Brain Research. 531{ 1990) 1¢~-24 Elsevier
16
BRES 15950
GABAA/benzodiazepine receptor localization in the circadian timing system K.M. Michels 1, L.P. M o r i n 2'3 a n d R.Y. M o o r e ~'4 Departments of 1Neurology, 2psychiatry, 3psychology and 4Neurobiology and Behavior, State University of New York, Stony Brook, NY (U.S.A.) (Accepted 24 April 1990)
Key words: 7-Aminobutyric acid; y-Aminobutyric acid A/Benzodiazepine receptor; Glutamic acid decarboxylase; Benzodiazepine binding; Suprachiasmatic nucleus; Intergeniculate leaflet; Raphe nucleus
y-Aminobutyric acid (GABA) and exogenous benzodiazepines are thought to play a role in the neural regulation of circadian rhythms. Because binding sites for the benzodiazepines and GABA A iigands are functionally coupled as part of the GABAA/benzodiazepine receptor complex (GABAA/BZR), we analyzed the localization of GABA neurons and GABAA/BZR within 3 nuclei involved in circadian rhythm regulation using autoradiographic and immunohistochemical techniques. Glutamic acid decarboxylase-immunoreactive axons are present in the suprachiasmatic nuclei (SCN), intergeniculate leaflet (IGL), and dorsal raphe nucleus (DR). Immunoreactivity for the GABAA/BZ receptor complex is absent from the SCN and the IGL whereas the DR shows a dense, uniform immunoreactivity. Semiquantitative analysis of autoradiograms for [3H]diazepam and [3H]flunitrazepam binding reveals a moderate level of binding in the SCN, a low level of binding in the IGL, and the highest level of the DR. Based on both the pattern of benzodiazepine binding and of receptor immunoreactivity the DR would appear to be a likely target site for GABAA and benzodiazepine action. The SCN would also appear to be a possible target site. The results suggest the IGL is not a site for direct GABAA and benzodiazepine action, but do not exclude a role for the IGL in the neural circuitry mediating GABA and benzodiazepine interactions with the circadian system.
INTRODUCTION Exogenous benzodiazepines potentiate many of the effects of the inhibitory neurotransmitter y-aminobutyric acid ( G A B A ) within the central nervous system. The benzodiazepine (BZ) binding site is associated with the G A B A A receptor and a chloride ionophore to form the G A B A A / B Z receptor complex ( G A B A A / B Z R ) 41. The participation of G A B A A / B Z R in the regulation of circadian function is suggested by pharmacological studies of rodent activity rhythms. Bicuculline, a G A B A A antagonist, and the benzodiazepine agonist, diazepam, block light-induced phase delays and advances, respectively, in hamsters 44-46. The short-acting B Z agonist triazolam (TZ) causes either phase advances or delays of golden hamster activity rhythms dependent upon the time of administration 59, accelerates re-entrainment after an 8-h photoperiod shift 63 and entrains the activity rhythm 64. The location of the receptor complex responsible for mediating G A B A and B Z effects on circadian rhythmicity is not known, but several sites merit consideration. The first is the suprachiasmatic nucleus (SCN) which is
the principal circadian pacemaker in mammals 33'47"49, and effects on entrainment, whether direct or indirect, must be expressed through it 15. Regions which project to the SCN are also candidates for mediating T Z effects on circadian rhythm phase. There is a direct retinal input to the SCN 34 and the pineal gland releases melatonin which may act at melatonin receptors within the SEN 19'68. However, neither blinding nor pinealectomy blocks the phase-shifting effects of T Z 62 and message for G A B A A / B Z R is not present in the pineal 52. The intergenicutate leaflet (IGL) receives direct retinal afferents and projects to the SCN through a neuropeptide-Y (NPY)-containing pathway the geniculo-hypothalamic tract ( G H T ) 6A4'32. Activation of the G H T 16"26'48 and infusion of N P Y into the SCN 1 produce a phase-response curve that is very similar to that for T Z 59. Ablation of the I G L eliminates the phase-shifting effects of T Z 17. Therefore, the I G L may be one site at which T Z acts to modify circadian rhythm phase. The raphe nuclei represent another potential site since they project to both the SCN and the I G L 3,35,60. In the present study we investigated the localization of
Correspondence: K.M. Michels. Present address: NIMH-LCS, Section on Pharmacology, Bldg. 10, Rm. 2D-45, Bethesda, MD 20892, U.S.A. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
17 the G A B A - s y n t h e s i z i n g enzyme, glutamic acid decarboxylase ( G A D ) within the SCN, I G L and dorsal raphe nucleus ( D R ) by immunocytochemistry and the localization of G A B A A / B Z R using immunocytochemistry and autoradiographic d e m o n s t r a t i o n of benzodiazepine binding. A preliminary report of these results has b e e n previously presented 27.
MATERIALS AND METHODS
Animals Adult Sprague-Dawley rats and outbred golden hamsters were maintained in standard laboratory conditions (light:dark (L:D) 12:12 h for rats and 14:10 h for hamsters), food and water available continuously.
Materials [3H]Flunitrazepam ([3H]FLU) and [3H]diazepam ([3H]DZP) were obtained from Amersham at a specific activity of 85 Ci/mmol. Clonazepam and DZP were obtained from Sigma. TZ was kindly provided by the Upjohn Company, Kalamazoo, Michigan. NPY antiserum was from Peninsula; serotonin (5-HT) antiserum was from Immunonuclear; glutamic acid decarboxylase (GAD) antiserum 4° was the gift of Dr. D.E. Schmechel (Duke Univ., Durham, NC); glial fibrillary acidic protein (GFAP) antiserum was supplied by ACS and the GABAA/BZR monoclonal antibody 62-3G1t~ was the gift of Dr. Angel deBlas (SUNY, Stony Brook, NY).
Immunocytochemistry Animals were sacrificed under deep anesthesia (25 mg/kg ketamine/Rompun) and perfused transcardially with 0.9% saline followed by periodate-lysine-paraformaldehyde fixative (PLp)24. The brains were posffixed for 1 h and then sequentially transferred through sucrose solutions of 10, 20 and 30%, allowing the brains to sink in each solution. The brains were sectioned coronally through the SCN, LGN and DR at 30/zm on a freezing microtome and stored in 0.01 M phosphate-buffered saline. The sections were then processed for immunocytochemistry using the avidin-biotin complex procedure. Throughout the SCN, IGL and DR sections adjacent to those immunostained for GAD or GABAA/BZR were incubated with NPY, GFAP or 5-HT antisera. NPY and GFAP immunoreactivity was used to localize the SCN and IGL. Immunoreactivity for 5-HT was used to define the boundaries of the DR. Sections prepared for GAD immunocytochemistry were stored in 0.05 M Tris-buffered saline and pretreated with H20 z and Triton X-100 as described by Oertel et al.4o. Antisera dilutions were 1:2000 for GAD, NPY and 5-HT antisera and 1:15 for mAb62-3G1. The 5-HT and NPY antisera were raised in rabbit, mAb62-3G1 in mouse and the GAD antiserum in sheep. A total of 7 rats and 6 hamsters were used for all immunocytochemistry.
lmmunocytochemistry on sections used for autoradiography Slide mounted sections previously used for receptor binding and autoradiography were fixed with paraformaldehyde fumes for 15 min and washed in phosphate-buffered saline. The sections were processed for NPY or GFAP immunocytochemistry by the avidinbiotin complex procedure or the peroxidase-antiperoxidase procedure. These sections were then used to locate the IGL for image analysis of BZ binding. Dilutions were 1:1000 for NPY and GFAP antisera.
Receptor binding and autoradiography Tissue preparation. A total of 6 hamsters and 6 rats were deeply anesthetized with ketamine/Rompun solution (25 mg/kg) and then perfused transcardially with 0.1% paraformaldehyde in 0.01 M phosphate-buffered saline. The brains were removed and frozen in
isopentane cooled to -70 °C. Frozen sections were taken at 10 am on a Hacker cryostat and thaw-mounted on gelatin-subbed slides. The slides were stored at 4 *C in boxes with desiccant until ready for use. [~H]Flunitrazepam and f lIfdiazepam binding. Binding was carded out according to the conditions of Young and Kuhar72 and Mohler and Okada3°. Slides with mounted tissue sections were incubated at 4 *C in 0.170 M Tris-HCI (pH 7.4) containing 1-2 nM [3H]FLU for 40 rain or 1.5-3.0 nM [3H]DZP for 35 rain (both from Amersham, 85 Ci/mmole). Clonazepam (Upjohn, 1/zM) was added to the incubation solution for some sections to determine nonspecific binding. The slides were then washed in ice-cold buffer for 3 min and quickly dipped in distilled water before drying the section with a stream of cold dry air. The slides were then used for autoradiography. Autoradiography. Autoradiography was carried out with tritiumsensitive film according to the method of Palacios et al.42. The slide-mounted tissue sections were placed in 10 x 12 cassettes in rows and opposed to the emulsion side of a sheet (24 x 3 cm) of [3H]Ultrofilm (LKB) before closing the cassettes. After 3 weeks exposure at 4 °C the film was developed for 5 min at 22 °C in Kodak D-19 developer rinsed in acetic acid-water then fixed in Kodak rapid fix for 5 min. In some cases, sections were also exposed to Kodak NTB-2 emulsion-coated coverslips for 2 weeks. This allowed greater resolution and direct visualization of silver grains over immunocytochemically stained regions. The coverslips was then developed as described above for the film. GFAP staining reliably defines the IGL in hamster but not rat 36 and stains slide-mounted sections more robustly than does NPY. Therefore, sections containing IGL were immunocytochemically stained for NPY (rats) or GFAP (hamsters) and all other sections were Nissl stained. Semiquantitative analysis of regional silver grain density in the film autoradiograms was carded out with the Drexel University image analysis system BRAIN 1.01. Relative optical density was converted to fmol/mg tissue equivalents from a standard curve generated using tritium plastic standards (American Radiochemicals) exposed along with the tissue. Specific binding was determined as the difference between total binding and non-specific binding obtained by direct density measurements on the autoradiograms. Binding within discrete areas was determined by superimposing the autoradiogram onto the immunocytochemically or Nissl-stained tissue section.
RESULTS
Immunocytochemistry G A D - l i k e immunoreactivity is present throughout the SCN of both hamster and rat (Fig. 1A,B). There are some stained perikarya in both species but the n u m b e r is variable and the staining light. The principal staining is of n u m e r o u s small punctate structures present throughout the entire nucleus but c o n c e n t r a t e d somewhat more in the dorsomedial region. The staining overlaps with the distribution of both N P Y and G F A P staining (data not shown). In both hamster and rat I G L , there is dense G A D - l i k e i m m u n o s t a i n i n g of punctate structures, from the rostral portion of the geniculate complex to the caudal zone lateral and ventral to the medial geniculate (Fig. 1C,D). This staining overlaps with the i m m u n o staining for both NPY and G F A P throughout the extent of the nucleus (data not shown). The raphe nuclei, both dorsal raphe and median raphe, contain a dense plexus of GAD-immunoreactive fibers and
18
Fig. 1. Photomicrographs showing distribution of GAD in coronal sections through the SCN of hamster (A) and rat (B), the IGL of hamster (C, note the blood vessel which is a characteristic feature of the IGL; arrowheads indicate dorsal border of IGL) and rat (D) and the dorsal raphe of hamster (E) and rat (F). IIIV, third ventricle; DLG, dorsal lateral geniculate; IGL, intergeniculate leaflet; OC, optic chiasm; VLG. ventral lateral geniculate. Bar = 0.1 mm.
punctate structures (Fig. 1E,F) which overlap the distribution of serotonin-containing neurons (data not shown).
G A B A A / B Z R - l i k e immunoreactivity d e m o n s t r a t e d by the monoclonat antibody, m A b 6 2 - 3 G 1 , is present in the
19
D
m
Fig. 2. Photomicrographs showing distribution of mAb 62-3G1 immunoreactivity in coronal sections through the SCN of hamster (A) and rat (B), the IGL of hamster (C) and rat (D) and dorsal raphe of hamster (E) and rat (F). Abbreviations as in Fig. 1. Bar -- 0.1 m.
raphe nuclei but not in the SCN or I G L (Fig. 2). The SCN in both rat and hamster is entirely free of immunoreactive structures (Fig. 2A,B). In the lateral genicu-
late complex ( L G N ) , the dorsal L G N and lateral area of the ventral L G N contain GABAA/BZR staining while the IGL, as defined by NPY immunostaining (not shown), is
20
Fig. 3. Darkfield photomicrograph of the autoradiographic image of 1.5 nM [3H]FLU binding to a 12/am coronal section through the hamster SCN. The arrowheads indicate the lateral borders of the nucleus. Abbreviations as in Fig. 1. Bar = 0.1 mm.
entirely free of staining (Fig. 2C,D). Some of the dense immunoreactivity in the raphe nuclei appears to be localized to small punctate structures but much of it is associated with relatively long, linear structures (Fig. 2E,F).
[3H]FLU and [3H]DZP binding and autoradiography The density of binding of [3H]FLU and [3H]DZP in selected areas of rat and hamster brain at the level of the SCN, the I G L and the D R is summarized in Table I. Binding in the region of a representative hamster SCN is shown for [3H]FLU (Fig. 3). The binding of both [3H]FLU and [3H]DZP is moderate within the SCN (an average of 45.7 + 1.4 fmol/mg tissue for 1.5 nM F L U and 23.5 + 2.8 fmol/mg tissue for 1.5 nM DZP).
Fig. 5. Photograph of the computer-enhanced darkfield autoradiographic image of 1.5 nM [3H]FLU binding to a 12 !lm coronal section at the level of the hamster DR. Bar = 0.5 mm.
Binding in the region of a representative hamster L G N is shown for [3H]FLU (Fig. 4). The binding of both [3H]FLU and [3H]DZP were low within the I G L (an average of 20.3 _+ 1.2 fmol/mg tissue for 1.5 nM F L U and 13.7 _+ 1.8 fmol/mg tissue for 1.5 nM D Z P ) compared to the surrounding areas, in particular the lateral region of the ventral L G N (an average of 55.1 _+ 1.2 fmol/mg tissue for 1.5 nM F L U and 36.1 _+ 2.5 fmol/mg tissue for 1.5 nM DZP). Binding in the region of a representative hamster D R
TABLE I
Density of specific [3H]FLU and [3H]DZP binding sites in selected regions of hamster and rat brain The binding of 1.5 nM [3H]FLU and [3H]DZP was to 12 /am cryostat-cut slide-mounted coronal sections through the SCN, IGL and DR. Values represent the mean + S.E.M., the number of determinations given in parentheses. These values are based on densitometric measurements with specific binding determined as the difference between total and non-specific binding densities. SCN, suprachiasmatic nucleus; All, anterior hypothalamus; IGL, intergeniculate leaflet; dLGN, dorsal lateral geniculate nucleus; vLGN, ventral lateral geniculate nucleus; DG, dentate gyrus; DR, dorsal raphe nucleus.
i,
~~i~ Region
Receptordensity (fmol/mg tissue) Hamster
2
Fig. 4. Darkfield photomicrograph of the autoradiographic image of 1.5 nM [3H]FLU binding to a 12/am coronal section through the hamster IGL (about midway through the rostral-caudal extent of the nucleus). OPT, optic tract; other abbreviations as in Fig. 1. Bar = 0.1 mm.
SCN AH IGL dLGN vLGN DG DR
Rat
[3HIFLU
[3nlnZP fH]FLV
[~t-tlOZp
45.7+1.4(5) 59.6+1.8(3) 20.3+1.2(4) 35.6+1.8(4) 55.1+1.2(4) 97.9+1.2(4) 66.8+3.5(7)
23.5+2.8(5) 29.8+2.7(3) 13.7+1.8(4) 29.7+1.0(4) 36.1+2.5(4) 53.3+2.8(4) 33.7+2.8(4)
10.7+1.4(5) 21.0+1.6(4) 5.1+1.6(4) 19.9+1.3(4) 24.6+1.4(4) 36.9+1.8(4) 35.5+0;5(4)
35.0+1.2(5) 47.4+1.6(5) 12.2+1.2(4) 22.1___1.2(4) 39.7___0.9(4) 74.8+1.6(4) 43.3+1.6(6)
21 TABLE II GABA-BZ markers in 3 nuclei of the circadian rhythm generating system
Density of immunoreactivestructures or autoradiographiclabeling is designated in a roughly linear subjective scale from - (absent) to + + + + (very dense). Nucleus
GAD-IR
GABAA/BZR-1R BZbinding
SCN IGL DR
++++ ++ + +++
++
++ + ++++
is shown for [3H] (Fig. 5). The binding of both [3H]FLU and [3H]DZP are higher in the DR than in the IGL or SCN (an average of 66.8 + 3.5 fmol/mg tissue for 1.5 nM FLU and 33.7 + 2.8 fmol/mg tissue for 1.5 nM DZP). DISCUSSION A summary of the results obtained with G A B A neuron and G A B A receptor markers used is shown in Table II. Immunocytochemical staining for the GABAsynthesizing enzyme, GAD, was observed in fibers and punctate structures in SCN, IGL and D R of both hamster and rat. Previous electron microscopic studies have described GAD-like immunoreactivity within axon terminals in the S E N 61 and D R 38'39 and we have previously observed GAD-like immunoreactivity in the hamster IGL 37. This pattern of immunoreactivity in the SCN, IGL and D R suggests that one or both of the known G A B A receptor subtypes ( G A B A A or GABAB) is present in these regions. The G A B A A receptor subtype is associated with a chloride channel and several modulatory binding sites, among them a benzodiazepine binding site through which benzodiazepines can potentiate the inhibitory effects of G A B A 11'56. The G A B A B receptor subtype, which is not associated with a chloride channel or a known benzodiazepine binding site, has not been as well studied but appears to have both a presynaptic and postsynaptic localization and may be associated with calcium channels, potassium channels and/or a cyclic nucleotide second messenger system 2'4'5'2°'54. In the present study immunoreactivity for the GABAA/ BZR monoclonal antibody, mAb62-3G1, correlated well with [3H]BZ binding in the LGN and DR but not in the SCN of rat or hamster. MAb62-3G1 immunoreactivity was absent from the IGL and dense in the dLGN and lateral region of the vLGN. Similarly, the binding of the [3H]BZs FLU and DZP within the IGL was low compared to most other regions including adjacent regions. For example binding with the lateral region of the ventral LGN was approximately 2.5-fold higher and within the dentate gyrus approximately 5-fold higher than
in the IGL. There was also a good correlation between receptor immunoreactivity and BZ binding in the region of the DR. Receptor immunoreactivity was dense and BZ binding very high (3-4-fold higher than in the IGL and 1.5-2-fold higher than in the SCN). In the SCN a moderate level of [3H]BZ binding is present but no receptor immunoreactivity is observed. The patterns of GABAA/BZR immunoreactivity and [3H]BZ binding strongly suggest that the IGL contains a very low level of G A B A A / B Z R , while the DR contains a high level of such receptors. Their presence in the SCN remains uncertain since ligand binding for the receptor is present but we could not demonstrate receptor immunocytochemically. These autoradiographic and immunocytochemical patterns were similar for both hamster and rat. The ability of lesions of the IGL to block the phase-shifting effects of T Z 17 implicates the IGL as a primary site for TZ action. The present results do not support such a hypothesis. Nevertheless, the present data are consistent with the view that TZ acts at a site in a neural circuit that includes the IGL as a necessary component. In light of the low levels of markers for the GABAA/BZR in the IGL the dense GAD-like staining may reflect the presence of G A B A B receptors in this region. The GABAB agonist, baclofen, has been shown to block both phase delays and advances induced by light 46. The SCN and DR are also possible sites for interactions of TZ and other BZs with the circadian system. The SCN is a logical possibility because it is the accepted location for the circadian clock33 and its rhythm generation is phasically responsive to a variety of drugs 58. In addition the SCN sends an efferent projection to the IGL that could provide reciprocal control of the G H T input to the S C N 7'67. However, the presence of GABAA/BZR in the SCN remains to be clarified. Although ligands for the BZ site of the receptor complex bind at moderate levels in the SCN (present study), binding of [3H]muscimol, a ligand for the G A B A site, is very low in the SCN 42 and receptor immunoreactivity is absent. In contrast, the unequivocal high density of GABAA/BZR in the DR combined with its anatomical connections to both the SCN and the IGL would suggest the DR as a likely site for G A B A and benzodiazepine modulation of circadian rhythms. However, in a recent study 28'53 neurotoxic lesions of the hamster serotonergic system did not impair the ability of TZ to accelerate re-entrainment to an altered light-dark cycle. Other, more minor, inputs into the SCN include inputs from other hypothalamic areas 9'1°'33. However, these areas, like the SCN, contain low to moderate levels of benzodiazepine binding sites and do not exhibit immunoreactivity for the G A B A A / B Z R . The subicular cortex
22 of the h i p p o c a m p u s is rich in both m a r k e r s for G A B A A / B Z R and sends a p r o j e c t i o n to the region surrounding the SCN 25. H o w e v e r , the projection does not enter the SCN so is unlikely to m e d i a t e G A B A and B Z effects on circadian rhythms. Both [3H]FLU binding13,57,71,72 and [3H]DZP binding 13'29'3°'55 to G A B A A / B Z R have been well-characterized in the brain. T h e specificity of the monoclonal a n t i b o d y m A b 6 2 - 3 G 1 for the G A B A A / B Z R has also been well-defined. T h e antibody was raised to the G A B A A / B Z R purified on benzodiazepine affinity columns and i m m u n o p r e c i p i t a t e s a r e c e p t o r complex which retains binding activities for the benzodiazepines, for muscimol and for chloride channel m a r k e r s 66. H o w e v e r , the distribution of mAb62-3G1 immunoreactivity colocalizes b e t t e r with [3H]muscimol ( G A B A A site) than with [3H]FLU ( b e n z o d i a z e p i n e site) binding 12. Biochemical and immunological evidence suggest that G A B A A / B Z R is c o m p o s e d of at least two subunit types, a, which binds the B Z s , and fl, which exhibits G A B A binding s'23m. Subtypes of the a-subunit have been cloned which, when expressed with the fl-subunit, form functional r e c e p t o r subtypes 21'22. These r e c e p t o r subtypes have distinct distributions in the brain t8"51"69'7°. T h e r e are ' p e r i p h e r a l ' B Z binding sites which are not linked to G A B A receptors but these receptors are present in very limited areas of the brain such as the e p e n d y m a , choroid plexus and olfactory neurons 5°'65. Therefore, the B Z sites shown
by [3H]BZ binding in the SCN m a y represent a G A B A A / B Z R subtype or conformation which binds G A B A A ligands with very low affinity and is not recognized by mAb62-3G1. It has been suggested that some actions of BZs may be e x e r t e d at as yet unidentified binding sites possibly linked to potassium channels 43. In situ hybridization using c D N A ' s encoding the different G A B A A / B Z R subunits m a y help to clarify the distribution of these r e c e p t o r subtypes with the circadian system. In conclusion, based on both the pattern of B Z binding and of r e c e p t o r immunoreactivity the D R would a p p e a r to be a likely target site for G A B A A and B Z action. A l t h o u g h the SCN a p p e a r s to be a possible target site as well, the characteristics of the B Z binding sites in the SCN remain tO be elucidated. The results suggest the I G L is not a site for direct G A B A A and B Z action, but do not exclude a role for the I G L in the neural mechanisms mediating G A B A and b e n z o d i a z e p i n e interactions with the circadian system. F u r t h e r studies are necessary to clarify the function of G A B A neurons and exogenous B Z s in the regulation of the circadian timing system.
Acknowledgements. This work was supported by NIH Grants NS-16304 to R.Y.M. and NS-22168 to L.P.M. We are grateful to Dr. Angel DeBlas for providing the antibody to the GABAA/BZR, to Ms. Joan Speh for her excellent technical advice and assistance and to Ms. Priscilla Wu, Ms. Jane Blanchard and Ms. Alice Borella.
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receptors, Annu. Rev. Pharmacol. Toxicol., 29 (1989) 307-322. 66 Vitorica, J., Park, D., Chin, G. and Deblas, A.L., Monoclona[ antibodies and conventional antisera to the GABA A receptor/ benzodiazepine receptor/Cl-channel complex, J. Neurosci., 8 (1988) 615-622. 67 Watts, A.G., Swanson, L.W. and Sanchez-Watts, G., Efferent Projections of the suprachiasmatic nucleus: I. Studies using anterograde transport of phaseolus vulgaris leucoagglutinin in the rat, J. Comp. Neurol., 258 (1987) 204-229. 68 Weaver, D.R., Rivkees, S.A. and Reppert, S.M., Localization and characterization of melatonin receptors in rodent brain by in vitro autoradiography, J. Neurosci., 9 (1989) 2581-2590. 69 Wisden, W., Morris, B.J., Darlison, M.G., Hunt, S.P. and Barnard, E.A., Distinct GABA A receptor a subunit mRNAs show differential patterns of expression in bovine brain, Neuron, 1 (1988) 937-947. 70 Wisden, W., Morris, B.J., Darlison, M.G., Hunt, S.P. and Barnard, E.A., Differential distribution in bovine brain of distinct gamma-aminobutyric acid A receptor a-subunit mRNAs, Biochem. Soc, Trans., 17 (1989) 566-567. 71 Young, W.S. and Kuhar, M.J., Autoradiographic localisation of benzodiazepine receptors in the brains of humans and animals, Nature, 280 (1979) 391-394. 72 Young, W.S. III. and Kuhar, M.J., Radiohistochemical localization of benzodiazepine receptors, J. Pharmacol. Exp. Ther., 212 (1980) 337-346.
16
BRES 15950
GABAA/benzodiazepine receptor localization in the circadian timing system K.M. Michels 1, L.P. M o r i n 2'3 a n d R.Y. M o o r e ~'4 Departments of 1Neurology, 2psychiatry, 3psychology and 4Neurobiology and Behavior, State University of New York, Stony Brook, NY (U.S.A.) (Accepted 24 April 1990)
Key words: 7-Aminobutyric acid; y-Aminobutyric acid A/Benzodiazepine receptor; Glutamic acid decarboxylase; Benzodiazepine binding; Suprachiasmatic nucleus; Intergeniculate leaflet; Raphe nucleus
y-Aminobutyric acid (GABA) and exogenous benzodiazepines are thought to play a role in the neural regulation of circadian rhythms. Because binding sites for the benzodiazepines and GABA A iigands are functionally coupled as part of the GABAA/benzodiazepine receptor complex (GABAA/BZR), we analyzed the localization of GABA neurons and GABAA/BZR within 3 nuclei involved in circadian rhythm regulation using autoradiographic and immunohistochemical techniques. Glutamic acid decarboxylase-immunoreactive axons are present in the suprachiasmatic nuclei (SCN), intergeniculate leaflet (IGL), and dorsal raphe nucleus (DR). Immunoreactivity for the GABAA/BZ receptor complex is absent from the SCN and the IGL whereas the DR shows a dense, uniform immunoreactivity. Semiquantitative analysis of autoradiograms for [3H]diazepam and [3H]flunitrazepam binding reveals a moderate level of binding in the SCN, a low level of binding in the IGL, and the highest level of the DR. Based on both the pattern of benzodiazepine binding and of receptor immunoreactivity the DR would appear to be a likely target site for GABAA and benzodiazepine action. The SCN would also appear to be a possible target site. The results suggest the IGL is not a site for direct GABAA and benzodiazepine action, but do not exclude a role for the IGL in the neural circuitry mediating GABA and benzodiazepine interactions with the circadian system.
INTRODUCTION Exogenous benzodiazepines potentiate many of the effects of the inhibitory neurotransmitter y-aminobutyric acid ( G A B A ) within the central nervous system. The benzodiazepine (BZ) binding site is associated with the G A B A A receptor and a chloride ionophore to form the G A B A A / B Z receptor complex ( G A B A A / B Z R ) 41. The participation of G A B A A / B Z R in the regulation of circadian function is suggested by pharmacological studies of rodent activity rhythms. Bicuculline, a G A B A A antagonist, and the benzodiazepine agonist, diazepam, block light-induced phase delays and advances, respectively, in hamsters 44-46. The short-acting B Z agonist triazolam (TZ) causes either phase advances or delays of golden hamster activity rhythms dependent upon the time of administration 59, accelerates re-entrainment after an 8-h photoperiod shift 63 and entrains the activity rhythm 64. The location of the receptor complex responsible for mediating G A B A and B Z effects on circadian rhythmicity is not known, but several sites merit consideration. The first is the suprachiasmatic nucleus (SCN) which is
the principal circadian pacemaker in mammals 33'47"49, and effects on entrainment, whether direct or indirect, must be expressed through it 15. Regions which project to the SCN are also candidates for mediating T Z effects on circadian rhythm phase. There is a direct retinal input to the SCN 34 and the pineal gland releases melatonin which may act at melatonin receptors within the SEN 19'68. However, neither blinding nor pinealectomy blocks the phase-shifting effects of T Z 62 and message for G A B A A / B Z R is not present in the pineal 52. The intergenicutate leaflet (IGL) receives direct retinal afferents and projects to the SCN through a neuropeptide-Y (NPY)-containing pathway the geniculo-hypothalamic tract ( G H T ) 6A4'32. Activation of the G H T 16"26'48 and infusion of N P Y into the SCN 1 produce a phase-response curve that is very similar to that for T Z 59. Ablation of the I G L eliminates the phase-shifting effects of T Z 17. Therefore, the I G L may be one site at which T Z acts to modify circadian rhythm phase. The raphe nuclei represent another potential site since they project to both the SCN and the I G L 3,35,60. In the present study we investigated the localization of
Correspondence: K.M. Michels. Present address: NIMH-LCS, Section on Pharmacology, Bldg. 10, Rm. 2D-45, Bethesda, MD 20892, U.S.A. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
17 the G A B A - s y n t h e s i z i n g enzyme, glutamic acid decarboxylase ( G A D ) within the SCN, I G L and dorsal raphe nucleus ( D R ) by immunocytochemistry and the localization of G A B A A / B Z R using immunocytochemistry and autoradiographic d e m o n s t r a t i o n of benzodiazepine binding. A preliminary report of these results has b e e n previously presented 27.
MATERIALS AND METHODS
Animals Adult Sprague-Dawley rats and outbred golden hamsters were maintained in standard laboratory conditions (light:dark (L:D) 12:12 h for rats and 14:10 h for hamsters), food and water available continuously.
Materials [3H]Flunitrazepam ([3H]FLU) and [3H]diazepam ([3H]DZP) were obtained from Amersham at a specific activity of 85 Ci/mmol. Clonazepam and DZP were obtained from Sigma. TZ was kindly provided by the Upjohn Company, Kalamazoo, Michigan. NPY antiserum was from Peninsula; serotonin (5-HT) antiserum was from Immunonuclear; glutamic acid decarboxylase (GAD) antiserum 4° was the gift of Dr. D.E. Schmechel (Duke Univ., Durham, NC); glial fibrillary acidic protein (GFAP) antiserum was supplied by ACS and the GABAA/BZR monoclonal antibody 62-3G1t~ was the gift of Dr. Angel deBlas (SUNY, Stony Brook, NY).
Immunocytochemistry Animals were sacrificed under deep anesthesia (25 mg/kg ketamine/Rompun) and perfused transcardially with 0.9% saline followed by periodate-lysine-paraformaldehyde fixative (PLp)24. The brains were posffixed for 1 h and then sequentially transferred through sucrose solutions of 10, 20 and 30%, allowing the brains to sink in each solution. The brains were sectioned coronally through the SCN, LGN and DR at 30/zm on a freezing microtome and stored in 0.01 M phosphate-buffered saline. The sections were then processed for immunocytochemistry using the avidin-biotin complex procedure. Throughout the SCN, IGL and DR sections adjacent to those immunostained for GAD or GABAA/BZR were incubated with NPY, GFAP or 5-HT antisera. NPY and GFAP immunoreactivity was used to localize the SCN and IGL. Immunoreactivity for 5-HT was used to define the boundaries of the DR. Sections prepared for GAD immunocytochemistry were stored in 0.05 M Tris-buffered saline and pretreated with H20 z and Triton X-100 as described by Oertel et al.4o. Antisera dilutions were 1:2000 for GAD, NPY and 5-HT antisera and 1:15 for mAb62-3G1. The 5-HT and NPY antisera were raised in rabbit, mAb62-3G1 in mouse and the GAD antiserum in sheep. A total of 7 rats and 6 hamsters were used for all immunocytochemistry.
lmmunocytochemistry on sections used for autoradiography Slide mounted sections previously used for receptor binding and autoradiography were fixed with paraformaldehyde fumes for 15 min and washed in phosphate-buffered saline. The sections were processed for NPY or GFAP immunocytochemistry by the avidinbiotin complex procedure or the peroxidase-antiperoxidase procedure. These sections were then used to locate the IGL for image analysis of BZ binding. Dilutions were 1:1000 for NPY and GFAP antisera.
Receptor binding and autoradiography Tissue preparation. A total of 6 hamsters and 6 rats were deeply anesthetized with ketamine/Rompun solution (25 mg/kg) and then perfused transcardially with 0.1% paraformaldehyde in 0.01 M phosphate-buffered saline. The brains were removed and frozen in
isopentane cooled to -70 °C. Frozen sections were taken at 10 am on a Hacker cryostat and thaw-mounted on gelatin-subbed slides. The slides were stored at 4 *C in boxes with desiccant until ready for use. [~H]Flunitrazepam and f lIfdiazepam binding. Binding was carded out according to the conditions of Young and Kuhar72 and Mohler and Okada3°. Slides with mounted tissue sections were incubated at 4 *C in 0.170 M Tris-HCI (pH 7.4) containing 1-2 nM [3H]FLU for 40 rain or 1.5-3.0 nM [3H]DZP for 35 rain (both from Amersham, 85 Ci/mmole). Clonazepam (Upjohn, 1/zM) was added to the incubation solution for some sections to determine nonspecific binding. The slides were then washed in ice-cold buffer for 3 min and quickly dipped in distilled water before drying the section with a stream of cold dry air. The slides were then used for autoradiography. Autoradiography. Autoradiography was carried out with tritiumsensitive film according to the method of Palacios et al.42. The slide-mounted tissue sections were placed in 10 x 12 cassettes in rows and opposed to the emulsion side of a sheet (24 x 3 cm) of [3H]Ultrofilm (LKB) before closing the cassettes. After 3 weeks exposure at 4 °C the film was developed for 5 min at 22 °C in Kodak D-19 developer rinsed in acetic acid-water then fixed in Kodak rapid fix for 5 min. In some cases, sections were also exposed to Kodak NTB-2 emulsion-coated coverslips for 2 weeks. This allowed greater resolution and direct visualization of silver grains over immunocytochemically stained regions. The coverslips was then developed as described above for the film. GFAP staining reliably defines the IGL in hamster but not rat 36 and stains slide-mounted sections more robustly than does NPY. Therefore, sections containing IGL were immunocytochemically stained for NPY (rats) or GFAP (hamsters) and all other sections were Nissl stained. Semiquantitative analysis of regional silver grain density in the film autoradiograms was carded out with the Drexel University image analysis system BRAIN 1.01. Relative optical density was converted to fmol/mg tissue equivalents from a standard curve generated using tritium plastic standards (American Radiochemicals) exposed along with the tissue. Specific binding was determined as the difference between total binding and non-specific binding obtained by direct density measurements on the autoradiograms. Binding within discrete areas was determined by superimposing the autoradiogram onto the immunocytochemically or Nissl-stained tissue section.
RESULTS
Immunocytochemistry G A D - l i k e immunoreactivity is present throughout the SCN of both hamster and rat (Fig. 1A,B). There are some stained perikarya in both species but the n u m b e r is variable and the staining light. The principal staining is of n u m e r o u s small punctate structures present throughout the entire nucleus but c o n c e n t r a t e d somewhat more in the dorsomedial region. The staining overlaps with the distribution of both N P Y and G F A P staining (data not shown). In both hamster and rat I G L , there is dense G A D - l i k e i m m u n o s t a i n i n g of punctate structures, from the rostral portion of the geniculate complex to the caudal zone lateral and ventral to the medial geniculate (Fig. 1C,D). This staining overlaps with the i m m u n o staining for both NPY and G F A P throughout the extent of the nucleus (data not shown). The raphe nuclei, both dorsal raphe and median raphe, contain a dense plexus of GAD-immunoreactive fibers and
18
Fig. 1. Photomicrographs showing distribution of GAD in coronal sections through the SCN of hamster (A) and rat (B), the IGL of hamster (C, note the blood vessel which is a characteristic feature of the IGL; arrowheads indicate dorsal border of IGL) and rat (D) and the dorsal raphe of hamster (E) and rat (F). IIIV, third ventricle; DLG, dorsal lateral geniculate; IGL, intergeniculate leaflet; OC, optic chiasm; VLG. ventral lateral geniculate. Bar = 0.1 mm.
punctate structures (Fig. 1E,F) which overlap the distribution of serotonin-containing neurons (data not shown).
G A B A A / B Z R - l i k e immunoreactivity d e m o n s t r a t e d by the monoclonat antibody, m A b 6 2 - 3 G 1 , is present in the
19
D
m
Fig. 2. Photomicrographs showing distribution of mAb 62-3G1 immunoreactivity in coronal sections through the SCN of hamster (A) and rat (B), the IGL of hamster (C) and rat (D) and dorsal raphe of hamster (E) and rat (F). Abbreviations as in Fig. 1. Bar -- 0.1 m.
raphe nuclei but not in the SCN or I G L (Fig. 2). The SCN in both rat and hamster is entirely free of immunoreactive structures (Fig. 2A,B). In the lateral genicu-
late complex ( L G N ) , the dorsal L G N and lateral area of the ventral L G N contain GABAA/BZR staining while the IGL, as defined by NPY immunostaining (not shown), is
20
Fig. 3. Darkfield photomicrograph of the autoradiographic image of 1.5 nM [3H]FLU binding to a 12/am coronal section through the hamster SCN. The arrowheads indicate the lateral borders of the nucleus. Abbreviations as in Fig. 1. Bar = 0.1 mm.
entirely free of staining (Fig. 2C,D). Some of the dense immunoreactivity in the raphe nuclei appears to be localized to small punctate structures but much of it is associated with relatively long, linear structures (Fig. 2E,F).
[3H]FLU and [3H]DZP binding and autoradiography The density of binding of [3H]FLU and [3H]DZP in selected areas of rat and hamster brain at the level of the SCN, the I G L and the D R is summarized in Table I. Binding in the region of a representative hamster SCN is shown for [3H]FLU (Fig. 3). The binding of both [3H]FLU and [3H]DZP is moderate within the SCN (an average of 45.7 + 1.4 fmol/mg tissue for 1.5 nM F L U and 23.5 + 2.8 fmol/mg tissue for 1.5 nM DZP).
Fig. 5. Photograph of the computer-enhanced darkfield autoradiographic image of 1.5 nM [3H]FLU binding to a 12 !lm coronal section at the level of the hamster DR. Bar = 0.5 mm.
Binding in the region of a representative hamster L G N is shown for [3H]FLU (Fig. 4). The binding of both [3H]FLU and [3H]DZP were low within the I G L (an average of 20.3 _+ 1.2 fmol/mg tissue for 1.5 nM F L U and 13.7 _+ 1.8 fmol/mg tissue for 1.5 nM D Z P ) compared to the surrounding areas, in particular the lateral region of the ventral L G N (an average of 55.1 _+ 1.2 fmol/mg tissue for 1.5 nM F L U and 36.1 _+ 2.5 fmol/mg tissue for 1.5 nM DZP). Binding in the region of a representative hamster D R
TABLE I
Density of specific [3H]FLU and [3H]DZP binding sites in selected regions of hamster and rat brain The binding of 1.5 nM [3H]FLU and [3H]DZP was to 12 /am cryostat-cut slide-mounted coronal sections through the SCN, IGL and DR. Values represent the mean + S.E.M., the number of determinations given in parentheses. These values are based on densitometric measurements with specific binding determined as the difference between total and non-specific binding densities. SCN, suprachiasmatic nucleus; All, anterior hypothalamus; IGL, intergeniculate leaflet; dLGN, dorsal lateral geniculate nucleus; vLGN, ventral lateral geniculate nucleus; DG, dentate gyrus; DR, dorsal raphe nucleus.
i,
~~i~ Region
Receptordensity (fmol/mg tissue) Hamster
2
Fig. 4. Darkfield photomicrograph of the autoradiographic image of 1.5 nM [3H]FLU binding to a 12/am coronal section through the hamster IGL (about midway through the rostral-caudal extent of the nucleus). OPT, optic tract; other abbreviations as in Fig. 1. Bar = 0.1 mm.
SCN AH IGL dLGN vLGN DG DR
Rat
[3HIFLU
[3nlnZP fH]FLV
[~t-tlOZp
45.7+1.4(5) 59.6+1.8(3) 20.3+1.2(4) 35.6+1.8(4) 55.1+1.2(4) 97.9+1.2(4) 66.8+3.5(7)
23.5+2.8(5) 29.8+2.7(3) 13.7+1.8(4) 29.7+1.0(4) 36.1+2.5(4) 53.3+2.8(4) 33.7+2.8(4)
10.7+1.4(5) 21.0+1.6(4) 5.1+1.6(4) 19.9+1.3(4) 24.6+1.4(4) 36.9+1.8(4) 35.5+0;5(4)
35.0+1.2(5) 47.4+1.6(5) 12.2+1.2(4) 22.1___1.2(4) 39.7___0.9(4) 74.8+1.6(4) 43.3+1.6(6)
21 TABLE II GABA-BZ markers in 3 nuclei of the circadian rhythm generating system
Density of immunoreactivestructures or autoradiographiclabeling is designated in a roughly linear subjective scale from - (absent) to + + + + (very dense). Nucleus
GAD-IR
GABAA/BZR-1R BZbinding
SCN IGL DR
++++ ++ + +++
++
++ + ++++
is shown for [3H] (Fig. 5). The binding of both [3H]FLU and [3H]DZP are higher in the DR than in the IGL or SCN (an average of 66.8 + 3.5 fmol/mg tissue for 1.5 nM FLU and 33.7 + 2.8 fmol/mg tissue for 1.5 nM DZP). DISCUSSION A summary of the results obtained with G A B A neuron and G A B A receptor markers used is shown in Table II. Immunocytochemical staining for the GABAsynthesizing enzyme, GAD, was observed in fibers and punctate structures in SCN, IGL and D R of both hamster and rat. Previous electron microscopic studies have described GAD-like immunoreactivity within axon terminals in the S E N 61 and D R 38'39 and we have previously observed GAD-like immunoreactivity in the hamster IGL 37. This pattern of immunoreactivity in the SCN, IGL and D R suggests that one or both of the known G A B A receptor subtypes ( G A B A A or GABAB) is present in these regions. The G A B A A receptor subtype is associated with a chloride channel and several modulatory binding sites, among them a benzodiazepine binding site through which benzodiazepines can potentiate the inhibitory effects of G A B A 11'56. The G A B A B receptor subtype, which is not associated with a chloride channel or a known benzodiazepine binding site, has not been as well studied but appears to have both a presynaptic and postsynaptic localization and may be associated with calcium channels, potassium channels and/or a cyclic nucleotide second messenger system 2'4'5'2°'54. In the present study immunoreactivity for the GABAA/ BZR monoclonal antibody, mAb62-3G1, correlated well with [3H]BZ binding in the LGN and DR but not in the SCN of rat or hamster. MAb62-3G1 immunoreactivity was absent from the IGL and dense in the dLGN and lateral region of the vLGN. Similarly, the binding of the [3H]BZs FLU and DZP within the IGL was low compared to most other regions including adjacent regions. For example binding with the lateral region of the ventral LGN was approximately 2.5-fold higher and within the dentate gyrus approximately 5-fold higher than
in the IGL. There was also a good correlation between receptor immunoreactivity and BZ binding in the region of the DR. Receptor immunoreactivity was dense and BZ binding very high (3-4-fold higher than in the IGL and 1.5-2-fold higher than in the SCN). In the SCN a moderate level of [3H]BZ binding is present but no receptor immunoreactivity is observed. The patterns of GABAA/BZR immunoreactivity and [3H]BZ binding strongly suggest that the IGL contains a very low level of G A B A A / B Z R , while the DR contains a high level of such receptors. Their presence in the SCN remains uncertain since ligand binding for the receptor is present but we could not demonstrate receptor immunocytochemically. These autoradiographic and immunocytochemical patterns were similar for both hamster and rat. The ability of lesions of the IGL to block the phase-shifting effects of T Z 17 implicates the IGL as a primary site for TZ action. The present results do not support such a hypothesis. Nevertheless, the present data are consistent with the view that TZ acts at a site in a neural circuit that includes the IGL as a necessary component. In light of the low levels of markers for the GABAA/BZR in the IGL the dense GAD-like staining may reflect the presence of G A B A B receptors in this region. The GABAB agonist, baclofen, has been shown to block both phase delays and advances induced by light 46. The SCN and DR are also possible sites for interactions of TZ and other BZs with the circadian system. The SCN is a logical possibility because it is the accepted location for the circadian clock33 and its rhythm generation is phasically responsive to a variety of drugs 58. In addition the SCN sends an efferent projection to the IGL that could provide reciprocal control of the G H T input to the S C N 7'67. However, the presence of GABAA/BZR in the SCN remains to be clarified. Although ligands for the BZ site of the receptor complex bind at moderate levels in the SCN (present study), binding of [3H]muscimol, a ligand for the G A B A site, is very low in the SCN 42 and receptor immunoreactivity is absent. In contrast, the unequivocal high density of GABAA/BZR in the DR combined with its anatomical connections to both the SCN and the IGL would suggest the DR as a likely site for G A B A and benzodiazepine modulation of circadian rhythms. However, in a recent study 28'53 neurotoxic lesions of the hamster serotonergic system did not impair the ability of TZ to accelerate re-entrainment to an altered light-dark cycle. Other, more minor, inputs into the SCN include inputs from other hypothalamic areas 9'1°'33. However, these areas, like the SCN, contain low to moderate levels of benzodiazepine binding sites and do not exhibit immunoreactivity for the G A B A A / B Z R . The subicular cortex
22 of the h i p p o c a m p u s is rich in both m a r k e r s for G A B A A / B Z R and sends a p r o j e c t i o n to the region surrounding the SCN 25. H o w e v e r , the projection does not enter the SCN so is unlikely to m e d i a t e G A B A and B Z effects on circadian rhythms. Both [3H]FLU binding13,57,71,72 and [3H]DZP binding 13'29'3°'55 to G A B A A / B Z R have been well-characterized in the brain. T h e specificity of the monoclonal a n t i b o d y m A b 6 2 - 3 G 1 for the G A B A A / B Z R has also been well-defined. T h e antibody was raised to the G A B A A / B Z R purified on benzodiazepine affinity columns and i m m u n o p r e c i p i t a t e s a r e c e p t o r complex which retains binding activities for the benzodiazepines, for muscimol and for chloride channel m a r k e r s 66. H o w e v e r , the distribution of mAb62-3G1 immunoreactivity colocalizes b e t t e r with [3H]muscimol ( G A B A A site) than with [3H]FLU ( b e n z o d i a z e p i n e site) binding 12. Biochemical and immunological evidence suggest that G A B A A / B Z R is c o m p o s e d of at least two subunit types, a, which binds the B Z s , and fl, which exhibits G A B A binding s'23m. Subtypes of the a-subunit have been cloned which, when expressed with the fl-subunit, form functional r e c e p t o r subtypes 21'22. These r e c e p t o r subtypes have distinct distributions in the brain t8"51"69'7°. T h e r e are ' p e r i p h e r a l ' B Z binding sites which are not linked to G A B A receptors but these receptors are present in very limited areas of the brain such as the e p e n d y m a , choroid plexus and olfactory neurons 5°'65. Therefore, the B Z sites shown
by [3H]BZ binding in the SCN m a y represent a G A B A A / B Z R subtype or conformation which binds G A B A A ligands with very low affinity and is not recognized by mAb62-3G1. It has been suggested that some actions of BZs may be e x e r t e d at as yet unidentified binding sites possibly linked to potassium channels 43. In situ hybridization using c D N A ' s encoding the different G A B A A / B Z R subunits m a y help to clarify the distribution of these r e c e p t o r subtypes with the circadian system. In conclusion, based on both the pattern of B Z binding and of r e c e p t o r immunoreactivity the D R would a p p e a r to be a likely target site for G A B A A and B Z action. A l t h o u g h the SCN a p p e a r s to be a possible target site as well, the characteristics of the B Z binding sites in the SCN remain tO be elucidated. The results suggest the I G L is not a site for direct G A B A A and B Z action, but do not exclude a role for the I G L in the neural mechanisms mediating G A B A and b e n z o d i a z e p i n e interactions with the circadian system. F u r t h e r studies are necessary to clarify the function of G A B A neurons and exogenous B Z s in the regulation of the circadian timing system.
Acknowledgements. This work was supported by NIH Grants NS-16304 to R.Y.M. and NS-22168 to L.P.M. We are grateful to Dr. Angel DeBlas for providing the antibody to the GABAA/BZR, to Ms. Joan Speh for her excellent technical advice and assistance and to Ms. Priscilla Wu, Ms. Jane Blanchard and Ms. Alice Borella.
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