Costimulatory Effects of Interferon-γ and Interleukin-1β or Tumor Necrosis Factor α on the Synthesis of Aβ1-40 and Aβ1-42 by Human Astrocytes

Chronic inflammation and astrocytosis are characteristic histopathological features of Alzheimer's Disease (AD). Astrocytes are one of the predominant cell types in the brain. In AD they are activated and produce inflammatory components such as complement components, acute phase proteins, and cytokines. In this study we analyzed the effect of cytokines on the production of amyloid β (Aβ) in the astrocytoma cell line U373 and in primary human astrocytes isolated postmortem from healthy aged persons as well as from patients with AD. Astrocytes did not produce Aβ in the absence of stimuli or following stimulation with IL-1β, TNFα, IL-6, and TGF-β1. Neither did combinations of TNFα and IL-1β, IL-6 or TGF-β1, or the coadministration of IFNγ and IL-6 or TGF-β1 induce Aβ production. In contrast, pronounced production of Aβ1-40 and Aβ1-42 was observed when primary astrocytes or astrocytoma cells were stimulated with combinations of IFNγ and TNFα or IFNγ and IL-1β. Induction of Aβ production was accompanied by decreased glycosylation of APP as well as by increased secretion of APPsβ. Our results suggest that astrocytes may be an important source of Aβ in the presence of certain combinations of inflammatory cytokines. IFNγ in combination with TNFα or IL-1β seems to trigger Aβ production by supporting β-secretase cleavage of the immature APP molecule.     

Extrahypophyseal distribution of α-melanocyte stimulating hormone (α-MSH)-like immunoreactivity in postmortem brains from normal subjects and Alzheimer-type dementia patients

Using a sensitive double-antibody solid-phase enzyme immunoassay method α-melanocyte stimulating hormone-like immunoreactivity (α-MSH-LI) was measured in 21 regions of postmortem brains from 8 normal subjects and 5 patients with Alzheimer-type dementia (ATD). In the brains from the normal subjects, the highest concentration of α-MSH-LI was found in the hypothalamus. Relatively high concentration were also measured in the locus coeruleus, substantia innominata, substantia nigra, amygdala and medial nucleus of thalamus. α-MSH-LI in other regions was approximately1/100 of the hypothalamic content. This data is consistent with the existence of α-MSH in extrahypophyseal regions and indicates its regional distribution in the human brain. In the Alzheimer brains, although the temporal cortex and hippocampus had normal concentrations of α-MSH-LI, the cingulate cortex, caudate and substantia nigra showed significantly lower concentrations of α-MSH-LI than those of the control brains. This data suggests that further studies of α-MSH content in a larger number of ATD brains would be useful.     

Effects of TNFα on immature and mature oligodendrocytes and their progenitors in vitro

Tumor necrosis factor α (TNFα) appears to take part in the pathogenesis of multiple sclerosis and to contribute to the degeneration of oligodendrocytes as well as neurons. TNFα is produced by microglia and astrocytes, which also produce hormones and cytokines that influence its biological activity. Thus, in mixed cultures the effects of exogenous TNFα might be modified by products of astrocytes and microglia. The effects of TNFα in oligodendrocyte-enriched cultures are reported below. We prepared the cultures by shaking oligodendrocytes off primary mixed glial-cell cultures from brains of 2-day-old rats at 7 days in vitro and plating them (0 days post-shake, DPS). Platelet-derived growth factor and fibroblast growth factor were included in the media at 1–5 DPS in order to encourage proliferation. At 2 DPS media were added with no TNFα (controls) or 1000, 2000 or 5000 U/ml of TNFα, and at 5 DPS media were replaced with fresh serum-free media. Cultures were fixed with 4% paraformaldehyde at 5, 7, 9 and 12 DPS and immunostained. Oligodendrocyte progenitors were not reduced in numbers immediately after the incubation with TNFα (i.e. at 5 DPS). However, after an additional 4 days in culture fewer progenitors remained in the cultures that had been treated with TNFα than in the untreated cultures. In the absence of the growth factors there were fewer progenitors, but their numbers also were reduced by TNFα. Maturation to the myelin basic protein (MBP)-positive stage was inhibited by about 36% at 9 DPS by 1000–2000 U/ml of TNFα, while numbers of O4+/MBP− precursors were unaffected. It is interesting that the steady-state number of O4-positive precursors was unchanged by TNFα at 9 DPS, when there were reductions in the numbers of A2B5-positive progenitors and MBP-positive mature oligodendrocytes. That observation suggests that the rates of proliferation, death and maturation are controlled by multiple factors, with a particularly vulnerable time at the maturation to the MBP-positive stage. At 5000 U/ml TNFα the specific effect on maturation was overtaken cytotoxicity. These data and a summary of the literature suggest that inhibition of MBP expression is sensitive to lower TNFα concentrations and incubation times than is cell survival. Specific effects on numbers of MBP-positive cells, morphology and MBP expression occur at 1000–2000 U/ml for 48–72 h or at up to 10 000 U/ml for≤24 h, and the deficits remain after removal of the TNFα.     

The role of interleukin-1 and tumor necrosis factor α in the neurochemical and neuroendocrine responses to endotoxin

Both interleukin-1 (IL-1) and endotoxin (lipopolysaccharide, LPS) are potent activators of the hypothalamo-pituitary-adrenal (HPA) axis, and they also increase cerebral norepinephrine metabolism and tryptophan. Injections of cause macrophages to synthesize and release various cytokines, including IL-1 and tumor necrosis factor α (TNFα). The hypothesis that macrophage production of IL-1 mediates the HPA-activating effect of LPS was tested in mice using the IL-1-receptor antagonist protein (IRAP). Administration of IRAP largely prevented the effects of IL-1α or IL- 1β on the elevation of plasma corticosterone and the concomitant increase in hypothalamic norepinephrine metabolism, but failed to alter the responses to LPS. IRAP did not prevent the increases in brain tryptophan that occurred after treatment with IL-1 or LPS. Recombinant human TNFa, TNFβ, IL-6, and interferon-a injected intraperitoneally failed to activate the HPA axis, but mouse TNFa was effective by this route, and human TNFα, TNFβ, and IL-6 were effective intravenously. None of these cytokines was as potent as IL-1. Pretreatment with an antibody specific for mouse TNFα, either alone or in combination with IRAP, also failed to prevent the elevation of plasma corticosterone by LPS. Thus, either IL-1 and TNFα are not involved in the HPA and noradrenergic responses to LPS, or there are alternative (redundant) pathways by which LPS can activate the HPA axis.     

Tumor Necrosis Factor α Is Toxic to Embryonic Mesencephalic Dopamine Neurons

Levels of the proinflammatory cytokine tumor necrosis factor α (TNFα) are increased in postmortem brain and cerebral spinal fluid from patients with Parkinson's disease (PD). This observation provides a basis for associating TNFα with neurodegeneration, but a specific toxicity in dopamine (DA) neurons has not been firmly established. Therefore, we investigated TNFα-induced toxicity in DA neurons by utilizing primary cultures of embryonic rat mesencephalon. Exposure to TNFα resulted in a dose-dependent decrease in DA neurons as evidenced by decreased numbers of tyrosine hydroxylase-immunoreactive (THir) cells. TNFα toxicity was selective for DA neurons in that neither glial cell counts nor the total number of neurons was decreased and no general cytotoxicity was evidenced by lactate dehydrogenase assay. Many of the cells which remained immunoreactive for TH had shrunken and rounded cell bodies with broken, blunted, or absent processes. However, TNFα-treated cultures also contained some THir cells which appeared to be undamaged and possibly resistant to TNFα-induced toxicity. Additionally, immunocytochemistry revealed basal expression of TNFα receptor 1 (p55, R1) and TNFα receptor 2 (p75, R2) on all cells within the mesencephalic cultures to some degree, even though only DA neurons were affected by TNFα treatment. These data strongly suggest that TNFα mediates cell death in a sensitive population of DA neurons and support the potential involvement of proinflammatory cytokines in the degeneration of DA neurons in PD.     

Cellular signaling roles of TGFβ, TNFα and βAPP in brain injury responses and Alzheimer's disease

β-Amyloid precursor protein (βAPP), transforming growth factor β (TGFβ), and tumor necrosis factor-α (TNFα) are remarkably pleiotropic neural cytokines/neurotrophic factors that orchestrate intricate injury-related cellular and molecular interactions. The links between these three factors include: their responses to injury; their interactive effects on astrocytes, microglia and neurons; their ability to induce cytoprotective responses in neurons; and their association with cytopathological alterations in Alzheimer's disease. Astrocytes and microglia each produce and respond to TGFβ and TNFα in characteristic ways when the brain is injured. TGFβ, TNFα and secreted forms of βAPP (sAPP) can protect neurons against excitotoxic, metabolic and oxidative insults and may thereby serve neuroprotective roles. On the other hand, under certain conditions TNFα and the fibrillogenic amyloid β-peptide (Aβ) derivative of βAPP can promote damage of neuronal and glial cells, and may play roles in neurodegenerative disorders. Studies of genetically manipulated mice in which TGFβ, TNFα or βAPP ligand or receptor levels are altered suggest important roles for each factor in cellular responses to brain injury and indicate that mediators of neural injury responses also have the potential to enhance amyloidogenesis and/or to interfere with neuroregeneration if expressed at abnormal levels or modified by strategic point mutations. Recent studies have elucidated signal transduction pathways of TGFβ (serine/threonine kinase cascades), TNFα (p55 receptor linked to a sphingomyelin-ceramide-NFκB pathway), and secreted forms of βAPP (sAPP; receptor guanylate cyclase-cGMP-cGMP-dependent kinase-K⁺ channel activation). Knowledge of these signaling pathways is revealing novel molecular targets on which to focus neuroprotective therapeutic strategies in disorders ranging from stroke to Alzheimer's disease.     

Tumor necrosis factor-α in immune-mediated demyelination and Wallerian degeneration of the rat peripheral nervous system

This study reports on the immunocytochemical localization of tumor necrosis factor-alpha (TNFα) in immune-mediated demyelination and Wallerian degeneration of the rat peripheral nervous system (PNS) using teased nerve fiber preparations. In experimental autoimmune neuritis induced by active immunization (EAN) or by adoptive transfer of autoreactive T cells (AT-EAN), macrophages passing blood vessels as well as macrophages adherent to nerve fibers were TNFα-positive. Large post-phagocytic macrophages at later stages of demyelination were TNFα-negative. Intraperitoneal application of an anti-TNFα antibody to EAN rats significantly reduced the degree of inflammatory demyelination, suggesting a pathogenic role for TNFα. After nerve transection only macrophages located within degenerating nerve fibers were TNFα-positive, while those entering and leaving nerves were negative. TNFα produced by macrophages seems to bevolved in immune-mediated demyelination and non-immune myelin degradation after axotomy. While interferon-gamma (IFNγ) is present in EAN nerves and may act as a local stimulus for TNF expression, the nature of this signal in Wallerian degeneration in the absence of IFNγ is unknown.     

Neurons of the chick brain and retina expressing both α-bungarotoxin-sensitive and α-bungarotoxin-insensitive nicotinic acetylcholine receptors: an immunohistochemical analysis

Immunohistochemical methods were used to study the possible co-localization of two α-bungarotoxin-sensitive (α7 and α8) and two α-bungarotoxin-insensitive (β2 and α3) subunits of the nicotinic acetylcholine receptors in neurons of the chick brain and retina. Several structures contained neurons that were doubly-labeled with antibodies against the α7 subunit and the β2 subunit. These structures included, for example, the interpeduncular nucleus, nucleus spiriformis lateralis, optic tectum, pretectal visual nuclei, and the lateral hypothalamus. Double-labeling with antibodies against the α7 and α8 subunits was also seen in several regions, which included the interpeduncular nucleus, visual pretectum, lateral hypothalamus, dorsal thalamus, and the habenular complex. In the retina, many cells in the inner nuclear layer were observed to contain α8 and α3 subunits, whereas neurons in the ganglion cell layer were seen to contain α7 and α8 or, less frequently, α7 and α3 subunits. These results indicate that α-bungarotoxin-sensitive and α-bungarotoxin-insensitive subunits of the nicotinic receptors are co-expressed by neurons of the chick brain and retina.     

Tumor necrosis factor-α attenuates N-methyl-d-aspartate-mediated neurotoxicity in neonatal rat hippocampus

Tumor necrosis factor-α (TNFα) has been implicated in the pathophysiology of acute neonatal brain injury. We hypothesized that acute brain injury would induce TNFα expression and that exogenous TNFα would influence the severity of N-methyl-d-aspartate-induced tissue damage. We performed two complementary groups of experiments to evaluate the potential role(s) of TNFα in a neonatal rodent model of excitotoxic injury, elicited by intracerebral injection of N-methyl-d-aspartate. We used immunohistochemistry and ELISA to evaluate N-methyl-d-aspartate-induced changes in TNFα expression, and we co-injected TNFα with N-methyl-d-aspartate, to evaluate the effect of this cytokine on the severity of tissue injury. Both intra-hippocampal and intra-striatal injection of N-methyl-d-aspartate (5 nmol) stimulated TNFα expression. Increased TNFα expression was detected 3–12 h after lesioning; TNFα was localized both in glial cells in the corpus callosum, and in cells with the morphology of interneurons in the ipsilateral hippocampus, striatum, cortex and thalamus. Intra-hippocampal or intra-striatal administration of TNFα (50 ng) alone did not elicit neuropathologic damage. In the hippocampus, when co-injected with N-methyl-d-aspartate (5 or 10 nmol), TNFα (50 ng) attenuated excitotoxic injury by 35%–57%, compared to controls co-injected with heat-treated TNFα. In contrast, in the striatum, co-injection of TNFα with N-methyl-d-aspartate had no effect on the severity of the ensuing damage. The data indicate that TNFα is rapidly produced in glial cells and neurons after an excitotoxic insult in the neonatal rat brain, and that administration of exogenous TNFα results in region-specific attenuation of excitotoxic damage. We speculate that endogenous TNFα may modulate the tissue response to excitotoxic injury in the developing brain.     

TNFα reduces glutamate induced intracellular Ca increase in cultured cortical astrocytes

The proinflammatory cytokine TNFα is locally released during various inflammatory CNS diseases and high cerebrospinal fluid (CSF) titers of TNFα were found in meningitis patients. We know from previous studies that TNFα also depolarizes astrocytes by reducing their inwardly rectifying K⁺ currents. We have now investigated the effect of TNFα on the glutamate induced intracellular Ca²⁺ increase in astrocytes, a process which seems to be involved in glial mediated modulation of neuronal synaptic transmisssion. Incubation with TNFα (50–1000 U/ml for 60 min) reduces the glutamate induced intracellular Ca²⁺ increase in astrocytes but not in neurons and this seems to be a phenomenon secondary to the TNFα induced depolarization. While other proinflammatory cytokines (interleukin 1β, IL-2, IL-6) did not interfere with the astrocytic glutamate response, incubation in CSF from septic meningitis patients (CSF–SM) also reduced the glutamate induced intracellular Ca²⁺ increase. The application of a neutralizing anti-TNFα antibody to the CSF–SM prior to cell incubation partially restored the glutamate response. Our data suggest that inflammatory molecules such as TNFα impair astrocytes’ response to glutamate and this may indirectly affect neuronal synaptic transmission.     

Brain-derived TNFα: involvement in neuroplastic changes implicated in the conscious perception of persistent pain

The pleiotropic cytokine tumor necrosis factor-alpha (TNFα) is implicated in the development of persistent pain through its actions in the periphery and in the central nervous system (CNS). Activation of the α₂-adrenergic receptor is associated with modulation of pain, possibly through its autoregulatory effect on norepinephrine (NE) release in the CNS. The present study employs a chronic constriction nerve injury (CCI) pain model to demonstrate the interactive role of presynaptic sensitivity to TNFα and the α₂-adrenergic autoreceptor in the pathogenesis of neuropathic pain. Accumulation of TNFα is increased initially in a region of the brain containing the locus coeruleus (LC) at day 4 post-ligature placement, followed by an increase in TNFα in the hippocampus at day 8 post-ligature placement, coincident with hyperalgesia. Levels of TNFα in the thoraco-lumbar spinal cord are also increased at day 8 post-ligature placement. Concurrently, α₂-adrenergic receptor and TNFα-induced inhibition of NE release are increased, and stimulated NE release is decreased in superfused hippocampal slices isolated at day 8 post-ligature placement. Stimulated NE release is also decreased in spinal cord slices (lumbar region) from animals undergoing CCI, although in contrast to that which occurs in the hippocampus, α₂-adrenergic receptor inhibition of NE release is not changed. These results indicate an important role that TNFα plays in adrenergic neuroplastic changes in a region of the brain that, among its many functions, appears to be a crucial link in the conscious perception of pain. We predict that neuroplastic changes, involving increased functional responses of α₂-adrenergic autoreceptors and increased presynaptic sensitivity to TNFα, culminate in decreased NE release in the CNS. These neuroplastic changes provide a mechanism for the role of CNS-derived TNFα in the pathogenesis of persistent pain.     

Brain β-endorphin-like immunoreactivity in adult rats given β-endorphin neonatally

β-endorphin-like immunoreactivity was measured by radioimmunoassay in the brains of adult rats treated neonatally with β-endorphin, naloxone, or vehicle. After treatment with β-endorphin, the decreases observed in β-endorphin-like immunoreactivity in the hypothalamus, pineal, midbrain, pons-medulla, hippocampus, striatum, frontal cortex, occipital cortex, and posterior cortex were highly significant but the 23% decrease in the thalamus was not significantly different from that of control rats. Neonatal administration of naloxone only resulted in a significant decrease in β-endorphin-like immunoreactivity in the hypothalamus. In contrast, no differences were discernible in content of either β-endorphin-like immunoreactivity or ACTH-like immunoreactivity in the pituitary of rats treated with β-endorphin, naloxone, or vehicle in the neonatal period. These same rats had shown an increased threshold to painful thermal stimulation by the tail-flick test after administration of either β-endorphin or naloxone at birth. The results suggest that neonatally injected β-endorphin may alter the levels of β-endorphin-like immunoreactivity in rat brain as well as the response to pain.     

Glial cell-specific mechanisms of TGF-β1 induction by IL-1 in cerebral cortex

Transforming growth factor beta-1 (TGF-β1) immunoreactive product (IRP) has recently been detected in autopsied brains of individuals who died with central nervous system diseases and/or fever but not in normal individuals or in normal rodent brain. However, the mechanism(s) of induction of TGF-β1 in brain and the identity of cells expressing TGF-β1 need to be understood before a role, if any, for this potent pleiotropic cytokine in neuropathogenesis can be discerned. Towards this end we determined that IL-1 stimulated the production of TGF-β1 IRP in cells and TGF-β1 activity in culture fluids of all glial cells, astrocytes, microglial cells, and oligodendrocytes, derived from neonatal rat cortex and grown in cell type-enriched cultures. TGF-β1 production in vitro varied with the cell type and isoform of IL-1. Oligodendrocytes produced the most and astrocytes the least amount of TGF-β1. IL-1α stimulated TGF-β1 production in all glial cell types, whereas IL-1β did not. In vivo, TGF-β1 IRP was detected in human tissues from cerebral frontal cortex and subcortical white matter only when interleukin-1 (IL-1) was elevated in the same tissues. Moreover, the amount of detectable TGF-β1 was positively correlated with the amount of detectable IL-1 (rho = 0.605; P = 0.003), as determined by morphometry. Double-labelling of cells for their phenotypic markers and expression of TGF-β1 indicated that all glial cells, but not neurons, expressed TGF-β1. IL-1α and IL-1β IRPs were also detected in all three glial cell types, most frequently in astrocytes and least frequently in microglial cells. The cells containing both cytokine IRPs were also detected. These results indicate that TGF-β1 may be induced by IL-1 in all glial cells of the frontal cortex, by both autocrine and paracrine mechanisms.     

Interferon β-1a downregulates TNFα-induced intercellular adhesion molecule 1 expression on brain microvascular endothelial cells through a tyrosine kinase-dependent pathway

TNFα (100 U/ml, 24 h) upregulated intercellular adhesion molecule 1 (ICAM1) expression on brain microvascular endothelial cell (BMEC) culture. The tyrosine kinase (TK) inhibitor genestein (100 μg/ml), the protein kinase C (PKC) inhibitor staurosporin (1 nM), and interferon (IF) β-1a (1000 U/ml) antagonized TNFα effect. When an ineffective dose of IFβ-1a (100 U/ml) was challenged with ineffective doses of either genestein (10 μg/ml) or staurosporin (0.1 nM), the combination IFβ-1a–genestein significantly reduced TNFα-induced ICAM1 expression whereas IFβ-1a–staurosporin did not. These findings indicate that a TK- rather than a PKC-dependent mechanism is involved in the modulation of TNFα response by IFβ-1a on BMECs.     

Tumor necrosis factor α and interleukin-6 mRNA expression in neonatal Lewis rat Schwann cells and a neonatal rat Schwann cell line following interferon γ stimulation

There is increasing evidence that Schwann cells play an important role in the pathogenesis of autoimmune inflammatory peripheral nerve disease. Schwann cells have been reported to express major histocompatibility complex class I and II (MHC I and II) and intercellular adhesion molecule-1 (ICAM-1), and to produce interleukin-1 (IL-1), prostaglandin E2 and thromboxane A2. In this study we investigated freshly dissociated neonatal Lewis rat Schwann cells and a SV40 transfected neonatal rat Schwann cell line (Schwann cell line) for production of mRNA for the immunomodulatory cytokines IL-2, IL-4, IL-6, IL-10, interferon-gamma (IFNγ), and tumor necrosis factor-alpha (TNFα) employing RT-PCR. Primary Schwann cells and Schwann cell line were examined following IFNγ stimulation and were found to express TNFα and IL-6 mRNA. These results further support a role for Schwann cell participation in inflammatory responses within the peripheral nervous system (PNS).     

Characterization of α-melanocyte-stimulating hormone (α-MSH)-like peptides in discrete regions of the rat brain. In vitro release of α-MSH from perifused hypothalamus and amygdala

The neuropeptide α-melanocyte-stimulating hormone (α-MSH) is synthesized by discrete populations of hypothalamic neurons which project in different brain regions including the cerebral cortex, hippocampus and amygdala nuclei. The purpose of the present study was to identify the α-MSH-immunoreactive species contained in these different structures and to compare the ionic mechanisms underlaying α-MSH release at the proximal and distal levels, i.e. within the hypothalamus and amygdala nuclei, respectively. The molecular forms of α-MSH-related peptides stored in discrete areas of the brain were characterized by combining high-performance liquid chromatography (HPLC) separation and radioimmunoassay detection. In mediobasal and dorsolateral hypothalamic extracts, HPLC analysis confirmed the existence of a major immunoreactive peak which co-eluted with the syntheticdes-Nα-acetyl α-MSH standard. In contrast, 3 distinct forms of immunoreactive α-MSH, which exhibited the same retention times as synthetic des-, mono- and di-acetyl α-MSH, were resolved in amygdala nuclei, hippocampus, cortex and medulla oblongata extracts. The proportions of acetylated α-MSH (authentic α-MSH plus diacetyl α-MSH) contained in these extrahypothalamic structures were, respectively, 78, 80, 60 and 92% of the total α-MSH immunoreactivity. In order to compare the ionic mechanisms underlaying α-MSH release from hypothalamic and extrahypothalamic tissues, we have investigated in vitro the secretion of α-MSH by perifused slices of hypothalamus and amygdala nuclei. High potassium concentrations induced a marked increase of α-MSH release from both tissue preparations. However, a higher concentration of KCl was required to obtain maximal stimulation of amygdala nuclei (90 mM) than hypothalamic tissue (50 mM). The effect of depolarizing concentrations of KCl was totally suppressed in the absence of Ca²⁺, indicating that high-K⁺ induced the opening of voltage-operated Ca²⁺ (VOC) channels. Veratridine (50 μM), a depolarizing agent which activates Na⁺ conductances, caused a robust stimulation of α-MSH release from hypothalamic slices but had virtually no effect on amygdala nuclei. ω-Conotoxin (1 μM), a peptide toxin which blocks L- and N-type VOC channels, caused a slight reduction of K⁺-evoked α-MSH release from hypothalamic slices but induced a dramatic decrease of α-MSH release from amygdala nuclei. These data suggest that acetylation of α-MSH to generate the biologically active forms of the peptide is a slow process which occurs gradually during axonal transport. Our results also indicate that release of α-MSH at the hypothalamic level mainly results from activation of T-type VOC channels whereas, in the amygdala nuclei, L- and (or) N-type VOC channels are involved in the regulation of α-MSH secretion.     

Distribution of α-melanocyte-stimulating hormone in the rat brain: Evidence that α-MSH-containing cells in the arcuate region send projections to extrahypothalamic areas

The distribution and concentration of α-MSH in the rodent brain has been determined by radioimmunoassay. The limbic system contained substantial quantities of α-MSH. Forty per cent of the α-MSH present in the brain was localized in the hypothalamus, with the highest concentration of α-MSH in the arcuate nucleus. More than 40% of the extrahypothalamic α-MSH in the brain was found in the following areas: midbrain (16%), preoptic area (13%), septum (7%), and thalamus (7%). To determine the source of the hypothalamic and extrahypothalamic α-MSH, the anterior hypothalamic preoptic area of the brain was surgically separated from more caudal diencephalic structures, and the arcuate region of the hypothalamus was surgically isolated from the remainder of the brain. Following these deafferentations, no significant reduction in hypothalamic α-MSH levels was observed; however, a significant reduction in extrahypothalamic α-MSH levels was demonstrated. This dramatic decrease of α-MSH in extrahypothalamic areas of the rodent brain strongly suggests that the bulk of the extrahypothalamic α-MSH arises from neuronal perikarya in the arcuate region. These findings are consistent with the hypothesis that a population of neuronal cell bodies producing α-MSH originate in the arcuate region of the hypothalamus and that they send axonal projections to many areas of the limbic system and brain stem.     

Inhibition of astrocyte TNFα expression by extracellular potassium

TNFα and IL-6 are cytokines of great interest, given the numerous biological activities and the documented expression in several central nervous system (CNS) pathologies. In this report, we have examined cultures of IL-1- or IL-1/IFNγ-activated human fetal astrocytes as a model to study mechanisms of cytokine regulation in the inflamed CNS. Since one of the major functions of astrocytes is spatial buffering of K⁺ ions, we examined the effect of high extracellular KCl on astrocyte cytokine expression by ribonuclease protection assay and ELISA. Results demonstrate that astrocyte TNFα production was potently inhibited by K⁺ with 44 and 89% inhibition at 25 and 55 mM K+, respectively. In contrast, astrocyte IL-6 inhibition required higher concentrations of K+ (≥75 mM). These results demonstrate a novel role for astrocyte potassium channel activity in modulation of glial cytokine production.