Early neuroendocrine disruption in hypothalamus and hippocampus: developmental effects including female sexual maturation and implications for endocrine disrupting chemical screening.

The timing of puberty has been mainly studied in females for several reasons, including the possible evaluation of a precise timer (i.e. menarcheal age) and concerns with respect to the high prevalence of precocity in females as opposed to males. Human evidence of altered female pubertal timing after exposure to endocrine disrupting chemicals (EDCs) is equivocal. Among the limiting factors, most studies evaluate exposure to single EDCs at the time of puberty and hardly assess the impact of lifelong exposure to mixtures of EDCs. Some rodent and ovine studies indicate a possible role of foetal and neonatal exposure to EDCs, in accordance with the concept of an early origin of health and disease. Such effects possibly involve neuroendocrine mechanisms because the hypothalamus is a site where homeostasis of reproduction, as well as control of energy balance, is programmed and regulated. In our previous studies, pulsatile gonadotrophin-releasing hormone (GnRH) secretion control via oestrogen, glutamate and aryl hydrocarbon receptors was shown to be involved in the mechanism of sexual precocity after early postnatal exposure to the insecticide dichlorodiphenyltrichloroethane. Very recently, we have shown that neonatal exposure to the potent synthetic oestrogen diethylstilbestrol (DES) is followed by early or delayed puberty depending on the dose, with consistent changes in developmental increase of GnRH pulse frequency. Moreover, DES results in reduced leptin stimulation of GnRH secretion in vitro, an effect that is additive with prenatal food restriction. Thus, using puberty as an endpoint of the effects of EDC, it appears necessary to consider pre- and perinatal exposure to low doses and to pay attention to the other conditions of prenatal life, such as energy availability, keeping in mind the possibility that puberty could not only be advanced, but also delayed through neuroendocrine mechanisms.

Neuromuscular electrical stimulation of completely paralyzed abdominal muscles in spinal cord-injured patients: a pilot study.

STUDY DESIGN: Prospective placebo-controlled. OBJECTIVE: The effect of abdominal neuromuscular electrical stimulation (NMES) in patients with spinal cord injury. The principal parameters observed in this study are lung capacity, colonic transit, patient satisfaction of used method and of aesthetics effect on abdominal wall. SETTINGS: Centre de Traumatologie et de Réadaptation, Brussels, Belgium. METHODS: A total of 10 volunteers participated in this study and were assigned to two groups-the effective electrical stimulation group (ESG) and the placebo-controlled group (PG). NMES of abdominal muscles was performed 25 min per day for 8 weeks. RESULTS: NMES significantly decreased forced vital capacity (FVC) in ESG but not in PG. In ESG, colonic transit was accelerated in ascending, transverse and descending colon but transit in rectosigmoideum was not affected. In PG, no variations in colonic transit were observed. Satisfaction scale shows a better influence on aesthetics effect in ESG than in PG. CONCLUSION: This pilot study shows that NMES of paralyzed abdominal muscles positively affects colonic transit except in rectosigmoideum segment and negatively affects FVC. It could be a simple self-used method to regulate colonic transfer with considerably good cosmetic effect on abdominal wall. However, regular verification of FVC will probably be necessary.

A contactin-receptor-like protein tyrosine phosphatase beta complex mediates adhesive communication between astroglial cells and gonadotrophin-releasing hormone neurones.

Although it is well established that gonadotrophin-releasing hormone (GnRH) neurones and astrocytes maintain an intimate contact throughout development and adult life, the cell-surface molecules that may contribute to this adhesiveness remain largely unknown. In the peripheral nervous system, the glycosylphosphatidyl inositol (GPI)-anchored protein contactin is a cell-surface neuronal protein required for axonal-glial adhesiveness. A glial transmembrane protein recognised by neuronal contactin is receptor-like protein tyrosine phosphatase beta (RPTP beta), a phosphatase with structural similarities to cell adhesion molecules. In the present study, we show that contactin, and its preferred in cis partner Caspr1, are expressed in GnRH neurones. We also show that the RPTP beta mRNA predominantly expressed in hypothalamic astrocytes encodes an RPTP beta isoform (short RPTP beta) that uses its carbonic anhydrase (CAH) extracellular subdomain to interact with neuronal contactin. Immunoreactive contactin is most abundant in GnRH nerve terminals projecting to both the organum vasculosum of the lamina terminalis and median eminence, implying GnRH axons as an important site of contactin-dependent cell adhesiveness. GT1-7 immortalised GnRH neurones adhere to the CAH domain of RPTPbeta, and this adhesiveness is blocked when contactin GPI anchoring is disrupted or contactin binding capacity is immunoneutralised, suggesting that astrocytic RPTP beta interacts with neuronal contactin to mediate glial-GnRH neurone adhesiveness. Because the abundance of short RPTP beta mRNA increases in the female mouse hypothalamus (but not in the cerebral cortex) before puberty, it appears that an increased interaction between GnRH axons and astrocytes mediated by RPTP beta-contactin is a dynamic mechanism of neurone-glia communication during female sexual development.

Effects of in vivo and in vitro administration of ghrelin, leptin and neuropeptide mediators on pulsatile gonadotrophin-releasing hormone secretion from male rat hypothalamus before and after puberty.

The present study aimed to investigate the effects of leptin and ghrelin on pulsatile pulsatile gonadotrophin-releasing hormone (GnRH) secretion in vitro with emphasis on neuropeptide mediators and changes between prepuberty (15 days) and sexual maturity (50 days) in the male rat. When hypothalamic explants were studied 90 min after an intraperitoneal injection of leptin, ghrelin or agouti-related protein (AgRP) at 15 days, the GnRH interpulse interval (IPI) was significantly increased by ghrelin and AgRP and decreased by leptin. At 50 days, an increase in GnRH IPI was also caused by ghrelin and AgRP. When the peptides were directly incubated with the explants, the effects of leptin and AgRP in vitro were consistent with those seen after in vivo administration. By contrast, ghrelin resulted in a reduction of GnRH IPI and this was observed at 15 days only. To delineate the neuropeptide mediators of leptin and the effects of ghrelin in the hypothalamus, various hypothalamic neuropeptides and antagonists were used in vitro. At 15 days, the GnRH IPI was significantly decreased after incubation with cocaine and amphetamine-regulated transcript (CART), alpha-melanocyte-stimulating hormone, corticotrophin-releasing factor (CRF) and neuropeptide Y (NPY). The reduction of GnRH IPI caused by leptin was partially prevented by either an anti-CART antiserum or SHU 9119, a melanocortin MC3/MC4 receptor antagonist or a CRF receptor antagonist. The NPY-Y5 receptor antagonist did not influence the effects of leptin whereas that antagonist totally prevented the decrease in GnRH IPI caused by ghrelin. The ghrelin-induced reduction of GnRH IPI was partially prevented by SHU 9119. When used alone, SHU 9119 or a CRF-receptor antagonist resulted in increased GnRH IPI at 50 days while they had no effects at 15 days. The NPY-Y5 receptor antagonist resulted in increased GnRH IPI at 15 and 50 days. In conclusion, leptin and ghrelin show opposing effects on pulsatile GnRH secretion after administration in vivo whereas they both have stimulatory effects in vitro. Such effects involve consistently the anorectic peptides CART and CRF for leptin that are mainly active at 15 days. The melanocortigenic system appears to mediate the effects of both leptin and ghrelin. The effects of ghrelin also involve NPY receptors and operate effectively before and at sexual maturity.

Leptin effects on pulsatile gonadotropin releasing hormone secretion from the adult rat hypothalamus and interaction with cocaine and amphetamine regulated transcript peptide and neuropeptide Y.

Leptin may act as a negative feedback signal to the hypothalamic control of appetite through suppression of neuropeptide Y (NPY) secretion and stimulation of cocaine and amphetamine regulated transcript (CART). We aimed at studying the effects of leptin, CART and NPY on the hypothalamic control of the pituitary-gonadal system. Pulsatile gonadotropin-releasing hormone (GnRH) secretion was studied in vitro using retrochiasmatic hypothalamic explants from adult rats. In the female, GnRH pulse amplitude was significantly increased by leptin (10(-7) M) and CART (10(-6) M) irrespective of the estrus cycle phase while no such effects were seen in the male. The GnRH interpulse interval was not affected in both sexes. Passive immunoneutralization against CART caused a reduction in GnRH pulse amplitude in the female. A slight but significant increase in GnRH pulse amplitude was caused by NPY (10(-7) M) in the female. However, GnRH pulse amplitude was not affected by a Y5-receptor antagonist (10(-6) M) while the interpulse interval was significantly increased as shown previously in the male. The increase in GnRH pulse amplitude caused by leptin was totally prevented by coincubation with an anti-CART antiserum whereas it was not affected by coincubation with the NPY Y5-receptor antagonist (10(-7) M). In conclusion, leptin and NPY show separate permissive effects on GnRH secretion in the adult rat hypothalamus. In both sexes, NPY is prominently involved in the control of the frequency of pulsatile GnRH secretion through the Y5 receptor subtype. Leptin causes a female-specific facilitatory effect on GnRH pulse amplitude which is mediated by CART and which occurs irrespective of the estrus cycle phase.

Cocaine and amphetamine-regulated-transcript peptide mediation of leptin stimulatory effect on the rat gonadotropin-releasing hormone pulse generator in vitro.

Pulsatile gonadotropin-releasing hormone (GnRH) secretion was studied in vitro using explants of the retrochiasmatic hypothalamus from prepubertal male and female rats. Leptin caused a dose-dependent reduction of the GnRH interpulse interval in both sexes. We studied the effects of cocaine- and amphetamine-regulated transcript (CART) since this peptide was shown recently to mediate the anorectic effects of leptin in the hypothalamus. CART caused a reduction of the GnRH interpulse interval. This effect was prevented using an anti-CART antiserum which could partially overcome leptin stimulatory effects as well. Using hypothalamic explants from Zucker rats homozygous for the leptin receptor mutation ( fa/fa), GnRH pulse frequency was not affected by leptin, while a significant acceleration was caused by the CART-peptide. In conclusion, leptin involves the hypothalamic CART-peptide to stimulate the prepubertal GnRH pulse generator in vitro.

In vitro stimulation of the prepubertal rat gonadotropin-releasing hormone pulse generator by leptin and neuropeptide Y through distinct mechanisms.

Leptin may act as a negative feedback signal to the brain in the control of appetite through suppression of neuropeptide Y (NPY) secretion and stimulation of cocaine- and amphetamine-regulated transcript (CART), a new anorectic peptide. We aimed at studying whether leptin, NPY, and CART have related effects on the hypothalamic control of the pituitary-gonadal system and the developmental changes in NPY and CART effects. Using retrochiasmatic hypothalamic explants from prepubertal 15-day-old male rats, the GnRH interpulse interval (mean +/- SD: 62 +/- 5 min) was significantly reduced by 10(-7) M of leptin (46 +/- 3.3 min) as well as 10(-7) M of NPY (47 +/- 4.4 min) and 10(-6) M of CART (46 +/- 2.7 min), whereas the GnRH pulse amplitude was not affected. The stimulatory effects of different NPY receptor agonists [human PYY 3-36, porcine NPY 13-36, human (D-Trp 32) NPY, porcine (Leu 31 Pro 34) NPY, human pancreatic polypeptide (PP)], as well as the absent effects of rat PP were consistent with the involvement of the Y5-receptor subtype in mediation of NPY effects. Incubation with 10(-7) M of a Y5-receptor selective antagonist prevented the effect of NPY (61 +/- 4 vs. 46 +/- 2 min), whereas leptin and CART effects were not (47 +/- 3 vs. 46 +/- 3 min and 46 +/- 3 vs. 46 +/- 2 min, respectively), suggesting that NPY was not involved in leptin and CART effects. Using an anti-CART antiserum (1:1000), the reduction of GnRH interpulse interval caused by leptin was partially prevented (56.2 +/- 4 vs. 47.9 +/- 3.8 min), whereas the reduction of GnRH interval caused by NPY was not affected (45.9 +/-2.5 vs. 47.8 +/- 3.7). The GnRH interpulse interval was decreased by 10(-7) M of NPY at 5 days (72 +/- 3.8 vs. 91.9 +/- 3.5) as well as at 15 days, whereas such an effect was not observed anymore at 25 and 50 days. Similar effects were observed using 10(-6) M of CART-peptide. Using 10(-6) M of the Y5-receptor antagonist, the GnRH interpulse interval was significantly increased at 15 days (66.6 +/- 2.7 min), 25 days (56.5 +/- 39.9 min), and 50 days (52.5 vs. 38.2 min), whereas no change was observed at 5 days. Using the anti-CART antiserum, a significant increase of GnRH interpulse interval was observed at 25 days only. In conclusion, the stimulatory effects of leptin and NPY on the frequency of pulsatile GnRH secretion before puberty involve two distinct mechanisms. NPY causes acceleration of GnRH pulsatility via the Y5-receptor subtype, which is not involved in leptin effects while the CART is involved in leptin effects on GnRH secretion but not in NPY effects. The reduction of pulsatility by the Y5 antagonist provides evidence of endogenous NPY involvement in the control of GnRH secretion from the time of onset of puberty.

The pallidofugal projection system in primates: evidence for neurons branching ipsilaterally and contralaterally to the thalamus and brainstem.

This paper summarizes the results of some of our previous neuroanatomical studies on the pallidofugal projections in squirrel monkeys and also reports more recent data obtained with double retrograde and single axon tracing methods. Injections of anterograde tracers in the internal pallidum label axons that reach the ventral tier, centromedian and lateral habenular thalamic nuclei, as well as the pedunculopontine tegmental nucleus. The pallidofugal projections are composed of axons that branch to the ventral tier and pedunculopontine nuclei, and to ventral tier and centromedian nuclei. Double retrograde labeling with fluorescent tracers and single axon tracing confirm this high degree of collateralization. Furthermore, some pallidal labeled axons cross the midline and arborize contralaterally in the major pallidal targets. Double retrograde fluorescent labeling experiments support these findings. Pallidal axons that branch ipsilaterally as well as contralaterally to the thalamus and brainstem could play a crucial role in the functional organization of primate basal ganglia.

Calretinin-immunoreactive neurons in primate pedunculopontine and laterodorsal tegmental nuclei.

Single- and double-antigen localization procedures were used to study the distribution, morphological characteristics and chemical phenotype of neurons containing the calcium-binding protein calretinin in the pedunculopontine and laterodorsal tegmental nuclei of the cynomolgus monkey (Macaca fascicularis). Calretinin was detected in neurons that belonged to a highly heteromorphic and widely distributed subpopulation of the pedunculopontine and laterodorsal tegmental nuclei in the cynomolgus monkey. Double-immunostaining experiments revealed that about 12% of these calretinin-containing neurons displayed immunoreactivity for another calcium-binding protein, Calbindin-D28k. The calretinin/Calbindin-D28k double-labeled neurons had small to medium-sized perikarya, from which emerged a bipolar or multipolar dendritic arborization. Calretinin was also present in approximately 8% of the cholinergic neurons of the pedunculopontine/laterodorsal nuclear complex, as visualized on single sections immunostained for both calretinin and choline acetyltransferase. These calretinin/choline acetyltransferase double-labeled neurons displayed markedly different sizes and shapes, and occurred preferentially in the pars compacta and dissipata of the pedunculopontine tegmental nucleus. Numerous calretinin-immunoreactive fibers were also present within and around the superior cerebellar peduncle. Some of these varicose fibers closely surrounded large non-immunoreactive neurons, as well as large neurons staining positively for choline acetyltransferase. This study provides the first evidence for the existence of calretinin-immunoreactive neurons within the primate pedunculopontine and laterodorsal tegmental nuclei. Our data suggest that calretinin may play a role in the function of the pedunculopontine/laterodorsal nuclear complex by acting either alone or in conjunction with acetylcholine or Calbindin-D28k.

Axonal arborization of corticostriatal and corticothalamic fibers arising from prelimbic cortex in the rat.

This study aimed at elucidating the branching pattern of striatal and thalamic projections arising from prelimbic (Cg3) cortex in the rat. Small pools (5-15 cells) of neurons were microiontophoretically injected with biotin-dextran or biocytin and their labeled axons were individually reconstructed from serial horizontal sections immunostained for calbindin-D28k to delineate striatal patch/matrix compartments. Reconstruction of > 40 axons shows that all Cg3 corticofugal fibers, including corticothalamic axons from layer VI, course through the patch network in the rostromedial sector of the striatum. Corticostriatal projections arise from two types of layer V cells: (i) long-range corticofugal neurons, whose main axons reach the brainstem and/or spinal cord, and (ii) neurons arborizing into both striatum and claustrum, either ipsi-, contra- or bilaterally. The axons of these two types of neurons arborize profusely in striatal patches and only sparsely in the matrix. Layer VI neurons do not arborize in the striatum but target principally the thalamus. The same corticothalamic axon can innervate the anterior, rostral intralaminar and mediodorsal thalamic nuclei. These findings support the concept that no corticofugal fiber system exists that is solely devoted to the striatum. They also shed new light on how neural information from prelimbic cortex is conveyed to various subcortical limbic structures in the rat.

Chemoarchitecture of the primate dorsal raphe nucleus.

The dorsal raphe nucleus (DR) harbours the largest single collection of serotonin (5-HT)-containing neurons in the brain but also comprises other types of chemospecific neurons. The aim of the present study was to characterise morphologically and immunohistochemically the DR in the squirrel monkey (Saimiri sciureus). The morphology of the DR 5-HT-immunoreactive (ir) neurons was analysed and their distribution compared to that of neurons displaying immunoreactivity for either tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA), substance P (SP), calbindin-D28k (CB), calretinin (CR) or parvalbumin (PV). The 5-HT-ir neurons were distributed in a highly heterogeneous manner throughout the rostrocaudal extent of the DR. The morphology and density of the 5-HT neurons were found to vary significantly in the major subdivisions of the primate DR, that is, the median, ventral, dorsal, ventrolateral, lateral and caudal subnuclei. Numerous SP-, GABA- and PV-ir neurons occurred in all six subnuclei of the DR. The distribution of SP-ir neurons was largely in register with that of 5-HT-ir neurons. Neurons expressing the other neuronal markers (TH, CB, CR) were not present in all six DR subnuclei and their distribution was either complementary to, or in register with, that of 5-HT-ir neurons. The median subnucleus was unique because it contained all the different types of chemospecific neurons. This study has revealed that the primate DR is chemically highly heterogeneous, a finding that may explain the multifarious influence that this nucleus exerts upon various forebrain structures.

Calretinin-immunoreactive neurons in the human thalamus.

The distribution of the calcium-binding protein calretinin in the thalamus of normal human individuals was studied with immunohistochemistry. Calretinin immunoreactivity was weak in the geniculate bodies and in nuclei of the ventral and posterior groups, moderate in the reticular nucleus and in nuclei of the anterior, medial, and lateral groups, and strong in nuclei of the midline group and anterior intralaminar nuclei. The mediodorsal nucleus was unique among thalamic nuclei because it contained a wide variety of intensely immunostained perikarya embedded in a moderately-labelled neuropil. The reticular nucleus displayed several small and uniformly distributed neuronal clusters composed of immunostained perikarya lying in a moderately-labelled neuropil. Intense and uniform immunostaining was observed in all midline nuclei and in the anterior intralaminar nuclei, including the paracentral and central lateral nuclei. These nuclei, which harboured numerous intensely-stained perikarya lying in a dense immunoreactive neuropil, were the most strongly-immunoreactive structures of the entire human thalamus. At the level of the posterior intralaminar nuclei, the central median nucleus was virtually free of immunostaining whereas the parafascicular nucleus was moderately labelled. The nucleus submedius located just beneath the central median/parafascicular complex displayed a very intense calretinin immunostaining. This study has provided evidence for the presence of the protein calretinin in the human thalamus. The pattern of distribution of calretinin, as delineated in the present study, suggests that this calcium-binding protein may participate in various subcortical and cortical thalamic systems involved in the modulation of emotional and motivational states.

Calretinin gene expression in the human thalamus.

The localization and levels of expression of the calcium-binding protein calretinin (CR) in the human thalamus was studied with an in situ hybridization method applied to formalin-fixed postmortem material from normal individuals. The riboprobe used was generated from a specific fragment of human CR cDNA. As visualized on X-ray film autoradiographs, high levels of CR gene transcript occurred in several thalamic nuclei, including the reticular nucleus, mediodorsal nucleus, rostral intralaminar nuclei (paracentral, central medial and central lateral) and several midline nuclei (paraventricular, reuniens and medioventral nuclei). In the reticular nucleus, neurons expressing CR mRNA were few in number but formed dense and widely distributed clusters. In contrast, virtually all neurons in the rostral intralaminar and midline nuclei expressed very high levels of CR mRNA and formed a prominent rim around the mediodorsal nucleus, which contained scattered clusters of labeled neurons. The caudal intralaminar nuclei, principally the centromedian nucleus, displayed very few neurons expressing CR mRNA. Only the medial part of the parafascicular nucleus expressed moderate levels of CR mRNA. The nuclei of the ventral group (ventral anterior, lateral and posterior nuclei) were virtually devoid of CR gene transcript. This highly heterogeneous pattern of mRNA expression suggests that CR may be heavily involved in the function of the so-called non-specific nuclei, but not in that of the specific relay nuclei of the human thalamus. The data also demonstrate that the presence of CR gene transcript can easily be detected on formalin-fixed sections of the human brain.

Parvalbumin and calbindin D-28k in the entopeduncular nucleus, subthalamic nucleus, and substantia nigra of the rat as revealed by double-immunohistochemical methods.

The cellular localization of calbindin D-28k (CB) and parvalbumin (PV) was analyzed by means of double-immunohistochemical techniques applied to single sections in the entopeduncular nucleus (EP), the subthalamic nucleus (STh), and the substantia nigra (SN) of the rat. In EP, PV-positive cells abounded centrally, where CB immunostaining was minimal. The medial and ventral sectors of EP were markedly enriched with CB neuropil but devoid of PV-positive cells. CB-positive neurons abounded particularly in the rostral pole of EP. In STh, PV-positive neurons and neuropil were concentrated in the lateral two thirds of this nucleus. Only a few PV-positive cells were detected in sectors of STh devoid of PV-positive neuropil. The STh was completely devoid of CB immunostaining. In the rostral two thirds of SN, PV-positive neurons were largely confined to the lateral half of the pars reticulata (SNR), and occurred more ventrally and medially in the caudal third. Intense CB-immunoreactive neuropil was found in medial and dorsal parts of rostral SNR, and CB-positive cells were observed in the SN pars compacta and the ventral tegmental area. PV and CB cells were also observed in the pars lateralis of SN. The markedly heterogeneous pattern of distribution of PV and CB in EP, STh, and SN suggests that these two calcium-binding proteins may label distinct functional domains in each of these three components of the rat basal ganglia.

Distribution of calretinin, calbindin-D28k and parvalbumin in the hypothalamus of the squirrel monkey.

The immunohistochemical approach was used to study the distribution of three calcium-binding proteins of the 'EF hand' family, namely calretinin, calbindin-D28k and parvalbumin, in the preoptico-hypothalamic complex of the squirrel monkey (Saimiri sciureus). These three calcium-binding proteins were found to be heterogeneously distributed in the primate hypothalamus. Neurons expressing high levels of calretinin immunoreactivity were particularly abundant in the infundibular (arcuate) nucleus, the suprachiasmatic nucleus, the lateral area and the dorsomedial nucleus of the hypothalamus. Neurons displaying immunoreactivity for calbindin-D28k were especially numerous in the medial preoptic area and diagonal band nucleus, as well as in the magnocellular subdivision of the paraventricular nucleus, the suprachiasmatic nucleus, the supraoptic nucleus, the infundibular nucleus, the ventromedial nucleus and the mammillary bodies of the hypothalamus. Fibers displaying intense immunoreactivity for either calretinin or calbindin-D28k were very abundant in the median eminence of the hypothalamus. In contrast to calretinin- and calbindin-D28k, parvalbumin was largely absent from the primate preoptico-hypothalamic complex. Parvalbumin-immunoreactive neurons occurred in significant number only in the most lateral portion of the medial mammillary nucleus in the squirrel monkey. The results of the present study suggest that calretinin and calbindin-D28k may act, either in concert or in a complementary manner, so as to participate in some specific aspects of the multifarious role of the hypothalamus in primates. In contrast to the other two calcium-binding proteins, parvalbumin is unlikely to be involved in a significant manner in hypothalamic functions in primates.

Axonal arborizations of corticostriatal and corticothalamic fibers arising from the second somatosensory area in the rat.

Corticostriatal and corticothalamic projections arising from the second somatosensory area in the rat were studied after labeling small pools of neurons in laminae V and VI with biocytin. Tracing the axon of single neurons revealed the following principles of organization: (i) all corticostriatal and corticothalamic projections arising from layer V cells are collaterals of long-range corticofugal axons that also project below the thalamic level; (ii) all layer V cells that project to the thalamus also project to the striatum; (iii) all layer VI corticothalamic cells project exclusively to the thalamus; (iv) cells of the upper part of lamina VI send collaterals to the thalamic reticular nucleus and arborize in the ventrobasal complex forming rod-like terminal fields; and (v) cells of the lower part of lamina VI also send collaterals to the thalamic reticular nucleus, give off few branches in the ventrobasal complex and terminate principally in the caudal part of the posterior thalamic group. On the basis of these findings, and in the light of previous anatomical studies, it is proposed that the above mentioned organizing principles represent general rules that also apply to corticostriatal and corticothalamic pathways arising from other areas of the neocortex.

Calcium-binding proteins in primate basal ganglia.

This paper describes the distribution of the calcium-binding proteins calbindin-D28k. Parvalbumin and calretinin in primate basal ganglia. The data derive from immunocytochemical studies undertaken in squirrel monkeys (Saimiri sciureus) and in normal human individuals. In the striatum, calbindin labels medium-sized spiny projection neurons whereas parvalbumin and calretinin mark two separate classes of aspiny interneurons. The striatal matrix compartment is markedly enriched with calbindin while striatal patches (striosomes) display a calretinin-rich neuropil. In the pallidum, virtually all neurons contain parvalbumin but none express calbindin. Calretinin occurs only in a small subpopulation of both large and small pallidal neurons. In the subthalamic nucleus, there exists a multitude of parvalbumun-positive cells and fibers but the number of calretinin and calbindin-positive neuronal elements is small. In the substantia nigra/ventral tegmental area complex, calbindin and calretinin occur principally in dopaminergic neurons of the dorsal tier of the pars compacta and in those of the ventral tegmental area. Parvalbumin is strictly confined to the GABAergic neurons of the pars reticulata and lateralis. Calbindin-rich fibers abound in the pars reticulata and lateralis, while calretinin-positive axons are confined to the pars compacta. These results indicate that calbindin and parvalbumin are distributed according to a strikingly complementary pattern in primate basal ganglia. Calretinin is less ubiquitous but occurs in all basal ganglia components where it labels distinct subsets of neurons. Such highly specific patterns of distribution indicate that calbindin, parvalbumin and calretinin may work in synergy within primate basal ganglia.

Determination of oenothein B as the active 5-alpha-reductase-inhibiting principle of the folk medicine Epilobium parviflorum.

Several extracts from Epilobium parviflorum, a plant used in Central Europe for the treatment of prostate disorders, were evaluated in a biochemical assay with 5-alpha-reductase. The aqueous extract displaying inhibition of the enzyme was analyzed, the fraction responsible for this activity was purified, and the active compound identified as a macrocyclic tannin, oenothein B (1).

Calretinin immunoreactivity in the thalamus of the squirrel monkey.

The distribution of the calcium-binding protein, calretinin, in the thalamus of the squirrel monkey (Saimiri sciureus) was studied with immunohistochemical methods. Calretinin was found to be heterogeneously distributed in the primate thalamus and to occur only in specific neuronal populations of certain thalamic nuclei. Neuronal cells and fibers in midline nuclei and their dorsolateral extension, which includes the parataenial and central superior lateral nuclei, displayed the most intense calretinin immunoreactivity. The immunoreactivity for cells and fibers in the intralaminar nuclei was moderate rostrally but very weak caudally. The centre mèdian nucleus, together with the medial habenular nucleus, were virtually devoid of calretinine immunostaining. The mediodorsal nucleus displayed a markedly heterogeneous staining, with numerous clusters of labeled cells and fibers in its central parvicellular part. Cell and fiber immunoreactivity ranged from moderate to high in the nuclei of the anterior and lateral groups, but was very weak in the nuclei of the ventral and posterior groups. There was a small to moderate number of heterogeneously distributed calretinin-immunoreactive cells and fibers in the lateral and medial geniculate bodies, as well as in the reticular nucleus. The present study provides the first evidence for the existence of calretinin in primate thalamus, where this protein is distributed according to a highly heterogeneous pattern. This specific pattern of distribution suggests that calretlnin may play a role that is complementary to those of the other calcium-binding proteins parvalbumin and calbindin D-28k in the thalamus of primates.

Corticostriatal projections from layer V cells in rat are collaterals of long-range corticofugal axons.

Corticostriatal projections arising from the infragranular layers of the motor and second somatosensory cortices were studied in rats after labeling small pools of neurons with biocytin. Camera lucida reconstruction of 263 fibers arising from laminae V and VI revealed that all corticostriatal projections derive from collaterals of lamina V cells whose main axons descend into the cerebral peduncle. In contrast, lamina VI cells do not branch upon the striatum, but upon the thalamus. Together with the results obtained in previous tracing studies, the present data raise the possibility that no neuron is exclusively corticostriatal. We therefore propose that all corticostriatal projections are collaterals given off by the axons of two types of neurons: layer V cells whose main axon project to the brainstem and/or spinal cord, and layer III cells that project to the contralateral hemisphere.