Ovine Brain Research Articles

Induction of immunoreactive prostaglandin H synthases 1 and 2 and fos in response to cerebral hypoperfusion in late-gestation fetal sheep

The goal of the present study was to localize and quantify immunoreactive prostaglandin H synthase 1 (PGHS-1), PGHS-2, and Fos expression by immunohistochemistry in the fetal brain 30 minutes and 2 hours after the onset of a 10-minute period of cerebral hypoperfusion in barodenervated and chemodenervated fetal sheep. Fetal sheep of known gestational age were studied intact or sinoaortic denervated. Fetuses were sacrificed and tissues recovered for immunohistochemistry or real-time polymerase chain reaction measurements of protein and mRNA, respectively. Some fetuses were subjected to brachiocephalic occlusion, produced by inflation of an extravascular balloon occluder around the brachiocephalic artery. Immunohistochemistry results were quantified using image analysis, and mRNA was quantified by estimation of cycle threshold in generation of PGHS-1 or PGHS-2 amplicons. Sinoaortic denervation by itself did not alter the abundance of PGHS-1 or PGHS-2 protein in any brain region, although the denervation did reduce the abundance of PGHS-1 mRNA in hypothalamus. We assessed PGHS-1, PGHS-2, and Fos immunoreactive protein abundance by image analysis of histologic sections stained for the respective proteins using immunohistochemistry. Cerebral hypoperfusion increased the intensity of staining of immunoreactive PGHS-1, PGHS-2, and Fos in the anterior pituitary, hippocampus, and cerebellum. In the cerebral microvasculature, the intensity of PGHS-1 and Fos was significantly greater, and in the cerebral cortex, the intensity of PGHS-2 was significantly greater. Changes in the amount of immunostaining in the nucleus of tractus solitarius and paraventricular nucleus were not statistically significant. Cerebral hypoperfusion altered the expression and distribution of prostaglandin biosynthetic enzymes in ovine fetal brain by a mechanism that is independent of baroreceptor and chemoreceptor afferent activity.

Immunoreactive forms of natriuretic peptides in ovine brain: response to heart failure

In order to elucidate how brain natriuretic peptides (NPs) are affected by experimentally induced heart failure, we have measured the immunoreactive (IR) levels of the NP in extracts from 10 regions of ovine brain, including pituitary, and clarified their molecular forms using high performance liquid chromatography (HPLC). Using species-specific radioimmunoassay (RIA), atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were all detected in extracts taken from control animals and sheep that had undergone rapid ventricular pacing for 7 days to induce heart failure. CNP was the most abundant NP as assessed by specific RIA, and the pituitary contained the highest IR levels for all three NP. Compared with control animals, the pituitary content of BNP in animals with heart failure was reduced by 40% (control, 0.26±0.02 pmol/g wet weight versus heart failure 0.16±0.01; P<0.01, n=7). No other significant changes were observed. The molecular forms of ANP and CNP in whole brain extracts as assessed by HPLC were proANP and CNP22, CNP53 and proCNP, respectively. BNP in pituitary extracts was assessed to be primarily proBNP with a minor component of mature BNP26.

Progesterone receptor, estrogen receptor alpha, and the type II glucocorticoid receptor are coexpressed in the same neurons of the ovine preoptic area and arcuate nucleus: a triple immunolabeling study.

The neuroendocrine reproductive and stress axes are known to be closely linked, but the mechanisms underlying these links remain poorly understood. In the ovine brain, GnRH neurons do not contain type II glucocorticoid (GR), progesterone (PR), or alpha estrogen (ERalpha) receptors. We sought to determine whether PR, ERalpha, and GR coexist within the same hypothalamic neurons. A triple immunocytochemical study, involving antisera raised in three different species, was performed on cryostat sections from ovariectomized ewes treated either with estradiol and progesterone or with progesterone alone. All PR-immunoreactive neurons contained ERalpha, and about 95% of ERalpha were PR immunoreactive in the preoptic area and arcuate nucleus. Although the PR with ERalpha colocalization ratio was not affected by the steroid treatments, immunolabeling for PR was weaker in animals that did not receive estradiol. Numerous PR- and ERalpha-immunoreactive cells contain GR. PR+ERalpha+GR-immunoreactive cells represent 70% of PR, 65% of ERalpha, and 72% of GR in the preoptic area and 70% of PR, 66% of ERalpha, and 63% of GR in the arcuate nucleus. These results suggest that estrogen, progesterone, and glucocorticoids may influence the activity of the same neurons to modulate both reproductive and stress axes.

Sampling of the ovine brain stem for tse-testing

Sampling of the ovine brain stem for tse-testing

PACAP and its receptors exert pleiotropic effects in the nervous system by activating multiple signaling pathways.

Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from the ovine brain in 1989 as a novel hypothalamic hormone that potently activates adenylate cyclase to produce cyclic AMP in pituitary cells. This neuropeptide belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) superfamily, and exists in two amidated forms as PACAP38 (38-amino acid residues) and PACAP27 derived from the same precursor. The primary structure of PACAP has been remarkably conserved throughout evolution among tunicata, ichthyopsida, amphibia and mammalia, and a PACAP-like neuropeptide has also been determined in Drosophila. Both PACAP and its receptors are mainly distributed in the nervous and endocrine systems showing pleiotropic functions with high potency. There are three types of receptors with high PACAP-binding affinity and with different tissue-distribution patterns. All of them belong to G-protein-coupled receptor superfamily with seven transmembrane domains. PAC(1) is the PACAP-specific receptor and exists in at least eight splice variants which couple to different intracellular signal transduction pathways. VPAC(1) and VPAC(2) are the common receptors for both PACAP and VIP, which are coupled to adenylate cyclase. This review article presents and discusses an update on PACAP research and its pleiotropic physiological functions based on multiple receptor-mediated signaling mechanisms in both the central and peripheral nervous system, including the regulation of hypothalamic neurosecretion, homeostatic control of circadian clock and behavioral actions, involvement in learning and memory processes, neuroprotective effects such as anti-apoptosis and response to injury and inflammation, and neural ontogenetic functions on proliferation/differentiation processes from early stages.

Prostaglandin D synthase in the prenatal ovine brain and effects of its inhibition with selenium chloride on fetal sleep/wake activity in utero.

It has been proposed that prostaglandin (PG) D(2) induces physiological sleep in mammals by acting on sleep centers located in the anterior hypothalamus. In fetal sheep, definitive rapid-eye-movement and non-rapid-eye-movement sleep states appear at approximately 125 d gestation (term is approximately 147 d). In adult animals, PGD synthase (PGDS) (functionally and structurally homologous to beta-trace protein) is secreted into CSF with a circadian pattern, with the highest concentrations present during sleep. In this study we show that PGDS/beta-trace protein is present in fetal sheep CSF at 125 and 135 d gestation but not at 90 d gestation. SeCl(4), a specific inhibitor of PGDS, was given to unanesthetized fetal sheep (130-140 d gestation) by intracerebroventricular infusion at a dose of 25, 100, 500, or 1000 pmol/min for 4 hr. Artificial CSF was infused in control experiments. Arousal behavior, defined as the presence of nuchal muscle electromyogram activity, electro-ocular activity, and breathing movements during low-amplitude electrocortical activity, increased from 3.8 +/- 1 min/hr to 6.6 +/- 0.5 and 7.0 +/- 0.3 min/hr at doses of 100 and 500 pmol/min, respectively (p < 0.05). SeCl(4) at 25 and 1000 pmol/min had no significant effect on arousal activity. Infusion of PGD(2) at 500 pmol/min intracerebroventricularly for 4 hr decreased the incidence of arousal from 3.8 +/- 0.5 min/hr to 0.7 +/- 0.3 min/hr (p < 0.05). When 500 pmol/min PGD(2) was infused immediately after a 4 hr infusion of SeCl(4) (500 pmol/min), the SeCl(4)-induced increase in arousal behavior was abolished. Together, the presence of PGDS/beta-trace protein in fetal CSF in late gestation and the effects of SeCl(4) in increasing the incidence of arousal-like behavior suggest that PGD(2) has a role in the induction and maintenance of prenatal sleep.

Prostaglandin D Synthase in the Prenatal Ovine Brain and Effects of Its Inhibition with Selenium Chloride on Fetal Sleep/Wake ActivityIn Utero

It has been proposed that prostaglandin (PG) D₂ induces physiological sleep in mammals by acting on sleep centers located in the anterior hypothalamus. In fetal sheep, definitive rapid-eye-movement and non-rapid-eye-movement sleep states appear at ∼125 d gestation (term is ∼147 d). In adult animals, PGD synthase (PGDS) (functionally and structurally homologous to β-trace protein) is secreted into CSF with a circadian pattern, with the highest concentrations present during sleep. In this study we show that PGDS/β-trace protein is present in fetal sheep CSF at 125 and 135 d gestation but not at 90 d gestation. SeCl₄, a specific inhibitor of PGDS, was given to unanesthetized fetal sheep (130–140 d gestation) by intracerebroventricular infusion at a dose of 25, 100, 500, or 1000 pmol/min for 4 hr. Artificial CSF was infused in control experiments. Arousal behavior, defined as the presence of nuchal muscle electromyogram activity, electro-ocular activity, and breathing movements during low-amplitude electrocortical activity, increased from 3.8 ± 1 min/hr to 6.6 ± 0.5 and 7.0 ± 0.3 min/hr at doses of 100 and 500 pmol/min, respectively (<i>p</i> &lt; 0.05). SeCl₄ at 25 and 1000 pmol/min had no significant effect on arousal activity. Infusion of PGD₂ at 500 pmol/min intracerebroventricularly for 4 hr decreased the incidence of arousal from 3.8 ± 0.5 min/hr to 0.7 ± 0.3 min/hr (<i>p</i> &lt; 0.05). When 500 pmol/min PGD₂ was infused immediately after a 4 hr infusion of SeCl₄ (500 pmol/min), the SeCl₄-induced increase in arousal behavior was abolished. Together, the presence of PGDS/β-trace protein in fetal CSF in late gestation and the effects of SeCl₄ in increasing the incidence of arousal-like behavior suggest that PGD₂ has a role in the induction and maintenance of prenatal sleep.

Apoptosis in the preterm and near term ovine fetal brain and the effect of intermittent umbilical cord occlusion

Programmed cell death or apoptosis plays a central role during the development of the brain, but can also be activated by hypoxic/ischemic insult. The purpose of the present study was to determine the regional distribution of apoptotic cells in the preterm and near term ovine fetal brain and thus in relation to the maturation of neurobehavioural activity, and the effect of intermittent umbilical cord occlusion (UCO), which might then contribute to adverse neurodevelopment. Fetal sheep (control and experimental groups at 0.75 and 0.90 of gestation) were studied over 4 days with UCOs performed in the experimental group animals by complete inflation of an occluder cuff for 90 s every 30 min for 3 to 5 h each day. Animals were then euthanized and the fetal brain perfusion-fixed and prepared for subsequent histology and apoptosis staining using the TUNEL assay method. The number of TUNEL positive cells for both the preterm and near term control group animals was low but with a significant regional hierarchy whereby values were higher in the cerebellar peduncle and cortex and lower in the cortical grey and white matter, hippocampus, and pons. While the apoptotic indices (expressed as TUNEL positive cells/1000 cells or high powered field) for most brain regions were not significantly changed between the preterm and near term control group animals, that for the hippocampus and pons were increased ∼5- and 4-fold, respectively, (both P<0.05), in the near term animals. Intermittent UCO with severe but limited hypoxemia and no cumulative acidosis to ensure longer term survival, had no significant effect on apoptotic indices in the brains of either the preterm or near term animals, although hippocampal values for both occlusion groups were increased ∼2–3-fold. Levels of apoptosis noted for the ovine fetal brain at 0.75 to 0.90 of gestation are thus low and likely approaching the basal levels of later life, but there are regional differences and changes over this period although little change in response to intermittent cord occlusion as studied, with implications for behavioural state activity and antenatal hypoxic insults in the brain’s development.

Importance of Hyaluronan Length in a Hyaladherin-Based Assay for Hyaluronan

Specific hyaladherin-based assays have been set up to measure the concentration of hyaluronan in biological fluids. Hyaluronectin (HN; a hyaladherin extracted from ovine brain) binds to hyaluronan (HA) that must be 10 units (HA10) or more long. It was therefore of interest to determine whether HN would continue to bind to HA10 in full-length HA since conformational changes might mask potential binding sites. We used the enzyme-linked sorbent assay (ELSA) to assay HA and hyaluronan-derived oligosaccharides, with different standard HAs, and the results were compared to results obtained with the carbazole technique. Oligosaccharide length was calculated from the ratio glucuronic acid/reducing N-acetylglucosamine in fractions of hyaluronidase-digested macromolecular hyaluronan prepared by chromatography; the size of the HA12 oligosaccharide was confirmed by matrix-assisted laser desorption ionization mass spectrometry. During the digestion of macromolecular HA with hyaluronidase, the binding of HN to HA first increased and then decreased as shown using the ELSA. The concentration of HA fragments of HA60 and below was overestimated when intact macromolecular HA was used as the reference for the ELSA, while the concentration of HA100 and above was underestimated when HA10 was used as the reference. The binding of HN to HA20, HA40, and HA60 saccharides was consistent with binding to multiples of HA10 sites. In conclusion, the level of HN binding is determined by the conformation of HA, which may mask binding sites. Hence, calibration HA used in the ELSA must be adapted to the size of HA to assay.

Estrogen receptor β is the predominant isoform expressed in the brain of adult and fetal sheep

Objective: The objective of this study was to examine the expression of estrogen receptors α and β in the cerebral cortex of the adult and fetal sheep. Study Design: A reverse transcriptase–polymerase chain reaction–based approach was used to examine the expression of ovine estrogen receptor α and estrogen receptor β in the cerebral cortex of 4 adult and 2 fetal sheep. Results: Estrogen receptor β was expressed in the 4 adult and 2 fetal brain samples. Estrogen receptor α expression was seen in only 1 adult brain and 1 fetal brain. Conclusion: Estrogen receptor β is the predominant isoform expressed in the cerebral cortex of both adult and fetal sheep. These data may have implications for the many important actions of estrogen in the adult and developing ovine brain. (Am J Obstet Gynecol 2001;184:1077-9.)

Antenatal betamethasone treatment reduces synaptophysin immunoreactivity in presynaptic terminals in the fetal sheep brain

Knowledge of morphofunctional effects on the fetal brain induced by exogenous glucocorticoids is limited. Recently, we reported alterations of both the neuronal cytoskeleton and electrocortical function in the ovine fetal brain after antenatal betamethasone treatment in doses used in perinatal medicine. In the present study we examined whether these changes are accompanied by morphological alterations of synapses. Chronically instrumented fetal sheep at 0.87 of gestation were treated either with isotonic saline ( n=7) or 10 μg/h betamethasone ( n=7) over 48 h administered directly to the fetal jugular vein. Paraffin sections of the frontal neocortex, caudate putamen and hippocampus were stained with a monoclonal antibody against synaptophysin, a specific membrane protein of presynaptic vesicles and quantified morphometrically. Synaptophysin-like immunoreactivity (synaptophysin-LI) showed a widespread granular pattern in the neuropil. Betamethasone exposure reduced synaptophysin-LI in the frontal neocortex, caudate putamen and hippocampus by 46.9, 41.0 and 55.4%, respectively, ( P<0.05) that was not accompanied by irreversible neuronal damage. These results suggest that clinical doses of betamethasone have acute effects on presynaptic terminals in the fetal sheep brain that could contribute to the altered complexity of electrocortical function that we have shown previously to occur following fetal exposure to betamethasone.

Adaptation of laser-Doppler flowmetry to measure cerebral blood flow in the fetal sheep.

The purpose of this study was to devise a means to use laser-Doppler flowmetry to measure cerebral perfusion before birth. The method has not been used previously, largely because of intrauterine movement artifacts. To minimize movement artifacts, a probe holder was molded from epoxy putty to the contour of the fetal skull. A curved 18-gauge needle was embedded in the holder. At surgery, the holder, probe, and skull were fixed together with tissue glue. Residual signals were recorded after fetal death and after maternal death 1 h later. These averaged <5% of baseline flow signals, indicating minimal movement artifact. To test the usefulness of the method, cerebral flow responses were measured during moderate fetal hypoxia induced by giving the ewes approximately 10% oxygen in nitrogen to breathe. As fetal arterial PO(2) decreased from 21.1 +/- 0.5 to 10.7 +/- 0.4 Torr during a 30-min period, cerebral perfusion increased progressively to 56 +/- 8% above baseline. Perfusion then returned to baseline levels during a 30-min recovery period. These responses are quantitatively similar to those spot observations that have been recorded earlier using labeled microspheres. We conclude that cerebral perfusion can be successfully measured by using laser-Doppler flowmetry with the unanesthetized, chronically prepared fetal sheep as an experimental model. With this method, relative changes of perfusion from a small volume of the ovine fetal brain can be measured on a continuous basis, and movement artifacts can be reduced to 5% of measured flow values.

Characterisation of GABA A receptors in fetal, neonatal and adult ovine brain: region and age related changes and the effects of allopregnanolone

Progesterone metabolites acting via GABA A receptors suppress central nervous system (CNS) activity. The aim of the present study was to examine binding characteristics of GABA A receptors in fetal, newborn and adult sheep brains using [ 35S]TBPS, and to determine the effects of allopregnanolone on this binding. Receptor affinity ( K D) and density ( B MAX) in the brainstem were not different in fetal, newborn (1–2 days old) and adult brains. In the hypothalamus K D and B MAX increased significantly in the fetus between 85 and 128 days gestation, and were then similar to postnatal and adult values. In the frontal cortex K D and B MAX increased progressively between 85 days and term (∼147 days gestation), and were then not different from postnatal and adult values. The K i values for the GABA A receptor antagonist picrotoxin was similar at all ages. Allopregnanolone inhibited [ 35S]TBPS binding in the presence of 5 μM GABA, but enhanced binding in the absence of GABA. These results show that (i), functional GABA A receptors are present in the fetal brain from at least 85 days gestation; (ii), 3α-pregnane steroids modify receptor affinity in the late gestation fetal brain; and (iii) there are region-specific changes in GABA A receptor binding parameters. Steroid modulation of the GABA A receptor in the fetal brain is likely to influence fetal CNS activity in late gestation.

Ovine Fetal Estrogen Sulfotransferase in Brain Regions Important for Hypothalamus-Pituitary-Adrenal Axis Control

Ovine parturition is initiated by increases in fetal hypothalamus-pituitary-adrenal (HPA) axis activity which, in turn, increase placental estrogen biosynthesis and ultimately increase uterine contractility. In addition to their action in the uterus, estrogens in fetal plasma augment fetal corticotropin (ACTH) secretion. In late gestation, estrone sulfate is more abundant in fetal plasma than unconjugated estrone and it is possible that there is interconversion of sulfoconjugated and unconjugated steroids within the fetal brain. We studied hypothalamus and brainstem tissue from fetal, neonatal, and adult sheep to test the hypothesis that the ovine brain contains estrogen sulfotransferase. Although no significant ontogenic pattern was revealed, the presence of estrogen sulfotransferase within the hypothalamus and brainstem was detectable. Immunohistochemistry revealed the presence of estrogen sulfotransferase in the paraventricular nucleus of the hypothalamus, the nucleus of the tractus solatarius, and the rostral ventral lateral medulla. We conclude that ovine fetal hypothalamus and brainstem contain estrogen sulfotransferase in brain regions important for HPA axis control.

Urocortin is the principal ligand for the corticotrophin-releasing factor binding protein in the ovine brain with no evidence for a sauvagine-like peptide.

To purify novel ligands for the corticotrophin-releasing factor binding protein (CRF-BP) from ovine brain, whole brain was homogenised in methanol and the supernatant extracted on Sep-pak C18 cartridges followed by a preliminary HPLC step. Three peaks of ovine CRF-BP ligand activity were detected in the HPLC fractions, the first two of which were also detected by a specific corticotrophin-releasing factor two-site immunoradiometric assay, the third peak being detected by a human CRF-BP ligand assay, which will not detect ovine CRF. Human CRF-BP ligand-containing fractions were further purified by affinity chromatography on a human recombinant CRF-BP column with two additional HPLC steps. The human CRF-BP ligand was found to: (a) possess a molecular mass of 4707 Daltons, (b) have an N-terminal amino acid sequence (5 residues) identical to rat urocortin, (c) be detected by a specific urocortin radioimmunoassay, (d) have high affinity for both the human and ovine CRF-BPs and (e) be present in many regions of the ovine brain. Additionally, a 300 bp cDNA fragment sharing 83% homology with the rat urocortin gene was cloned from ovine brain, the product of which was predicted to have an identical amino acid sequence to that of rat urocortin. These pieces of information confirmed the identity of the human CRF-BP ligand as an ovine urocortin. The specially developed CRF-BP ligand assays showed that the rank orders of affinity of the CRF family members for human CRF-BP were: carp urotensin-1>>human CRF=rat/ovine urocortin>human urocortin>>frog sauvagine>>ovine CRF, and those for the ovine CRF-BP were: carp urotensin-1> human CRF=rat/ovine urocortin>human urocortin> frog sauvagine>>ovine CRF. This study describes a successful technique for the purification and detection of peptide ligands for the CRF-BP. We conclude that urocortin is the principal ligand for the CRF-BP in ovine brain and we could find no evidence for a centrally located mammalian sauvagine-like peptide.

The ontogeny of glutamate receptors and d-aspartate binding sites in the ovine CNS

The ontogeny of ligand binding to N-methyl- d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate (KA) receptors and to the high affinity, sodium-dependent d-aspartate binding site in prenatal and postnatal ovine brains was studied using quantitative in vitro autoradiography. In general, the binding density for each of the excitatory amino acid receptors peaked during late prenatal and early postnatal development. In contrast, binding density for d-aspartate remained low during late prenatal and early postnatal development and peaked in the adult. These data suggest that an excess number of excitatory amino acid receptors and/or a relative deficiency of transporters may make the immature brain more vulnerable to the pathologic effects of glutamate and other related excitatory amino acids.

Role of adenosine in regulation of brain temperature in fetal sheep

Objective: The purpose of this study was to test adenosine’s possible suppression of heat production by the ovine fetal brain during acute hypoxia. Study Design: Hypoxia was induced by occluding the umbilical cord for 5 minutes in 8 fetal sheep with and without an adenosine receptor blocker, theophylline, and fetal brain and core temperatures were recorded. Results: In 8 untreated fetuses, cord occlusion induced severe but reversible hypoxemia (decrease in carotid arterial P O 2 from 23 ± 1 to 5 ± 1 mm Hg ( P < .01), a 2.3-fold increase in plasma adenosine, and an increase in body core temperature of 0.19°C ± 0.03°C ( P < .01), yet brain temperature remained constant. However, after cord occlusion with prior and continuing administration of theophylline, brain temperature did not increase as hypothesized but rather decreased, suggesting marked reductions in cerebral metabolic rate. This response occurred despite similar degrees of hypoxemia and similar increases in plasma adenosine and body core temperature. Conclusion: We conclude that the temperature of the fetal ovine brain remains constant or decreases during severe reversible hypoxemia by mechanisms other than those dependent on adenosine. (Am J Obstet Gynecol 1999;181:681-7.)

GLT1, glial glutamate transporter, is transiently expressed in neurons and develops astrocyte specificity only after midgestation in the ovine fetal brain

Glutamate transport is a primary mechanism for regulating extracellular levels of glutamate in the central nervous system. GLT1, the most abundant of the known high-affinity glutamate transporters, is found exclusively in astrocytes in adult brain of several species, but we and others have recently identified neurons that transiently express GLT1 protein in the developing brain. We now demonstrate the development of cell type specificity for GLT1 expression at 60, 71, and 136 days' gestation in the developing sheep brain (term = 145 days). At 60 and 71 days of gestation, GLT1 colocalizes with calbindin in Purkinje cells in the cerebellum, and this expression pattern has a novel distribution that is reminiscent of the parasagittal zebrin-like bands. GLT1 immunoreactivity simultaneously occurs in periventricular white matter, anterior commissure, and striatal white matter, dissipating by 136 days. GLT1 protein expression within astrocytes is developmentally regulated, appearing first in vimentin positive radial glia at 60 and 71 days and then switching to GFAP positive parenchymal and perivascular astrocytes at 136 days. Expression of GLT1 in subsets of vimentin-positive astrocytes persists in white matter but not in cortex. These results identify a novel compartmentation within cerebellar cortex and neuronal and axonal pathway localization of GLT1, suggesting the participation of this glutamate transporter in the development of the topographic organization of cerebellar cortex and a transient neuronal function for GLT1 in developing brain. In addition, GLT1 expression is highly plastic, being neither exclusively astroglial nor uniformly expressed in different populations of astrocytes during brain development. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 515–526, 1999

Ontogeny of estrogen sulfatase activity in ovine fetal hypothalamus, hippocampus, and brain stem.

Ovine parturition is initiated by increases in fetal hypothalamus-pituitary-adrenal (HPA) axis activity, which in turn increase placental estrogen biosynthesis and ultimately increase uterine contractility. In addition to the action in the uterus, estrogens augment fetal ACTH secretion. In late gestation, estrone sulfate is more abundant in fetal plasma than is unconjugated estrone. We studied hypothalamus, hippocampus, and brain stem tissue from fetal, neonatal, and adult sheep to test the hypothesis that the ovine brain contains estrogen sulfatase activity. We found that the activity in the hippocampus was significantly increased in late-gestation fetuses compared with both younger and older animals. No significant change in either hypothalamus or brain stem was revealed; however, the activity in all brain areas was high. Immunohistochemistry revealed the presence of estrogen sulfatase in the paraventricular nucleus of the hypothalamus, the nucleus of the solitary tract, and the rostral ventrolateral medulla. We conclude that ovine fetal hypothalamus, hippocampus, and brain stem contain estrogen sulfatase activity and that the activity in the hippocampus is developmentally regulated.

GLT1, glial glutamate transporter, is transiently expressed in neurons and develops astrocyte specificity only after midgestation in the ovine fetal brain

Glutamate transport is a primary mechanism for regulating extracellular levels of glutamate in the central nervous system. GLT1, the most abundant of the known high-affinity glutamate transporters, is found exclusively in astrocytes in adult brain of several species, but we and others have recently identified neurons that transiently express GLT1 protein in the developing brain. We now demonstrate the development of cell type specificity for GLT1 expression at 60, 71, and 136 days' gestation in the developing sheep brain (term = 145 days). At 60 and 71 days of gestation, GLT1 colocalizes with calbindin in Purkinje cells in the cerebellum, and this expression pattern has a novel distribution that is reminiscent of the parasagittal zebrin-like bands. GLT1 immunoreactivity simultaneously occurs in periventricular white matter, anterior commissure, and striatal white matter, dissipating by 136 days. GLT1 protein expression within astrocytes is developmentally regulated, appearing first in vimentin positive radial glia at 60 and 71 days and then switching to GFAP positive parenchymal and perivascular astrocytes at 136 days. Expression of GLT1 in subsets of vimentin-positive astrocytes persists in white matter but not in cortex. These results identify a novel compartmentation within cerebellar cortex and neuronal and axonal pathway localization of GLT1, suggesting the participation of this glutamate transporter in the development of the topographic organization of cerebellar cortex and a transient neuronal function for GLT1 in developing brain. In addition, GLT1 expression is highly plastic, being neither exclusively astroglial nor uniformly expressed in different populations of astrocytes during brain development.