Regulation of catecholamine release from the adrenal medulla is altered in deer mice (Peromyscus maniculatus) native to high altitudes.

High-altitude natives have evolved to overcome environmental hypoxia and provide a compelling system to understand physiological function during reductions in oxygen availability. The sympathoadrenal system plays a key role in responses to acute hypoxia, but prolonged activation of this system in chronic hypoxia may be maladaptive. Here, we examined how chronic hypoxia exposure alters adrenal catecholamine secretion and how adrenal function is altered further in high-altitude natives. Populations of deer mice (Peromyscus maniculatus) native to low and high altitudes were each born and raised in captivity at sea level, and adults from each population were exposed to normoxia or hypobaric hypoxia for 5 mo. Using carbon fiber amperometry on adrenal slices, catecholamine secretion evoked by low doses of nicotine (10 µM) or acute hypoxia (Po2 ∼15-20 mmHg) was reduced in lowlanders exposed to hypobaric hypoxia, which was attributable mainly to a decrease in quantal charge rather than event frequency. However, secretion evoked by high doses of nicotine (50 µM) was unaffected. Hypobaric hypoxia also reduced plasma epinephrine and protein expression of 3,4-dihydroxyphenylalanine (DOPA) decarboxylase in the adrenal medulla of lowlanders. In contrast, highlanders were unresponsive to hypobaric hypoxia, exhibiting typically low adrenal catecholamine secretion, plasma epinephrine, and DOPA decarboxylase. Highlanders also had consistently lower catecholamine secretion evoked by high nicotine, smaller adrenal medullae with fewer chromaffin cells, and a larger adrenal cortex compared with lowlanders across both acclimation environments. Our results suggest that plastic responses to chronic hypoxia along with evolved changes in adrenal function attenuate catecholamine release in deer mice at high altitude.

Social environment affects central distribution of estrogen receptor-α in Peromyscus californicus.

Social environment has well-established effects on an animal's social behavior and associated neuroendocrine responses. The presence of estrogen receptor alpha (ERα) in limbic system brain regions is related to the expression of a variety of social, reproductive and aggressive behaviors. We hypothesized that alterations to the social environment, specifically social isolation, would cause changes in ERα throughout the limbic system. The number of ERα immunoreactive (ERα-ir) cells within specific limbic system brain regions was quantified in male and female California mice (Peromyscus californicus), isolated or same sex pair-housed for 4 or 24 days. Peromyscus californicus is a highly social rodent species (monogamous and bi-parental) and therefore, may be particularly sensitive to manipulations of its social environment. Isolated males had a significantly greater number of ERα-ir cells in the ventromedial nucleus of the hypothalamus (VMH) and similar patterns within the bed nucleus of the stria terminalis (BST) and medial preoptic area (MPOA). Males housed for 24 days had a significantly greater number of ERα-ir cells in the BST, VMH, MPOA when compared with males housed for 4 days. Females housed for 24 days had significantly greater ERα-ir in the dentate gyrus of the hippocampus (DG) when compared with females housed for 4 days. No differences were found in the medial amygdala (MeA). These data demonstrate that social environment has region and sex specific effects on ERα-ir cells in this species. These results add to the comparative evidence regarding ERα, demonstrating a consistent role for ERα in species specific responsiveness to changes in the social environment.

Estrogen Receptor Alpha Distribution and Expression in the Social Neural Network of Monogamous and Polygynous Peromyscus.

In microtine and dwarf hamsters low levels of estrogen receptor alpha (ERα) in the bed nucleus of the stria terminalis (BST) and medial amygdala (MeA) play a critical role in the expression of social monogamy in males, which is characterized by high levels of affiliation and low levels of aggression. In contrast, monogamous Peromyscus males display high levels of aggression and affiliative behavior with high levels of testosterone and aromatase activity. Suggesting the hypothesis that in Peromyscus ERα expression will be positively correlated with high levels of male prosocial behavior and aggression. ERα expression was compared within the social neural network, including the posterior medial BST, MeA posterodorsal, medial preoptic area (MPOA), ventromedial hypothalamus (VMH), and arcuate nucleus in two monogamous species, P. californicus and P. polionotus, and two polygynous species, P. leucopus and P. maniculatus. The results supported the prediction, with male P. polionotus and P. californicus expressing higher levels of ERα in the BST than their polygynous counter parts, and ERα expression was sexually dimorphic in the polygynous species, with females expressing significantly more than males in the BST in both polygynous species and in the MeA in P. leucopus. Peromyscus ERα expression also differed from rats, mice and microtines as in neither the MPOA nor the VMH was ERα sexually dimorphic. The results supported the hypothesis that higher levels of ERα are associated with monogamy in Peromyscus and that differential expression of ERα occurs in the same regions of the brains regardless of whether high or low expression is associated with social monogamy. Also discussed are possible mechanisms regulating this differential relationship.

Genetic variation in total number and locations of GnRH neurons identified using in situ hybridization in a wild-source population.

The evolution of brain function in the regulation of physiology may depend in part upon the numbers and locations of neurons. Wild populations of rodents contain natural genetic variation in the inhibition of reproduction by winter-like short photoperiod, and it has been hypothesized that this functional variation might be due in part to heritable variation in the numbers or location of gonadotropin releasing hormone (GnRH) neurons. A naturally variable wild-source population of white-footed mice was used to develop lines artificially selected for or against mature gonads in short, winter-like photoperiods. We compared a selection line that is reproductively inhibited in short photoperiod (Responsive) to a line that is weakly inhibited by short photoperiod (Nonresponsive) for differences in counts of neurons identified using in situ hybridization for GnRH mRNA. There was no effect of photoperiod, but there were 60% more GnRH neurons in total in the Nonresponsive selection line than the Responsive selection line. The lines differed specifically in numbers of GnRH neurons in more anterior regions, whereas numbers of GnRH neurons in posterior areas were not statistically different between lines. We compare these results to those of an earlier study that used immunohistochemical labeling for GnRH neurons. The results are consistent with the hypothesis that the selection lines and natural source population contain significant genetic variation in the number and location of GnRH neurons. The variation in GnRH neurons may contribute to functional variation in fertility that occurs in short photoperiods in the laboratory and in the wild source population in winter.

The effects of exogenous melatonin and melatonin receptor blockade on aggression and estrogen-dependent gene expression in male California mice (Peromyscus californicus).

Photoperiodic regulation of aggression has been well established in several vertebrate species, with rodents demonstrating increased aggression in short day photoperiods as compared to long day photoperiods. Previous work suggests that estrogens regulate aggression via rapid nongenomic pathways in short days and act more slowly in long days, most likely via genomic pathways. The current study therefore examines the role of melatonin in mediating aggression and estrogen-dependent gene transcription. In Experiment 1, male California mice were housed under long day photoperiods and were treated with either 0.3 µg/g of melatonin, 40 mg/kg of the melatonin receptor antagonist luzindole, or vehicle for 10 days. We found that melatonin administration significantly increased aggression as compared to mice receiving vehicle, but this phenotype was not completely ameliorated by luzindole. In Experiment 2, male California mice were injected with either 1mg/kg of the aromatase inhibitor letrozole or vehicle, and oxytocin receptor (OTR), estrogen receptor alpha (ERα), and c-fos gene expression was examined in the bed nucleus of the stria terminalis (BNST) and medial preoptic area (MPOA). In the BNST, but not MPOA, OTR mRNA was significantly downregulated following letrozole administration, indicating that OTR is an estrogen-dependent gene in the BNST. In contrast, ERα was not estrogen dependent in either brain region. In the MPOA, OTR mRNA was inhibited by melatonin, and luzindole suppressed this effect. C-fos and ERα did not differ between treatments in any brain region examined. These results suggest that it is unlikely that melatonin facilitates aggression via broad spectrum regulation of estrogen-dependent gene expression. Instead, melatonin may act via regulation of other transcription factors such as extracellular signal regulated kinase.

Nitric oxide production by Peromyscus yucatanicus (Rodentia) infected with Leishmania (Leishmania) mexicana

Peromyscus yucatanicus (Rodentia: Cricetidae) is a primary reservoir of Leishmania (Leishmania) mexicana (Kinetoplastida: Trypanosomatidae). Nitric oxide (NO) generally plays a crucial role in the containment and elimination of Leishmania. The aim of this study was to determine the amount of NO produced by P. yucatanicus infected with L. (L.) mexicana. Subclinical and clinical infections were established in P. yucatanicus through inoculation with 1 x 10 2 and 2.5 x 10 6 promastigotes, respectively. Peritoneal macrophages were cultured alone or co-cultured with lymphocytes with or without soluble Leishmania antigen. The level of NO production was determined using the Griess reaction. The amount of NO produced was significantly higher (p ≤ 0.0001) in co-cultured macrophages and lymphocytes than in macrophages cultured alone. No differences in NO production were found between P. yucatanicus with subclinical L. (L.) mexicana infections and animals with clinical infections. These results support the hypothesis that the immunological mechanisms of NO production in P. yucatanicus are similar to those described in mouse models of leishmaniasis and, despite NO production, P. yucatanicus is unable to clear the parasite infection.

Cutaneous B-cell lymphoma in a Perdido Key Beach mouse (Peromyscus poliontus trissyllepsis).

The Perdido Key beach mouse (Peromyscus poliontus trissyllepsis) is an endangered mammal indigenous to the panhandle beaches of Northwest Florida. A captive 3.5-y-old female mouse was evaluated because of severe pruritus, diffuse alopecia, skin reddening, and ulcerations over the dorsum of her body. Initial skin biopsy of the affected area suggested bacterial dermatitis but was inconclusive. Despite empiric antibiotic, anthelmintic, and antihistamine treatments, she continued to decline and developed severe ulcerations over the majority of her body. Postmortem histopathologic evaluation led to a tentative diagnosis of epitheliotropic lymphoma, suggestive of a mycosis fungoides T-cell-type cutaneous lymphoma. However, immunohistochemistry results challenged this diagnosis, indicating that the lesion was actually an epidermotropic B-cell lymphoma. Spontaneous cutaneous B-cell lymphomas are rare in rodents and had not previously been reported to occur in Perdido Key beach mice. This case report provides initial evidence that the Perdido Key beach mouse is susceptible to cutaneous B-cell lymphoma.

Cortico-striatal oxidative status, dopamine turnover and relation with stereotypy in the deer mouse.

The deer mouse presents with spontaneous stereotypic movements that resemble the repetitive behaviours of obsessive-compulsive disorder (OCD), and demonstrates a selective response to serotonin reuptake inhibitors. OCD has been linked to altered redox status and since increased dopamine signalling can promote stereotypies as well as oxidative stress, we investigated whether the severity of deer mouse stereotypy may be associated with altered dopamine turnover and cortico-striatal redox status. Deer mice were separated into high (HSB), low (LSB) and non-stereotypy (NS) groups. Frontal cortical and striatal dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), as well as superoxide dismutase (SOD) activity, reduced (GSH) and oxidised (GSSG) glutathione and glutathione redox index, were analysed as markers for regional dopamine turnover and oxidative stress, respectively. Dopamine and its metabolites and SOD activity did not differ across the stereotypy groups. Significantly reduced GSH and GSSG and increased glutathione redox index were only observed in the frontal cortex of HSB animals. Frontal cortical GSH and GSSG were inversely correlated while glutathione redox index was positively correlated with stereotypy. Deer mouse stereotypy is thus characterised by a deficient glutathione system in the frontal cortex but not striatum, and provides a therapeutic rationale for using glutathione-active antioxidants in OCD. The evidence for a primary frontal lesion has importance for future OCD research.

Estrogen receptor alpha and vasopressin in the paraventricular nucleus of the hypothalamus in Peromyscus.

The purpose of this study was to determine the presence of estrogen receptor alpha (ERalpha) and the relationship between neurons that express ERalpha and produce vasopressin (AVP) in the paraventricular nucleus of the hypothalamus (PVN) in new world mice of the genus Peromyscus. Brains were collected from male and female Peromyscus californicus, Peromyscus leucopus, Peromyscus maniculatus, and Peromyscus polionotus, and double labeled for the expression of ERalpha and AVP immunoreactivity (IR). The number of cells expressing ERalpha-IR and AVP-IR was determined in the medial and posterior region of the PVN. The results indicate that Peromyscus is the first taxonomic group reported to have ERalpha widely distributed in the PVN, occurring in both medial and posterior regions of the PVN. While estrogen can regulate the production of AVP, AVP and ERalpha were rarely colocalized. There was, however, a significant inverse relationship between the number of cells that expressed ERalpha-IR and the number expressing AVP-IR. There were no sex differences in the expression of ERalpha-IR or AVP-IR.

Response to selection for photoperiod responsiveness on the density and location of mature GnRH-releasing neurons.

Natural variation in neuroendocrine traits is poorly understood, despite the importance of variation in brain function and evolution. Most rodents in the temperate zones inhibit reproduction and other nonessential functions in short winter photoperiods, but some have little or no reproductive response. We tested whether genetic variability in reproductive seasonality is related to individual differences in the neuronal function of the gonadotropin-releasing hormone network, as assessed by the number and location of mature gonadotropin-releasing hormone-secreting neurons under inhibitory and excitatory photoperiods. The experiments used lines of Peromyscus leucopus previously developed by selection from a wild population. One line contained individuals reproductively inhibited by short photoperiod, and the other line contained individuals nonresponsive to short photoperiod. Expression of mature gonadotropin-releasing hormone (GnRH) immunoreactivity in the brain was detected using SMI-41 antibody in the single-labeled avidin-biotin-peroxidase-complex method. Nonresponsive mice had 50% more immunoreactive GnRH neurons than reproductively inhibited mice in both short- and long-day photoperiods. The greatest differences were in the anterior hypothalamus and preoptic areas. In contrast, we detected no significant within-lines differences in the number or location of immunoreactive GnRH neurons between photoperiod treatments. Our data indicate that high levels of genetic variation in a single wild population for a specific neuronal trait are related to phenotypic variation in a life history trait, i.e., winter reproduction. Variation in GnRH neuronal activity may underlie some of the natural reproductive and life history variation observed in wild populations of P. leucopus. Similar genetic variation in neuronal traits may be present in humans and other species.

Modulation of endocrine pathways by 4,4'-DDE in the deer mouse Peromyscus maniculatus.

4,4'-DDT and 4,4'-DDE are widespread environmental contaminants that cause eggshell thinning in birds, altered sex ratios in the American alligator, and changes in the anal-genital distance in rodents. These contaminants are known to cause some of their toxicity by altering steroid receptor-mediated mechanisms. However, chemical-specific alterations in the expression of hormone-metabolizing enzymes may also be a mechanism for endocrine disruption, by altering the half-life of hormones in critical tissues. Previously, we showed that 4,4'-DDE causes a dose-dependent increase in ethoxyresorufin-O-deethylase (EROD) activity, but not pentoxyresorufin-O-dealkylase (PROD) activity, in the deer mouse. In this study, we demonstrated that 4,4'-DDE elicited a corresponding increase in CYP1A protein expression but not CYP2B using Western blotting and immunoprecipitation. 4,4'-DDE-mediated changes in phase II conjugating enzymes; UDP-glucuronosyltransferase (UGT) and phenolsulfotransferase (ST), were also investigated for the first time. Prepubescent female deer mice were dosed with 4,4'-DDE by gavage on days 1 and 2, then euthanized on day 4. As anticipated, dose-dependent increases in hepatic EROD and MROD activities, but not PROD or BROD, were observed. UGT activity was monitored by incubating liver microsomes and 14C-UDP-GA with potential substrates and measuring incorporation of radioactivity into TLC-resolved glucuronides. Dose-dependent increases in conjugation were observed with p-nitrophenol (a general UGT substrate) but not testosterone. Interestingly, a biphasic dose-response curve was observed for ST activity, with a peak at the 3 mg/kg dose. Dose-dependent increases in CYP1A1 and UGT-specific immunoreactive proteins were observed, suggesting de novo synthesis as a consequence of 4,4'-DDE exposure. We also measured Phase I and II enzymes in deer mouse platelets. Preliminary results indicate that the 4,4'-DDE-induced changes in liver Phase I and II enzyme activity were similar, but not identical, to those found in platelets. These results indicate that environmentally-relevant levels of 4,4'-DDE modulate the activity and expression of CYP1A1 and phase II enzymes in the deer mouse and that certain changes may be measured non-lethally.

Molecular basis of the alcohol dehydrogenase-negative deer mouse. Evidence for deletion of the gene for class I enzyme and identification of a possible new enzyme class.

The molecular basis of the alcohol dehydrogenase (ADH)-negative deer mouse (Peromyscus maniculatus) has been investigated. Several classes of mammalian ADHs have been recognized based upon biochemical and structural properties. ADH cDNA clones identified by hybridization to a mouse class I ADH cDNA clone were obtained from a deer mouse ADH-positive liver cDNA library. This cDNA has been identified as being a class I sequence and represents the deer mouse Adh-1 gene. An additional cDNA sequence identified in both the ADH-positive and -negative deer mouse cDNA libraries was identified by weak cross-hybridization to the mouse cDNA. This cDNA encodes an amino acid sequence representing a new class of mammalian ADH, and the deer mouse gene for this ADH is named Adh-2. ADH-negative deer mice do not produce mRNA, that is detected by the Adh-1 cDNA probe. However, both stocks of deer mice produce high levels of Adh-2 mRNA in liver. Southern analysis using an essentially full-length Adh-1 cDNA probe has shown that the Adh-1 gene is deleted in the ADH-negative mice. Biochemical analysis of enzyme activity suggests at least three ADH polypeptides are expressed in different tissues and have somewhat different substrate specificities, as in the mouse.

Photoperiodic adjustments in hypothalamic amines, gonadotropin-releasing hormone, and beta-endorphin in the white-footed mouse.

This study was undertaken to examine short photoperiod (SD; 8 h of light, 16 h of darkness)-induced alterations in reproductive endocrine and neuroendocrine parameters in the male white-footed mouse, Peromyscus leucopus. Exposure to SD for 8 weeks caused dramatic reductions in testis and seminal vesicle weights, decreased circulating LH and testosterone levels, and lowered the content of LH in the pituitary gland relative to those in mice under long photoperiod (LD; 16 h of light, 8 h of darkness). These changes were associated with significant increases in content of radioimmunoassayable GnRH in the mediobasal hypothalamus (MBH) and anterior hypothalamus at two time points in the light/dark cycle: 2100 h (dark phase) and 0900 h (light phase), respectively. Exposure to SD also caused an increase in radioimmunoassayable beta-endorphin in the MBH and preoptic area of the hypothalamus (POA) at 2100 h, but not at 0900 h. Mice exposed to SD also had a significantly higher metabolism of serotonin in the MBH at 0900 and 2100 h compared to mice under LD. The concentration of noradrenaline in the hypothalamus was unaffected by exposure to SD. However, the metabolism of dopamine (DA) in the POA at 0900 h was significantly increased relative to that in mice maintained under LD at this time. This increase in DA metabolism was associated with enhanced immunocytochemical staining for tyrosine hydroxylase in nerve fibers of the POA. Conversely, staining for tyrosine hydroxylase in tuberoinfundibular DA cell bodies of the arcuate nucleus was less intense under SD exposure. From these data it is concluded that exposure to SD caused regional and time-dependent alterations in the activities of hypothalamic amines (serotonin and DA) and neuropeptides (beta-endorphin and GnRH). These changes may be part of the neuroendocrine mechanism for SD-induced seasonal adaptations.

Non-alcohol dehydrogenase-mediated metabolism of methylazoxymethanol in the deer mouse, Peromyscus maniculatus.

The concept that alcohol dehydrogenase (ADH) is involved in the metabolism of methylazoxymethanol (MAM) was examined in a model consisting of two strains of the deer mouse, Peromyscus maniculatus, one of which has a normal complement of the enzyme [ADH(+)], and the other, which completely lacks it [ADH(-)]. Both the ADH(+) and the ADH(-) strains rapidly metabolized [14C]MAM, administered in the form of the acetic acid ester, [14C] MAMOAc , to 14CO2, and the rates and extents of metabolism were virtually identical. Determination of O6-methylguanine and 7-methylguanine in liver DNA 6 and 24 hr after MAMOAc (25 mg/kg) administration showed that the levels of DNA methylation induced by the carcinogen were not significantly different in the two strains, indicating that both are capable of the metabolic activation of MAM to methylating species. Pyrazole, a potent inhibitor of ADH, inhibited MAM metabolism as well as liver DNA methylation in the ADH(+) strain; however similar inhibition of these processes also occurred in the ADH(-) strain. 3-Methylpyrazole, a weak or noninhibitor of ADH, also decreased the levels of MAM metabolism in both the ADH(+) and the ADH(-) strains. From these results, we conclude that ADH is not obligatory either in the metabolism or in the metabolic activation of MAM. As a possible alternative to ADH, liver microsomes were examined for their ability to metabolize MAM. In the presence of a NADPH-generating system, liver microsomes from both strains converted [14C]MAM to 14CH3OH and 14CH2O , although liver microsomes from the ADH(-) strain were more active in this respect. The microsomal metabolism was sensitive to inhibition by CO as well as to inhibition by pyrazole and 3-methylpyrazole.

Development and characterization of a homologous radioimmunoassay for deer mouse (Peromyscus maniculatus bairdii) prolactin.

A highly specific and sensitive homologous radioimmunoassay has been developed for the secreted form of prolactin from the deer mouse Peromyscus maniculatus bairdii. Peromyscus serum and pituitary homogenates displayed parallel dilution response curves, and no cross reaction was seen with either mouse prolactin, mouse growth hormone or rat prolactin. The assay was sensitive to 25 picograms per tube and the intra- and inter-assay coefficients of variation were 5 and 3.6%, respectively. In addition, we have demonstrated that Peromyscus prolactin does not show parallel displacement in a homologous radioimmunoassay utilized for measuring prolactin in the common laboratory mouse.

Isolation, purification, and characterization of deer mouse (Peromyscus maniculatus bairdii) prolactin.

Prolactin (PRL) secreted by Peromyscus maniculatus bairdii anterior pituitaries was purified by gel filtration on Sephadex G-100 and ion-exchange chromatography on DEAE-cellulose. Peromyscus PRL (pmPRL) eluted from Sephadex G-100 with an elution-to-void volume ratio of 1.9 and at a salt concentration of 100 mM NaCl on DEAE-cellulose. Electrophoretic homogeneity of the hormone was demonstrated in several gel systems. On 7 1/2% alkaline polyacrylamide gels, pmPRL migrated with an Rf of 0.65. The molecular weight of pmPRL was estimated at 24,000 to 26,000 by sodium dodecyl sulfate electrophoresis and molecular exclusion chromatography. The amino acid composition of pmPRL was similar to other mammalian PRLs including two tryptophan residues and three disulfide bonds. Leucine was found to be the NH2-terminal residue. Circular dichroism (CD) spectra indicated an alpha-helix content of 55 +/- 5%, a value typical for prolactin molecules. However, in the region of side-chain absorption, the CD spectrum displayed a weak, asymmetric, negative band near 299 nm, with the CD returning to slightly positive values at 292 nm. This CD pattern is not typical of other secreted or stored prolactins. In the pigeon crop-sac assay, pmPRL showed a prolactin-like activity of 21 IU/mg.

Baseline levels of selected trace elements in Colorado oil shale region animals.

Baseline levels of boron, fluorine, molybdenum, and copper are described for 18 mule deer (Odocoileus hemionus) and for 45 composite samples of deer mice (Peromyscus maniculatus) from the Piceance Creek Basin, Rio Blanco County, Colorado. These data were collected before oil shale mining took place, and can be used to compare with levels found after mining is initiated. The data can thus be used to monitor changes in levels in animal tissues and as a basis for mitigating possible harmful effects due to the mining. Mean ppm (+/- S.D.) dry basis of each element is presented for selected tissues of each species. Results are also presented by habitat type for deer mice and by age for mule deer. Significant differences (P < 0.05) in molybdenum levels in deer mice were found between habitats. Significant differences (P < 0.05) were found between fawns and adult mule deer for boron levels, but not for the other elements. A need to standardize bone selection for analysis of fluorine was indicated. Kidneys appeared to be the organ of choice for baseline sampling of molybdenum and copper, and livers may be the organ of choice when toxic levels are suspected.