Novel target sites for estrogen action in the dorsal hippocampus: an examination of synaptic proteins.

Structural studies have shown that estrogens increase dendritic spine number in the dorsal CA1 field of rat hippocampus using Golgi impregnation as well as the number of dorsal CA1 synapses visualized via electron microscopy. The present study was carried out to further these findings by examining changes in the levels of pre- and postsynaptic proteins using radioimmunocytochemistry (RICC). In this study, 2 days of estradiol-benzoate treatment produced significant and comparable increases in synaptophysin, syntaxin, and spinophilin immunoreactivity (IR) in the CA1 region of the dorsal hippocampus of ovariectomized female rats. For spinophilin, IR was also increased in the hilar region of the dentate gyrus as well as CA3. In all cases, the nonsteroidal estrogen antagonist CI628, which has been previously shown to block spine formation, inhibited the effects of estrogen. However, these protein differences were not detected in whole hippocampus using Western blots. These findings add to a growing body of evidence that estrogens increase synapses in the CA1 region of hippocampus along with changes in previously unidentified sites. These results also suggest that RICC is a rapid and sensitive method for examining molecular changes in synaptic profiles in anatomically distinct brain regions.

Mitogen-stimulated lymphocyte proliferation and pituitary hormones in major depression.

To assess cellular immune status and the hypothalamic-pituitary (HP) axis in patients with major depression, we examined peripheral blood mononuclear cells (PBMC) and measured the plasma levels of cortisol, adrenocorticotropin hormone (ACTH), growth hormone (GH), and prolactin (PRL). Twenty patients with major depression were compared with 20 control subjects matched for age, sex, and race. The dose-response curves for concanavalin-A (Con-A) and phytohemagglutinin (PHA) stimulation were not significantly different between the two groups. The patients had decreased Con-A-stimulated T-lymphocyte proliferation when compared to the control subjects, but only at the lowest suboptimal concentration of Con-A. None of the four concentrations of PHA-stimulated proliferation were different between the two groups, neither was PHA-induced interleukin-2 production. Within the patient group only, plasma prolactin (PRL) correlated significantly with stimulated lymphocyte proliferation using two optimal concentrations of PHA and one optimal concentration of Con-A, when the proliferation was expressed using the stimulation index.

Autonomic nervous system innervation of thymic-related lymphoid tissue in wildtype and nude mice.

A study was undertaken to determine the source and terminal distribution of the autonomic nervous system (ANS) innervation of lymphoid tissue in the mediastinum of male and female B10 and Balb/c wildtype and syngeneic nude (nu/nu) mice. Acetylcholinesterase (AChE) histochemistry was used to localize this hydrolytic enzyme in neural tissue and glyoxylic acid fluorescent histochemistry was used to characterize catecholaminergic (CA) innervation. The thymus was innervated by AChE-positive fibers of the vagus, the recurrent laryngeal, and the phrenic nerves. Catecholaminergic innervation was derived from the stellate ganglia and other small ganglia of the thoracic sympathetic nervous system chain. Whereas intrinsic AChE-positive innervation of the thymus was evident at the cortico-medullary boundaries and under the capsule, CA innervation was observed along the trabeculae with perivascular plexuses at the cortico-medullary boundaries and interlobular septa. Free CA fibers were noted in the medulla and in the cortex, where they were often proximal to cortical autofluorescent (CAF) cells. The rudimentary thymus of the adult nude mouse received limited ANS innervation. Only a few CA or AChE-positive fibers were evident, with the majority of fibers associated with the acinar cells of the gland. Mediastinal lymph nodes in male and female wildtype mice varied in their distribution and were frequently found clustered around nerves and ganglia. The intrinsic innervation of lymph nodes by AChE-positive fibers was confined to the subcapsular marginal sinus, with a few vascular-associated fibers in the parenchyma. Catecholaminergic perivascular plexuses were present in the hilar zone, whereas some free fibers were noted under the capsule and in the parenchyma. The mediastinal lymph nodes of male and female nude mice when compared to the lymph nodes of male and female wildtype mouse were found to be increased in number and in distribution. Clusters of these lymph nodes were found in close proximity to ANS tissue. The intrinsic innervation of the nude mediastinal lymph nodes was comparable to that present in the wildtype mouse tissue. The functional implication of these data is discussed with regard to nervous:immune system interactions.

Ovarian steroids and the brain: implications for cognition and aging.

Ovarian steroids have many effects on the brain throughout the lifespan, beginning during gestation and continuing into senescence. These hormones affect areas of the brain that are not primarily involved in reproduction, such as the basal forebrain, hippocampus, caudate putamen, midbrain raphe, and brainstem locus coeruleus. Here we discuss three effects of estrogens and progestins that are especially relevant to memory processes and identify hormonal alterations associated with aging and neurodegenerative diseases. First, estrogens and progestins regulate synaptogenesis in the CA1 region of the hippocampus during the 4- to 5-day estrous cycle of the female rat. Formation of new excitatory synapses is induced by estradiol and involves N-methyl-D-aspartate (NMDA) receptors, whereas synaptic downregulation involves intracellular progestin receptors. Second, there are developmentally programmed sex differences in the hippocampal structure that mat help explain why male and female rats use different strategies to solve spatial navigation problems. During the period of development when testosterone is elevated in the male, aromatase and estrogen receptors are transiently expressed in the hippocampus. Recent data on behavior and synapse induction strongly suggest that this pathway is involved in the masculinization or defeminization of hippocampal structure and function. Third, ovarian steroids have effects throughout the brain, including effects on brainstem and midbrain catecholaminergic neurons, midbrain serotonergic pathways, and the basal forebrain cholinergic system. Regulation of the serotonergic system appears to be linked to the presence of estrogen- and progestin-sensitive neurons in the midbrain raphe, whereas the ovarian steroid influence on cholinergic function involves induction of choline acetyltransferase and acetylcholinesterase according to a sexually dimorphic pattern. Because of these widespread influences on these various neuronal systems, it is not surprising that ovarian steroids produce measurable cognitive effects after ovariectomy and during aging.

The effects of cortisone on acetylcholinesterase (AChE) in the neonatal and aged thymus.

Acetylcholinesterase (AChE) histochemistry and biochemistry was used to characterize the distribution and species of this enzyme within the developing thymus gland of the mouse. The results indicate that AChE-positive nerves and related structures are involved in a steroid-induced mechanism for regulating thymocyte populations. Low doses of cortisone injected into mice produce an activation of quiescent cholinergic nerves and the appearance of several new molecular forms of AChE within areas of the thymus where thymocyte death is prevalent. The action of cortisone on AChE is age dependent. In neonates, AChE activity is extremely high in the cortex of the gland, and cortisone causes little or no increase in AChE activity. In mice three to six weeks old, cortisone exerts its most profound effect on the AChE activity within the thymus. In mice eight months old and older, the AChE activity of the normal thymus is restricted to nerves and nerve-related structures at the cortical-medullary boundaries, with little or no activity observed in the cortex. Injections of cortisone in these mice does not cause an increase in AChE activity in the cortex and only slightly enhances activity within the cortico-medullary boundaries.

Nerve-related 3S acetylcholinesterase in murine thymus.

One form of acetylcholinesterase (AChE), 3S, has been identified in the thymus of normal mice as the predominant species. Histochemical studies show that the AChE is localized to nerves or to nerve-related tissues. The form isolated is composed of a salt and a detergent-sensitive fraction. Since the sedimentation values and the kinetics of the two fractions are identical, it is proposed that only one gene encodes for this globular 3S species of AChE.

Calcitonin gene-related peptide in the developing and aging thymus. An immunocytochemical study.

Calcitonin gene-related peptide (CGRP) is known to block Con A and PHA induced T cell proliferation. As a first step in determining the role of this peptide in T cell education and function we have studied the distribution of CGRP within the developing mouse thymus using immunocytochemistry. CGRP-like immunoreactivity (CGRP-IR) was found in the thymic nerves in close proximity to blood vessels in the 17-day-old embryonic mouse thymus. A discrete population of small cells at the cortico-medullary junction also stained intensely for CGRP. As the mouse thymus reached maturity (three to eight weeks) CGRP innervation became more dense, with fibers running along the vasculature at the cortico-medullary boundary, then branching into the cortical and medullary regions. Some fibers were invested in the blood vessels while a large portion formed varicosities among the cells of the thymus. In the mature thymus, the small CGRP-IR cortico-medullary cells were more numerous, and CGRP-IR was also found in subcapsular and trabecular mast cells. The pattern of innervation remained the same in the aging mouse thymus (six months), but there appeared to be somewhat fewer cortico-medullary cells and an increase in mast cell number. In the aged (eighteen months) thymus, the small CGRP-IR cortico-medullary cells were rarely seen, but mast cells were more numerous, most of which stained positively for CGRP, in the connective tissue. Nerves containing CGRP-IR generally had the same distribution as in the younger mice but appeared somewhat truncated. The distribution of CGRP-IR nerves in the mouse thymus at different stages of development was similar to that reported for cholinergic (AChE-positive) nerves. Since the brain-stem vagal nuclei have been shown by retrograde transport studies to project to the thymus as well as to contain CGRP-IR neurons, our findings suggest that CGRP-IR thymic nerves may be derived from the vagus complex.

Selective regulation of T-cell development and function by calcitonin gene-related peptide in thymus and spleen. An example of differential regional regulation of immunity by the neuroendocrine system.

In the course of our studies, we have shown the presence of calcitonin gene related peptide (CGRP) by immunocytochemistry in cell bodies and nerve fibers of the murine thymus and in a sparse innervation of the spleen. Receptors for CGRP have been characterized within these glands, and their activation by physiological levels of CGRP was found to suppress Con A-stimulated proliferation of thymocytes and splenic T cells as well as antigen-specific T-cell proliferation. This suppression is blocked by the antagonist for CGRP (CGRP 8-37). Within the thymus cultures, the antagonist CGRP (8-37) alone enhanced proliferation of thymocytes during Con A stimulation, most likely by inhibiting the endogenous release of CGRP into the culture medium by resident thymocytes. Some of the CGRP-induced suppression of mitogenic stimulation of thymocytes, but not of splenocytes, was due to apoptosis. The antagonist, CGRP(8-37), did not block apoptosis caused by Con A or CGRP but rather enhanced it. Flow cytometric analysis of CGRP-treated cultures using antibodies to cluster determinates (CD) showed that the majority of thymocytes undergoing apoptosis induced by CGRP were of the CD4/CD8 double-positive type. These data indicate that apoptosis in the thymocytes is mediated by a CGRP receptor not sensitive to the antagonist CGRP(8-37). Because proliferation of thymocytes and splenocytes induced by Con A is blocked by this antagonist and splenocytes are refractory to CGRP induced apoptosis, CGRP appears to mediate at least two separate functions on subpopulations of thymocytes and T cells via two different CGRP receptors within the gland. These effects of a neuropeptide exemplify the phenomenon of differential regional regulation of immunity by the autonomic and neuroendocrine systems.

The role of calcitonin gene-related peptide in the mouse thymus revisited.

Calcitonin gene-related peptide has been identified by immunocytochemistry within the thymus of fetal through aged adult mice. Calcitonin gene-related peptide positive nerves are observed from embryonic day 17 throughout the lifespan of the mouse. A sparse cell population positive for CGRP is first observed during the late embryonic period at the corticomedullary boundary and the medulla, and it becomes more densely distributed in this region in the adult. In the thymus of the aged mouse the number of CGRP-positive cells diminishes. Pharmacologic studies demonstrated that fresh thymocytes display a receptor Kd for CGRP of 1.17 +/- 0.06 x 10(-10)M and a Bmax of 12.7 +/- 4.7 fmol/mg protein. Functional studies indicate that CGRP is a potent inhibitor of mitogen and antigen-stimulated proliferation of T cells and that it inhibits IL-2 production in cloned splenic T cells. Recent studies suggest that endogenous CGRP may serve as a natural inhibitor of inappropriate induction of mature, antigen-sensitive cells in the thymus as well as play a role in thymocyte education. These findings are discussed in terms of the distribution of CGRP cells and nerve terminals within the thymus and their relationship to positive and negative selection of the T-cell repertoire.

Calcitonin gene-related peptide level in the rat dentate gyrus increases after damage.

Calcitonin gene related peptide-like immunoreactivity (CGRP-LI) was examined in rat dentate gyrus (DG) following damage to granule cells by adrenalectomy or intrahippocampal colchicine injections. In normal DG, CGRP-LI was present in a diffuse hand within the inner third of the molecular layer (ITML) and in hilar cells. Following the experimental procedures, levels of CGRP-LI increased bilaterally in the ITML and in hilar interneurons resembling mossy cells. Ultrastructural analysis of the ITML revealed that CGRP-LI is associated with large, dense-core vesicles within axon terminals which form asymmetrical synapses with dendritic spines, and within dendritic spines. The increase in CGRP-LI level following granule cell damage suggests a protective role for CGRP in the response to hippocampal injury.

The derivation and characterization of neuronal cell lines from rat and mouse brain.

This study shows that permanent cell lines can be established from rat and mouse brain by direct tissue culture methodology without the aid of exogenous chemical or viral transforming agents. These cells were derived from specific areas of the brain, such as the cerebellum and hippocampus, at chosen times during fetal and neonatal development. Success in establishing neuronal cell lines was dependent upon the use of selection pressures designed to keep the background of glial cells and fibroblasts at a minimum. These manipulations included care in the choice and processing of the original tissue, utilization of cytotoxic anti-glial sera, and continuous manual isolation of cells with neuronal morphology. Slow-growing nerve cells were thus allowed to adapt spontaneously to culture with a minimum of competition from faster-adapting cell types. Many of these cell lines are judged to be neuronal on the basis of their electrical excitability and their characteristic surface antigens. The cells respond positively in a sodium flux assay which has been shown to correlate well with the ability to generate an action potential, and also express one or more of three antigens previously found to be specific for nerve cells.

Volumetric structural magnetic resonance imaging (MRI) of the rat hippocampus following kainic acid (KA) treatment.

An in vivo MRI study employing a high field (7T) magnet and a T1- and T2-weighted imaging sequence with subsequent histopathological evaluations was undertaken to develop and evaluate MRI-based volumetric measurements in the rat. The brain structures considered were the hippocampus, the cingulate cortex, the retrosplenial granular cortex and the ventricles. Control (n=3) and kainic acid (KA; n=4) treated rats were scanned 10 days following the manifestation of stage four seizures. The MRI images exhibited anatomical details (125 microm in-plane resolution) that enabled volumetric analysis with high intra-rater reliability. Volumetric analysis revealed that KA-treated rats had significantly smaller hippocampi, and a significant increase in ventricular size. The cingulate cortex and the retrosplenial granular cortex did not differ in volume between the two groups. The histological observations supported the MRI data showing neuronal loss and neuronal degeneration in CA1 and CA3 of the hippocampus, which was accompanied by strong microglia activation. These data demonstrate a reliable and valid method for the measurement of the rat hippocampus in vivo using MRI with a high field magnet, thereby providing a useful tool for future studies of rodent models of neuro-degenerative diseases.

Volumetric measurement of the hippocampus, the anterior cingulate cortex, and the retrosplenial granular cortex of the rat using structural MRI.

MRI imaging of the rodent brain is a rapidly growing field in the neurosciences. Relatively limited information is available for regional volume determination. The present paper describes a reliable method for the assessment of the hippocampus, the anterior cingulate cortex, the retrosplenial granular cortex and the ventricles in rats. MRI scans were acquired using a 7 T magnet. The anatomical sampling method was found to be highly reliable with an intra-rater reliability of greater than 0.93. The current protocol should facilitate future in vivo neuroimaging research using animal models of neurodegenerative diseases.

Immune cells and the hypothalamic-pituitary axis in major depression.

To explore changes in immune cell status with changes in the hypothalamic-pituitary (HP) axis in 20 patients with major depression as compared with 20 age-, sex-, and race-matched control subjects, we examined peripheral blood mononuclear cells (PBMC) for total T-cells (T3), total B-cells (B1), two T-cell subsets (T4 and T8), and natural killer cells (NKH1), and we measured the plasma level of cortisol, adrenocorticotropic hormone (ACTH), growth hormone (GH), and prolactin (PRL). The ratio of T4/T8 was increased in the patients. Within the group of control subjects only, increasing age correlated significantly with decreasing plasma PRL. Within the group of patients only, GH positively correlated significantly with T8 and NKH1, as did PRL with NKH1. No between-groups difference was found for T3, B1, T4, T8, NKH1, cortisol, ACTH, GH, or PRL.

A comparative study of the autonomic nervous system innervation of the thymus in the mouse and chicken.

It is well-established that the thymus of rats and mice are directly innervated prenatally by neurons of the central nervous system (CNS). Evidence now suggests that this innervation is involved in determining the neuroendocrine-immune functions of the gland. In this study an analysis of CNS innervation of the chicken thymus was carried out using the horseradish peroxidase (HRP) retrograde transport method to establish the phylogenetic integrity of CNS-thymic innervation and to gain insight into its function. Day-old chicks received HRP injections into upper of lower thymic lobes. Lower injections resulted in HRP-labeled neurons in the ventral horn of the cervical spinal cord and in the dorsal and ventral brainstem complex of the nucleus of X. Injections into upper thymic lobes resulted in similar spinal cord labeling and in labeling of neurons in the nuclei of both IX and X. These injections produced more labeled neurons in the ventral complex of X than did injections into lower thymic lobes. Unilateral HRP injections into upper or lower thymic lobes produced predominantly ipsilateral labeling in the appropriate areas. The results of this study indicate that chick thymuses are innervated by the CNS during embryogenesis. Furthermore, there appears to be a topographical distribution of nerve fibers suggesting discrete functions for individual lobes.

Dual-label retrograde transport: CNS innervation of the mouse thymus distinct from other mediastinum viscera.

Two different retrogradely transported tracers were identified and used to distinguish between the innervation of the thymus gland and the innervation of adjacent tissues residing within the mouse mediastinum. To determine the best procedure for peripheral dual-labeled retrograde transport, several different tracers, vehicles, and injection protocols were developed and/or tested. In the periphery, it proved ineffective to use Rhodamine latex microspheres (Luma-Fluor) as a retrograde transport agent. Horseradish peroxidase (HRP) suspended in the water-soluble carrier KY Jelly and Fluoro-Gold (FG) in an aqueous solution was the most effective combination of retrograde transport tracers. Equivalent doses of each of these tracers were determined, and injections of HRP or FG into the thymus and the esophagus produced distinct and separate patterns of labeled neurons in the spinal cord or brain stem. Throughout these CNS areas, some neurons contained both tracers suggesting that 1) these areas are innervated by neurons that give rise to collaterals to these tissues, 2) that the retrograde tracers are transported by nerves that innervate the lymphatics of the mediastinum, and/or 3) that some diffusion of the tracers occurs from the test organs and tissues to other organs and tissues of the mediastinum.

Characterization of choline O-acetyltransferase (ChAT) in the BALB/C mouse spleen.

The synthesizing enzyme, Choline-O-acetyl transferase (ChAT) (EC 2.3.1.6) and the degradation enzyme, acetylcholinesterase (EC 3.1.1.7) for the neurotransmitter acetylcholine, have been anatomically and biochemically characterized in the thymus of the BALB/C mouse. In the present study we continue to analyze the possibility of cholinergic immunomodulation of immune tissues by determining if ChAT is present in the BALB/C mouse spleen. Our enzymatic evaluation of ChAT activity in splenic extracts revealed .05 nmoles/min/mg protein as compared to .1 nmoles/min/mg of protein activity in controls prepared from whole brain extracts. No detectable levels of ChAT activity were observed in the serum. Immunoblotting and immunoprecipitating using the anti ChAT monoclonal antibody, MB16, demonstrated two bands in the brain and one band in the spleen. Membrane bound ChAT in the brain was composed of two subunits with apparent molecular weights of 28 and 50 kDa. The spleen demonstrated only one form of ChAT with an apparent molecular weight of 28 kDa. Immunoprecipitation of the enzyme from both the brain and spleen resulted in a recovery of 59% and 60% of the activity respectively.

Innervation of the thymus gland by brain stem and spinal cord in mouse and rat.

Central nervous system (CNS) projections to the thymus were studied in the mouse and rat using the horseradish peroxidase (HRP)-retrograde transport method. With discrete HRP injections localized to the thymus, labeled neurons are evident in both medulla and spinal cord. In the medulla the largest population of labeled neurons is present in the retrofacial nucleus. Within this cytoarchitectonically distinct nucleus, the majority of neurons are labeled with large HRP injections in the thymus. In addition to retrofacial nucleus, scattered labeled neurons are found throughout the rostrocaudal extent of the nucleus ambiguus and in the dorsal medullary tegmentum adjacent to the dorsal motor vagus nucleus. With HRP injections restricted to thymus parenchyma, no labeled neurons are evident in the dorsal motor vagus nucleus. Three groups of spinal cord neurons are labeled. In segments C2-C4, neurons localized to the ventral horn are labeled in two distinct columns, one located lying laterally in the ventral horn and the other located medially. Labeling of neurons in these segments is distinct from that of large motor neurons located medially in the ventral horn extending from the level of the decussation of the pyramids through the C1 segment. The location and sizes of neurons labeled in these areas following HRP injection in the thymus are identical in the mouse and rat. These observations provide evidence for previously unknown projections from spinal cord and brain stem to the thymus which may play an important role in the regulation of thymic function.