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Thyrotropin-releasing hormone (TRH)-containing neurons have been implicated in the central control of body temperature. TRH-containing neurons are located in brain areas known to influence body temperature, and TRH injected into these areas can produce changes in body temperature. While these lines of evidence support the view that central TRH is involved in thermoregulation, it has been difficult to confirm that TRH-containing neurons of the preoptic area are involved in this process. We used a different approach to test this hypothesis, based on recent evidence that changes in cellular levels of neuropeptide mRNA are linked to changes in neurosecretory processes. Hence, we predicted that if TRH neurons of the preoptic area are involved in body temperature regulation, cellular levels of TRH mRNA would be altered in animals in which body temperature had been experimentally altered. TRH mRNA levels were measured by in situ hybridization histochemistry in neurons of the preoptic area (POA) of animals that had been exposed to cold (5°C) or that had been given a hypothermic dose of ethanol. Cellular levels of TRH mRNA were reduced by both treatments. However, cellular levels of the mRNA-encoding gastrin-releasing peptide were not affected by these treatments in neurons of the POA, indicating that hypothermia exerted selective effects on TRH neurons in this brain region. Considering that both cold exposure and ethanol administration increase blood pressure, that the POA contains neurons which are both thermosensitive and barosensitive, and that TRH has been implicated in the control of blood pressure, we manipulated arterial blood pressure pharmacologically without changing body temperature to determine whether TRH neurons were also responsive to cardiovascular changes. Infusions with either nitroprusside, a vasodilator, or phenylephrine, a vasoconstrictor, produced significant changes in arterial blood pressure and heart rate, but did not affect TRH mRNA in the POA. These findings demonstrate that TRH neurons of the POA are thermoresponsive, supporting the view that they play a role in the central control of body temperature.

Background Hypercholesterolemia is thought to be a significant risk factor for coronary vasculopathy in cardiac transplant recipients. Methods and Results We examined the development of arteriosclerosis in mouse carotid artery loops allografted from B.10A(2R) (H-2 h2 ) donors to normocholesterolemic C57BL/6J (H-2 b ) recipients and hypercholesterolemic C57BL/6J recipients in which the apolipoprotein (apo) E gene had been knocked out. Luminal occlusion and cross-sectional neointimal area were greater in arteries allografted into hypercholesterolemic recipients at 15 and 30 days after transplantation. We also measured cellular and extracellular matrix components of the neointima by computerized planimetry of the fractional areas subtended by smooth muscle cells (anti–α-actin stain), collagen (Masson’s trichrome), lipid (oil red O), and leukocytes (anti-CD45). The neointimal area stained for smooth muscle cells was significantly greater in hypercholesterolemic recipients than in normocholesterolemic recipients at 15 and 30 days after allografting. Lipid contributed to neointimal area to a lesser degree, and there was no significant increase in the contribution of collagen or leukocytes. Conclusions Smooth muscle cell accumulation appears to be the principal contributor to the increase in neointimal area observed in arteries allografted into hypercholesterolemic mice.

We predicted that a significant source of background labeling after in situ hybridization (ISH) using 35S-labeled probes is attributable to a chemical reaction between the phosphorothioate moiety of the probe [O3 P=S] and disulfides in tissue. These covalent bonds would immobilize probe in the tissue, thereby increasing background labeling. On the basis of this view, we have explored the use of N-ethylmaleimide (NEM) to irreversibly alkylate the phosphorothioate moiety of the probe and/or to alkylate free sulfhydryls in tissue to block the formation of disulfides as a method of reducing background labeling. We report that NEM can significantly decrease background labeling of 35S-labeled oligodeoxy-nucleotide or cRNA probes but does not affect specific labeling. We conclude that the use of NEM in ISH protocols, as outlined here, may be an additional element researchers may consider to improve the signal-to-noise ratio.

The hypothalamic-pituitary-thyroid (HPT) axis functions abnormally in man and animals chronically exposed to ethanol. The most consistent observation in humans is that the thyrotropin response to thyrotropin-releasing hormone (TRH) is blunted. We have tested the hypothesis that chronic ethanol treatment in rats leads to a diminished responsiveness of the hypothalamus to central stimulation. Animals were maintained on 1 of 3 diets for 4 weeks: (1) laboratory chow and water provided ad libitum (chow-fed), (2) Sustacal chocolate liquid diet with vitamin mixture containing 5% (w/v) ethanol provided ad libitum (ethanol), or (3) Sustacal chocolate liquid diet with vitamin mixture containing sucrose substituted isocalorically (35%) for ethanol and provided in amounts matched to a weight-paired, ethanol-treated animal (pair-fed). At the end of 4 weeks, the animals were evaluated for their response to a single injection of ethanol (3 g/kg, ip) and/or exposure to 5 degrees C. Chronic ethanol treatment produced an increase in TRH mRNA in neurons of the paraventricular nucleus and fully blocked the thyrotropic response to cold exposure. However, chronic ethanol-treated animals did not exhibit altered basal levels of triiodothyronine or thyrotropin, nor did they have an altered response to a single injection of ethanol. These data demonstrate that chronic alcohol exposure alters functioning of the hypothalamic-pituitary-thyroid axis at least in part by affecting TRH neurons of the paraventricular nucleus.

Although immunosuppressive therapy minimizes the risk of graft failure due to acute rejection, transplant-associated arteriosclerosis of the coronary arteries remains a significant obstacle to the long-term survival of heart transplant recipients. The participation of specific inflammatory cell types in the genesis of this lesion was examined in a mouse model in which carotid arteries were transplanted across multiple histocompatibility barriers into seven mutant strains with immunologic defects. An acquired immune response--with the participation of CD4+ (helper) T cells, humoral antibody, and macrophages--was essential to the development of the concentric neointimal proliferation and luminal narrowing characteristic of transplant arteriosclerosis. CD8+ (cytotoxic) T cells and natural killer cells were not involved in the process. Arteries allografted into mice deficient in both T-cell receptors and humoral antibody showed almost no neointimal proliferation, whereas those grafted into mice deficient only in helper T cells, humoral antibody, or macrophages developed small neointimas. These small neointimas and the large neointimas of arteries grafted into control animals contained a similar number of inflammatory cells; however, smooth muscle cell number and collagen deposition were diminished in the small neointimas. Also, the degree of inflammatory reaction in the adventitia did not correlate with the size of the neointima. Thus, the reduction in neointimal size in arteries allografted into mice deficient in helper T cells, humoral antibody, or macrophages may be accounted for by a decrease in smooth muscle cell migration or proliferation.

Loss of intracellular calcium homeostasis has been regarded an important factor underlying neuron cell death after cerebral ischemic insult. In the brain, a major mechanism for regulation of intracellular calcium is through the signal transduction pathway involving hydrolysis of poly-phosphoinositides and release of the second messenger, inositol 1,4,5-trisphosphate (IP3). IP3 mobilizes calcium by interacting with an intracellular receptor. Upon its release after agonist stimulation, this second messenger is catabolized by a 3-kinase and a 5-phosphatase. In this study, in situ hybridization was carried out to examine the mRNA expression of IP3, receptor (IP3R) and IP3 3-kinase (IP3K) in rat brain cortex after transient focal cerebral ischemia induced by temporary occlusion of the middle cerebral artery (MCA) and the common carotid arteries (CCAs). Results indicate a large decrease (52%) in IP3R mRNA levels in the ischemic cortex as compared to that in the contralateral side at 4 h after a 45 min ischemic insult. By 16 h, practically no IP3R mRNA could be detected in the ischemic cortex. On the other hand, IP3K mRNA levels remained unaltered until 16 h after reperfusion, during which time, expression in the infarct core decreased but that surrounding the core area increased instead. Hybridization of adjacent brain sections with probes for neuron specific enolase (NSE) and beta-actin indicated also a time-dependent decrease in mRNA levels after ischemia, but these changes were less dramatic as compared to IP3R. At 16 and 24 h after reperfusion, there was an increase in beta-actin mRNA in cortical areas outside the MCA cortex, suggesting of reactive gliosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Experiments were designed to test the hypothesis that ethanol exposure during development can selectively affect the expression of specific isoforms of myelin protein gene expression in the rat cerebellum. We focused on myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) gene expression. Both of these genes are alternatively spliced to yield 4 (MBP) or 2 (MAG) mRNA isoforms. Prenatal ethanol exposure, delivered to the dams in a liquid diet, did not significantly alter the expression of MBP or MAG gene expression in the cerebellums of 15-day-old pups, as measured by quantitative in situ hybridization using specific oligodeoxynucleotide probes. In contrast, postnatal ethanol exposure delivered directly to the pups over a 6-day period by gastrostomy tube (PN days 4-10) reduced the expression of specific MBP and MAG isoforms in the cerebellum of animals in adulthood. These data demonstrate that ethanol exposure, especially during the period of rapid myelination, has selective effects on mRNA isoforms encoding specific MBPs and MAG.

We have previously demonstrated that a single administration of ethanol induces the expression of c- fos mRNA in the hypothalamic paraventricular nucleus (PVN). However, Fos protein must interact with a member of the Jun family to form functional heterodimers. To determine whether ethanol may have differential effects on c- fos and c- jun expression, we injected male rats acclimated to a 25°C environment with thanol (3 g/kg b.wt.) or saline. Using in situ hybridization histochemistry with oligonucleotide probes, we found that ethanol increased c- fos mRNA in the PVN, but decreased c- jun mRNA both in the PVN and in hippocampus. Considering that ethanol produces hypothermia and that the PVN contains neurons activated during hypothermia, we evaluated the effect of cold on c- fos and c- jun mRNA. Both cold and ethanol increased c- fos mRNA, and the effects were additive. However, c- jun mRNA levels in both PVN and hippocampus were unaffected by temperature. Finally, c- jun mRNA levels in the hippocampus were significantly reduced by chronic ethanol exposure, and this trend was also observed in the PVN. These findings demonstrate that a single injection of ethanol has opposite effects on the expression of nuclear transcription factors which interact to regulate gene expression in the nervous system.