Combined analysis of pharmacokinetic and efficacy data of preclinical studies with statins markedly improves translation of drug efficacy to human trials.

Correct prediction of human pharmacokinetics (PK) and the safety and efficacy of novel compounds based on preclinical data, is essential but often fails. In the current study, we aimed to improve the predictive value of ApoE*3Leiden (E3L) transgenic mice regarding the cholesterol-lowering efficacy of various statins in humans by combining pharmacokinetic with efficacy data. The efficacy of five currently marketed statins (atorvastatin, simvastatin, lovastatin, pravastatin, and rosuvastatin) in hypercholesterolemic patients (low-density lipoprotein ≥ 160 mg/dl) was ranked based on meta-analysis of published human trials. Additionally, a preclinical combined PK efficacy data set for these five statins was established in E3L mice that were fed a high-cholesterol diet for 4 weeks, followed by 6 weeks of drug intervention in which statins were supplemented to the diet. Plasma and tissue levels of the statins were determined on administration of (radiolabeled) drugs (10 mg/kg p.o.). As expected, all statins reduced plasma cholesterol in the preclinical model, but a direct correlation between cholesterol lowering efficacy of the different statins in mice and in humans did not reach statistical significance (R(2) = 0.11, P < 0.57). It is noteworthy that, when murine data were corrected for effective liver uptake of the different statins, the correlation markedly increased (R(2) = 0.89, P < 0.05). Here we show for the first time that hepatic uptake of statins is related to their cholesterol-lowering efficacy and provide evidence that combined PK and efficacy studies can substantially improve the translational value of the E3L mouse model in the case of statin treatment. This strategy may also be applicable for other classes of drugs and other preclinical models.

Altered hematopoiesis, behavior, and sexual function in mu opioid receptor-deficient mice.

The mu opioid receptor is thought to be the cellular target of opioid narcotics such as morphine and heroin, mediating their effects in both pain relief and euphoria. Its involvement is also implicated in a range of diverse biological processes. Using a mouse model in which the receptor gene was disrupted by targeted homologous recombination, we explored the involvement of this receptor in a number of physiological functions. Mice homozygous for the disrupted gene developed normally, but their motor function was altered. Drug-naive homozygotes displayed reduced locomotor activity, and morphine did not induce changes in locomotor activity observed in wild-type mice. Unexpectedly, lack of a functional receptor resulted in changes in both the host defense system and the reproductive system. We observed increased proliferation of granulocyte-macrophage, erythroid, and multipotential progenitor cells in both bone marrow and spleen, indicating a link between hematopoiesis and the opioid system, both of which are stress-responsive systems. Unexpected changes in sexual function in male homozygotes were also observed, as shown by reduced mating activity, a decrease in sperm count and motility, and smaller litter size. Taken together, these results suggest a novel role of the mu opioid receptor in hematopoiesis and reproductive physiology, in addition to its known involvement in pain relief.

Life in the fat lane: seasonal regulation of insulin sensitivity, food intake, and adipose biology in brown bears.

Grizzly bears (Ursus arctos horribilis) have evolved remarkable metabolic adaptations including enormous fat accumulation during the active season followed by fasting during hibernation. However, these fluctuations in body mass do not cause the same harmful effects associated with obesity in humans. To better understand these seasonal transitions, we performed insulin and glucose tolerance tests in captive grizzly bears, characterized the annual profiles of circulating adipokines, and tested the anorectic effects of centrally administered leptin at different times of the year. We also used bear gluteal adipocyte cultures to test insulin and beta-adrenergic sensitivity in vitro. Bears were insulin resistant during hibernation but were sensitive during the spring and fall active periods. Hibernating bears remained euglycemic, possibly due to hyperinsulinemia and hyperglucagonemia. Adipokine concentrations were relatively low throughout the active season but peaked in mid-October prior to hibernation when fat content was greatest. Serum glycerol was highest during hibernation, indicating ongoing lipolysis. Centrally administered leptin reduced food intake in October, but not in August, revealing seasonal variation in the brain's sensitivity to its anorectic effects. This was supported by strong phosphorylated signal transducer and activator of transcription 3 labeling within the hypothalamus of hibernating bears; labeling virtually disappeared in active bears. Adipocytes collected during hibernation were insulin resistant when cultured with hibernation serum but became sensitive when cultured with active season serum. Heat treatment of active serum blocked much of this action. Clarifying the cellular mechanisms responsible for the physiology of hibernating bears may inform new treatments for metabolic disorders.

A protocol for the isolation and cultivation of brown bear (Ursus arctos) adipocytes.

Brown bears (Ursus arctos) exhibit hyperphagia each fall and can become obese in preparation for hibernation. They do this without displaying the physiological problems typically seen in obese humans, such as Type 2 diabetes and heart disease. The study of brown bear hibernation biology could therefore aid in the development of novel methods for combating metabolic diseases. To this end, we isolated mesenchymal stem cells from subcutaneous fat biopsies, and culture methods were developed to differentiate these into the adipogenic lineage. Biopsies were taken from 8 captive male (N = 6) and female (N = 2) brown bears, ages 2-12 years. Plastic adherent, fibroblast-like cells were proliferated and subsequently cryopreserved or differentiated. Differentiation conditions were optimized with respect to fetal bovine serum content and time spent in differentiation medium. Cultures were characterized through immunostaining, RT-qPCR, and Oil red O staining to quantify lipid accumulation. Adiponectin, leptin, and glycerol medium concentrations were also determined over the course of differentiation. The culturing protocol succeeded in generating hormone-sensitive lipase-expressing, lipid-producing white-type adipocytes (UCP1 negative). Serum concentration and time of exposure to differentiation medium were both positively related to lipid production. Cells cultured to low passage numbers retained similar lipid production and expression of lipid markers PLIN2 and FABP4. Ultimately, the protocols described here may be useful to biologists in the field investigating the health of wild bear populations and could potentially increase our understanding of metabolic disorders in humans.

Generation of Bayesian prediction models for OATP-mediated drug-drug interactions based on inhibition screen of OATP1B1, OATP1B1∗15 and OATP1B3.

Human organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3 are important hepatic uptake transporters. Early assessment of OATP1B1/1B3-mediated drug-drug interactions (DDIs) is therefore important for successful drug development. A promising approach for early screening and prediction of DDIs is computational modeling. In this study we aimed to generate a rapid, single Bayesian prediction model for OATP1B1, OATP1B1∗15 and OATP1B3 inhibition. Besides our previously generated HEK-OATP1B1 and HEK-OATP1B1∗15 cells, we now generated and characterized HEK-OATP1B3 cells. Using these cell lines we investigated the inhibitory potential of 640 FDA-approved drugs from a commercial library (10µM) on the uptake of [(3)H]-estradiol-17ß-d-glucuronide (1µM) by OATP1B1, OATP1B1∗15, and OATP1B3. Using a cut-off of ⩾60% inhibition, 8% and 7% of the 640 drugs were potent OATP1B1 and OATP1B1∗15 inhibitors, respectively. Only 1% of the tested drugs significantly inhibited OATP1B3, which was not sufficient for Bayesian modeling. Modeling of OATP1B1 and OATP1B1∗15 inhibition revealed that presence of conjugated systems and (hetero)cycles with acceptor/donor atoms in- or outside the ring enhance the probability of a molecule binding these transporters. The overall performance of the model for OATP1B1 and OATP1B1∗15 was ⩾80%, including evaluation with a true external test set. Our Bayesian classification model thus represents a fast, inexpensive and robust means of assessing potential binding of new chemical entities to OATP1B1 and OATP1B1∗15. As such, this model may be used to rank compounds early in the drug development process, helping to avoid adverse effects in a later stage due to inhibition of OATP1B1 and/or OATP1B1∗15.

Dopaminergic A14/A15 neurons are activated during estradiol negative feedback in anestrous, but not breeding season, ewes.

A major factor responsible for seasonal anestrus in sheep is a striking increase in the ability of estradiol (E) to inhibit pulsatile GnRH and LH secretion. Previous studies suggest that dopaminergic neurons in the A14 and A15 groups of the ovine hypothalamus play a key role in conveying the inhibitory effects of E in anestrous ewes. The present study tested the hypothesis that A14/A15 neurons in anestrous ewes are activated in response to E, and that this activation is specifically related to seasonal changes in E negative feedback. Expression of the immediate early gene products, Fos and the Fos-related antigens (FRAs), was used as a marker of neuronal activation. Ovariectomized anestrous ewes received either blank implants (no E) or 0.5-cm long E implants sc and were killed 6 h later (E+6h) or 7 days later (E+7d and no E groups). During the breeding season, two additional groups of ovariectomized ewes were perfused 7 days after insertion of either blank or E implants. During anestrus, E completely suppressed LH pulses in the E+7d group, but had no effect in the E+6h group. In the E+7d anestrous group, there was also a significant increase in the mean percentage of tyrosine hydroxylase (TH)-positive cells that expressed nuclear Fos/FRAs in A14 and A15 areas compared to that in either the no E or E+6h group. By contrast, during the breeding season, E had no effect on LH pulse frequency, and there were relatively few TH-positive cells in A14 and A15 that coexpressed Fos/FRAs in either the no E or E+7d group. No significant steroidal or seasonal differences in Fos/FRA expression were seen in other hypothalamic dopaminergic cell groups (A12 and A13) or in the preoptic area-anterior hypothalamus or suprachiasmatic nucleus. Furthermore, E did not alter the total number of TH-positive neurons in A14/A15 or other cell groups. There were seasonal differences in the number of TH-positive neurons, with a significantly greater number of cells in the A13 and A15 of breeding season animals compared to anestrous ewes. Thus, E increased Fos/FRA expression in A14/A15 neurons only during anestrus at a time when it also inhibited LH pulse frequency. These findings are consistent with the view that activation of dopaminergic cells in A14 and A15 is a critical link in the chain of events leading to seasonal shifts in sensitivity to E negative feedback in the ewe.

Thyroid hormone receptor (alpha) distribution in hamster and sheep brain: colocalization in gonadotropin-releasing hormone and other identified neurons.

Thyroid hormones appear to play an important role in the seasonal reproductive transitions of a number of mammalian and avian species. These seasonal transitions as well as the effects of thyroid hormones on the reproductive neuroendocrine axis are mediated by the GnRH system. How thyroid hormones affect the GnRH system is unclear. Double label immunocytochemistry was used to examine GnRH- and other neurotransmitter/neuropeptide-containing neurons for thyroid hormone receptor (alphaTHR) colocalization in two seasonal breeders, the golden hamster and the sheep. AlphaTHR was identified in hamster and sheep brain by Western blot analysis. Furthermore, alphaTHR immunoreactivity was widely distributed in brain and was colocalized in identified populations: GnRH neurons (hamster, 28%; sheep, 46%); dopaminergic neurons of the A14 (hypothalamic) and A16 (olfactory bulb) cell groups, but not in the hypothalamic A13 cell group; and neurophysin-immunoreactive neurons of the supraoptic and paraventricular nuclei. The finding of alphaTHR in GnRH and A14 dopamine neurons provides an anatomical substrate for direct thyroid hormone action on the reproductive neuroendocrine system of these two seasonally breeding species. It remains to be determined whether the GnRH gene itself or the gene of another constituent within the same GnRH neuron is responsive to thyroid hormones.

The ability of estradiol to induce Fos expression in a subset of estrogen receptor-alpha-containing neurons in the preoptic area of the ewe depends on reproductive status.

In the ewe, seasonal anestrus results from a change in the hypothalamic responsiveness to estradiol (E2) negative feedback. Considerable evidence has implicated a specific group of dopaminergic neurons (the A15 group) in this seasonally dependent E2 effect, but these neurons do not appear to contain estrogen receptor-alpha (ERalpha). This apparent discrepancy raises the possibility that at least one other neural system is also involved in mediating E2 inhibition. The purpose of this study was to determine whether ERalpha-containing neurons are activated by the negative feedback action of E2 in anestrus. In Exp 1, we examined the effects of E2 on expression of the immediate early gene products, Fos and Fos-related antigens, in ERalpha-positive cells in anestrous ewes. ERalpha and Fos/Fos-related antigens were colocalized using a dual immunofluorescence procedure in sections throughout the hypothalamus from ovariectomized and E2-treated ovariectomized anestrous ewes. A low dose E2 treatment that inhibited LH pulse frequency and induced Fos in A15 dopaminergic neurons in a previous study significantly increased the percentage of ERalpha-containing neurons expressing Fos (17.8% vs. 1.7%) in the medial preoptic area, but not in other hypothalamic areas. In Exp 2, we determined whether there was a seasonal difference in the effect of E2 on Fos/ERalpha colocalization in this region. E2 treatment produced a 3-fold increase in the percentage of ERalpha-positive cells expressing Fos (15.1% vs. 3.4%) in anestrus, but failed to increase ERalpha/Fos colocalization (1.8% vs. 3.5%) during the breeding season. These data raise the possibility that a subset of ERalpha-containing neurons in the medial preoptic area plays a role in the seasonal change in response to E2 negative feedback in the ewe.

Potential for polysialylated form of neural cell adhesion molecule-mediated neuroplasticity within the gonadotropin-releasing hormone neurosecretory system of the ewe.

The GnRH neurosecretory system undergoes marked structural and functional changes throughout life. The initial goal of this study was to examine the neuroanatomical relationship between GnRH neurons and a glycoprotein implicated in neuroplasticity, the polysialylated form of neural cell adhesion molecule (PSA-NCAM). Using dual label immunocytochemistry in conjunction with confocal microscopy, we determined that fibers, terminals, and perikarya of GnRH neurons in adult ovariectomized ewes are intimately associated with PSA-NCAM. In the preoptic area, intense PSA-NCAM immunoreactivity was evident around the periphery of GnRH cell bodies. The second goal of this study was to determine whether PSA-NCAM expression associated with GnRH neurons varies in conjunction with seasonal changes in the activity of the GnRH neurosecretory system in ovariectomized ewes treated with constant release implants of estradiol. During the breeding season when reproductive neuroendocrine activity was enhanced, the expression of PSA-NCAM immunoreactivity associated with GnRH neurons was significantly greater than that during anestrus when GnRH secretion was reduced. This difference, which occurred despite an unchanging ovarian steroid milieu, was not observed in preoptic area structures devoid of GnRH immunoreactivity, suggesting that the seasonal change is at least partially specific to the GnRH system. The close association between PSA-NCAM and GnRH neurons and the change in this relationship in conjunction with seasonal alterations in GnRH secretion provide anatomical evidence that this molecule may contribute to seasonal remodeling of the GnRH neurosecretory system of the adult.

Retinal degeneration in cone photoreceptor cell-ablated transgenic mice.

PURPOSE: To examine the effect of loss of cone photoreceptor cells on retinal degeneration. METHODS: We previously identified a cone photoreceptor cell-specific promoter of human cone transducin a-subunit (GNAT2) gene. In this report, a minigene, Trc-Tox176, that contains the GNAT2 promoter, an attenuated diphtheria toxin A-chain gene, and an enhancer element from human interphotoreceptor retinoid-binding protein (IRBP) was used to generate coneless transgenic mice. Transgenic mice were identified by PCR and the copy number of the transgene was determined by Southern hybridization, and examined by histology. RESULTS: The results of immunostaining with anti-mouse GNAT2 antibodies and reverse transcription-PCR (RT-PCR) analysis with mRNA from the retinas of transgenic mice showed that cone photoreceptor cells were ablated in one of four transgenic mouse lines. The ablation of cone cells began at postnatal day 8, at the same time as the expression of endogenous GNAT2. An age-related rod degeneration was also found in this cone-ablated mouse line, beginning at postnatal day 9, proceeding from the central retina to the peripheral retina. CONCLUSIONS: Cone photoreceptor cells may play an important role in the survival of rod photoreceptor cells during mouse retina development.

Cold water swim stress increases the expression of neurotensin mRNA in the lateral hypothalamus and medial preoptic regions of the rat brain.

Stress-induced analgesia is a well-documented phenomenon that occurs in all mammalian species. Forced cold water swim produces a type of stress-induced analgesia that is independent of mu opioid receptors. The neuropeptide neurotensin (NT) has been implicated in mu opioid-independent analgesia (MOIA), but the circuitry of this system is largely unknown. The medial preoptic area (MPO) and lateral hypothalamus (LH) are two regions that are known to modulate pain processing. These two regions also contain neurotensinergic projections to the periaqueductal gray, a region that has been shown to produce MOIA upon injection of NT. The goal of this study was to determine if cold water swim (CWS) stress, which produces MOIA, activates the NT-ergic systems in these two regions. In situ hybridization results indicate that CWS increases the level of NT mRNA within neurons in the MPO and LH, suggesting that these two regions are activated during this process.

Disruption of reproductive rhythms and patterns of melatonin and prolactin secretion following bilateral lesions of the suprachiasmatic nuclei in the ewe.

To determine whether the photoperiodic responses of reproductive and prolactin (PRL) rhythms in the ewe requires an intact suprachiasmatic nucleus (SCN) driving the pineal rhythm of melatonin secretion, four groups of ovary-intact ewes over a 6-year period were subjected to bilateral (n = 40) or sham lesions (n = 15) of the SCN. Animals were exposed to an alternating 90-120 day photoregimen of 9L:15D and 16L:8D photoperiods. Blood samples taken twice weekly were assayed for prolactin and for progesterone to monitor oestrous cycles. On several occasions blood samples also were taken at hourly intervals for 24 h and analyzed for melatonin. Melatonin concentrations in sham lesioned ewes were basal during the lights-on period and rose robustly during darkness. Those sheep bearing unilateral lesions of the SCN (n = 13) or where the lesion spared the SCN entirely (n = 8) had patterns of melatonin secretion similar to sham ewes. The remaining ewes, having complete (n = 9) or incomplete bilateral (n = 8) destruction of the SCN, with one exception, had disrupted patterns of melatonin secretion. The nature of this disruption varied from complete suppression to continuously elevated levels. In lesioned ewes where melatonin secretion was not affected the onset and cessation of ovarian cycles were similar to sham ewes; stimulation of oestrous cycles under 9L:15D and cessation of oestrous cycles under 16L:8D. In contrast, 13 of 17 ewes with disrupted melatonin secretion also exhibited disrupted patterns of ovarian activity. In these animals oestrous cycles were no longer entrained by photoperiod but still occurred in distinct clusters, that is, groups of cycles began and ended spontaneously. Sheep with normal melatonin patterns showed low levels of PRL secretion during short days and elevated PRL levels during long days. However, 8 of 13 ewes with disrupted melatonin showed patterns of PRL secretion that were no longer entrained by photoperiod. A minority of ewes with disrupted melatonin patterns still showed reproductive (n = 4) and PRL (n = 5) responses similar to those of sham-lesioned ewes. These results show that bilateral destruction of the SCN in the ewe disrupts the circadian pattern of melatonin secretion and that this disruption usually, but not always, is associated with altered photoperiodic responses. These results strongly suggest that the SCN are important neural elements within the photoperiod time-keeping system in this species. A role for the SCN in the generation of endogenous transitions in reproductive activity (refractoriness) and prolactin secretion is not supported.

A subset of estrogen receptor-containing neurons project to the median eminence in the ewe.

The neural pathways responsible for conveying the steroid feedback signals that ultimately affect reproductive neuroendocrine function remain largely undefined. One possibility involves a direct projection from estrogen receptor (ER)-containing neurons to the median eminence (ME), a site of neuroendocrine peptide release. To examine this possibility, 8 ewes received stereotaxic injections of the retrograde neuronal tract-tracing compound cholera toxin-beta subunit (CT beta) into the ME. Neurons sending projections to the ME and containing ER were identified using a dual-label immunoperoxidase method. Double-labeled cells were found in distinct regions: (1) the ER-rich arcuate nucleus (ARC) that contained the greatest number of double-labeled cells, and (2) the organum vasculosum of the lamina terminalis (OVLT) which contained a very consistent, but low, number of double-labeled cells. While a fairly large number of retrogradely-labeled ARC neurons containing ER were identified, the majority of ER-containing ARC neurons were unlabeled and thus send projections elsewhere. Other regions containing high concentrations of ER-positive cells such as the medial preoptic area (MPOA), anterior hypothalamic area, and ventrolateral portion of the ventromedial hypothalamic nucleus, were devoid of double-labeled cells. Similarly, regions rich in neuroendocrine neurons such as the periventricular hypothalamus and paraventricular and supraoptic hypothalamic nuclei contained no double-labeled cells. These results suggest that modulation of neuroendocrine secretory activity may occur directly at the level of the ME by ER-containing neurons located within restricted regions of the hypothalamus and forebrain. However, the relatively low proportion of ER-containing neurons projecting to the ME suggests that the influence of estradiol upon neuroendocrine function also may include target sites other than the ME.

Multiple regulatory elements result in regional specificity in circadian rhythms of neuropeptide expression in mouse SCN.

It is well established that the mammalian circadian system consists of pacemaker cells in the suprachiasmatic nuclei (SCN). The mouse has become increasingly important in understanding the circadian timing system, due to the availability of mutant animals with abnormal circadian rhythms. In the present paper, we describe the organization of the mouse SCN, comparing the wild type and Clock mutant animal, with a special focus on those peptides bearing an upstream E-box element (vasopressin, vasoactive intestinal peptide, cholecystokinin and substance P). To this end, we describe the distribution of the foregoing SCN peptidergic cell types as well as gastrin-related peptide, calretinin, calbindin, somatostatin, neurotensin and retinal input to the SCN (determined by both tract tracing and fos-immunoreactivity in response to a light pulse). The Clock mutant mouse has decreased expression of vasopressin mRNA and protein in the SCN, with normal patterns of expression elsewhere in the brain. No other differences were detected between the Clock mutant and the wild type mouse. The results are consistent with the hypothesis that there are multiple regulatory elements of clock-controlled genes in the SCN.

A new method for simultaneous demonstration of anterograde and retrograde connections in the brain: co-injections of biotinylated dextran amine and the beta subunit of cholera toxin.

In studying reciprocally connected brain networks, it is advantageous to use techniques that allow simultaneous visualization of both efferent and afferent connections from a single injection site. We report on a new technique to achieve this using pressure injections of a mixture of biotinylated dextran amine (BDA) and the beta subunit of cholera toxin (Ctb). Adult male hamsters (n = 12) received 20-30-nl injections of either a 1:1 mixture of BDA (Sigma, 10%) and Ctb (List Biological, 0.5%), or each tracer by itself, into the medial amygdala. Adult female sheep (n = 4) received 200-300 nl of the combined tracer into the A15 region of the hypothalamus. After 1 (hamster) or 2 weeks' (sheep) survival, animals were perfused with 4% paraformaldehyde. Sections were double-labeled, first for BDA histochemistry using nickel-enhanced DAB, then for Ctb using a PAP technique and unenhanced DAB. In all animals, combined injections resulted in clear and consistent patterns of both anterograde and retrograde labeling. Ctb immunoreactivity was distinct and easily distinguished from BDA labeling. There was no evidence for loss of sensitivity of either tracer due to the combined delivery; no differences were seen between combined or single tracer injections in numbers of retrogradely-labeled cells or in the distribution of anterogradely-labeled fibers. In summary, the combined delivery of BDA and Ctb is an easy and reliable technique for simultaneous afferent and efferent tract tracing in both small and large animals; it could potentially be combined with immunocytochemistry to determine the neurochemical content of labeled cells or fibers.

Single- and double-label immunocytochemical study of the ovine suprachiasmatic nucleus (SCN): GABAergic and peptidergic relationships.

This study evaluated the neuropeptide and neurotransmitter content of the ovine suprachiasmatic nucleus (SCN) using both single- and double-label immunocytochemical methods. Single-label immunocytochemistry identified a few lightly labeled gamma aminobutyric acid (GABA) cells within the SCN as well as a dense plexus of fibers staining positive for the GABA biosynthetic enzyme, glutamic acid decarboxylase (GAD). Vasoactive intestinal polypeptide (VIP) fibers exhibited a similar distribution to GAD fibers; VIP cells were found throughout the SCN, as well as in the paraventricular (PVN) and supraoptic nuclei. Both GAD and VIP fibers exited dorsally from the SCN towards the PVN. Neurophysin (NP) and neuropeptide-Y (NPY) fibers were sparsely distributed throughout the SCN. Double-label immunocytochemistry revealed that GAD varicosities were often in close apposition to VIP cells. These results confirm the presence of GABAergic elements within the sheep SCN. Furthermore, they raise the possibility of a GABAergic modulation of VIP neuronal activity within the ovine SCN.

Central connections of the ovine olfactory bulb formation identified using wheat germ agglutinin-conjugated horseradish peroxidase.

Pheromonal stimuli elicit rapid behavioral and reproductive endocrine changes in the ewe. The neural pathways responsible for these effects in sheep are unknown, in part, because the olfactory bulb projections have not been examined in this species. Using the anterograde and retrograde neuronal tracer, wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP), we describe the afferent and efferent olfactory bulb connections of the Suffolk ewe. Injections of WGA-HRP limited to the main olfactory bulb resulted in retrograde labeling of cells in numerous telencephalic, diencephalic, and metencephalic regions. Terminal labeling was limited to layer la of ipsilateral cortical structures extending rostrally from the anterior olfactory nucleus (AON), piriform cortex, anterior-, and posterolateral-cortical amygdaloid nuclei to lateral entorhinal cortex caudally. Injections involving the accessory olfactory bulb and AON produced additional labeling of cells within the bed nucleus of the stria terminalis (BNST), medial nucleus of the amygdala, and a few cells in the posteromedial cortical nucleus of the amygdala. Terminal labeling included a small dorsomedial quadrant of BNST and also extended to the far lateral portions of the supraoptic nucleus. A clearly defined accessory olfactory tract and nucleus was not evident, perhaps due to limitations in the sensitivity of the method. With this possible exception, the afferent and efferent olfactory connections in the sheep appear similar to those reported for other species.

A re-evaluation of the effects of gonadal steroids on neuronal activity in the male rat.

Single unit activity (SUA) was recorded from 77 cells located in the arcuate nucleus (ARC) and medial preoptic area (MPA) of anesthetized, intact male rats. Animals were administered vehicle, testosterone (T; 5 or 50 micrograms) or 17 beta-estradiol (E; 0.5 microgram) intravenously and SUA was monitored for 8-12 min. T (50 micrograms) reduced SUA in 50% of ARC units and 44% of MPA units within 2.1 +/- 0.46 and 3.3 +/- 0.92 min, respectively. Inhibition of ARC SUA was more pronounced than MPA SUA. A small percentage (9%) of ARC units were excited by T. E reduced SUA in 29% of ARC units and 27% of MPA units. Single doses of 5 micrograms T did not affect ARC activity. However, when followed within 10 min by an additional dose of 5 or 50 micrograms T, 30% and 43% of ARC units were inhibited, respectively. Doses (10 micrograms) of T produced plasma T concentrations within physiological limits, although 50 micrograms doses produced supraphysiological T levels. Neither dose affected circulating LH concentrations. We conclude that physiological and supraphysiological concentrations of T can rapidly affect SUA within the ARC.

The GnRH system of seasonal breeders: anatomy and plasticity.

Seasonal breeders, such as sheep and hamsters, by virtue of their annual cycles of reproduction, represent valuable models for the study of plasticity in the adult mammalian neuroendocrine brain. A major factor responsible for the occurrence of seasonal reproductive transitions is a striking change in the responsiveness of gonadotropin-releasing hormone (GnRH) neurons to the inhibitory effects of gonadal steroids. However, the neural circuitry mediating these seasonal changes is still relatively unexplored. In this article, we review recent findings that have begun to define that circuitry and its plasticity in a well-studied seasonal breeder, the ewe. Tract tracing studies and immunocytochemical analyses using Fos and FRAs as markers of activation point to a subset of neuroendocrine GnRH neurons in the MBH as potential mediators of pulsatile GnRH secretion. Because the vast majority of GnRH neurons lack estrogen receptors, seasonal changes in responsiveness to estradiol are most probably conveyed by afferents. Two possible mediators of this influence are dopaminergic cells in the A14/A15 cell groups of the hypothalamus, and estrogen receptor-containing cells in the arcuate nucleus that project to the median eminence. The importance of GnRH afferents in the regulation of season breeding is underscored by observations of seasonal changes in the density of synaptic inputs onto GnRH neurons. Thyroid hormones may participate in this remodeling, because they are important in seasonal reproduction, influence the morphology of other brain systems, and thyroid hormone receptors are expressed within GnRH neurons. Finally, in the hamster, neonatal hypothyroidism affects the number of caudally placed GnRH neurons in the adult brain, suggesting that thyroid hormones may influence development of the GnRH system as well as its reproductive functions in the adult brain.