Predisposition to Alcohol Drinking and Alcohol Consumption Alter Expression of Calcitonin Gene-Related Peptide, Neuropeptide Y, and Microglia in Bed Nucleus of Stria Terminalis in a Subnucleus-Specific Manner.

Excessive alcohol consumption is often linked to anxiety states and has a major relay center in the anterior part of bed nucleus of stria terminalis (BNST). We analyzed the impact of (i) genetic predisposition to high alcohol preference and consumption, and (ii) alcohol intake on anterior BNST, namely anterolateral (AL), anteromedial (AM), and anteroventral (lateral + medial subdivisions: AVl, AVm) subnuclei. We used two rat lines selectively bred for low- and high-alcohol preference and consumption, named Sardinian alcohol-non preferring (sNP) and -preferring (sP), respectively, the latter showing also inherent anxiety-related behaviors. We analyzed the modulation of calcitonin gene-related peptide (CGRP; exerting anxiogenic effects in BNST), neuropeptide Y (NPY; exerting mainly anxiolytic effects), and microglia activation (neuroinflammation marker, thought to increase anxiety). Calcitonin gene-related peptide-immunofluorescent fibers/terminals did not differ between alcohol-naive sP and sNP rats. Fiber/terminal NPY-immunofluorescent intensity was lower in BNST-AM and BNST-AVm of alcohol-naive sP rats. Activation of microglia (revealed by morphological analysis) was decreased in BNST-AM and increased in BNST-AVm of alcohol-naive sP rats. Prolonged (30 consecutive days), voluntary alcohol intake under the homecage 2-bottle "alcohol vs. water" regimen strongly increased CGRP intensity in BNST of sP rats in a subnucleus-specific manner: in BNST-AL, BNST-AVm, and BNST-AM. CGRP area sum, however, decreased in BNST-AM, without changes in other subnuclei. Alcohol consumption increased NPY expression, in a subnucleus-specific manner, in BNST-AL, BNST-AVl, and BNST-AVm. Alcohol consumption increased many size/shapes parameters in microglial cells, indicative of microglia de-activation. Finally, microglia density was increased in ventral anterior BNST (BNST-AVl, BNST-AVm) by alcohol consumption. In conclusion, genetic predisposition of sP rats to high alcohol intake could be in part mediated by anterior BNST subnuclei showing lower NPY expression and differential microglia activation. Alcohol intake in sP rats produced complex subnucleus-specific changes in BNST, affecting CGRP/NPY expression and microglia and leading to hypothesize that these changes might contribute to the anxiolytic effects of voluntarily consumed alcohol repeatedly observed in sP rats.

Calcitonin gene-related peptide decreases IL-1beta, IL-6 as well as Ym1, Arg1, CD163 expression in a brain tissue context-dependent manner while ameliorating experimental autoimmune encephalomyelitis.

Activation states of immune cells (among them, the so-called pro- or anti-inflammatory states) influence the pathogenesis of multiple sclerosis (MS). The neuropeptide calcitonin gene-related peptide (CGRP) can exert a pro- or anti-inflammatory role in a context-dependent manner. In mice CGRP was found to attenuate the development of experimental autoimmune encephalomyelitis (EAE, a common MS animal model). We analyzed CGRP effects on the expression of cytokines and markers of activation states, as well as its intracellular cascade, following intrathecal administration during EAE immunization. Real Time quantitative-PCR (RT-PCR) showed that IL-1beta and IL-6 (associated to a pro-inflammatory state in EAE), but also Ym1 (also known as Chil3), Arg1 and CD163 (associated to an anti-inflammatory state in EAE) were decreased in the encephalon (devoid of cerebellum). In the cerebellum itself, IL-1beta and Ym1 were decreased. TNF-alpha (associated to a pro-inflammatory state in EAE), but also IL-10 (associated to another type of anti-inflammatory state) and BDNF were unchanged in these two regions. No changes were detected in the spinal cord. Additional tendencies toward a change (as revealed by RT-PCR) were again decreases: IL-10 in the encephalon and Arg1 in the spinal cord. CGRP decreased percentage of Ym1+/CD68+ immunoreactive cells and cell density of infiltrates in the cervical spinal cord pia mater. Instead, Ym1 in the underlying parenchyma and at thoracic and lumbar levels, as well as Arg1, were unchanged. In cultured microglia the neuropeptide decreased Ym1, but not Arg1, immunoreactivity. Inducible NOS (iNOS) was unchanged in spinal cord microglia and astrocytes. The neuropeptide increased the activation of ERK1/2 in the astrocytes of the spinal cord and in culture, but did not influence the activation of ERK1/2 or p38 in the spinal cord microglia. Finally, in areas adjacent to infiltration sites CGRP-treated microglia showed a larger ramification radius. In conclusion, CGRP-induced EAE amelioration was associated to a concomitant, context-dependent decrease in the expression of markers belonging to both pro- or anti-inflammatory activation states of immune cells. It can be hypothesized that CGRP-induced EAE attenuation is obtained through a novel mechanism that promotes down-regulation of immune cell activation that facilitates the establishment of a beneficial environment in EAE provided possibly also by other factors.

Microglia-Induced Maladaptive Plasticity Can Be Modulated by Neuropeptides In Vivo.

Microglia-induced maladaptive plasticity is being recognized as a major cause of deleterious self-sustaining pathological processes that occur in neurodegenerative and neuroinflammatory diseases. Microglia, the primary homeostatic guardian of the central nervous system, exert critical functions both during development, in neural circuit reshaping, and during adult life, in the brain physiological and pathological surveillance. This delicate critical role can be disrupted by neural, but also peripheral, noxious stimuli that can prime microglia to become overreactive to a second noxious stimulus or worsen underlying pathological processes. Among regulators of microglia, neuropeptides can play a major role. Their receptors are widely expressed in microglial cells and neuropeptide challenge can potently influence microglial activity in vitro. More relevantly, this regulator activity has been assessed also in vivo, in experimental models of brain diseases. Neuropeptide action in the central nervous system has been associated with beneficial effects in neurodegenerative and neuroinflammatory pathological experimental models. This review describes some of the mechanisms of the microglia maladaptive plasticity in vivo and how neuropeptide activity can represent a useful therapeutical target in a variety of human brain pathologies.

Involvement of calcitonin gene-related peptide and receptor component protein in experimental autoimmune encephalomyelitis.

Calcitonin Gene-Related Peptide (CGRP) inhibits microglia inflammatory activation in vitro. We here analyzed the involvement of CGRP and Receptor Component Protein (RCP) in experimental autoimmune encephalomyelitis (EAE). Alpha-CGRP deficiency increased EAE scores which followed the scale alpha-CGRP null>heterozygote>wild type. In wild type mice, CGRP delivery into the cerebrospinal fluid (CSF) 1) reduced chronic EAE (C-EAE) signs, 2) inhibited microglia activation (revealed by quantitative shape analysis), and 3) did not alter GFAP expression, cell density, lymphocyte infiltration, and peripheral lymphocyte production of IFN-gamma, TNF-alpha, IL-17, IL-2, and IL-4. RCP (probe for receptor involvement) was expressed in white matter microglia, astrocytes, oligodendrocytes, and vascular-endothelial cells: in EAE, also in infiltrating lymphocytes. In relapsing-remitting EAE (R-EAE) RCP increased during relapse, without correlation with lymphocyte density. RCP nuclear localization (stimulated by CGRP in vitro) was I) increased in microglia and decreased in astrocytes (R-EAE), and II) increased in microglia by CGRP CSF delivery (C-EAE). Calcitonin like receptor was rarely localized in nuclei of control and relapse mice. CGRP increased in motoneurons. In conclusion, CGRP can inhibit microglia activation in vivo in EAE. CGRP and its receptor may represent novel protective factors in EAE, apparently acting through the differential cell-specific intracellular translocation of RCP.

Calcitonin gene-related peptide (CGRP) stimulates purkinje cell dendrite growth in culture.

Previous reports described the transient expression during development of Calcitonin Gene-Related Peptide (CGRP) in rodent cerebellar climbing fibers and CGRP receptor in astrocytes. Here, mixed cerebellar cultures were used to analyze the effects of CGRP on Purkinje cells growth. Our results show that CGRP stimulated Purkinje cell dendrite growth under cell culture conditions mimicking Purkinje cell development in vivo. The stimulation was not blocked by CGRP8-37, a specific antagonist, suggesting the activation of other related receptors. CGRP did not affect survival of Purkinje cells, granule cells or astrocytes. The selective expression of Receptor Component Protein (RCP) (a component of CGRP receptor family) in astrocytes points to a role of these cells as mediators of CGRP effect. Finally, in pure cerebellar astrocyte cultures CGRP induced a transient morphological differentiation from flat, polygonal to stellate form. It is concluded that CGRP influences Purkinje cell dendrite growth in vitro, most likely through the involvement of astrocytes.

Calcitonin gene-related peptide (CGRP) triggers Ca2+ responses in cultured astrocytes and in Bergmann glial cells from cerebellar slices.

The neuropeptide calcitonin gene-related peptide (CGRP) is transiently expressed in cerebellar climbing fibers during development while its receptor is mainly expressed in astrocytes, in particular Bergmann glial cells. Here, we analyzed the effects of CGRP on astrocytic calcium signaling. Mouse cultured astrocytes from cerebellar or cerebral cortex as well as Bergmann glial cells from acutely isolated cerebellar slices were loaded with the Ca(2+) sensor Fura-2. CGRP triggered transient increases in intracellular Ca(2+) in astrocytes in culture as well as in acute slices. Responses were observed in the concentration range of 1 nm to 1 mm, in both the cell body and its processes. The calcium transients were dependent on release from intracellular stores as they were blocked by thapsigargin but not by the absence of extracellular calcium. In addition, after CGRP application a further delayed transient increase in calcium activity could be observed. Finally, cerebellar astrocytes from neonatal mice expressed receptor component protein, a component of the CGRP receptor, as revealed by immunofluorescence and confocal microscopy. It is thus proposed that the CGRP-containing afferent fibers in the cerebellum (the climbing fibers) modulate calcium in astrocytes by releasing the neuropeptide during development and hence possibly influence the differentiation of Purkinje cells.