Tight junction proteins of cerebral endothelial cells in early postnatal development

The formation and functional plasticity of the blood–brain barrier are inextricably linked to the molecular events occurring in cerebral neurovascular unit during the embryonic and early postnatal development of the organism. To study the features of the barrier genesis under physiological conditions, as well as after perinatal hypoxia and stress in early life, tight junction proteins of cerebral endothelial cells (the number of JAM-, ZO1-, and CLDN5-positive cells) in rats at the age of 7 (P₇), 28 (P₂₈), and 70 (P₇₀) days were investigated. It was found that, under physiological conditions, the number of cells expressing JAM, ZO1, and CLDN5 slightly increase in the period from P₇ to P₇₀ in the cortex, hippocampus, and amygdala of the brain. After perinatal hypoxia, the number of cells expressing the proteins of tight junctions (JAM, CLDN5) is significantly increased to the age of P₂₈–P₇₀, while the number of ZO1⁺ cells in the same period of time is reduced. Early life stress causes an imbalance between the expression of ZO1 and other proteins of tight junctions, but these changes are opposite in direction.     

Early Postnatal In Vivo Gliogenesis From Nestin-Lineage Progenitors Requires Cdk5

The early postnatal period is a unique time of brain development, as diminishing amounts of neurogenesis coexist with waves of gliogenesis. Understanding the molecular regulation of early postnatal gliogenesis may provide clues to normal and pathological embryonic brain ontogeny, particularly in regards to the development of astrocytes and oligodendrocytes. Cyclin dependent kinase 5 (Cdk5) contributes to neuronal migration and cell cycle control during embryogenesis, and to the differentiation of neurons and oligodendrocytes during adulthood. However, Cdk5’s function in the postnatal period and within discrete progenitor lineages is unknown. Therefore, we selectively removed Cdk5 from nestin-expressing cells and their progeny by giving transgenic mice (nestin-CreERT2/R26R-YFP/CDK5^flox/flox [iCdk5] and nestin-CreERT2/R26R-YFP/CDK5^wt/wt [WT]) tamoxifen during postnatal (P) days P2-P 4 or P7-P 9, and quantified and phenotyped recombined (YFP+) cells at P14 and P21. When Cdk5 gene deletion was induced in nestin-expressing cells and their progeny during the wave of cortical and hippocampal gliogenesis (P2-P4), significantly fewer YFP+ cells were evident in the cortex, corpus callosum, and hippocampus. Phenotypic analysis revealed the cortical decrease was due to fewer YFP+ astrocytes and oligodendrocytes, with a slightly earlier influence seen in oligodendrocytes vs. astrocytes. This effect on cortical gliogenesis was accompanied by a decrease in YFP+ proliferative cells, but not increased cell death. The role of Cdk5 in gliogenesis appeared specific to the early postnatal period, as induction of recombination at a later postnatal period (P7-P9) resulted in no change YFP+ cell number in the cortex or hippocampus. Thus, glial cells that originate from nestin-expressing cells and their progeny require Cdk5 for proper development during the early postnatal period.     

HSP70 constitutive expression in rat central nervous system from postnatal development to maturity.

We studied the level of the basal (constitutive) HSP70 expression (inducible and constitutive forms) in the central nervous system (CNS) of male and female rats from the postnatal period to maturity. HSP70 levels were analyzed by immunoblotting in five different areas (cortex, hippocampus, hypothalamus, cerebellum, and spinal cord). The highest levels of HSP70 were found in juvenile rats and decreased progressively until reaching baseline levels between 2 and 4 months. A slight and nonsignificant increase in aged (2-year-old) rats compared with adult subjects was observed in some cerebral areas (cerebral cortex, hippocampus, and cerebellum). In the first weeks of postnatal development, HSP70 immunoreactivity was distributed throughout CNS sections and no specific immunopositive cells could be clearly determined. In adult animals, strong immunostaining was observed in some large neurons (Purkinje neurons and mesencephalic and spinal cord motor neurons), some perivascular and subpial astrocytes, and ependymocytes. Immunoelectron microscopy revealed that HSP70 in these cells is located in the perinuclear area and in mitochondria, rough endoplasmic reticulum, and microtubules. In neurons, strong immunolabeling was also observed in synaptic membranes. The postnatal time course of HSP70 levels and the location and size of HSP70-immunopositive cells suggest that HSP70 constitutively expressed in the rat CNS may be mainly determined by the degree of development and metabolic activity of the neural cells.     

Development of the various glial cell types in the cerebral cortex of postnatal rats

The present quantitative study in the postnatal rats showed the rapid growth of the various glial cell types in the cerebral cortex. Among them, the increase of microglia was most dramatic. The increase was about 15 times, covering a period of 15 days extending from 5 days of age to 20 days. The majority of the microglia observed were in the outer third of the cortex. During the same period, the number of oligodendrocytes and astrocytes also showed a steady but moderate increase. The increase of oligodendrocytes was most significant between 5 and 10 days. Their density was greater in the inner third of the cortex. Astrocytes were distributed uniformly throughout. Examination of the cerebral cortex in 1- to 3-day-old rats by electron microscopy showed sporadic ameboid microglia cells and glioblasts. The possibility that they served as the precursor cells of microglia and macroglia (astrocytes and oligodendrocytes), respectively, was considered.     

Deficits in cerebellar granule cell development and social interactions in CD47 knockout mice

CD47 is involved in neurite differentiation in cultured neurons, but the function of CD47 in brain development is largely unknown. We determined that CD47 mRNA was robustly expressed in the developing cerebellum, especially in granule cells. CD47 protein was mainly expressed in the inner layer of the external granule layer (EGL), molecular layer, and internal granule layer (IGL), where granule cells individually become postmitotic and migrate, leading to neurite fasciculation. At postnatal day 8 (P8), CD47 knockout mice exhibited an increased number of proliferating granule cells in the EGL, whereas the CD47 agonist peptide 4N1K increased the number of postmitotic cells in primary granule cells. Knocking out the CD47 gene and anti‐CD47 antibody impaired the radial migration of granule cells from the EGL to the IGL individually in mice and slice cultures. In primary granule cells, knocking out CD47 reduced the number of axonal collaterals and dendritic branches; by contrast, overexpressing CD47 or 4N1K treatment increased the axonal length and numbers of axonal collaterals and dendritic branches. Furthermore, the length of the fissure between Lobules VI and VII was decreased in CD47 knockout mice at P21 and at 14 wk after birth. Lastly, CD47 knockout mice exhibited increased social interaction at P21 and depressive‐like behaviors at 10 wk after birth. Our study revealed that the cell adhesion molecule CD47 participates in multiple phases of granule cell development, including proliferation, migration, and neurite differentiation implying that aberrations of CD47 are risk factors that cause abnormalities in cerebellar development and atypical behaviors.© 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 463–484, 2015     

Expression of CD95 and CD95L on astrocytes in the CA1 area of the immature rat hippocampus after hypoxia-ischemia injury

The immature brain is affected profoundly by hypoxia-ischemia (HI) injury, which can lead to permanent neurologic sequelae in survivors. Neuronal degeneration after HI injury usually is achieved through apoptosis. Both CD95 and its natural ligand, CD95L, which are key molecules in the regulation of apoptosis, are constitutively expressed by neurons and astrocytes during embryonic and early postnatal stages. Further, CD95 or CD95L may have a functional relationship in glial cells and lead to apoptosis of these cells. The hippocampus, especially the CA1 area, is particularly susceptible to HI injury. We therefore investigated the temporal and spatial alterations in CD95 and CD95L expression in the CA1 area of 7-d-old rats after unilateral ligation of the carotid artery. Using immunohistochemistry and Western blotting, we showed that expression of CD95 and CD95L in the hippocampus peaked at 12 h and then decreased. In addition, we used terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick end-labeling to demonstrate apoptosis among CD95- and CD95L-reactive cells. Our findings show that increases in the expression of CD95 and CD95L after HI injury may involve astrocytic apoptosis in the 7-d-old rat hippocampus, and these molecules may act as targets or inducers of cell death.     

Distribution of neuronal nitric oxide synthase-immunoreactive neurons in the cerebral cortex and hippocampus during postnatal development

Although many reports have argued a role for nitric oxide (NO) during postnatal development, there has been no combined demonstration in the cerebral cortex and hippocampus. We have investigated the distribution and morphology of neurons and fibers expressing neuronal NO synthase (nNOS) in the cerebral cortex and hippocampal formation of rats during the postnatal development, and correlated these findings with developmental events taking place in these regions. In the cerebral cortex, the nNOS-immunoreactive cells could be divided into two classes : heavily stained neurons and lightly stained neurons. For the lightly stained nNOS-positive neurons, only the cell bodies were observed, whereas for the heavily stained neurons, the cell bodies and their dendrites were visible. During the postnatal days, heavily stained neurons reached their typical morphology in the second week and appeared in all layers except for layer I. In the hippocampus, there was a transient expression of nNOS in the pyramidal cell layer at P3â€P7, and this expression disappeared during following days. The adult pattern of staining developed gradually during the postnatal period. This study suggested that these alterations might reflect a region-specific role of NO and a potential developmental role in the postnatal cerebral cortex and hippocampus     

Expression of TRPC6 in renal cortex and hippocampus of mouse during postnatal development

TRPC6, a member of the TRPC family, attracts much attention from the public because of its relationship with the disease. In both the brain and kidney, TRPC6 serves a variety of functions. The aim of the present study was to observe the expression and effects of TRPC6 in renal cortex and hippocampus during early?postnatal development of?the mouse. In the present study, immunohistochemistry and Western blotting were used to detect the expression of TRPC6 in the mouse kidney and hippocampus of postnatal day 1, 3, 5, 7, 14, 21, 28 and 49 (P1, P3, P5, P7, P14, P21, P28 and P49). Results showed that the expression of TRPC6 was increased in the mouse hippocampus, and there was a significant increase between P7 and P14 during the postnatal development. Meanwhile, the expression of TRPC6 was also detected in glomerulus and tubules, and a decreased expression was found during postnatal maturation of mouse renal cortex. From these in vivo experiments, we concluded that the expression of TRPC6 was active in the developing mouse kidney cortex, and followed a loss of expression with the development of kidney. Meanwhile, an increased expression was found in the hippocampus with the development. Together, these data suggested that the developmental changes in TRPC6 expression might be required for proper postnatal kidney cortex development, and played a critical role in the hippocampus during development, which formed the basis for understanding the nephrogenesis and neurogenesis in mice and provided a practically useful knowledge to the clinical and related research.     

Fetuin‐A in the developing brain

The serum protein fetuin‐A is essential for mineral homeostasis and shows immunomodulatory functions, for example by binding to TGF superfamily proteins. It proved neuroprotective in a rat stroke model and reduced lethality after systemic lipopolysaccharide challenge in mice. Serum fetuin‐A concentrations are highest during intrauterine life. Different species show intrauterine cerebral fetuin‐A immunoreactivity, suggesting a contribution to brain development. We therefore aimed at specifying fetuin‐A immunoreactivity in brains of newborn rats (age P0–P28) and human neonates (20–40 weeks of gestation). In humans and rats, fetuin‐A was found in cortex, white matter, subplate, hippocampus, subventricular zone, and ependymal cells which supports a global role for brain function. In rats, overall fetuin‐A immunoreactivity decreased with age. At P0 fetuin‐A immunoreactivity affected most brain structures. Thereafter, it became increasingly restricted to distinct cells of the hippocampus, cingular gyrus, periventricular stem cell layer, and ependyma. In ependymal cells the staining pattern complied with active transependymal transport from cerebrospinal fluid. Double immunofluorescence studies revealed colocalization with NeuN (mature neurons), beta III tubulin (immature neurons), GFAP (astrocytes), and CD68 (activated microglia). This points to a role of fetuin‐A in different brain functional systems. In human neonatal autopsy cases, frequently affected from severe neurological and non‐neurological diseases, fetuin‐A immunoreactivity was heterogeneous and much less associated with age than in healthy tissues studied earlier, suggesting an impact of exogeneous noxious factors on fetuin‐A regulation. Further research on the role of fetuin‐A in the neonatal brain during physiological and pathological conditions is recommended. © 2012 Wiley Periodicals, Inc. Develop Neurobiol 73: 354–369, 2013     

Estrogen formation in the mammalian brain: Possible role of aromatase in sexual differentiation of the hippocampus and neocortex

Recent studies suggest that sex differences in cognitive function may involve effects of circulating androgens on the developing cerebral cortex and hippocampus. The mechanism of these effects is not understood. In rhesus monkeys, aromatase activity is present in the hippocampus and several areas of the cerebral cortex during late fetal and early postnatal life. Similarly, work in rats and mice indicates that the hippocampus and cerebral cortex may be capable of estrogen biosynthesis during early development. These results are consistent with the hypothesis that the actions of androgens on the developing cerebral cortex and hippocampus may involve local estrogen-mediated effects similar to those responsible for differentiation of the hypothalamic mechanisms controlling reproductive function.     

Induction of Vascular Endothelial Growth Factor and Matrix Metalloproteinase-9 via CD47 Signaling in Neurovascular Cells

Neurovascular injury comprises a wide spectrum of pathophysiology that underlies the progression of brain injury after cerebral ischemia. Recently, it has been shown that activation of the integrin-associated protein CD47 mediates the development of blood–brain barrier injury and edema after cerebral ischemia. However, the mechanisms that mediate these complex neurovascular effects of CD47 remain to be elucidated. Here, we compare the effects of CD47 signaling in brain endothelial cells, astrocytes, and pericytes. Exposure to 4N1 K, a specific CD47-activating peptide derived from the major CD47 ligand thrombospondin-1, upregulated two major neurovascular mediators, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9), in brain endothelial cells and astrocytes. No changes were detected in pericytes. These findings may provide a potential mechanism for CD47-induced changes in blood–brain barrier homeostasis, and further suggest that CD47 may be a relevant neurovascular target in stroke.     

Loss of Neurons in the Hippocampus and Cerebral Cortex of AMSH-Deficient Mice

AMSH, a molecule that associates with STAM1, is involved in the in vitro cell growth signaling mediated by interleukin 2 and granulocyte-macrophage colony-stimulating factor. To investigate the in vivo functional role of AMSH, we have generated AMSH-deficient mice by gene targeting. The AMSH-deficient mice were morphologically indistinguishable from their littermates at birth, and histopathological examinations revealed normal morphogenesis in all tissues tested. However, all the AMSH-deficient mice exhibited postnatal growth retardation and died between postnatal day 19 (P19) and P23. Examination of brain sections at P6 demonstrated significant loss of neurons and apoptotic cells in the CA1 subfield of the hippocampus. Brain atrophy developed by P16 and was accompanied by complete loss of the CA1 neurons in the hippocampus and marked atrophy of the cerebral cortex. Furthermore, AMSH-deficient hippocampal neuronal cells were unable to survive in vitro, even in the presence of several stimulatory cytokines, while AMSH-deficient cerebellar neurons, thymocytes, and embryonic fibroblasts survived normally. Taken together, these observations indicate that AMSH is an essential molecule for the survival of neuronal cells in early postnatal mice.     

Fluoro Jade stains early and reactive astroglia in the primate cerebral cortex.

The fluorescent agent Fluoro Jade was applied to cortical brain sections obtained from human patients at early postnatal ages and in patients with Alzheimer's disease, and from a Cebus apella monkey after mechanical lesioning of the cerebral cortex. Fluoro Jade labeled reactive astrocytes and early differentiating astroglial cells.     

成年大鼠神经元和星形胶质细胞细胞周期蛋白表达的差异研究

目的比较研究成年大鼠细胞周期蛋白在神经元和星形胶质细胞的表达差异。方法应用免疫荧光和激光扫描共聚焦显微镜观察成年大鼠生理状态下大脑皮层或海马CA1区神经元和星形胶质细胞细胞周期素D1、E、A、B1、(CyclinD1、E、A、B1)的表达。结果成年大鼠海马CA1区和大脑皮层的神经元有Cyclin D1、E、A和B1的表达,细胞核和细胞浆均有表达,以胞核为主;星形胶质细胞也有上述细胞周期蛋白的表达但细胞数目较少,并且表达这些指标的星形胶质细胞多聚集在海马CA1区。结论成年大鼠大脑皮层和海马区的神经元和星形胶质细胞均表达细胞周期蛋白,而其在神经元的表达较星形胶质细胞更为普遍。     

Distribution of mGluR1α and SMI 311 immunoreactive Lugaro cells in the kitten cerebellum

The Lugaro cell is a feedback interneuron of the cerebellar cortex, recognizable by its characteristic morphology. Postnatal neuronal migration to the cortex has been described for several cerebellar interneurons. Since in our previous studies we observed Lugaro-like cells (LCs) in the white matter (WM) and internal granular layer (IGL) of the cerebellum of young cats, we assumed that a proportion of these cells migrate also postnatally to their destination. In the present study using and immunostaining for the metabotropic glutamate receptor mGluR1α and neurofilament protein SMI 311 the number and spatial distribution of LCs at different postnatal days were investigated. We found that the number and distribution of both mGluR1a-immunoreactive (ir) and of SMI 311-ir LCs changed with age in the developing cerebellar cortex of kittens: developing LCs express mGluR1α already in the newborn, while expression of SMI 311-ir in LCs appears only about a week later. At postnatal day 1 (P1) relatively few mGluR1-ir LCs were detected in the WM and at the border of WM and IGL. Later, their number increased sharply until P15 (6–7 fold) and decreased continuously between P15 and P135. SMI 311-ir LCs were not present at P1 and even at P8 only a few were observed in the WM or in infraganglionic positions. Their number increased gradually (12–14 fold) until adulthood when their number was stabilized at 8.000–10.000/cerebellum. At the same time the number of probably ectopic SMI 311-ir LCs decreased with age: at P22 about one third of them was found in “ectopic” position, whereas in the adult cat only about 10–12% of LCs's was either in the WM or scattered in the whole depth of the granular layer. These results suggest that: (1) most LCs appear in the cerebellar cortex postnatally; and (2) postnatal migration and incorporation of LCs to the cortex is a much longer process than previously expected, occurring even after the cytoarchitectonic built-up (about P65–P70 in cat) of the cerebellum.     

Regional changes in CNS levels of the S-100 and 14-3-2 proteins during development and aging of the mouse

The levels of the S-100 and 14-3-2 proteins were determined in a number of regions of mouse brain at intervals from 1 day to 30 months of age. Both S-100 and 14-3-2 were found in measurable amounts as early as the first day of postnatal age but did not begin to accumulate rapidly in the forebrain, brain stem and cerebellum of the mouse brain until some time between the 7th and 14th days. From days 14 to 28 the levels of S-100 and 14-3-2 in each region continued to increase rapidly with the exception of the forebrain where the rate of accumulation of S-100 appeared to lag considerably behind that in the other regions. The proteins continued to accumulate at a rapid rate until approximately 6 months of age. From 6 to 30 months of age, the levels of 14-3-2 remained relatively stable in cerebellum, hippocampus and hypothalamus and appeared to decrease slightly in striatum and cerebral cortex. In the case of S-100, the level of the protein increased in all regions of brain from 6 to 30 months but the increase was most pronounced in the hippocampus, hypothalamus and striatum. The principal conclusion derived from this study is that the biochemical development and aging of the central nervous system are regionally selective processes.     

A regional study of developing rat brain: The accumulation and distribution of proteolipid proteins

The accumulation and distribution of proteolipid proteins in rat brain and selected brain regions (cerebellum, cerebral cortex, basal ganglia, and hippocampus) were studied during early postnatal development. In whole brain an eightfold increase of proteolipid was observed between ten and 33 days after birth. This was reflected in the separate regions examined where the proteolipid protein content increased six- to ten-fold during the same period. The basal ganglia and cerebral cortex contributed the greatest amount to the total proteolipid present. However, at 28–33 days the greatest concentration (mg/g tissue) was observed in the basal ganglia and hippocampus. When the proteolipid protein preparations were examined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, distinctive, heterogeneous patterns for each brain region were obtained. Proteolipid from basal ganglia (the region richest in white matter) consisted primarily of two major protein bands with apparent molecular weights of approximately 21,500 and 26,000. Both of these bands dramatically increased in quantity during myelination, and the larger protein coelectrophoresed with isolated myelin proteolipid protein. Both bands were also found present in proteolipid preparations from the other brain regions but in varying amounts relative to the total. The data suggest that the increase in proteolipid observed during this developmental period was due in large measure to the accumulation of myelin-specific proteolipids, but also that a significant proportion of the increase was due to the accumulation of nonmyelin components.     

Expression of mRNAs Encoding Subunits of the NMDA Receptor in Developing Rat Brain

Abstract: Developmental changes in the levels of N -methyl- d -aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low-affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5' insert and with or without both 3' inserts. In cerebellum, however, the major NR1 variants were those containing the 5' insert and lacking both 3' inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur during development.     

Ethanol exposure alters neurotrophin receptor expression in the rat central nervous system: Effects of neonatal exposure

The detrimental effects of ethanol exposure during nervous system development have been well established. The cellular mechanisms of ethanol neurotoxicity, however, have not been clearly defined. Recent studies suggest that neurotrophin signaling pathways may be involved in ethanol-mediated neuronal death. The present investigation, therefore, was designed to examine ethanol-induced alterations in neurotrophin receptor protein levels in the developing central nervous system (CNS) following chronic ethanol treatment administered during the early neonatal period. For this study, rats were exposed to ethanol via vapor inhalation from postnatal day 4 (P4) to P10. Brains were then dissected on P10 or P21, and Western blots used to quantify expression of neurotrophin receptors TrkA, TrkB, TrkC, and p75. This early postnatal ethanol treatment produced significant alterations in receptor levels in hippocampus, septum, cerebral cortex, and cerebellum. The alterations seen were variable, with decreases generally found in hippocampus and cerebellum, increases noted in septum, and changes in both directions occurring in cortex. These alterations were generally more prevalent in males than in females. While most of the receptor changes observed were transient, sustained or delayed alterations were occasionally seen in hippocampus, cortex, and cerebellum. These results suggest that developmental ethanol exposure modulates expression of these neurotrophin receptors throughout the CNS, alterations which could have wide-ranging effects on functional CNS development. The possible linkage between such changes and abnormalities encountered in the fetal alcohol syndrome are considered.     

Enhanced Brain Cell Proliferation Following Early Adrenalectomy in Rats

We have previously demonstrated an increase in adult brain DNA content in rats adrenalectomized on postnatal day 11. The present studies examined cell proliferation in cerebral cortex, cerebellum, hippocampus, and midbrain-diencephalon following adrenalectomy at this age. Compared to sham-operated controls, adrenalectomized animals showed increased [3H]thymidine incorporation into DNA (measured at 1 h following a pulse injection) in all brain regions at 7 and 14 days postsurgery. In some areas, the effect was already present as early as 2 days following adrenalectomy. Chronic replacement with corticosterone prevented this increase in DNA labelling in a dose-dependent manner. When cell proliferation in the cerebral cortex and cerebellum was independently assessed by measuring changes in thymidine kinase activity, enzyme activity was significantly elevated in both areas at 7 and 14 days postsurgery. Finally, histological examination of the cerebellar cortex suggested a delayed disappearance of the external granular layer in several cerebellar lobules of adrenalectomized animals. Overall, these findings indicate that day-11 adrenalectomy leads to a prolonged stimulation of mitotic activity in areas where cell formation at this time is exclusively glial (i.e., cerebral cortex and mid-brain-diencephalon) as well as in areas where postnatal neurogenesis is also occurring (cerebellum and hippocampus). It is hypothesized that this stimulation results from the removal of a tonic inhibitory effect exerted by circulating glucocorticoids in the normal intact animal.