In vivo inhibition of hippocampal Ca2+/calmodulin-dependent protein kinase II by RNA interference.

Hippocampal alpha-Ca2+/calmodulin-dependent protein kinase II (alpha-CaMKII) has been implicated in spatial learning, neuronal plasticity, epilepsy, and cerebral ischemia. In the present study, an adeno-associated virus (AAV) vector was designed to express green fluorescent protein (GFP) from the CBA promoter and a small hairpin RNA targeting alpha-CaMKII (AAV-shCAM) driven from the U6 promoter. The AAV-shCAM or control vector was microinfused into the rat hippocampus and behavioral testing conducted 19-26 days following surgery. Expression of the marker gene and alpha-CaMKII was evaluated 31 days following AAV infusion. GFP expression was localized to the hippocampus and extended +/-2 mm rostral and caudal from the injection site. Hippocampal alpha-CaMKII was significantly reduced following AAV-shCAM treatment as demonstrated using immunohistochemical and Western analysis. This suppression of alpha-CaMKII was associated with changes in exploratory behavior (open field task) and impaired place learning (water maze task). These results demonstrate the efficacy of a viral-based delivered shRNA to produce gene suppression in a specific circuit of the brain.

Upregulation of the p75 low-affinity neurotrophin receptor by phagocytically active perivascular active cells in the rat neural lobe.

Expression of the p75 low-affinity neurotrophin receptor (p75NTR) was investigated immunocytochemically at the light and ultrastructural level during the axonal degeneration that follows partial denervation of the rat neural lobe (NL) and following systemic administration of lipopolysaccharide (LPS). A significant increase in the intensity and extent of p75NTR immunoreactivity in the NL of partially denervated animals compared with age-matched, sham-operated controls was observed at 5-10 days postdenervation, with immunoreactivity returning to control values by 35 days. Dual-label confocal comparison of p75NTR localization with that of the C3bi complement receptor, a microglial marker, and S100, an astrocyte-specific Ca2+-binding protein, revealed no colocalization. Immunoelectron-microscopic examination demonstrated that the p75NTR immunoreactivity is present in a subpopulation of cells located within the extensive perivascular space of the NL. No examples of p75NTR-immunoreactive pituicytes or endothelia were observed at the light or ultrastructural level. Dense p75NTR immunoreactivity was frequently observed surrounding endocytotic omega profiles of plasmalemma engulfing extracellular debris as well as lining vacuoles within the cytoplasm of perivascular cells. The association of p75NTR with phagocytosis was confirmed by confocal microscopy, showing the presence of p75NTR in all cells expressing the ED-1 antigen, which is restricted to the lysosomal membrane of phagocytes (Damoiseaux et al. 1994). Likewise, a marked increase in p75NTR and ED-1 immunoreactivity was observed in the NL following systemic administration of LPS. These results suggest a strong correlation between modulation of p75NTR immunoreactivity and conditions that induce high levels of phagocytic activity by perivascular cells in the NL of the rat. Implications for understanding the mechanisms by which phagocytes may support compensatory responses to neuronal injury are discussed.

Evidence that IGF-I acts as an autocrine/paracrine growth factor in the magnocellular neurosecretory system: neuronal synthesis and induction of axonal sprouting.

The ability of mature oxytocinergic (OT) and vasopressinergic (VP) neurons of the magnocellular neurosecretory system (MNS) to undergo axonal growth implies that one or more growth factors may be active in the adult MNS, yet little is known regarding their possible identity. One such potential factor is insulin-like growth factor I (IGF-I). We have examined the expression of IGF-I mRNA and IGF-I-immunoreactivity (IGF-I-ir) in the mature MNS and have also determined the in vivo response of OT and VP neurons to hypothalamic implants of IGF-I. In situ hybridization revealed moderate labeling of IGF-I mRNA in both the supraoptic (SON) and the paraventricular (PVN) nuclei of adult male rats. RT-PCR analysis confirmed the presence of authentic IGF-I mRNA in extracts of the basal hypothalamus. Faint IGF-I-ir was detected in scattered magnocellular neurons within both the PVN and the SON of normal rats, but IGF-I-ir was much more intense and the majority of MNS neurons including those in the accessory nuclei were immunoreactive in sections from rats given colchicine, as were some parvocellular neurons in the PVN. Confocal microscopy revealed that IGF-I-ir was present in both OT and VP neurons, but VP neurons contained the most intense IGF-I-ir. Finally, a dramatic growth response of OT but not of VP fibers was observed following implantation of polymer rods containing IGF-I into the hypothalamus. A dense OT fiber plexus grew along the cannula track and OT fibers invaded the leptomeninges ventral to the SON and encircled the rostral cerebral artery. To our knowledge this is the first demonstration of axonal sprouting by mature OT neurons in response to an identified growth factor and the first direct demonstration of sprouting in response to exogenous IGF-I in the adult CNS. These findings suggest that IGF-I is synthesized and transported by adult MNS neurons where it may act as an autocrine and/or paracrine growth factor.

In vivo glutamate neurotoxicity is associated with reductions in calcium/calmodulin-dependent protein kinase II immunoreactivity.

Calcium/calmodulin-dependent protein kinase II (CaM kinase) activity is inhibited in cultured hippocampal cells following direct application of glutamate. The goal of the present study was to determine if hippocampal regions that undergo delayed cell death following glutamate microinfusion would exhibit changes in CaM kinase immunoreactivity. Gerbils received bilateral intra-hippocampal infusions of L-glutamate (34 microg/microl), or control treatments of D-glutamate or saline. Animals were sacrificed at 12 or 24 hr to assess cell loss and determine changes in CaM kinase-like immunoreactivity. Hippocampi of gerbils euthanized 12 hr following L-glutamate, or 24 hr following D-glutamate, did not exhibit cell death in the hippocampal CA1 region. Animals injected with L-glutamate and sacrificed 24 hr after infusion had extensive cell damage that was restricted to the hippocampal CA1 region. CaM kinase-like immunoreactivity was absent in the hippocampal CA1 region of all L-glutamate treated animals sacrificed at 12 hr. In these same sections, CaM kinase immunoreactivity was evident in the subiculum, CA2 and CA3 regions. Reduction in CaM kinase immunoreactivity following L-glutamate were also observed using Western analysis. The results confirm and extend the findings of earlier cell culture studies by demonstrating a reduction in CaM kinase immunoreactivity that occurred prior to cell death.

Central peptidergic neurons are hyperactive during collateral sprouting and inhibition of activity suppresses sprouting.

Little is known regarding the effect of chronic changes in neuronal activity on the extent of collateral sprouting by identified CNS neurons. We have investigated the relationship between activity and sprouting in oxytocin (OT) and vasopressin (VP) neurons of the hypothalamic magnocellular neurosecretory system (MNS). Uninjured MNS neurons undergo a robust collateral-sprouting response that restores the axon population of the neural lobe (NL) after a lesion of the contralateral MNS (). Simultaneously, lesioned rats develop chronic urinary hyperosmolality indicative of heightened neurosecretory activity. We therefore tested the hypothesis that sprouting MNS neurons are hyperactive by measuring changes in cell and nuclear diameters, OT and VP mRNA pools, and axonal cytochrome oxidase activity (COX). Each of these measures was significantly elevated during the period of most rapid axonal growth between 1 and 4 weeks after the lesion, confirming that both OT and VP neurons are hyperactive while undergoing collateral sprouting. In a second study the hypothesis that chronic inhibition of neuronal activity would interfere with the sprouting response was tested. Chronic hyponatremia (CH) was induced 3 d before the hypothalamic lesion and sustained for 4 weeks to suppress neurosecretory activity. CH abolished the lesion-induced increases in OT and VP mRNA pools and virtually eliminated measurable COX activity in MNS terminals. Counts of the total number of axon profiles in the NL revealed that CH also prevented axonal sprouting from occurring. These results are consistent with the hypothesis that increased neuronal activity is required for denervation-induced collateral sprouting to occur in the MNS.

Axons containing the growth associated protein GAP-43 specifically target rat corticotrophs following adrenalectomy.

An extensive network of nerve fibers immunoreactive for the neuronal growth associated protein GAP-43 (GAP-43-IR) is present within the anterior pituitary (AP) of the rat, and the density of these fibers has been reported to increase 4 days after adrenalectomy (ADX). In the present study, we employed confocal dual-label immunofluorescence microscopy to determine whether GAP-43-IR fibers are specifically associated with corticotrophs at various intervals after ADX. A dramatic increase in the density of GAP-43-IR was apparent 4 days after ADX, and this increase was sustained at 7 and 14 days post-ADX. The percentage of corticotrophs in apparent contact with GAP-43-IR axons was 87% at 4 days after ADX and 92% at 14 days. In addition, fewer than 15% of GAP-43-IR terminals were associated with cells other than corticotrophs in either group. This highly specific targeting of corticotrophs during a period in which these cells are undergoing both hypertrophy and hyperplasia indicates that axonal sprouting is occurring in response to ADX. While the less intense GAP-43-IR in the AP of intact rats precluded precise quantitative analysis, the majority of corticotrophs also appeared to be selectively innervated in these animals. The observations that GAP-43-IR axons selectively contact corticotrophs, and that both the specificity and thoroughness of innervation are maintained by targeted growth of GAP-43-IR axons following ADX, strongly suggest that these fibers are of functional significance.

Expression of insulin-like growth factor binding protein-2 during postnatal development of the rat neurointermediate pituitary.

The expression of insulin-like growth factor binding protein-2 (IGFBP-2) during postnatal development of the rat neurointermediate pituitary was characterized using immunocytochemistry and in situ hybridization. Glial cells in the neural lobe (NL) showed robust expression of IGFBP-2 throughout the postnatal period and continuing into adulthood. These were identified as pituicytes by the presence of S-100 protein; IGFBP-2 was not present in microglia in the NL. IGFBP-2 immunoreactivity was more punctate in mature pituicytes, suggesting a possible association with the cell membrane. No expression of IGFBP-2 by brain astrocytes was observed at any age examined, even in regions such as the median eminence and subfornical organ that lack a blood-brain barrier. IGFBP-2 was also localized immunocytochemically in melanotropes and glial-like cells of the intermediate lobe (IL). Positive cells were most numerous in neonates and declined thereafter, with immunoreactivity undetectable by 65 days of age. IGFBP-2 mRNA was detected in the IL only at 1 day of age. These findings are consistent with a potential role for IGFBP-2 in modulating effects of the insulin-like growth factors during development of the neurointermediate lobe. The constitutive expression of high levels of IGFBP-2 by mature pituicytes also suggests that this protein could be secreted and/or influence a variety of processes in the mature NL, such as glial proliferation, axonal growth, or morphological plasticity of pituicytes.

Transient cerebral ischemia decreases calcium/calmodulin-dependent protein kinase II immunoreactivity, but not mRNA levels in the gerbil hippocampus.

During transient cerebral ischemia, intracellular calcium increases initiating a cascade of events which leads to the delayed death of neurons located in the hippocampus. Coupled to this calcium disturbance is the rapid decrease of calcium/calmodulin kinase II (CaM kinase) activity, a protein kinase critical to neuronal functioning. The present study correlated the increased locomotor activity following ischemic insult with alterations in CaM kinase mRNA levels and immunocytochemical labeling of alpha and beta CaM kinase subunits in the hippocampus. The protective effect of hypothermia was also compared with CaM kinase mRNA levels and immunoreactivity. Levels of CaM kinase message for either alpha or beta subunits was not altered in ischemic gerbils compared to sham or hypothermic ischemic conditions. Immunoreactivity for both the alpha and beta subunits was markedly reduced in the vulnerable CA1 region of ischemic animals compared to sham controls. Gerbils that underwent the ischemic insult while hypothermic showed no decrement in staining. CaM kinase-like immunoreactivity in the ischemia-resistant CA3 sector was not altered following ischemia. These data suggest that the loss of hippocampal CaM kinase immunoreactivity observed at 24 h following ischemia is not associated with a reduction in CaM kinase mRNA levels and support the notion that the rapid decline in CaM kinase activity following ischemic insult is a result of a posttranslational modification and/or translocation of the enzyme.

Distribution of growth-associated class I alpha-tubulin and class II beta-tubulin mRNAs in adult rat brain.

A comprehensive survey of class I alpha-tubulin (alpha 1) and class II beta-tubulin (beta II) mRNAs was performed using in situ hybridization in order to determine the extent of continued expression of these immature tubulin isotype mRNAs in the adult rat brain. Qualitatively similar distributions of the two isotype mRNAs were observed, with marked variations in hybridization intensity of both probes apparent across different brain regions. Neurons in a wide variety of structures throughout the brain exhibited intense hybridization signals. While the presence of large numbers of neurons with a moderate hybridization intensity could account for the relatively high level of total binding in some regions such as the cerebellar and dentate granule layers, in most cases higher regional mRNA levels reflected greater hybridization intensity per neuron. Little variability in hybridization intensity was typically seen between individual cells within specific nuclei throughout the brain. The presence of occasional intensely labeled neurons scattered throughout the basal ganglia provided the most striking exception to this pattern. While no qualitative differences between the distributions of alpha 1-tubulin and beta II-tubulin mRNAs were observed, consistent differences in the relative intensity of hybridization for alpha 1-tubulin versus beta II-tubulin mRNA were apparent in a few brain regions. Expression by glia did not appear to contribute significantly to detectable levels of either alpha 1-tubulin or beta II-tubulin mRNA. These findings suggest that continued expression of growth-associated tubulin isotype mRNAs may have functional significance in specific neuronal populations of the adult brain. Partial overlap between the distributions of alpha 1- and beta II-tubulin mRNAs and that of GAP-43 mRNA is discussed, as are potential roles for growth-associated tubulin gene expression in supporting cytoskeletal turnover, reactive axonal growth, and dendritic remodeling in the adult brain.

Coordinated upregulation of alpha 1- and beta II-tubulin mRNAs during collateral axonal sprouting of central peptidergic neurons.

An in situ hybridization study was performed to determine the relationship between levels of mRNAs for the axonal growth-associated alpha 1-tubulin and beta II-tubulin isotypes and the process of collateral axonal sprouting by identified central nervous system (CNS) neurons. A unilateral hypothalamic knife-cut was used to hemisect the hypothalamoneurohypophysial tract, which results in a robust collateral sprouting response by the uninjured neurons of the contralateral supraoptic nucleus (SON) (Watt and Paden: Exp Neurol 111:9-24, 1991). At 10 and 30-35 days after the lesion, cryosections of the SON were obtained and hybridized with 35S-labeled cDNA probes specific to alpha 1- and beta II-tubulin mRNAs. Quantitative evaluation of the resulting autoradiographs revealed that alpha 1-tubulin mRNA levels were significantly increased by 10 days in SON neurons that were undergoing collateral sprouting compared to controls and that this increase was sustained at 30-35 days post-lesion. Less marked increases in hybridization intensity of the beta II-tubulin probe were also apparent in sprouting neurons at both 10 and 30-35 days after the lesion, but were statistically significant only at 10 days. The measured increases in intensity of hybridization of alpha 1- and beta II-tubulin probes are likely to be conservative estimates of the underlying increase in alpha 1- and beta II-tubulin mRNA levels because sprouting SON neurons undergo significant hypertrophy. High levels of both alpha 1- and beta II-tubulin mRNAs were also seen in surviving axotomized SON neurons ipsilateral to the hypothalamic lesion. We conclude that the pattern of regulation of alpha 1- and beta II-tubulin mRNAs in CNS neurons which are capable of supporting new axonal growth includes three elements: maintenance of significant basal alpha 1- and beta II-tubulin mRNA pools in mature neurons, rapid increases in the pool size of the mRNAs following stimulation of collateral sprouting, and sustained elevation of mRNA levels during the period of axonal sprouting.

Microglia in the rat neurohypophysis increase expression of class I major histocompatibility antigens following central nervous system injury.

An immunocytochemical study of the expression of major histocompatibility complex (MHC) class I and II antigens by glial cells of the rat neurohypophysis was performed. Numerous cells with the appearance of microglia were found to constitutively express class I MHC antigens, while only rare cells expressed class II (Ia) antigens. Stereological analysis revealed that expression of class I MHC antigens increased significantly within 10 days after a unilateral hypothalamic lesion known to cause axonal degeneration and compensatory collateral axonal sprouting within the neurohypophysis. In addition, however, a brain lesion which did not affect the axonal population of the neurohypophysis also produced a significant increase in microglial expression of class I MHC antigens in this structure. Neither lesion affected the expression of class II MHC antigens in the neurohypophysis. Simultaneous immunofluorescent labeling for MHC I antigens and glial fibrillary acidic protein (GFAP, a pituicyte marker) or for MHC I and the C3bi complement receptor (a microglial marker) confirmed that the MHC class I-reactive cells were microglia. MHC I-positive cells also bound Griffonia simplicifolia B4 isolectin (GSA I-B4), consistent with their identification as microglia. The majority of MHC class I-reactive microglia were located in close apposition to blood vessels. These results indicate that an unusually large proportion of microglia within the neurohypophysis constitutively express MHC I antigens. In addition, neurohypophysial microglia are capable of responding to penetrating brain injury by upregulation of MHC I antigens in the absence of local tissue degeneration, possibly because of the absence of a blood-brain barrier.

Innervation of the rat anterior and neurointermediate pituitary visualized by immunocytochemistry for the growth-associated protein GAP-43.

Immunocytochemical localization of the neuronal growth associated protein GAP-43 revealed a dense axonal plexus throughout the neurointermediate lobe of the rat pituitary. These axons were fine, presumably monoaminergic fibers, whereas magnocellular neurosecretory axons did not appear to contain detectable GAP-43. These experiments also revealed the presence of an extensive nerve plexus within the anterior lobe. Fine beaded fibers were present throughout the parenchyma of the anterior lobe, and punctate staining suggestive of nerve terminals was seen surrounding numerous endocrine cells. Nerve fibers did not appear to cross directly between the intermediate and anterior lobes, but rather entered the anterior lobe directly from its margins or in association with blood vessels. Preabsorption of antisera with GAP-43 purified from neonatal rat brain completely eliminated immunoreactivity. These findings confirm the existence of a direct innervation of the anterior pituitary of the rat; moreover, the presence of GAP-43 in these fibers suggests that they may be capable of growth and terminal reorganization in the adult animal.

Expression of a novel nuclear protein is correlated with neuronal differentiation in vivo.

We report the production of a monoclonal antibody (MAb 526) that recognizes a novel, developmentally regulated nuclear protein expressed in neurons throughout the rat nervous system. Analysis of whole brain and cell nuclear extracts by SDS-PAGE and immunoblotting determined that MAb 526 recognizes a single nuclear protein (np) of apparent molecular weight 42 kD, designated np526, as well as a slightly larger (ca. 44 kD) cytoplasmic protein. Light microscopic immunocytochemistry showed np526 to be present in neurons of all types throughout the central and peripheral nervous systems. Nuclei of both fibrous and protoplasmic astrocytes were also immunoreactive, but oligodendrocyte nuclei were negative. Positive, but highly variable immunocytochemical staining of nonneural cell nuclei in a variety of other tissues was also observed. Electron microscopic (EM) immunocytochemistry using pre-embedding peroxidase methods revealed that np526 is associated with euchromatin or with the edges of condensed chromatin bundles in neurons, indicating that it is likely to be a chromosomal protein. Most interestingly, the expression of np526 was found to be developmentally regulated in brain. Immunocytochemical analysis of the developing cerebral cortex from embryonic day (E) 16 to postnatal day (P) 4 and cerebellum from P4 to P18 revealed that np526 first appears in central neurons following the cessation of mitosis and that the intensity of nuclear staining increases during subsequent neuronal maturation. To our knowledge, np526 is the first presumptive chromosomal protein whose expression has been precisely correlated with the early postmitotic differentiation of mammalian neurons.

Monoclonal antibodies identify two novel proteins associated with vasopressin secretory granules of the rat neurohypophysis.

Immunocytochemical and immunoblotting technique have been used to characterize the antigens recognized by two monoclonal antibodies (MAbs C6 and D5) produced against dissociated cells from punches of neonatal supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei of the rat. Peroxidase immunocytochemistry revealed that both MAbs label magnocellular perikarya in the adult and neonatal SON and PVN as well as smaller neurons in the suprachiasmatic nucleus. Axons of the hypothalamo-neurohypophysial tract are also immunolabeled within the hypothalamus and zona interna of the median eminence, and C6 and D5 each bind specifically to both the adult and neonatal neurohypophysis. Dual-label immunofluorescence experiments employing C6 or D5 simultaneously with rabbit antisera specific for either oxytocin, neurophysin or vasopressin neurophysin revealed that C6 binds only to vasopressinergic magnocellular perikarya in the SON, while D5 labels both vasopressinergic and a small subset of oxytocinergic magnocellular neurons. Post-embedding immunogold analysis of MAb binding to the neurohypophysis at the ultrastructural level showed that both C6 and D5 recognize antigens associated with large dense core neurosecretory granules in a subset of neurosecretory axons. Initial biochemical characterization of the antigens recognized by C6 and D5 was performed using SDS-PAGE and Western immunoblotting. MAbs C6 and D5 label single protein bands with apparent molecular weights of 38 and 68 kDa, resp., in blots of reduced extracts from the adult neurointermediate lobe. No cross-reactivity between C6 and D5 and the neurophysins was apparent, nor did anti-neurophysin sera recognize the bands identified by C6 and D5. We have therefore designated these novel antigens as VPGP38 and VPGP68 for VasoPressin Granule Proteins.

Compensatory sprouting of uninjured magnocellular neurosecretory axons in the rat neural lobe following unilateral hypothalamic lesion.

Axonal sprouting of intact neurons of the magnocellular neurosecretory system was investigated using a unilateral hypothalamic knife cut of the hypothalamoneurohypophysial tract to partially denervate the rat neural lobe (NL). Densitometric, morphometric, ultrastructural, and metabolic measures were utilized to demonstrate the compensatory response to denervation in this system. Densitometric analysis revealed a transient reduction in the intensity of vasopressin staining in the NL at 10 days postsurgery (PS) with a subsequent recovery by 20 days PS. There was a comparable initial reduction in the cross-sectional area of the NL followed by a more gradual recovery to normal by 90 days PS. Ultrastructural investigation revealed a reduction in total axon number in the NL at 10 days PS similar to the declines in vasopressin immunoreactivity and size of the NL. A subsequent partial recovery of axon number occurred, paralleling the return to normal NL size between 30 and 90 days PS. Hypertrophy of both somata and cell nuclei of magnocellular neurons in the supraoptic and paraventricular hypothalamic nuclei contralateral to the lesion was also apparent during this period. Daily measurements of urine osmolality revealed an initial transient hypoosmolality followed by a chronic hyperosmolality which persisted throughout the 90 day postsurgical period. There was a concomitant chronic decrease in both daily drinking and urine excretion volumes which began immediately following surgery. These results suggest that intact, contralateral magnocellular vasopressinergic efferents undergo compensatory sprouting as a result of partial denervation of the NL in the absence of a functional deficit in vasopressin.

Heterogeneous distribution and upregulation of mu, delta and kappa opioid receptors in the amygdala.

Levels of mu, delta and kappa opioid receptors in 4 subnuclei of the rat amygdala were determined by quantitative autoradiography following chronic treatment with naloxone or saline. A different distribution of each receptor subtype was observed, with mu binding greatest in the lateral nucleus (La), delta greatest in the basolateral (Bl), and kappa greatest in the medial (Me). Levels of all 3 receptors were very low in the central nucleus. Receptor upregulation following chronic naloxone treatment was also anatomically heterogeneous. Increases in mu receptors were statistically significant in the Me, Bl and La, while increases in delta and kappa receptors were significant only in the Bl.

Modulation of aromatase activity by testosterone in transplants of fetal rat hypothalamus-preoptic area.

Conversion of androgens to estrogens by neural aromatase appears to be a prerequisite for a variety of effects of androgens on brain function, including sexual differentiation. Activity of aromatase is modulated by its substrate testosterone (T) in adult hypothalamus-preoptic area (HPOA), resulting in significantly higher levels in the male. Perinatal sex differences in activity have also been observed in hypothalamus, POA and/or amygdala. However, it is not known if higher levels in the perinatal male occur in response to circulating androgens, nor whether early exposure to gonadal steroids is necessary to establish either basal levels or the androgen sensitivity of aromatase activity in the adult brain. In order to investigate the influence of early steroid exposure on the development of neural aromatase activity, embryonic day (E)17 fetal HPOA was transplanted onto the choroidal pia overlying the superior colliculus of adult ovariectomized-adrenalectomized (OVX-ADX) Holtzman female hosts. In the first experiment, the effect of androgen exposure on aromatase activity in mature HPOA transplants was determined. Hosts received T-filled silastic capsules or underwent sham surgery 7 weeks after transplantation and were sacrificed 7 days later. Aromatase activity was determined in vitro using the stereospecific production of 3H2O from [1 beta-3H]androstenedione as an index of estrogen formation. Aromatase activity was significantly greater in T-treated HPOA versus controls (P less than 0.005). Activity was not affected by the sex of the donor fetus. In the second experiment, the effect of androgen exposure during the first 6 days following transplantation of E17 HPOA (corresponding to the last gestational week) was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

A flow cytometric method for intracellular labeling and purification of rare neuronal populations: isolation of fixed neurophysin neurons.

A method is described for flow cytometric analysis and fluorescence-activated cell sorting of small populations of neurons following dissociation of fixed brain tissue and immunofluorescent labeling of intracellular antigens. This method has been successfully applied to neurophysin-containing magnocellular neurons of the rat supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. These neurons constitute a rare population in the context of flow cytometry, comprising less than 2% of all cells present in dissociated tissue punches of SON and PVN. Following labeling with anti-neurophysins sera and fluorescein-conjugated second antibody, a highly enriched population containing 80-85% neurophysin-positive neurons was isolated by fluorescence-activated cell sorting. Recovery of 29% of all neurophysin-containing neurons in the SON/PVN was achieved. Perikarya were recovered largely intact, frequently with attached proximal dendritic processes. Applications of this method include purification of specific neuronal types for use as immunogens in production of monoclonal antibodies to cell-type-specific antigens, and rapid surveys of fluorescent lectin or other ligand binding to cell populations identified by the presence of particular intracellular antigens.

Estrogen and progestin receptors appear in transplanted fetal hypothalamus-preoptic area independently of the steroid environment.

Sexual maturation and differentiation of the rat brain are believed to result from the interaction of gonadal steroids with specific neural receptors during late fetal and early postnatal life. A variety of evidence indicates that the first appearance of estrogen receptors in the hypothalamus-preoptic area (HPOA) during the perinatal period is a crucial evident underlying these processes. However, it is unknown to what extent the ontogeny of estrogen receptors is itself influenced by gonadal steroids present in the fetal environment. In order to address this question, estrogen receptors were assayed in HPOA 8 weeks after transplantation of the tissue from embryonic day 15 to 18 fetuses to either the choroidal pia overlying the superior colliculus or to the anterior chamber of the eye of adult female hosts. Host animals were either intact or ovariectomized and adrenalectomized, with or without estrogen replacement. The saturable binding of estradiol to cytosol of HPOA transplants exhibited the steroid specificity and high affinity characteristic of authentic estrogen receptors. No differences in the level of cytosol estrogen receptors in transplanted HPOA grown in the presence or absence of gonadal steroids were found. Receptor concentrations were also similar in HPOA taken from male or female fetuses. Autoradiography with [3H]estradiol revealed clusters of estrophilic cells in the transplants similar to those of the adult host hypothalamus, again regardless of whether the transplant developed in the presence of gonadal steroids. Estrogen receptors from both groups were also found to be biochemically functional as indicated by the ability of acute estrogen treatment to induce progestin receptors in the transplants.(ABSTRACT TRUNCATED AT 250 WORDS)