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. 2016 Aug;61(1):69-81.
doi: 10.1111/jpi.12328. Epub 2016 Apr 14.

GABAergic signaling in the rat pineal gland

Affiliations

GABAergic signaling in the rat pineal gland

Haijie Yu et al. J Pineal Res. 2016 Aug.

Abstract

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 GABAB 1 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 Ca(2+) 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.

Keywords: GABA; GABAA receptor; GAD67; bicuculline; melatonin secretion; membrane potential; pineal gland.

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Figures

Fig. 1
Fig. 1
GABA is synthesized in the adult rat pineal gland as revealed by immunoreactive GAD67 in Western blot and immunohistochemistry. (A) Representative blot of cytoplasmic extracts from cerebellum (Cer) and pineal gland (PG) pools collected at midday (D) and midnight (N). A 67 kDa band (white arrow) is prominent in pineal gland; cerebellum shows additional bands around 55 kDa (black arrowhead) and 110 kDa (black arrow). Actin (Act) served as a loading control. (B–K) Cerebellar and pineal gland sections immunolabeled for GAD67 (green), GABA (green or red), vimentin (VIM, red), Pax6 (red) and Iba1 (red); DAPI (blue) was used as general nuclear dye. (B, C) Cerebellar and pineal gland sections incubated with and without anti-GAD67 antibody, respectively. (B) GAD67-positive cells in the Purkinje cell layer (PkL) and molecular layer (ML) are indicated by white arrowheads. (C) Pinealocytes (Pc) and interstitial cells (Ic) are distinguished by their differential nuclear morphology and karyoplasm density. (D–G2, I–K) GABAergic nerve fibers (white arrows) in close proximity to blood vessels (v) and interstitial GABAergic cells (white arrowheads) positive for GAD67, GABA, and VIM. These cells are distinct from interstitial cells immunoreactive for Pax6 (yellow arrowheads) or Iba1 (red arrowhead). (H) The GABAergic cells represent ~5% of the total cells in the pineal gland. Abbreviations: GL, cerebellar granular layer; MW, molecular weight. (B) 4× digital zoom of a 40× image. (C) 1.1× magnification of a 60× image. (D) 2× digital zoom of a 100× image. (D′) 2× enlargement of the inset shown in D. (E–G2) 4× digital zooms from 60× images. (I–I2, J, K) 3×, 3.4× and 3.8× enlargements from 60× images.
Fig. 2
Fig. 2
Western blot and immunohistochemistry reveal the ionotropic receptor subunit GABAARα1 in the adult rat pineal gland. (A) A band around 51 kDa (white arrow) seen in whole extracts from cerebellum (Cer) and pineal gland (PG) pools at midday (D) and midnight (N). Omission of the primary antibody and actin (Act) served as negative and loading controls, respectively. (B–G′) Immunolabeling for GABAARα1 (green), vimentin (VIM, red), Tuj1 (red), GAD67 (red), and tyrosine hydroxylase (TH) (red); nuclei were counterstained with DAPI (blue). (B) Punctate pattern of the ionotropic subunit in the cytoplasm, surface and dendritic tree of Purkinje cells (PkL, white arrows). (C) Pineal gland without primary antibody. (D–G′) In pineal gland sections a specific signal for GABAARα1 is seen as extensions often interlocked with or parallel to VIM-positive blood vessels (v) and in a more discontinuous way, to sympathetic and GABAergic nerve fibers (white arrows). A signal not associated directly with pineal gland innervation is marked with yellow arrows. (D′, E′, F′, G′) Higher magnifications of the insets shown in D–G. Abbreviations: GL, cerebellar granular layer; Ic, interstitial cells; ML, cerebellar molecular layer; MW, molecular weight; Pc, pinealocytes. (B) 1.1× magnification of a 60× image. (D, E) 2× digital zooms of 60× images. (D′, E′) 2× enlargements of the insets shown in D and E. (F) 1.2× magnification of a 60× image. (G) 4× digital zoom of a 100× image. (F′, G′) 2× and 1.5× enlargements of the insets shown in F and G, respectively.
Fig. 3
Fig. 3
GABA evokes inward ionic currents blocked by GABAA antagonists in pinealocytes under whole-cell patch-clamp recording. (A) Representative trace of membrane currents evoked as increasing concentrations of GABA are perfused in the bath. (B) GABA concentration-response relation for peak evoked current. (n = 4–5 pinealocytes). (C) Sample trace of three coapplications of 20 μM GABA with 0, 3, or 30 μM bicuculline (BIC). (D) The inhibitory BIC concentration-response relation during coapplication with 20 μM GABA. (n = 4–5 pinealocytes). (E) Picrotoxin (PIC) blocks GABA-evoked currents. (n = 5 pinealocytes). Conditions for all panels: holding potential −100 mV; approximately equal chloride concentrations inside and outside. Bars show mean ± SEM. Age of rats: A, B, C, D, 7 weeks; E, 10 weeks.
Fig. 4
Fig. 4
GABAA agonists evoke current and a GABAB agonist does not. (A) Muscimol (MUS) evokes bicuculline (BIC)-sensitive inward currents. Individual recordings are shown as gray lines and the averaged record as a black line. (n = 3 pinealocytes) (B) Baclofen (BAC) evokes no current and does not antagonize muscimol-induced current. (n = 5 pinealocytes) (C) GABAB antagonist CGP 55845 (CGP) evokes no current and does not antagonize GABA-induced current. (n = 5 pinealocytes) (D) and (E) comparison of mean GABA current densities without and with two concentrations of CGP 55845 measured as marked by filled circles in C. Bars show mean ± SEM. * P < 0.05, ** P < 0.01. Conditions for all panels: whole-cell recording; holding potential −100 mV; approximately equal chloride concentrations inside and outside. Age of rats: A, B, 10 weeks; C, D, E, 8 weeks.
Fig. 5
Fig. 5
Current-voltage relations of GABA-induced current with different Cl concentration gradients across the membrane. (A) Whole-cell recording and nearly symmetrical high-Cl concentrations (red), block by 40 μM bicuculline (BIC, blue), and final wash (green). (n = 4 pinealocytes). (B) Gramicidin perforated-patch recording to preserve intracellular Cl levels. External solution is 169 mM Cl (red, reversal potential Vrev = −37.8±1.0 mV) and changed to 20 mM Cl (purple, Vrev = −12.4±2.0 mV). Chloride substitutes used are glutamate in pipette and gluconate in bath. (n = 5 pinealocytes). All currents shown are difference currents, with-GABA minus without-GABA or for the “wash” curve, final record minus initial baseline. Individual recordings are shown as pale lines and averaged records as heavy lines. Age of rats: A, 9 weeks; B, 10 weeks.
Fig. 6
Fig. 6
GABA suppresses KCl- and acetylcholine (ACh)-induced depolarization and calcium elevations. (A) Membrane potential recordings using perforated patch. KCl depolarizes the membrane, GABA partially repolarizes, and bicuculline (BIC) blocks the GABA effect. Five individual traces (gray) and their average (black). (n = 5 pinealocytes). (B, C, D) KCl- and ACh-induced calcium elevations in intact fura-2-loaded pinealocytes. (B) Five control applications of 30 mM KCl. (n = 22 pinealocytes). (C) Five applications of KCl variously mixed with GABA and bicuculline as marked showing strong suppression of Ca2+ response by 20 μM GABA and partial antagonism by 40 μM bicuculline. (D) Three applications of acetylcholine (ACh) showing suppression of Ca2+ response by 100 μM GABA. (n = 25 pinealocytes). In (B) (C), and (D), traces include mean (black) and SEM (gray) at each time point. Age of rats: A, 10 weeks; B, C, 6 weeks; D, 8 weeks.

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