Parallel development of noradrenergic innervation and cellular compartmentation in the rat spleen

By combining neurochemical measurement of norepinephrine (NE) with double-label immunocytochemistry for tyrosine hydroxylase-positive (TH+) noradrenergic nerves and specific lymphoid markers, we have examined the developmental compartmentation of noradrenergic nerves in the rat spleen. TH+ nerve fibers were present in the white pulp of the spleen at birth, among surface IgM-positive (sIgM+) B lymphocytes at the outer border of the periarteriolar lymphatic sheath (PALS), distant from the central artery. During the first 7 days, noradrenergic innervation developed rapidly, forming plexuses of nerve fibers along the central artery and its branches, among T and B lymphocytes of the PALS, and along the developing marginal sinus where ED3+ macrophages accumulate. The splenic concentration of NE (per mg wet wt.) and 3-methoxy-4-hydroxy-phenetheleneglycol (MHPG), a NE metabolite, increased rapidly during this period, suggesting that NE is available and released from these nerves. From 7-14 days, the white pulp expanded to include an inner PALS, outer PALS, marginal sinus, and marginal zone; during this period, TH+ fibers arborized principally among T lymphocytes of the inner PALS and adjacent to macrophages along the marginal sinus. By 14 days of age, NE concentration reached adult levels, although the MHPG/NE ratio (an index of NE turnover) remained higher throughout development than in adulthood. Finally, from 14-28 days, the outer PALS expanded to include follicles containing sIgM+ B lymphocytes. At the earliest stages of follicular development, a parafollicular rim of noradrenergic fibers was present, providing occasional branches which arborized within the follicle. No further changes were observed in either noradrenergic innervation or cellular compartmentation after 28 days of age. These findings suggest that noradrenergic fibers are present in developing compartments of the spleen at the earliest stages of their development, providing norepinephrine for interaction with a variety of adrenoceptor-bearing lymphoid and nonlymphoid cells.     

Autoimmune T cells retard the loss of function in injured rat optic nerves

We recently demonstrated that autoimmune T cells protect neurons from secondary degeneration after central nervous system (CNS) axotomy in rats. Here we show, using both morphological and electrophysiological analyses, that the neuroprotection is long-lasting and is manifested functionally. After partial crush injury of the rat optic nerve, systemic injection of autoimmune T cells specific to myelin basic protein significantly diminished the loss of retinal ganglion cells and conducting axons, and significantly retarded the loss of the visual response evoked by light stimulation. These results support our challenge to the traditional concept of autoimmunity as always harmful, and suggest that in certain situations T cell autoimmunity may actually be beneficial. It might be possible to employ T cell intervention to slow down functional loss in the injured CNS.     

Effects of streptozotocin diabetes on the noradrenergic innervation of the rat heart: A longitudinal histofluorescence and neurochemical study

FELTEN, S. Y., R. G. PETERSON, P. A. SHEA, H. R. BESCH, JR. AND D. L. FELTEN. Effects of Streptozotocindiabetes on the noradrenergic innervation of the rat heart: A longitudinal histofluorescence and neurochemical study. BRAIN RES. BULL. 8(6) 593–607, 1982.—The effects of the age of induction and total duration of Streptozotocin diabetes on the sympathetic noradrenergic innervation of the rat heart was examined with glyoxylic acid induced histofluorescence to demonstrate the distribution of noradrenergic fibers within the heart, and with high performance liquid chromatography with electrochemical detection to measure tissue levels of the neurotransmitter norepinephrine. Diabetes was induced in male Sprague-Dawley rats at 1, 2, and 4 months of age. Within each of these groups, diabetic rats survived for periods of 1, 2, and 4 months. Additional groups of diabetic rats survived to a chronological age of 8 months. Norepinephrine levels in the hearts of diabetic rats were increased over those of control rats in all groups at 1 month duration of diabetes. Ventricles were generally affected to a greater extent than atria. At 2 months duration of diabetes, ventricular levels remained elevated while atrial norepinephrine levels were at or below control levels. At 4 months duration of diabetes, and in all groups at 8 months of age, the norepinephrine levels were at or below control levels, except in the ventricles of rats induced at 4 months of age, which remained elevated. Histofluorescence studies demonstrated the presence of axon bundles and varicose noradrenergic profiles in the diabetic rat hearts, distributed in a pattern similar to that seen in controls. However, at 1 month duration of diabetes in all groups, the density of noradrenergic varicosities in diabetic rat hearts appeared increased with abundant branched profiles. These results are surprising, since studies on genetic models of diabetes have suggested decreased norepinephrine levels in the heart. The present study suggests that during the early phases of Streptozotocin induced diabetes, noradrenergic nerves are still intact and may be susceptible to pharmacologie manipulation. The later fall of norepinephrine levels back to or below control levels may indicate actual neuronal damage, suggesting that early intervention may be necessary to protect these nerves from degeneration. This issue is potentially important in view of the reported toxic effects of high NE levels on the heart, and the high incidence of death from myocardial infarct in diabetic humans with autonomie neuropathy.     

Biochemical mapping of noradrenergic nerves arising from the rat locus coeruleus

Norepinephrine (NE) concentration was measured in discrete brain regions of the rat following unilateral electrolytic lesions of the locus coeruleus, to determine the distribution of its noradrenergic neurons. Discrete brain nuclei and subdivisions were dissected from frozen sections, and norepinephrine was measured by a sensitive radio-isotopic assay.A significant reduction by 29–63% of control values in norepinephrine content was observed ipsilateral to the lesion in the following areas: all portions of the cerebral cortex examined (entorhinal, hippocampal, cingulate, parietal, and occipital areas), anterior half of the cerebellar cortex, hypothalamic periventricular and paraventricular nuclei, anterior ventral thalamic nucleus, ventral thalamic nucleus, and habenula. It appears that these regions receive unilateral innervation by axons from the locus coeruleus. In 3 regions (the medial geniculate body, inferior colliculus, and posterior half of the cerebellum), NE was reduced bilaterally in a pattern suggesting bilateral innervation from the locus coeruleus. Since no reduction in NE concentration occurred in the medial preoptic nucleus, nucleus interstitialis stria terminalis (ventralis), dorsomedial hypothalamic nucleus, or medial forebrain bundle, axons from noradrenergic neurons in the locus coeruleus do not appear to innervate these regions.The biochemical mapping of noradrenergic nerves from the locus coeruleus is discussed in relation to distribution studies based on the histofluorescence method.     

Effects of interleukin-2 on the expression of corticotropin-releasing hormone in nerves and lymphoid cells in secondary lymphoid organs from the Fischer 344 rat

This study examined the influence of interleukin (IL)-2 on corticotropin releasing hormone (CRH) immunoreactivity in the Fischer 344 (F344) rat spleen. Rats were given either vehicle or 1, 10, 25, 50, 100, or 200 ng of human recombinant (hr)IL-2 by intraperitoneal (i.p.) injection, and were sacrificed 0.5, 1, 4, 12, or 24 h after treatment. Spleens and mesenteric lymph nodes were prepared for immunocytochemistry to localize CRH. In spleens from vehicle-treated animals, CRH immunoreactivity was present in several types of cells of the immune system, but CRH(+) nerves were not observed in either spleens or lymph nodes from vehicle-treated animals. Treatment with IL-2 induced CRH expression in nerves in the spleen in a dose- and time-dependent manner. CRH(+) nerves were not found in the mesenteric lymph nodes after IL-2 treatment, instead a dramatic time- and dose-dependent accumulation of CRH(+) cells (resembling small lymphocytes and large granular mononuclear cells) in the cortex and medulla. These findings indicate that IL-2 stimulates the synthesis of CRH in nerves that innervate the F344 rat spleen, and promote the appearance of CRH(+) immunocytes into draining mesenteric lymph nodes.     

Age-related changes in noradrenergic sympathetic innervation of the rat spleen is strain dependent

Previous findings from our laboratory revealed an age-related decline in noradrenergic (NA) sympathetic innervation of the spleen in male Fischer 344 (F344) rats. The purpose of this study was to determine whether other rat strains also progressively lose NA sympathetic nerves in the aging spleen. Sympathetic innervation of spleens from 3- and 21-month-old male F344, Brown Norway (BN), BN X F344 (BNF(1)), and Lewis rats was examined using fluorescence histochemistry to localize catecholamines combined with morphometric analysis and using high-performance liquid chromatography with electrochemical detection for measuring norepinephrine (NE). Neurochemistry revealed a significant age-related decline in NE concentrations in spleens from F344 and Lewis rats. In contrast, there was no effect of age on splenic NE concentrations in BN or BNF(1) rats. Consistent with neurochemical analysis, fluorescence histochemistry revealed a striking decline in NA innervation of spleens from old F344 and Lewis rats not observed in the other two strains. However, in BN and BNF(1) rats, nerve fibers were diminished in distal portions of the spleen but not in the hilar regions. Morphometric analysis confirmed neurochemical and histological findings, revealing approximately 65-70% loss in NA nerve density in spleens from F344 and Lewis rats. These findings indicate that age-related changes in sympathetic innervation of the rat spleen are strain-dependent. Whether the loss of sympathetic nerves in spleens from F344 and Lewis rats is associated with age-related changes in the splenic microenvironment remains to be determined. The functional significance of altered sympathetic innervation of the spleen with advancing age is discussed.     

Origin of noradrenergic innervation of the spleen in rats

Noradrenergic (NA) sympathetic innervation of the spleen was examined in young adult Sprague-Dawley rats (200-250 g) following surgical removal of the superior mesenteric-celiac ganglia (SM-CG) and/or bilateral transection of the subdiaphragmatic vagus nerve. Sham-operated and unoperated rats served as controls. NA sympathetic innervation of spleens from sham-operated and unoperated controls, and from vagotomized rats, was qualitatively similar, with fibers distributing to the capsule, trabeculae, vasculature, and parenchyma of the white pulp. Complete ganglionic extirpation resulted in almost total denervation of NA fibers in all compartments of the spleen. High-performance liquid chromatography with electrochemical detection (LCEC) for catecholamines (CA) and quantitative morphometry of the density of NA varicosities confirmed these observations. LCEC revealed a greater than 85% depletion of norepinephrine (NE) in the spleen following superior mesenteric-celiac ganglionectomy. Stereological evaluation of NA varicosities with a point counting method revealed a decline of 99% in the volume density of NA terminals that occurred uniformly in all compartments of spleens from ganglionectomized rats. In addition, stereological analysis revealed a loss of total NA varicosities (approximately 31% decrease) in spleens from sham-operated rats. This loss in volume density occurred largely due to a loss in parenchymal fibers (approximately 45% decrease). Bilateral subdiaphragmatic vagotomy blocked the effect on NA innervation produced by the surgical stress of sham operation. Retrograde tracing following injection of either fluorogold or true blue into the spleen, coupled with immunocytochemical localization of tyrosine hydroxylase (TH), demonstrated abundant fluorogold (true blue)-labeled neurons in the SM-CG; many, but not all, of these neurons also were TH-positive. These findings indicate that the SM-CG neurons supply NA innervation to the spleen, providing sympathetic innervation as the second neuron in the classical two-neuron sympathetic chain, and suggest additional non-NA innervation of the spleen as well. This study also suggests that surgical stress of sham operation may alter directly the NA innervation of the spleen, possibly by inducing temporary retraction of NA fibers of the parenchymal compartment, which is likely to reduce the availability of NE for interaction with cells of the immune system that possess adrenoceptors and are present adjacent to NA varicosities in this region.4+ Bilateral vagotomy ameliorated the effects of sham operation on NA innervation; since the vagal nerve does not distribute fibers to the spleen, this effect is likely to occur through altered feedback circuits effecting sympathetic outflow, or through altered neuroendocrine outflow.(ABSTRACT TRUNCATED AT 400 WORDS)     

Most ganglion cells in the rat adrenal medulla are noradrenergic

Nerve cells are located in the rat adrenal medulla but their transmitter phenotype has not yet been determined. In-situ hybridization histochemistry was used to investigate ganglion cells with reference to their putative transmitter. High expression of dopamine beta-hydroxylase (DBH), neuropeptide tyrosine (NPY) and nerve growth factor receptor (NGF-R) mRNA was found in many of these neurons, whereas no detectable labelling was seen using a probe complementary to choline acetyltransferase mRNA. Chromaffin cells were also labelled with the DBH, NPY and NGF-R probes. We propose that the DBH/NPY/NGF-R-positive adrenal medullary ganglion cells are sympathetic postganglionic neurons producing noradrenaline. However, other types of ganglion neurons may also be present in the adrenal medulla.     

Involvement of noradrenergic nerves in the activation and clonal deletion of T cells stimulated by superantigen in vivo

Superantigens, like staphylococcal enterotoxin B (SEB), induce a strong proliferative response followed by clonal deletion of a substantial portion of defined Vbeta T cells. The remaining cells display in vitro anergy. We found that the immune response to SEB was paralleled by biphasic changes in the activity of the sympathetic nervous system. Furthermore, sympathetic denervation resulted in decreased SEB-induced cell proliferation and IL-2 production, and impeded the specific deletion of splenic CD4Vbeta8 cells observed in intact animals without affecting anergy. These studies provide the first evidence of an immunoregulatory cross-talk between sympathetic nerves and superantigen-activated immune cells.     

A study of axonal diameters and areas in lumbosacral roots and nerves in the rat

There has been debate as to whether there is a size difference between central and peripheral processes of dorsal root ganglion cells. In the present study, the mean areas of myelinated and unmyelinated fibers are measured as 27.8 micron2 and 0.55 micron2, respectively, in peripheral nerves and 13.72 micron2 and 0.14 micron2 in dorsal roots. Thus myelinated central processes of dorsal root ganglion cells have mean areas 50% less than the mean areas of the myelinated sensory axons in the same peripheral nerves, and the mean diameters of the central myelinated axons are 30% less than the peripheral myelinated axons. The mean areas of the unmyelinated sensory axons in the dorsal roots are 25% of the mean areas of the unmyelinated sensory unmyelinated axons are 50% of the mean diameters of the unmyelinated sensory axons in the same peripheral nerves. These data indicate that both myelinated and unmyelinated central processes of dorsal root ganglion cells are smaller than the peripheral processes of these same cells for lumbosacral segments in the rat. It is shown that axonal tapering is not responsible for these striking differences. Finally, documentation of differences in myelinated fiber histograms from dorsal roots of different segments in the rat is provided.     

Early experience, stressful dysregulation of the hormonal axis and age-related vulnerability to neurodegenerative processus: a longitudinal study in the rat

The deleterious effects of particular environmental situations have been suspected to augment the repercussions of cerebral injuries leading to increased vulnerability during ageing. The relationship between the hormones of the hypothalamo-pituitary-adrenal axis, mainly glucocorticosteroids, and cerebral structures like the hippocampus has been the subject of intense investigation in the recent years. Data suggest that long-term elevated blood levels of these hormones can induce neuronal alterations leading to cognitive dysfunction. This hypothesis has been tested with relevant animal models of normal/abnormal ageing. The models are based on the existence of considerable inter-individual differences in the degree of age-related cognitive impairments observed in rodents. Results show that long-term glucocorticosteroid exposure induces cerebral changes related to the action of these hormones on their central receptors. Experimental data are in accordance with clinical investigations suggesting that hormonal changes, and chronic life events, could be considered as a predictive factor of future cognitive dysfunction.     

Effect of oestrogen and progesterone on noradrenergic nerves and on nerves showing serotonin-like immunoreactivity in the basilar artery of the rabbit

The effects of oestrogen and progesterone on noradrenergic nerves and nerves with serotonin (5-HT)-like immunoreactivity in the basilar artery were investigated in the rabbit using whole-mount stretch preparations. The noradrenergic nerves were demonstrated by glyoxylic acid fluorescence histochemistry and nerves with 5-HT-like immunoreactivity by indirect immunofluorescence techniques. Quantitative image analysis of fluorescent nerve fibres revealed that nerve density and varicosity diameter of nerves with 5-HT-like immunoreactivity were significantly (P less than 0.01 and P less than 0.05 respectively) reduced after 4-week administration of oestrogen and the intensity of fluorescence was also reduced. However, there were no significant changes after progesterone. Neither oestrogen nor progesterone had any effect on noradrenergic innervation. The findings are discussed in relation to higher incidence of migraine headaches in females taking oral contraceptives.     

Autonomic nervous system innervation of lymphoid territories in spleen: a possible involvement of noradrenergic neurons for prion neuroinvasion in natural scrapie

In experimental as well as in natural scrapie, transmissible spongiform encephalopathies (TSEs), the infectious agent, closely related to PrPsc, an abnormal isoform of the cellular prion protein, invades and replicates in lymphoid organs such as spleen before affecting the brain. To clarify the cellular requirements for the possible neuroinvasion of scrapie agent from the spleen to the central nervous system, we have studied the sympathetic innervation within the lymphoid territories of the spleen. These noradrenergic fibers originating from the coeliomesenteric ganglia were examined with regard to PrPsc-associated cells in spleen of adult sheep severely affected with natural scrapie. Using a double immunolabelling strategy, we demonstrated the proximity of noradrenergic endings with PrPsc-accumulating cells, strengthening its possible implication in the neuroinvasion process.     

Long-term effects of prenatal stress and postnatal handling on age-related glucocorticoid secretion and cognitive performance: a longitudinal study in the rat.

There is growing evidence that stress during prenatal and postnatal periods of life can modify adaptive capacities in adulthood. The hypothalamo-pituitary-adrenal axis may mediate an animal's responses to perinatal stressful events and thus serve as a neurobiological substrate of the behavioural consequences of these early events. However, little is known about the long-term effects of prenatal stressors throughout the entire life of the animals. The focus of the present study was to examine the long-term influences of a prenatal and postnatal stress on glucocorticoid secretion and cognitive performance. Prenatal stress of rat dams during the last week of pregnancy and postnatal daily handling of rat pups during the first 3 weeks of life were used as stressors. The long-term effects of these manipulations were analysed using a longitudinal approach throughout the entire life of the animals, and were repeatedly tested in adulthood (4-7 months), middle age (13-16 months) and in later life (20-24 months). The study demonstrated that prenatal stress and postnatal handling induced opposite effects on both glucocorticoid secretion and cognitive performance. Prenatal stress accelerated the age-related hypothalamo-pituitary-adrenal axis dysfunctions; indeed, circulating glucocorticoids levels of prenatally stressed middle-aged animals are similar to old control ones, and also induced cognitive impairments. In contrast, postnatal handling protected from the age-related neuroendocrine and behavioural alterations. These results show that the altered glucocorticoid secretion induced by early environmental manipulations is primary to the cognitive alterations observed only later in life and could be one cause of age-related memory deficits.     

Prolactin-releasing peptide-immunoreactivity in A1 and A2 noradrenergic neurons of the rat medulla

Distribution of prolactin-releasing peptide-like immunoreactivity (PrRP-LI) was investigated in the rat medulla with the use of a rabbit polyclonal antiserum against the human PrRP-31 peptide. PrRP-positive neurons were noted mainly in two areas of the caudal medulla: ventrolateral reticular formation and commissural nucleus of the nucleus of the solitary tract (NTS), corresponding to the A1 and A2 areas. PrRP-LI neurons were absent in the medulla rostral to the area postrema. Double-labeling the sections with PrRP antisera and tyrosine hydroxylase (TH) monoclonal antibodies revealed extensive colocalization of PrRP- and TH-like immunoreactivity (TH-LI) in neurons of the A1 and A2 areas. Our results show that PrRP-LI is expressed in a population of A1 and A2 noradrenergic neurons of the rat caudal medulla.     

The noradrenergic system in cultured aggregates of fetal rat brain cells: Morphology of the aggregates and pharmacological indices of noradrenergic neurons

Spherical aggregates formed rapidly in culture by re-aggregation of trypsin-dissociated brain cells from the 17-day-old fetal rat. Over days 10 days an initially random distribution of cells evolved into a 30layered arrangement; cells with characteristics of neurons were found largely in the intermediate layer. The survival of neuronal and glial cell types was evaluated histologically and verified by electron microscopy, which revealed synaptic and myelin structures that rapidly increased in number after 18 days in culture. Levels of norepinephrine (NE) and dopamine (DA) reached peaks of 9.5 and 4.4 ng/mg protein, respectively, at culture day 21. Uptake of [³H]NE paralleled these amine levels and was blocked by desipramine or pretreatment with either reserpine or 6-OH-DA. Autoradiographs of aggregates labeled with [³H]NE showed a high density of silver grains over cells, apparently neurons, with branching processes traced for 120 μm. Previously accumulated [³H]NE was released under depolarizing conditions (high [K⁺] or vertridine) only in the presence of Ca²⁺. Release was induced to a lesser extent by kainic > glutamic acid. Thus, such aggregates appear to contain catecholaminergic neurons capable of synthesis, uptake and release of NE. The time course of development of these functions supports suggestions that aggregate preparations might be useful in studying neurochemical or morphological aspects of brain development and function in vitro.     

Afferent and efferent connections of the A5 noradrenergic cell group in the rat

The supraspinal afferent and efferent connections of the A5 noradrenergic cell group were examined in rats. Very small deposits of HRP-WGA were made in the rostral A5 area. Catecholamine histofluorescence techniques were used to confirm that the deposits overlapped the A5 column. Retrogradely labeled cells were present in the perifornical area and paraventricular nucleus of the hypothalamus, the K?lliker-Fuse nucleus, dorsal parabrachial area, intermediate and caudal portions of the nucleus of the solitary tract, and the ventral medullary reticular formation in the areas of the A1 and B1 cell groups. Anterograde HRP-WGA labeling was found in several areas of the subcortical CNS. The contribution of A5 neurons to this labeling was confirmed with retrogradely transported fluorescent latex microspheres combined with catecholamine histofluorescence techniques. The A5 cell group was found to have significant projections to the central nucleus of the amygdala, perifornical area of the hypothalamus, midbrain periaqueductal gray, parabrachial area, and the nucleus of the solitary tract. Other A5 projections include the paraventricular nucleus of the thalamus, the bed nucleus of the stria terminalis, and possibly the zona incerta and lateral and dorsal hypothalamic areas. In addition, A5 neurons may innervate the ventrolateral reticular formation of the medulla. Virtually all of the areas innervated by A5 noradrenergic neurons are involved in cardiovascular regulation. In addition, the A5 area receives afferent input from major cardiovascular regulatory centers of the supraspinal CNS. Thus the A5 cell group has the potential to exert a significant influence on the cardiovascular regulatory system.     

Cerebrovascular nerve fibers immunoreactive for tryptophan-5-hydroxylase in the rat: distribution, putative origin and comparison with sympathetic noradrenergic nerves

The distribution of serotonergic nerves in major basal and isolated small pial arteries (diameter > or = 50 microns) was investigated immunohistochemically using an antibody directed against tryptophan-5-hydroxylase (TPOH), the rate-limiting enzyme in the synthesis of 5-hydroxytryptamine (5-HT or serotonin), and compared to that of the noradrenergic system labeled for the selective noradrenaline (NA) synthesizing enzyme, dopamine-beta-hydroxylase (DBH). In addition, the possible peripheral and/or central origins of the cerebrovascular serotonergic (TPOH-positive) nerve fibers were examined. Strongly labeled TPOH-immunoreactive (TPOH-I) fiber bundles were observed in major basal arteries and gave rise to small varicose fibers organized in a meshwork pattern. The highest density of TPOH-I fibers was found in the middle cerebral artery followed by the anterior cerebral and the anterior communicating arteries, with a moderate to low density in the internal carotid and the vertebro-basilar trunk. Of the isolated pial arteries, only the larger ones (diameter > 75 microns) were significantly endowed with TPOH-I varicose fibers. However, free floating TPOH-I nerves were observed coursing through the pia-arachnoid membranes and reaching small pial vessels. In contrast, DBH-I nerve fibers were fine and were visualized primarily as numerous varicosities distributed in a circumferential manner around the vessel wall. A very high density of DBH-I varicosities was seen in the rostral part of the circle of Willis, with the internal carotid being the most richly supplied followed by the anterior cerebral and the anterior communicating arteries; comparatively, the middle cerebral artery was moderately innervated. The differences in distribution pattern and density between TPOH-I and DBH-I cerebrovascular fibers clearly suggest that these two innervation systems are not exactly superimposable. Superior cervical ganglionectomy caused an almost complete disappearance of TPOH-I nerves in all vascular segments, with some residual fibers in selected vessels. Lesion of the central serotonergic component with the neurotoxin 5,7-dihydroxytryptamine had virtually no effect on the TPOH-I fibers in the major basal and isolated pial arteries. These results strongly suggest that the serotonergic innervation of major cerebral as well as pial arteries has a prominent peripheral origin closely related to the sympathetic system. Processing of superior cervical ganglion slices for TPOH immunocytochemistry, however, failed to unequivocally detect TPOH-I neurons.