Thrombophilia and venous thromboembolism. International consensus statement. Guidelines according to scientific evidence.

Thrombophilia is the term now used to describe predisposition to increased risk of venous and occasionally arterial thromboembolism due to hematological abnormalities. It can be a multifactorial disorder where congenital defects of anticoagulant or procoagulant factors may be combined with acquired hematological abnormalities. It should be considered in patients with a documented unexplained thrombotic episode or a positive family history. The aim of this document is to provide guidelines for investigation and management of patients with thrombophilia in the presence or absence of venous thromboembolism (VTE).

Molecular and cellular properties of GnRH neurons revealed through transgenics in the mouse.

Recent advances in the use of gonadotropin-releasing hormone (GnRH) promoter-driven transgenics in the mouse are beginning to open up the once elusive GnRH neuronal phenotype to detailed molecular and cellular investigation. This review highlights progress in the development of GnRH promoter transgenic constructs and the understanding of murine gene sequences required for the correct temporal and spatial targeting of transgenes to the GnRH phenotype in vivo. Strategies enabling the identification of single, living GnRH neurons in the acute brain slice preparation are allowing gene profiling and electrophysiological experiments to be undertaken. Results so far indicate that, like other neurons, GnRH cells express a variety of sodium, potassium and calcium channels as well as GABAergic and glutamatergic receptors which are responsible for determining the membrane properties and firing characteristics of the GnRH neuron. Many of these receptors and channels appear to be expressed heterogeneously within the GnRH phenotype. Furthermore, several display distinct postnatal developmental expression profiles which are likely to be of consequence to the development of synchronized, pulsatile GnRH secretion in the adult animal.

Fluoroquinolone use and the change in incidence of tendon ruptures in the Netherlands.

INTRODUCTION: Shortly after their introduction, fluoroquinolones were associated with reports of tendinitis and tendon rupture. During the past years, the number of reports has risen, possibly because of an increased use of fluoroquinolones. In this study, we describe the use of fluoroquinolones in the Dutch community and the possible public health effects of an association between fluoroquinolone use and tendon ruptures. METHODS: In the PHARMO drug database we identified all prescriptions for fluoroquinolones in the period 1991-1996. The incidence of fluoroquinolone use was expressed as the number of fluoroquinolone episodes per 1000 inhabitants in one year, and extrapolated to the Dutch population after standardisation on age and gender. The annual incidence of non-traumatic tendon ruptures in the period 1991-1996 was calculated with data from the nation-wide hospital registry. The expected number of fluoroquinolone attributable tendon ruptures was calculated on the basis of the use of fluoroquinolones, the number of non-traumatic tendon ruptures and an assumed relative risk of 1.5-10. RESULTS: In 1996, approximately 251,000 patients experienced 318,000 episodes of fluoroquinolone use in the Netherlands. Females used more often fluoroquinolones than males, and the number of episodes increased exponentially with age. In the period 1991 through 1996, the absolute number of fluoroquinolone episodes increased by 160%, from 122,000 to 318,000. The absolute number of hospitalised tendon ruptures increased with 28%, from 768 in 1991 to 984 in 1996. Assuming a relative risk of 1.5 to 10.0, 1 to 15 tendon ruptures could be attributed to fluoroquinolone use in 1996. Only 7% of the observed increase could be attributed to the increased use of fluoroquinolones. If the total increase of hospitalised non-traumatic tendon ruptures would be attributable to the increase in fluoroquinolone use, this would mean that the risk of non traumatic tendon ruptures to fluoroquinolones would be more than 250 times the risk during non-use. CONCLUSION: In the Netherlands, a large simultaneous increase in non-traumatic tendon ruptures and fluoroquinolone use was observed in the period between 1991 to 1996. Assuming a relative risk of 1.5 to 10.0 for tendon ruptures during fluoroquinolone use, only 0.5 to 7% of the increase in non-traumatic tendon ruptures could be attributed to the increased fluoroquinolone use. The increase in the incidence of non-traumatic hospitalised tendon ruptures in the Netherlands is not likely to be explained solely by the increased use of fluoroquinolones.

Regulation of gonadotropin-releasing hormone (GnRH) gene expression during GnRH neuron migration in the mouse.

The mechanisms underlying the migration of the gonadotropin-releasing hormone (GnRH) neurons from the nose into the forebrain are not resolved. In an attempt to characterize further the migrating GnRH neurons, we have employed in situ hybridization techniques and transgenic mouse models to examine levels of GnRH mRNA and GnRH gene transcription in GnRH neurons during migration in the mouse. In the first experiment, cellular levels of GnRH mRNA in neurons located throughout the nose and forebrain were examined in embryonic day (E) 12.5, 14.5, 16.5 and 19.5 mice using in situ hybridization. The GnRH mRNA content of cells located in both the nose (p < 0.01) and forebrain (p < 0.05) was found to increase significantly from E12.5 to E19.5 and from E14.5 to E19.5, respectively. However, cellular levels of GnRH mRNA were not significantly different in neurons located in the nose compared with the brain at each developmental age. In the second experiment, levels of GnRH gene transcription were investigated at E14.5 using two different GNLZ transgenic mouse lines in which 13.5 kb of GnRH gene sequences direct the expression of the LacZ reporter to the nucleus of GnRH neurons. Migrating GnRH neurons displayed up to a 3-fold increase (p < 0.01) in transgene expression, an index of GnRH transcription, precisely as they approached and entered the forebrain. These results indicate that GnRH gene expression in migrating GnRH neurons is likely regulated by temporal as well as spatial factors and that, as found postnatally, this may involve both transcriptional and post-transcriptional regulatory mechanisms.

Utility of the Pediatric Symptom Checklist for behavioral screening of disadvantaged children.

Investigated the utility of a parent report measure designed specifically for behavior screening in the pediatric primary care setting for use with disadvantaged children and families. Results indicated that the Pediatric Symptom Checklist was significantly related to scores derived from the Child Behavior Checklist. Receiver Operator Characteristic analysis indicated the need for modification of the cutting score previously established with middle-class children. A model for screening children for psychopathology within the structural-organizational constraints of the pediatric primary care setting is presented.

Differing, spatially restricted roles of ionotropic glutamate receptors in regulating the migration of gnrh neurons during embryogenesis.

We have examined here the role of glutamate in regulating the process of tangential neuronal migration during embryogenesis by investigating the roles of AMPA and NMDA receptors in the migration of the gonadotropin-releasing hormone (GnRH) neurons from the nose to the hypothalamus. We first determined that GluR1-4 subunit mRNAs were present from embryonic day (E) 12.5 along the complete nose-brain migratory pathway of the GnRH neurons, whereas that of the obligatory NMDAR1 transcript was present only in brain regions of GnRH migration. In vivo studies revealed that AMPA receptor antagonism between E12.5 and E16.5 resulted in a significant (p < 0.05) accumulation of GnRH neurons in the nose adjacent to the cribiform plate. In contrast, NMDA receptor antagonism over E12.5-E16.5 or E13.5-E16.5 caused a selective increase (p < 0.05) in the number of GnRH neurons located in their final resting place within the diagonal band of Broca and preoptic area. Dual-labeling studies using GnRH promoter-LacZ transgenic mice, which facilitate the identification of receptors in GnRH neurons, identified the presence of NMDAR1 receptors in approximately 6% of embryonic GnRH neurons located throughout the migratory pathway. Postnatally, the percentage of GnRH neurons expressing NMDAR1 increased to 50%. These results indicate that tonic AMPA receptor activation enhances the migration of GnRH neurons from the nose into the brain, whereas that of NMDA receptor activation slows the final phase of GnRH migration within the forebrain. These in vivo observations demonstrate differing, spatially restricted roles for AMPA and NMDA receptor activation in the process of tangential neuronal migration.

Recognition of facial affect by children and adolescents diagnosed with social phobia.

This study compared the ability of children with social phobia and children with no psychiatric disorder to accurately judge facial affect. Fifteen children and adolescents with social phobia and 14 control children were asked to identify emotions depicted in slides from the Pictures of Facial Affect. In addition, they rated their level of anxiety on a pictorial Likert scale prior to and upon completion of the facial recognition task. The results indicated that children with social phobia had significantly poorer facial affect recognition skills than normal controls and reported greater anxiety upon completion of the recognition task. Multivariate analysis revealed significant differences between groups in the number of errors based on the type of facial affect. Posthoc analysis indicated that deficits were most pronounced for facial representations of happiness, sadness, and disgust. The results are discussed in relation to an integrated model of social skills training that includes facial affect recognition training as a integral component in treatment programs for children and adolescents with social phobia. Directions for future research with larger samples of more ethnically diverse children and adolescents are presented.

Role of the GABA(A) receptor gamma2 subunit in the development of gonadotropin-releasing hormone neurons in vivo.

We have employed transgenic mouse models to examine the functional significance of the gamma2 subunit of the GABA(A) (gamma-aminobutyric acid) receptor to the correct development of gonadotropin-releasing hormone (GnRH) neurons in vivo. In the first experiment, the expression of gamma2 subunit protein by the GnRH phenotype was determined using transgenic mice in which GnRH gene sequences direct the expression of the LacZ reporter to the nucleus of the GnRH neurons. This greatly facilitates the immunocytochemical identification of non-nuclear-located antigens within GnRH neurons and revealed that approximately 25% of juvenile GnRH neurons were immunoreactive for the gamma2 subunit and that this increased to 40% in pubertal mice. In the second experiment, GnRH mRNA expression was examined in the brains of gamma2 subunit knockout mice (gamma2(0/0)) and their wild-type (gamma2+/+) littermates at embryonic day 15 and postnatal days (P) 0 and 11-16 using in situ hybridization. The distribution and numbers of cells expressing GnRH mRNA in gamma2+/+ and gamma2(0/0) mice were not found to differ at any age. However, the GnRH mRNA content of medial septal cells was significantly lower in gamma2(0/0) compared with gamma2+/+ mice at P11-16 (P<0.05) and the same trend was observed for preoptic area neurons. These results demonstrate that while the gamma2 subunit of the GABA(A) receptor is expressed by postnatal GnRH neurons, their embryonic development does not require a functional gamma2 subunit. In contrast, postnatal GnRH mRNA expression was found to be dependent upon signalling through the GABA(A) receptor.

Oestrogen modulation of noradrenaline neurotransmission.

Noradrenaline (NA) exerts an important neuromodulatory role within diverse neuronal networks and is also likely to be a target for oestrogen in the brain. Distinct, highly organized sub-populations of brainstem NA neurons express oestrogen receptors (ERs) and some of these display species differences. A number of genes expressed by NA neurons, ranging from transcription factors to co-released neuropeptides, are influenced by oestrogen and may have roles in the predominant enhancement in NA activity in response to oestrogen. The effects of oestrogen on genes involved directly in NA biosynthesis are less clear, although promoter transgenic work suggests oestrogen to have a powerful influence upon tyrosine hydroxylase gene transcription. In addition to direct actions on NA neurons, evidence suggests that oestrogen also regulates adrenergic receptor expression and function within the ER-rich hypothalamus as well as the cerebral cortex. Together, these investigations point to a multifaceted pre- and postsynaptic regulation of NA transmission by oestrogen. While the hypothalamic neuronal networks controlling reproduction remain the principal site of investigation of oestrogen regulated NA transmission, the role of oestrogen and NA and their potential interactions in cortical functioning are becoming of equal interest.

[Critical review of anti-influenza drugs]. / Griepremmers in kritisch perspectief.

Neuraminidase inhibitors such as zanamivir and oseltamivir belong to a new class of antiviral drugs for the treatment and prevention of influenza. As yet however, the therapeutic efficacy of these drugs (shortening of recovery time by approximately one day) has only been demonstrated in healthy adults affected by influenza A, but not in risk groups and in influenza B disease, whereas studies of prophylactic efficacy are still going on. Neither do these drugs impact on viral spread, a public health risk against which the economic advantages of early work resumption have to be weighed. Since flu symptoms can be caused by other germs than the influenza A or B virus, caution in prescribing these drugs seems warranted, also to prevent the development of drug resistance. In addition, when designing therapeutic efficacy trials in risk groups, selecting the rate of secondary complications and death may be more adequate as clinical endpoint than (economically important) duration of illness.

Identification and characterization of estrogen receptor alpha-containing neurons projecting to the vicinity of the gonadotropin-releasing hormone perikarya in the rostral preoptic area of the rat.

Gonadal steroids exert a powerful regulatory influence upon the functioning of gonadotropin-releasing hormone (GnRH) neurons despite the apparent absence of gonadal steroid receptors in these cells. By using retrograde-tracing techniques combined with dual-labeling immunocytochemistry, we show here that distinct populations of estrogen receptor alpha (ERalpha)-containing neurons located in the hypothalamus and caudal brainstem project to the vicinity of the GnRH perikarya located in the rostral preoptic area (rPOA). The strongest estrogen-receptive afferent projection to this area originated from neurons located in the anteroventral periventricular and medial preoptic nuclei of the preoptic area. Approximately 50% of arcuate nucleus neurons projecting to the rPOA were demonstrated to synthesize either neuropeptide Y or beta-endorphin, but little evidence was found for ERalpha immunoreactivity in either of these specific subpopulations. Over 80% of all tyrosine hydroxylase-expressing neurons in the arcuate nucleus expressed ERalpha, but none projected to the rPOA. In the caudal brainstem, the A1 and A2 norepinephrine neurons comprised nearly all of the retrogradely labeled neurons. However, only the A2 afferents expressed ERalpha immunoreactivity, whereas the A1 afferents coexpressed neuropeptide Y. These observations, combined with the anterograde labeling data of others, provide neuroanatomical evidence for the existence of specific estrogen-receptive neuronal cell populations that project to the rPOA and may be involved in the estrogen-dependent transsynaptic regulation of GnRH neurons in the rat.

Fluctuating estrogen and progesterone receptor expression in brainstem norepinephrine neurons through the rat estrous cycle.

Norepinephrine (NE) neurons within the nucleus tractus solitarii (NTS; A2 neurons) and ventrolateral medulla (A1 neurons) represent gonadal steroid-dependent components of several neural networks regulating reproduction. Previous studies have shown that both A1 and A2 neurons express estrogen receptors (ERs). Using double labeling immunocytochemistry we report here that substantial numbers of NE neurons located within the NTS express progesterone receptor (PR) immunoreactivity, whereas few PRs are found in ventrolateral medulla. The evaluation of ERa and PR immunoreactivity in NE neurons through the estrous cycle revealed a fluctuating pattern of expression for both receptors within the NTS. The percentage of A2 neurons expressing PR immunoreactivity was low on metestrus and diestrus (3-7%), but increased significantly to approximately 24% on proestrous morning and remained at intermediate levels until estrus. The pattern of ERalpha immunoreactivity in A2 neurons was more variable, but a similar increment from 11% to 40% of NE neurons expressing ERa was found from diestrus to proestrus. Experiments in ovariectomized, estrogen-treated and estrogen-plus progesterone-treated rats revealed that PR immunoreactivity in A2 neurons was induced strongly by estrogen treatment, whereas progesterone had no significant effect. The numbers of ERalpha-positive NE neurons were not influenced by steroid treatment. These observations provide direct evidence for PRs in NE neurons of the brainstem and show that cyclical patterns of gonadal steroid receptor expression exist in A2, but not A1, neurons through the rat estrous cycle. The expression of PR in A2 neurons appears to be driven principally by circulating estrogen concentrations. The fluctuating levels of ERalpha and PR expression in these brainstem NE neurons may help generate cyclical patterns of biosynthetic and electrical activity within reproductive neural networks.

Correlation of hypothalamic somatostatin mRNA expression and peptide content with secretion: sexual dimorphism and differential regulation by gonadal factors.

Sex differences in growth hormone (GH) secretion in the rat are thought to be determined, to a large extent, by gonadal steroid-dependent sex differences in somatostatin (SRIH) secretion from neurones in the periventricular nucleus (PeN) which project to the median eminence (ME). The present study aimed to obtain direct evidence for sex differences and gonadal regulation of SRIH release within this pathway and to determine the relationships between SRIH mRNA expression, SRIH peptide content and SRIH secretion in the adult rat. Somatostatin mRNA expression in the PeN and peptide content in both PeN and ME were higher in males than females (P<0.05). However, both basal and 56 mM K+-stimulated SRIH release in vitro from hypothalamic explants incorporating the PeN-ME pathway were higher (P<0.01) in females. The gonadectomy of female rats resulted in significantly reduced basal levels of SRIH release equivalent to that of males but had no effect on SRIH mRNA/peptide content or K+-stimulated release. In contrast, gonadectomy of male rats reduced SRIH mRNA and peptide contents and elevated K+-stimulated secretion (P<0.01) to levels similar to that seen in intact females, without affecting basal release. In summary, these results demonstrate that in the PeN-ME of the adult rat: (1) SRIH mRNA and peptide content is well correlated and sexually dimorphic but dependent on gonadal factors in the male only; (2) SRIH secretion is sexually dimorphic and dependent on gonadal factors; but (3) differences in mRNA/peptide content do not reflect secretory capacity; and (4) gonadal factors differentially modulate SRIH secretory dynamics in males and females.

Ontogeny and sexual differentiation of somatostatin biosynthesis and secretion in the hypothalamic periventricular-median eminence pathway.

The biosynthesis of somatostatin (SRIH) in the hypothalamic periventricular nucleus (PeN) is sexually differentiated in neonatal and adult rats by virtue of the organizational and activational actions, respectively, of sex steroid hormones. Little information exists, however, on the normal pattern of maturation of these neurones or on how the sexually differentiated biosynthesis may relate to ontogenetic changes in somatostatin secretion during the neonatal and pubertal periods of development. Hence in the present study we determined the postnatal developmental profile of SRIH mRNA and peptide levels in the PeN-median eminence (ME) pathway as well as SRIH secretion, using an acute explant preparation, from the day of birth, through puberty and into adulthood in male and female rats. The results demonstrate that: (1) developmental sex differences in SRIH biosynthesis in PeN neurones occurred in an orderly cascade with differences observed for mRNA expression at postnatal day 5, for peptide content in the perikarya at postnatal day 10 and for peptide content in the nerve terminal (ME) by postnatal day 25; (2) sex differences in SRIH release were not evident prior to postnatal day 40; and (3) the developmental profile of SRIH biosynthesis in PeN neurones is unique compared with other hypothalamic (ventromedial nucleus) and extrahypothalamic (parietal cortex) populations. Specific developmental changes in the biosynthetic and secretory activity of the hypothalamic SRIH PeN-ME pathway may have a functional importance in the maturation of hypothalamic SRIH pathways involved in the regulation of GH secretion.

Sexually dimorphic ontogeny of GABAergic influences on periventricular somatostatin neurons.

The biosynthesis and secretion of somatostatin (SRIH) within the hypothalamic periventricular-median eminence (PeN-ME) pathway follows a sexually differentiated developmental pattern beginning in the early neonatal period. It is generally accepted that testosterone plays a role in these processes, but the mechanisms underlying the age and sex differences are poorly understood. The present study sought to investigate the hypothesis that gamma-aminobutyric acid (GABA) may play a role in determining sex differences in SRIH neuronal activity. Using an in vitro hypothalamic preparation where more than 97% of the immunoreactive SRIH is contained within the PeN-ME pathway, peptide release in response to the GABA(A) receptor antagonist, bicuculline, was followed through development. In the male a stimulatory response, indicative of an inhibitory GABAergic tone on SRIH secretion, was observed as early as postnatal day (P) 5. This persisted throughout juvenile development (P10, P17) and was present also in the adult male (P75), but in the peripubertal period the response to bicuculline was first lost (P25) and then reversed to an inhibition (P40), suggesting a transient switch to an apparent stimulatory GABAergic tone on SRIH release. By contrast, in the female, no bicuculline responsiveness was seen until P25 when it caused a decrease in SRIH release which persisted into adulthood. Using in situ hybridization studies we found no evidence to support the view that these age- and sex-dependent differences were due to changes in the expression of GABA(A) receptor alpha-subunits (alpha(1) and alpha(2)) which are colocalised in the PeN SRIH neurons. Following adult gonadectomy, the bicuculline response was abolished in the male, whereas, in the female it was reversed and identical in magnitude to the response in the intact male. These results demonstrate marked sex differences in GABA(A)-receptor-mediated influences on SRIH release which develop soon after birth and, in the adult, depend on gonadal factors. In the male these factors activate a primarily inhibitory influence, whereas in the female they facilitate an apparently stimulatory tone of GABA on SRIH secretion via the GABA(A) receptor. Our findings thus support the view that GABAergic transmission may play a key role in generating sex differences in the mode of SRIH secretion from the hypothalamus which has been shown to be a major factor in determining the sexually dimorphic patterns of growth hormone secretion.

Estrogen receptor expression in brainstem noradrenergic neurons of the sheep.

Noradrenergic neurons are implicated in the estrogen-dependent neural regulation of luteinizing hormone secretion in a variety of mammalian species. The current study has used immunocytochemical methods to determine whether estrogen receptors (ER) are expressed within the brainstem of the ewe and to establish their relationship to noradrenergic neurons. Using a monoclonal mouse antiserum directed against the N-terminal of ERa, four distinct populations of ER alpha-immunoreactive cells were identified in ovine medulla and pons. The largest population was found in the superficial laminae of the spinal nucleus of the trigeminal nerve, followed by the nucleus tractus solitarius, lateral area postrema, and ventrolateral medulla. Double-labelling immunocytochemistry using antisera directed against the ER alpha and dopamine-beta-hydroxylase revealed that noradrenergic neurons expressing ER immunoreactivity were only found in ventrolateral medulla (A1 cell group) and nucleus tractus solitarius (A2 cell group). No double-labelled cells were identified in the A5, A6, or A7 noradrenergic cell groups. ERs were expressed with a clear rostrocaudal topography within the A1 and A2 populations, with 80-90% of noradrenergic neurons expressing ERA alpha in the caudalmost medulla as compared with less than 5% rostral to the obex. Our findings demonstrate that, as in the rat, the ovine A1 and A2 neurons express ERs in a defined topographical manner, while, dissimilar to the rat, ER alpha is not synthesized by noradrenergic neurons in the other cell groups. These observations indicate that A1 and A2 noradrenergic neurons in the ovine brainstem are likely to be influenced by circulating estrogens and lay the neuroanatomical foundations for investigating the functional role of these cell populations within the gonadotropin-releasing hormone neuron network of the sheep.

The one-year morphometric and neurodevelopmental outcome of the offspring of women who continued to exercise regularly throughout pregnancy.

OBJECTIVE: Our purpose was to test the hypothesis that continuing regular exercise throughout pregnancy alters morphometric and neurodevelopmental outcome at 1 year. STUDY DESIGN: The offspring of 52 women who exercised were compared with those of 52 control subjects who were similar in terms of multiple prenatal and postnatal variables known to influence outcome. All women were enrolled before pregnancy and had clinically normal antenatal and postnatal courses. Neurodevelopment was assessed by blinded examiners at 1 year of age, and morphometrics were obtained at birth and at 1 year of age. RESULTS: At birth, the offspring of the exercising women weighed less (3.38 +/- 0.06 kg vs 3.58 +/- 0.07 kg) and had less body fat (9.5% +/- 0.8% vs 12.6% +/- 0.6%). However, at 1 year, all morphometric parameters were similar, and no clinically significant between-group differences were observed in performance on either the Bayley psychomotor (108 +/- 1 vs 101 +/- 2) or mental (120 +/- 1 vs 118 +/- 1) scales. CONCLUSIONS: These data indicate that the offspring of exercising mothers have normal growth and development during the first year of life.

Estrogen-dependent ontogeny of sex differences in somatostatin neurons of the hypothalamic periventricular nucleus.

The sexually dimorphic profile of GH secretion is thought to be engendered by gonadal steroids acting in part on hypothalamic periventricular somatostatin (SOM) neurons. The present study set out to examine and characterize the development of sex differences in these SOM neurons. In the first series of experiments, we used in situ hybridization to examine SOM messenger RNA (mRNA) expression within the periventricular nucleus (PeN) of male and female rats on postnatal day 1 (P1), P5, and P10. Cellular SOM mRNA content was found to increase from P1 to P10 in both sexes (P < 0.01), but was 24% (P < 0.05) and 38% (P < 0.01) higher in males on P5 and P10, respectively. A second series of experiments examined the SOM peptide content of the PeN in developing rats and found increasing levels from P1 to P10, with a 44% higher SOM content in males compared with females on P10 (P < 0.05). The third series of experiments questioned the role of gonadal steroids in engendering sex differences in SOM mRNA expression by determining the effects of neonatal gonadectomy (GDX) and replacement of dihydrotestosterone or estradiol benzoate. The SOM mRNA content of PeN neurons in P5 males gonadectomized on the day of birth was the same as that in P5 females and was significantly reduced compared with that in sham-operated P5 males (P < 0.05). Male rats GDX on P1 and treated with estradiol benzoate from P1 to P5 had cellular SOM mRNA levels similar to those in intact males on P5, whereas dihydrotestosterone treatment had no effect. Treatment of intact males with an androgen receptor antagonist, cyproterone acetate, on P1 had no effect on cellular SOM mRNA on P5, whereas male rats given the aromatase inhibitor 1,4,6-androstatriene-3,17-dione from P1 to P5 had lower (P < 0.05) SOM mRNA levels than controls. In the final set of experiments, dual labeling immunocytochemistry showed that SOM neurons in the PeN of P5 rats did not contain estrogen receptor-alpha, but expressed androgen receptors in a sexually dimorphic manner. These results demonstrate that a sex difference in SOM biosynthesis, which persists into adulthood, develops between P1 and P5 in PeN neurons. Despite the absence of estrogen receptor-alpha in these neurons, the organizational influence of testosterone only occurs after its aromatization to estrogen.

Identification of estrogen receptor-containing neurons projecting to the rat supraoptic nucleus.

Circulating estrogens influence the electrical and biosynthetic activity of the hypothalamic magnocellular neurons which synthesize vasopressin or oxytocin and regulate body fluid homeostasis and reproduction. As none of these magnocellular neurons express nuclear estrogen receptor in the rat, the present study has combined estrogen receptor immunocytochemistry with retrograde tracing techniques to examine whether the first-order neurons projecting to magnocellular neurons in the supraoptic nucleus may be receptive to estrogen. Green fluorescent latex microspheres (50 nl) were injected into the supraoptic nucleus of five ovariectomized rats. The largest numbers of retrogradely-labelled cells expressing estrogen receptor immunoreactivity were detected in the organum vasculosum of the lamina terminalis, anteroventral periventricular nucleus and medial preoptic nucleus where approximately 15% of all retrogradely-labelled cells were estrogen receptor-immunoreactive. Other prominent sites where double-labelled cells were detected were the median preoptic nucleus, subfornical organ, ventrolateral division of the hypothalamic ventromedial nucleus and the brainstem nucleus tractus solitarii. Triple labelling experiments in the caudal medulla revealed that the estrogen-receptive neurons of the nucleus tractus solitarii and ventrolateral medulla projecting to the supraoptic nucleus were not noradrenergic. These findings show that sub-populations of neurons projecting to the supraoptic nucleus express estrogen receptors. This provides immunocytochemical evidence that estrogen may regulate the activity of magnocellular oxytocin and vasopressin neurons in an indirect, trans-synaptic manner by influencing the activity of first-order neurons projecting to the supraoptic nucleus. The predominance of estrogen-receptive lamina terminalis and preoptic area inputs to the supraoptic nucleus suggests respective sites of estrogen action on magnocellular neurons in modulating fluid balance and reproductive function.