GABAergic signaling in the rat pineal gland
Pinealocytes secrete melatonin at night in response to norepinephrine released from sympathetic nerve terminals in the pineal gland. The gland also contains many other neurotransmitters whose cellular disposition, activity, and relevance to pineal function are not understood. Here, we clarify source...
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| Published in: | Journal of pineal research Vol. 61; no. 1; pp. 69 - 81 |
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| Main Authors: | , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
England
Blackwell Publishing Ltd
01-08-2016
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| Subjects: | |
| Online Access: | Get full text |
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| Summary: | Pinealocytes secrete melatonin at night in response to norepinephrine released from sympathetic nerve terminals in the pineal gland. The gland also contains many other neurotransmitters whose cellular disposition, activity, and relevance to pineal function are not understood. Here, we clarify sources and demonstrate cellular actions of the neurotransmitter γ‐aminobutyric acid (GABA) using Western blotting and immunohistochemistry of the gland and electrical recording from pinealocytes. GABAergic cells and nerve fibers, defined as containing GABA and the synthetic GAD67, were identified. The cells represent a subset of interstitial cells while the nerve fibers were distinct from the sympathetic innervation. The GABAA receptor subunit α1 was visualized in close proximity of both GABAergic and sympathetic nerve fibers as well as fine extensions among pinealocytes and blood vessels. The GABAB1 receptor subunit was localized in the interstitial compartment but not in pinealocytes. Electrophysiology of isolated pinealocytes revealed that GABA and muscimol elicit strong inward chloride currents sensitive to bicuculline and picrotoxin, clear evidence for functional GABAA receptors on the surface membrane. Applications of elevated potassium solution or the neurotransmitter acetylcholine depolarized the pinealocyte membrane potential enough to open voltage‐gated Ca2+ channels leading to intracellular calcium elevations. GABA repolarized the membrane and shut off such calcium rises. In 48–72‐h cultured intact glands, GABA application neither triggered melatonin secretion by itself nor affected norepinephrine‐induced secretion. Thus, strong elements of GABA signaling are present in pineal glands that make large electrical responses in pinealocytes, but physiological roles need to be found. |
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| Bibliography: | ark:/67375/WNG-8DKRJBW0-G Wayne E. Crill Endowed Professorship National Institute of General Medical Sciences - No. R01GM-083913 PIP-CONICET - No. 112-201101-00247 ArticleID:JPI12328 PICT - No. 2007-682 ANPCyT - No. 2012-174 Figure S1. The metabotropic GABAB1 receptor subunit characterized by Western blot and immunohistochemistry is present in the interstitial compartment of adult rat pineal gland (PG). (A) Representative blot of whole extracts from cerebellum (Cer) and pineal gland (PG) pools collected at ZT6 (midday; D) and ZT18 (midnight; N). A band of near 100 kDa is indicated by a white arrow. The last lane omits the primary antibody. Actin (Act) served as loading control. (B-G) Immunolabeling for GABAB1 (green), Pax6 (red), and the membrane-associated G-protein Go (red). DAPI (blue) and the isolectin IB4 (red) were used as nuclear and blood vessel (v) dyes. (B) Cerebellar section with highly immunoreactive Purkinje cells (PkL); their dendritic trees and cytoplasm are densely covered by the metabotropic subunit (white arrows). (C) Pineal gland section incubated without the anti-GABAB1 antibody. (D-F2) A subpopulation of pineal interstitial cells (Ic) positive for Pax6 and GABAB1 (yellow arrowheads) and an immunoreactive blood vessel are indicated (v). (G) GABAB1 in close proximity with the membrane-associated protein Go; pinealocyte (Pc) surface is negative for the receptor subunit. Abbreviations: GL, cerebellar granular layer; ML, cerebellar molecular layer; MW, molecular weight. (B, C-D1) 60x. (E-E1, F-F2, G) 2.5×, 2× and 1.8× digital zooms of 100× images, respectively. Insets in F-F2 and G, 2× and 2.5× magnifications of areas indicated by white arrowheads, respectively.Figure S2. Analysis by mass spectrometry of 5-HT, N-acetylserotonin (NAS), and melatonin (MEL) secretion during treatment with NE, GABA, and acetylcholine (ACh) in various combinations. The medium bathing intact pineal glands, one per well, was collected every 2 or 3 h, and the total secretion is expressed as nanograms of indoleamine secreted each hour by a single gland. Values are mean ± SEM (n = 3). The grey shading denotes medium with pharmacological agents as marked. In the unshaded periods there were no drugs. (A-C) An experiment with 6-week old rats started 72 h after dissection. (A) 2 μM NE. (B) 2 μM NE plus 40 μM GABA. (C) 40 μM GABA without NE. (D-F) An experiment with 8-week old rats started 48 h after dissection. (D) 0.5 μM NE. (E) 0.5 μM NE plus 100 μM GABA. (F) 0.5 μM NE plus 100 μM ACh.Table S1. Specific parameters of the Waters XEVO TQ-S MS/MS for the measured transitions. [Correction added after online publication on 14th April 2016; Supporting information legends Figure S1 and S2 were updated] istex:B41C82D335B1B0851295C22317EF970A985217DE ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 co last authors co first authors |
| ISSN: | 0742-3098 1600-079X |
| DOI: | 10.1111/jpi.12328 |