Isolation and restraint stress results in differential activation of corticotrophin-releasing hormone and arginine vasopressin neurons in sheep.

This study investigated sex differences in the stress-induced activation of neurons containing corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and enkephalin in the paraventricular nucleus (PVN) of gonadectomized male and female sheep. Groups (n=3) of both sexes were either subjected to 90 min isolation and restraint stress (stress group) or were not stressed. Blood samples were taken every 10 min for 90 min prior to and after stress to monitor cortisol levels in plasma. Brains were harvested after 90 min of stress. Stress caused elevation of plasma cortisol levels to a similar extent in both sexes. Double-labeling immunohistochemistry for Fos and either CRH, AVP or enkephalin was undertaken to quantify the numbers of neurons staining for CRH, AVP and enkephalin that also immunostained for Fos. Stress increased Fos immunostaining in all cell types. There was a greater proportion of CRH than AVP neurons activated in stressed animals. There were no sex differences in the activation of CRH and AVP neurons although females had a greater proportion of enkephalin cells staining for Fos than males in both control and stressed animals. There were no differences between control and stressed animals in the proportion of cells co-staining for CRH and AVP. We conclude that isolation and restraint stress activates neurons producing CRH, AVP and enkephalin in sheep and that CRH may play a greater role than AVP in regulating adrenocorticotrophic hormone secretion in response to this stressor in sheep. Finally, isolation and restraint stress does not influence co-localization of CRH and AVP in sheep.

Evaluation of hot saline solution and restriction endonuclease techniques in cytogenetic studies of Cycloneda sanguinea L. (Coccinellidae).

The goal of the present study was to determine if simple methods, especially hot saline solution (HSS) and MspI and HaeIII restriction endonucleases, which do not require special equipments, may be helpful in studies of genetic variability in the lady beetle, Cycloneda sanguinea. The HSS method extracted the heterochromatin region, suggesting that it is composed mostly of DNA rich in A-T base pairs. However, the X and y chromosomes were resistant to HSS banding. These bands facilitated the identification of each chromosome. In this study, we used the restriction endonucleases with different G-C base target sequences: MspI C/GGC and HaeIII GG/CC. The use of restriction enzyme MspI did not show an effect on the autosomal chromosomes. On the other hand, the sex pair showed a pale staining, to help in the recognition of these chromosomes. HaeIII produced characteristic bands which were identified all along the chromosomes, facilitating the identification of each chromosome. Based on these results, we can consider the heterochromatin being heterogeneous. The findings obtained here, using different chromosomal banding techniques, may be useful in the identification of intraspecific chomosome variability, specifically in Coccinellidae (Coleoptera) chromosomes, even without special equipment.

Colocalization of kisspeptin and gonadotropin-releasing hormone in the ovine brain.

Kisspeptin is a peptide that has been implicated in the regulation of GnRH cells in the brain. Immunohistochemical studies were undertaken to examine the distribution of kisspeptin-immunoreactive (IR) cells in the ovine diencephalon and determine the effect of ovariectomy in the ewe. We report that kisspeptin colocalizes to a high proportion of GnRH-IR cells in the preoptic area, which is a novel finding. A high level of colocalization of kisspeptin and GnRH was also seen in varicose neuronal fibers within the external, neurosecretory zone of the median eminence. Apart from the kisspeptin/GnRH cells, a population of single-labeling kisspeptin-IR cells was also observed in the preoptic area. Within the hypothalamus, kisspeptin-IR cells were found predominantly in the arcuate nucleus, and there was an increase in the number of immunohistochemically identified cell within this nucleus after ovariectomy. Kisspeptin-IR cells were also found in the periventricular nucleus of the hypothalamus, but the number observed was similar in gonad-intact and ovariectomized ewes. The colocalization of GnRH and kisspeptin within cells of the preoptic area and GnRH neurosecretory terminals of the median eminence suggests that the two peptides might be cosecreted into the hypophyseal portal blood to act on the pituitary gland. Effects of ovariectomy on the non-GnRH, Kisspeptin-IR cells of the hypothalamus suggest that kisspeptin production is negatively regulated by ovarian steroids.

Roles of peptides in transmission in the enteric nervous system.

Studies of the enteric nervous system have proved to be important in the development of new concepts of the chemical nature of transmission from neurons. In particular, they have revealed the multiplicity of influences that peptides can have on transmission, such as their action as primary transmitters, and the fact that they often act as co-transmitters in enteric neurons. However, in other cases no roles can be attributed to neuropeptides in enteric neurons, and their involvement in short-term changes in excitability seems minor.

Elevated KiSS-1 expression in the arcuate nucleus prior to the cyclic preovulatory gonadotrophin-releasing hormone/lutenising hormone surge in the ewe suggests a stimulatory role for kisspeptin in oestrogen-positive feedback.

Kisspeptins are encoded by the gene KiSS-1 and regulate gonadotrophin-releasing hormone (GnRH) and gonadotrophin secretion in various species, including humans. Here, we quantify gene expression of KiSS-1 in the arcuate nucleus (ARC) across the ovine oestrous cycle and demonstrate an increase in the caudal division of the ARC during the preovulatory period. These data strongly suggest that kisspeptins are involved in the generation of the preovulatory GnRH and luteinising hormone surge.

Evidence that projections from the bed nucleus of the stria terminalis and from the lateral and medial regions of the preoptic area provide input to gonadotropin releasing hormone (GNRH) neurons in the female sheep brain.

The arcuate nucleus/ventromedial hypothalamic nucleus (ARC/VMH) region is thought to relay estrogen feedback signals to gonadotropin-releasing hormone (GnRH) cells in the sheep brain. This region sends major projections to the lateral preoptic area (lPOA), ventral bed nucleus of the stria terminals (vBnST) and the ventro-caudal division of the median preoptic nucleus (vcMePON) with little direct input to GnRH cell bodies, suggesting interneuronal relay to GnRH neurons. The brain stem also provides input to the POA. The present study aimed to identify possible relay circuits in the POA and BnST to GnRH neurons. Biotinylated dextran amine (BDA) was injected into lPOA (n=6), vBnST (n=2), vcMePON (n=3) and periventricular nucleus (PeriV; n=1) of ewes for anterograde tracing. GnRH immunoreactive (IR) perikarya appearing to receive input from BDA-containing varicosities were identified by fluorescence microscopy, with further analysis by confocal microscopy. When BDA was injected into rostral and caudal regions of lPOA (n=3), no tracer-filled varicose fibers were found in contact with GnRH-IR perikarya. Injections into the center of the lPOA (n=3) indicated direct projections to GnRH-IR cells. Injections into the vBnST, vcMePON and PeriV indicated that cells of these regions also provide input to GnRH cells. BDA-containing varicosities found in the MPOA were immunoreactive for NPY or were GABAergic or glutamatergic when the tracer was injected into vBnST and lPOA, but not when injections were placed in the vcMePON. With injection into the PeriV, tracer-filled varicosities in the MPOA were not immunoreactive for somatostatin or enkephalin. Injection of FluoroGold into ventral POA retrogradely labeled cells in the above mentioned areas, but few were also immunoreactive for estrogen receptor-alpha. Thus, cells of the vBnST, lPOA, vcMePON and PeriV project to GnRH neurons. These cells may provide an interneuronal route to GnRH neurons from the ARC/VMH, the brain stem and other regions of the brain.

Localization of leptin receptor-like immunoreactivity in the corticotropes, somatotropes, and gonadotropes in the ovine anterior pituitary.

Leptin is a secreted product of the adipocytes that regulates a variety of functions. The presence of the leptin receptor (LR) has been demonstrated in the endocrine and neuroendocrine tissue, but only limited information is available regarding cell-specific expression in the anterior pituitary gland. We have used double-label immunofluorescence histochemistry to study the distribution of LR-like immunoreactivity (LR-ir) in the corticotropes, somatotropes, and gonadotropes of the ovine anterior pituitary. LR-ir was found in 34% of cells in the pars distalis and 94% of the cells in the pars tuberalis. In the pars distalis, LR-ir was present in 27% of corticotropes, 69% of somatotropes, and 29% of gonadotropes. In contrast, 90% of the gonadotropes in the pars tuberalis were immunopositive for LR. There was no alteration in the number of gonadotropes containing LR-ir during the various phases of the estrous cycle (n = 3/group) in the pars distalis (luteal phase, 36%; follicular phase, 32%; and estrous phase, 32%). In conclusion, we show that, in the pars distalis, LR-ir is expressed to a greater extent in the somatotropes than in the gonadotropes or corticotropes. This is in accordance with the documented effects of leptin on pituitary GH secretion. The differential expression of LR-ir between the gonadotropes of the pars distalis and pars tuberalis probably reflects the different phenotypes of the cells in these two regions. Lower levels of LR-ir expression in gonadotropes and corticotropes of the pars distalis may suggest that leptin does not substantially influence these particular cells, at least in this species.

Imitate to integrate: reviewing the pathway for B chromosome integration in Trypoxylon (Trypargilum) albitarse (Hymenoptera, Sphecidae).

B chromosomes are genomic "intruders" normally characterized by their total dispensability counteracted by a variety of drive mechanisms, which assures their presence regardless of their harmful effects on the host genome. From an evolutionary standpoint, the relationship between standard (A) and B chromosomes can go through different pathways, from an everlasting arms race to a cordial B integration. Examples underlying the first situation are fairly common; B integration, however, has been more a theoretical than a practical possibility. The B chromosome in the haplodiploid solitary wasp Trypoxylon albitarse is probably the first example of a "mimetic" B, which is being integrated into the A genome by limiting itself to one B per haploid genome, the same dosage as the A chromosomes. Here we review some of the findings underlying this hypothesis and discuss the T. albitarse B strategy as a possible mechanism for B chromosome integration as a regular member of the chromosome complement in haplodiploid organisms.

A RAPD marker associated with B chromosomes in Partamona helleri (Hymenoptera, Apidae).

The hymenopteran Partamona helleri is found in southwestern Brazil in the Mata Atlântica from the north of the state of Santa Catarina until the south of Bahia. This work shows that P. helleri can carry up to four B chromosomes per individual. In order to obtain more information about P. helleri B chromosomes, the RAPD technique was used to detect DNA fragments associated with these chromosomes. The results showed that the RAPD technique is useful to detect specific sequences associated with B chromosomes. One RAPD marker was identified, cloned and used as probe in a DNA blot analysis. This RAPD marker hybridized with sequences present only in individuals containing B chromosomes.

Long-term alteration in bodyweight and food restriction does not affect the gene expression of either preproorexin or prodynorphin in the sheep.

Various hypothalamic neuropeptides are involved in central regulation of food intake and expression of genes encoding these peptides changes with alterations in the bodyweight/metabolic status/nutritional status. Orexin(s) and dynorphin have been implicated in the regulation of appetite and neuroendocrine systems, but the function of these peptides is not well understood. We have employed in situ hybridization to examine the effects of long-term alterations in the bodyweight on expression of mRNA for preproorexin and prodynorphin in the putative feeding centers of the ovine hypothalamus. Expression of preproorexin was localized to the dorsomedial hypothalamic nucleus, perifornical area and lateral hypothalamic area. Cells expressing prodynorphin were localized to the periventricular, supraoptic, paraventricular, ventromedial hypothalamic nuclei and the thalamus. Small numbers of single scattered cells were seen in other brain areas. A few scattered prodynorphin-expressing cells were found in the lateral hypothalamic area but, in contrast to observations in the rat, there was no colocalization with preproorexin. Long-term alterations in the bodyweight did not influence the level of expression of preproorexin or prodynorphin. These findings suggest that orexin and dynorphin may not play a direct role in appetite regulation in sheep, although regulation at the level of the receptors for these peptides remains a possibility.

Long-term alterations in body weight do not affect the expression of melanocortin receptor-3 and -4 mRNA in the ovine hypothalamus.

The pro-opiomelanocortin-derived peptides and the melanocortin receptors are implicated in various functions within the CNS including the regulation of food intake. In the present study, we used in situ hybridization, with specific 35S-labelled ovine riboprobes to map the expression of melanocortin receptor-3 (MC3-R) and -4 (MC4-R) mRNA in the diencephalon and brainstem of normal female sheep. Furthermore, we examined the effect of long-term alterations in energy balance on the distribution and expression of MC3-R and MC4-R mRNA in food-restricted and ad libitum-fed ovariectomized female sheep. The distribution of melanocortin receptors generally resembled that of the rat. A high number of MC3-R-labelled cells were seen in the ventral division of the lateral septum and the medial preoptic area. In the hypothalamus, a moderate number of MC3-R-labelled cells was observed in the lateral hypothalamic area while other nuclear groups had low to intermediate numbers of MC3-R-labelled cells. The distribution of MC4-R mRNA was generally similar to that of MC3-R mRNA in the septal/preoptic and hypothalamic regions, with a high number of labelled cells present in the intermediate division of the lateral septum. Within the hypothalamus, no MC4-R mRNA expression was observed in the arcuate nucleus. There was more widespread distribution of moderate to low numbers of MC4-R mRNA-expressing cells in the brainstem compared to that of MC3-R mRNA. Unlike findings in the rat, only a low number of cells expressed melanocortin receptor mRNA in the ovine hypothalamic nuclei associated with feeding behavior. The number of melanocortin receptor-labelled cells and the level of expression (silver grains/cell) in the hypothalamic feeding centers was similar in food-restricted and ad libitum-fed animals. These findings suggest that long-term alterations in metabolic status do not change the melanocortin receptor mRNA distribution and/or expression in the sheep hypothalamus.

Integration of a B chromosome into the A genome of a wasp.

B chromosomes are genome symbionts, the presence of which in many eukaryote species is explained, in most cases, by their violation of Mendelian rules, usually based on meiotic or mitotic instability, leading to their accumulation in the germ line (drive). However, B chromosome integration into the genome as a regular member of the chromosome set should imply the loss of drive. A possible way of bypassing this difficulty is to regularize meiosis when the B chromosome is frequent in the population, in order to yield gametes with one B chromosome. In diploid organisms, this task needs to be achieved in the two sexes, but in haplodiploids the problem simplifies to only the diploid sex. We have found, to the authors' knowledge, the first evidence of a B chromosome that is regularizing its meiotic behaviour and limiting its number to one B chromosome per haploid genome, the same dosage as the standard (A) chromosomes, in the solitary wasp Trypoxylon albitarse. It suggests a possible mechanism for B chromosome integration as a regular member of the chromosome complement.

Integration of a B chromosome into the A genome of a wasp, revisited.

A previous study showed that in the haplodiploid solitary wasp Trypoxylon albitarse, most individuals carry one B chromosome per haploid genome, the same dosage as the standard (A) chromosomes, indicating a possible regularization of B-chromosome meiotic behaviour and its integration into the A genome. In a new sampling, we have analysed 15 populations (including 9 out of the 10 previously analysed) to test the evolution of this integration process. The new results provide a direct report of the invasion process in the Porto Firme population, where B frequency has dramatically increased in only four generations. In the populations from the Viçosa region, however, B frequency has remained stable, although the principal B type, the metacentric one, has increased in frequency at the expense of the acrocentric one in several populations. The implications of these new results on the hypothesis of the integration of these B chromosomes, as regular members of the A genome, are discussed.

The percentage of pituitary gonadotropes with immunoreactive oestradiol receptors increases in the follicular phase of the ovine oestrous cycle.

During the oestrous cycle, there is an alteration in gonadotrope responsiveness to gonadotropin releasing hormone (GnRH). One cellular mechanism that may be involved in these changes at the pituitary level is the hormonal regulation of oestrogen receptor (ER) expression. Using double-label immunohistochemistry, we examined the proportion of gonadotropes, lactotropes and somatotropes with immunoreactive (ir) oestrogen receptor alpha (ERalpha) in pituitary sections from ewes at three stages of the ovine oestrous cycle (n = 8 per group). The percentage of ERalpha positive cells that also stained positive for luteinizing hormone (LH) increased in the transition from the luteal phase to the follicular phase (n = 8), with no further increase at the time of oestrus (n = 8). In the pituitaries from the luteal phase sheep, only a small number (15%) of lactotropes and 4% of somatotropes were found to contain ir-ERalpha and there were no alterations across the oestrous cycle. When we examined pituitaries from ovariectomized (OVX) ewes treated (i.m.) with either oestradiol benzoate (50 microg) or oil vehicle for 2, 4, 6 or 16 h (n = 4 per group), there was no effect of treatment. In fact, the percentage of gonadotropes that were ERalpha-positive in OVX ewes was similar to that observed in the pituitaries from the follicular phase ewes, both of which display a high frequency of pulsatile GnRH secretion. We conclude that the number of gonadotropes that contain ir-ERalpha increases in the follicular phase of the oestrous cycle and this may enhance the responsiveness of these cells to oestrogen and GnRH. We suggest that this may be due to increased pulsatile GnRH input rather than rising oestrogen levels.

Seasonal changes in the inputs to gonadotropin-releasing hormone neurones in the ewe brain: an assessment by conventional fluorescence and confocal microscopy.

The seasonal pattern of breeding in sheep offers an opportunity to examine plasticity of neuronal inputs to gonadotropin-releasing hormone (GnRH) neurones. We used conventional fluorescence microscopy and confocal microscopy to compare the extent of input to GnRH neurones from various neuropeptide/neurotransmitter systems in ewes during the breeding and anestrous seasons. Using double-labelling immunohistochemistry, we counted close appositions between GnRH cells and varicosities that were immunoreactive for either glutamic acid decarboxylase (GAD; for gamma-amino butyric acid-GABA-neurones), dopamine beta hydroxylase (DBH; for noradrenergic neurones), vesicular glutamate transporter-1 (VGluT-1, for glutamatergic neurones), neuropeptide Y (NPY) and tyrosine hydroxylase (TH; for dopaminergic/noradrenergic neurones). The percentage of GnRH cells displaying close appositions to GABA-ergic varicosities was higher (P < 0.02) in anestrus than in the breeding season. The percentage of GnRH cells receiving input from varicosities that were positive for TH, DBH and VGluT-1 was similar in both seasons. Approximately 26-49% of GnRH neurones were seen to receive inputs from NPY, TH, GABAergic or noradrenergic neurones, while a larger number of GnRH cells (72-75%) received input from glutamatergic neurones. Conventional microscopy consistently overestimated the number of close contacts on GnRH neurones compared to confocal microscopy. For TH-immunoreactive varicosities in the preoptic area, only 16-35% were also immunoreactive for DBH, suggesting that the remainder are dopaminergic. Approximately half of the noradrenergic inputs in the preoptic area were also immunoreactive for NPY. In conclusion, we present numerical data on the consensus between light and confocal microscopy and the level of input of various neuronal systems to GnRH cells; the data indicate a seasonal change in the GABAergic input to GnRH neurones.

Evidence that orexin-containing neurones provide direct input to gonadotropin-releasing hormone neurones in the ovine hypothalamus.

Orexins A and B (ORX) have been added recently to the growing list of neuropeptides implicated in feeding and drinking behaviour as well as neuroendocrine function. In the present study, we have used single and dual labelling immunohistochemistry and a rabbit polyclonal anti-orexin-A antibody, which recognizes both ORX A and B, to examine ORX pathways in the sheep hypothalamus. ORX immunoreactive cells were distributed in the dorsomedial hypothalamic nucleus, lateral hypothalamic area, zona incerta and perifornical area; a few cells were also observed in the anterior hypothalamic area. In contrast to distribution in the rat brain, most of the ORX immunoreactive cells are localized to the dorsomedial hypothalamic nucleus and perifornical area; scattered cells are found in lateral hypothalamic area. ORX immunoreactive fibres were widely distributed throughout the hypothalamus and preoptic area with dense innervation of the medial preoptic area and bed nucleus of stria terminalis. Dual labelling demonstrated widespread expression of the long form of the leptin receptor within all ORX cells that were examined. Thirty percent of the gonadotropin releasing hormone (GnRH) cells that were examined had ORX immunoreactive terminals in close contact with no regional or sex differences. FluoroGold injections into the preoptic area retrogradely labelled a subpopulation of ORX cells in the lateral hypothalamic/perifornical area, showing ORX cells of this region project to the preoptic and could potentially provide input to GnRH cells. These findings suggest an integral role for ORX in the regulation of GnRH cells in the sheep and thus provide evidence of a novel mechanism whereby leptin can influence reproductive neuroendocrine function.

Neuropeptide Y (NPY) delays the oestrogen-induced luteinizing hormone (LH) surge in the ovariectomized ewe: further evidence that NPY has a predominant negative effect on LH secretion in the ewe.

Studies in rats suggest that neuropeptide Y (NPY) plays a stimulatory role in the generation of the preovulatory luteinizing hormone (LH) surge, via the Y1 receptor. We have investigated this issue using the oestradiol benzoate (EB)-treated ovariectomized (OVX) ewe which is a model for the preovulatory LH surge. A Y1 receptor antagonist (BIBO3304) was infused (25 microg/h) into the third cerebral ventricle (III-V) from 2 h before EB injection for 24 h, and had no effect on the ensuing LH surge. Using in situ hybridization, we then examined expression of NPY mRNA in the arcuate nucleus during the luteal, follicular and oestrous phases of the oestrous cycle, and found that levels were greatest during the luteal phase. Thus, reduced NPY synthesis might be an integral factor in the events leading to the cyclic preovulatory LH surge. This was tested by infusion of NPY (25 microg/h) into the III-V (as above). The NPY infusion delayed the LH surge until the infusion was ceased. High levels of NPY expression during the luteal phase of the oestrous cycle may be caused by progesterone. Thus, we determined whether NPY cells possess progesterone receptors (PR) and whether progesterone treatment up-regulates NPY mRNA expression in the arcuate nucleus. Immunohistochemistry for NPY and PR was performed in OVX, oestrogen-treated ewes, but no NPY cells of the arcuate nucleus were seen to colocalize PR. In situ hybridization for NPY was performed in OVX and OVX ewes treated with progesterone. There was no significant effect of progesterone treatment on NPY mRNA expression in the arcuate nucleus. We conclude that chronically elevated levels of NPY block the preovulatory surge of gonadotropin-releasing hormone/LH secretion in sheep, but high levels of NPY mRNA expression in the luteal phase of the oestrous cycle cannot be explained by an action of progesterone.

Seasonal changes in the expression of neuropeptide Y and pro-opiomelanocortin mRNA in the arcuate nucleus of the ovariectomized ewe: relationship to the seasonal appetite and breeding cycles.

Sheep experience well-documented seasonal changes in reproductive activity and voluntary food intake (VFI). Within the hypothalamus, neurones that express neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) have been implicated in the regulation of reproduction and appetite. In this study, we aimed to determine the extent to which the expression of these two neuronal systems is linked to the seasonal reproductive cycle and/or the seasonal appetite cycle. VFI in our sheep reaches a nadir in August with no difference occurring between December and February. We examined the brains of ovariectomized (OVX) female sheep (n=5-7) that were killed during the breeding season (February) or during the early or late nonbreeding season (August and December, respectively). The brains of these animals were perfused with paraformaldehyde and processed for in situ hybridization histochemistry, using ribonucleotide probes labelled with 35S. The number of NPY and POMC cells and the number of silver grains per cell were counted using an image analysis system. For NPY, the number of cells counted in the arcuate nucleus/median eminence region and the number of silver grains per cell was significantly lower in animals killed during August than in animals killed in February or December. The number of grains per cell over NPY cells was also significantly lower in animals killed during August. For POMC, the number of cells was lower in February than in August and December. Similarly, the number of grains per cell for POMC were lower in February than in August and December. VFI was significantly lower in animals during August than at other times of the year. We conclude that in OVX ewes: (i) NPY gene expression is lower at the time of the year when VFI is reduced and (ii) POMC gene expression is greater at the time of the nonbreeding season than during the breeding season. Because these results were obtained in OVX animals, the changes appear to be independent of alterations in the secretion and/or action of ovarian steroids. Thus, the activity of NPY neurones appears to relate to changes in appetite whereas changes in POMC expression may be relevant to the seasonal breeding cycle.

Cells of the arcuate nucleus and ventromedial nucleus of the ovariectomized ewe that respond to oestrogen: a study using Fos immunohistochemistry.

Oestrogen produces a positive feedback effect on the secretion of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) when implanted into the ventromedial/arcuate nucleus of the ovariectomized (OVX) ewe. This has led to the belief that it is in this area of the hypothalamus that oestrogen causes the preovulatory surge in GnRH/LH. To date, however, the cell types that are integral to this response have not been identified. The present study aimed to examine cellular responsiveness to oestrogen in this region of the brain using Fos immunohistochemistry and further aimed to determine the cell type that shows an acute response to oestrogen. OVX ewes (n = 4-6 per group) were given i.m. injections of oestradiol benzoate or oil (vehicle) and were killed 1-6 h later. Brains were perfused for immunohistochemistry. The number of cells in the arcuate nucleus which were immunopositive for Fos was greater (two- to fourfold) in the oestradiol benzoate-treated OVX ewes (n = 5) 1 h after injection. The number of Fos-positive cells in the ventromedial hypothalamic nucleus was 10-fold greater in the oestradiol benzoate-treated ewes 1 h after injection. Because there were high levels of Fos-immunoreactive cells in oil-treated ewes, we repeated the experiment with i.v. injection of 50 microg oestrogen or vehicle (n = 5). With this latter procedure, we found that oestrogen injection caused a significant increase in the number of Fos immunoreactive cells in the arcuate nucleus within 1 h, but there was no response in the ventromedial hypothalamus. To further characterize the types of cells that might respond to oestrogen, we double-labelled cells for Fos and either adrenocorticotropin hormone, neuropeptide Y or tyrosine hydroxylase (a marker for dopaminergic cells). These cell types could account for less than 30% of the total number of cells that were Fos-positive and oestrogen treatment did not cause an increase in the Fos labelling of any of these types of cell. These data show that oestrogen activates cells of the arcuate/ventromedial hypothalamus within 1 h of injection and that this response could relate to the feedback effects of this gonadal hormone. The majority of cells that produce Fos following oestrogen injection are of unknown phenotype. The data further suggest that induction of cells of the ventromedial hypothalamic nucleus require more prolonged oestrogen stimulus than cells of the arcuate nucleus.

Comparison of the presence and actions of substance P and neurokinin A in guinea-pig taenia coli.

The presence and sites of action of two closely related tachykinins, substance P (SP) and neurokinin A (NKA), were examined in the taenia coli of the guinea-pig. SP- and NKA-like immunoreactivity (LI) were demonstrated histochemically in nerve fibres supplying the taenia. Chromatographic characterization of aqueous acetic acid extracts of taenia showed only one peak of SP-LI, corresponding in retention time to authentic SP, whereas there were multiple peaks of NKA-LI, the major one of which corresponded to authentic NKA. SP-LI and NKA-LI, determined by radioimmunoassay, were in a molar ratio of SP equivalents to NKA equivalents of 8.5:1 in taenia extracts. Extrinsic denervation of the caecum had no significant effect on the concentration of either SP-LI or NKA-LI or on their immunohistochemical distributions. Both SP and NKA (10(-10) to 10(-5) M) caused contractions of the taenia that were unaffected by hyoscine (10(-6) M), mepyramine (10(-6) M) or tetrodotoxin (5 x 10(-7) M), indicating that both peptides act directly on the smooth muscle of the taenia. Contractions to SP occurred after a short, but concentration-dependent, delay, reached a peak quickly, and then decayed. In contrast, NKA caused contractions after longer latencies, the peak was reached more slowly, and the response was maintained for up to 10 min. (D-Pro2, D-Trp7,9)-SP (10(-5) M) antagonised responses to SP and NKA to a similar degree. It is concluded that both NKA and SP should be considered as transmitter candidates for non-cholinergic nerve-mediated excitation in the taenia.