation increased modification of VACM-1 by an ubiquitin-like protein, Nedd8, essential for cullin-dependent E3 ligase activity. Although water deprivation did not significantly change AQP1 levels in the mesenteric arteries, AQP1 protein concentrations were inversely correlated with the ratio of the VACM-1 to Nedd8-modified VACM-1. These results suggest that VACM-1/cul5 may regulate endothelial AQP1 concentration both in vivo and in vitro.

Developing a project-oriented introduction to neuroscience lab at hope college.

The Introduction to Neuroscience course at Hope College includes a three-hour laboratory period each week. Seven of the fifteen weeks of the lab are used for a lab project that is focused on understanding the effects of gonadal hormones on brain and behavior. Students perform ovariectomies and implant sham, estradiol, or testosterone capsules in rats and then carry out five experiments: 1) Sexual Behavior, 2) Spatial Learning using the Morris Water Maze, 3) The Size of the Sexually Dimorphic Nucleus, 4) Phosphorylation of NMDA Receptors, and 5) Long Term Potentiation in Hippocampal Slices. The experiments are designed to provide the students with experiences at different levels of neuroscience, while improving their skills in statistics, using the primary literature, and scientific writing. The students generate interesting and statistically significant data which they summarize in a journal style lab reports. Using a Self Assessment of Learning Gains tool, we learned that students perceive the lab project improves their ability to A) pose questions from more than one disciplinary perspective that can be addressed by collecting and evaluating scientific evidence, B) learn about complex science problems that require insight from more than one discipline, C) extract main points from a scientific article and develop a coherent summary, and D) write reports using scientific data as evidence. Based on our results, we believe an extended lab project in an introductory neuroscience course can be used to engage students in neuroscience topics and help them develop the skills and habits of neuroscientists.

Cultivation of an interdisciplinary, research-based neuroscience minor at hope college.

Hope College is an undergraduate liberal arts college with an enrollment of approximately 3,000 students. In the spring of 2005, we began to offer an interdisciplinary neuroscience minor program that is open to all students. The objective of this program is to introduce students to the field of neuroscience, and to do so in such a way as to broaden students' disciplinary perspectives, enhance communication and quantitative skills, and increase higher-level reasoning skills by encouraging collaboration among students who have different disciplinary backgrounds. This is a research-based program that culminates in a one-year capstone research course. Here we present the story of the program development at Hope College, including a description of our newly developed curriculum, our initial assessment data, and the lessons we have learned in developing this program.

T47D breast cancer cell growth is inhibited by expression of VACM-1, a cul-5 gene.

Vasopressin-activated calcium-mobilizing (VACM-1), a cul-5 gene, is localized on chromosome 11q22-23 close to the gene for Ataxia Telangiectasia in a region associated with a loss of heterozygosity in breast cancer tumor samples. To examine the biological role of VACM-1, we studied the effect of VACM-1 expression on cellular growth and gene expression in T47D breast cancer cells. Immunocytochemistry studies demonstrated that VACM-1 was expressed in 0.6-6% of the T47D cells and localized to the nucleus of mitotic cells. Overexpressing VACM-1 significantly attenuated cellular proliferation and MAPK phosphorylation when compared to the control cells. In addition, VACM-1 decreased egr-1 and increased Fas-L mRNA levels. Further, egr-1 protein levels were significantly lower in the nuclear fraction from VACM-1 transfected cells when compared to controls. These data indicate that VACM-1 is involved in the regulation of cellular growth.

Thermal dehydration-induced thirst in lithium-treated rats.

Lithium is used as the primary treatment for bipolar disorder but has the common side effects of diuresis and thirst. In the present study, the effects of lithium on water balance responses of male Sprague-Dawley rats to thermal dehydration were examined. Rats ate either unadulterated food or food containing 2 g/kg lithium carbonate for 10 days. Then the control and lithium-treated rats were exposed to either 25 or 37.5 degrees C without food or water for 4 h. The rats were then allowed access to water for 3 h at 25 degrees C or were anesthetized and blood samples were taken. Lithium treatment caused an initial decrease in food intake, a decrease in body weight, and an increase in urine output. Heat exposure caused similar increases in evaporative water loss in control and lithium-treated rats. Heat exposure led to changes in blood indicators of body water status indicative of dehydration, whereas lithium had no effects on blood indicators of body water status. Water intake was increased by both heat exposure and by lithium treatment with the lithium-treated rats being more responsive to the thirst-inducing effects of thermal dehydration. Lithium treatment does not appear to impair water balance responses to heat exposure.