Distribution of phosphatase inhibitor-1-immunoreactive neurons in the suprachiasmatic nucleus of the Syrian hamster

Brain Research, 623 (1993) 147-154

147

© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00 BRES 19228

Distribution of phosphatase inhibitor-l-immunoreactive neurons in the suprachiasmatic nucleus of the Syrian hamster Jens D. Mikkelsen

a

and Eric L. Gustafson b.,

a Institute of Medical Anatomy, Department B, Universityof Copenhagen, Copenhagen (Denmark) and b Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York (USA)

(Accepted 20 April 1993)

Key words: Phosphorylation; Circadian; Phosphoprotein; Suprachiasmatic nucleus; Immunohistochemistry

The protein phosphatase inhibitor-1 (I-l) is phosphorylated by a cyclic AMP-dependent protein kinase, and is itself involved in the regulation of phosphorylation of other proteins. The enzyme has been shown to he present in skeletal muscles and in distinct neuronal systems of the brain. The suprachiasmatic nucleus is essential in generation of circadian rhythms, but the cellular mechanisms by which the oscillator is entrained are not understood. Since cyclic AMP is known to phase shift the rhythm of electrical activity in SCN neurons in vitro, we aimed by an avidin-biotin immunohistochemical technique to localize I-l-containing neurons in the hamster suprachiasmatic nucleus and thereby identify potential target neurons for cyclic AMP effects. Numerous densely stained neurons were observed in the hamster SCN. The I-l-immunoreactive cell bodies were intermingled with non-immunoreactive neurons and occupied mostly the ventral half of the nucleus, but cell bodies were found in all compartments of the nucleus. The Ll-immunoreactive neurons located in the ventral SCN sent dendrite-like processes into the underlying optic chiasm, indicating that they are directly innervated from the retina, the intergeniculate leaflet of the thalamus, and/or the dorsal raphe. A few I-l-immunoreactive neurons were observed immediately outside the borders of the SCN, but their pronounced staining intensity and their similar morphology to those found inside the SCN indicate that they belong to the same type of neurons as found in the SCN. Delicate I-l-immunoreactive nerve fibers possessing boutons were found throughout the SCN. Furthermore, axonal fibers were followed dorsally into the subparaventricular area. These data demonstrate that the I-1 is highly concentrated in a large portion of neurons within the hamster SCN. The functional significance of the I-1 in SCN neurons remains to be established.

INTRODUCTION Protein p h o s p h o r y l a t i o n represents a p a t h w a y t h r o u g h which neurotransmitters p r o d u c e their biological effect in target cells 13. T h e elevation o f the intracellular messenger, c A M P , which after binding o f ligand to m e m b r a n e - b o u n d receptors results in increased phosphorylation of target proteins by the c A M P - d e p e n d e n t protein kinases represents o n e m e c h a n i s m of phosphorylation 9'2s. A n o t h e r route for increasing cellular protein phosphorylation is inhibition o f the protein phosphatases. This activity is regulated by a n u m b e r o f p h o s p h a t a s e inhibitors. T h e m e m b e r s o f the family o f inhibiting enzymes are all low molecular weight, acidsoluble, and heat-stable proteins exhibiting similarities in their amino acid compositions 1-3,29,41 (see refs. 13, 28). In particular the amino sequences a r o u n d the

phosphorylation sites of the protein p h o s p h a t a s e inhibitor-I, and inhibitor-2, G-substrate and D A R P P - 3 2 show substantial homology. Despite this h o m o l o g y the p h o s p h a t a s e inhibitors have b e e n f o u n d to have differences in their kinetics 12, and they are differently distributed in the brain 14. F o r example the D A R R P - 3 2 immunoreactive cells were f o u n d to be t h e D 1 d o p a m i n o r e c e p t i v e cells mainly in the striatum, whereas the G-substrate was f o u n d in the purkinje cells of the cerebellum 3°'35'4°. Thus, it m a y be that o t h e r p h o s p h o proteins are present in n e u r o n a l populations stimulated by specific neurotransmitters or h o r m o n e s as shown to be the case with D A R R P - 3 2 . T h e p h o s p h a t a s e inhibitors have b e e n isolated, their c D N A s cloned, and they have b e e n localized in specific ceils in the organism. A m o n g these is the p h o s p h a t a s e inhibitor-1 (I-1), which was isolated f r o m rabbit skele-

Correspondence: J.D. Mikkelsen, Institute of Medical Anatomy B, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. Fax: (45) (3) 5369612. * Present address: Synaptic Pharmaceutical Corporation, 215 College Road, Paramus, NJ 07652, USA.

148

Fig. 1. Five photomicrographs of frontal sections through the hamster SCN. Fig. A shows the prechiasmatic area, where a few positive cells are observed. The I-l-immunoreactive cell bodies are distributed mainly in the ventral part of the SCN. V, third ventricle. Bar = 200/~m.

149 tal muscle 29, and its primary sequence has been determined l's. Northern RNA hybridization analysis of rat tissue RNA showed that I-1 mRNA was present in high concentrations in brain, and skeletal muscles, but not in heart, lung, liver, or kidney 8. However, MacDougall et al. TM detected the protein by immunoblotting and found immunoreactivity to the 26 kDa protein in rat brain, uterus, adipose tissue, and skeletal muscle. The localization of I-1 has been investigated in the rat brain by use of immunohistochemistry 10. High numbers of I-l-immunoreactive cell bodies neurons were found in the cerebral cortex, the dentate gyrus of the hippocampal formation, the lateral hypothalamus, the horizontal limb of the diagonal band of Broca, the habenula, the superior colliculus, the caudate-putamen, the accumbens, and in the hypothalamic suprachiasmatic nucleus (SCN). The SCN is known to be an important nucleus in the generation of circadian rhythms 20'25'34. The presence of specific enzymes responsible for protein phosphorylation in neuronal signal transduction in these cells may help to understand the molecular mechanisms involved in entrainment processes of circadian rhythms. The presence of specific proteins known to mediate receptor transmission within the SCN will provide an important distinction of SCN in terms of function. In this context, we analyzed

the distribution of 1-1 in the SCN with emphasis on its distribution in relation to the subcompartments and the inputs of the nucleus. MATERIALS AND METHODS Tissue and fixation Ten adult male Syrian hamsters (Mesocriteus auratus) kept under a 12L:12D light-regimen were deeply anaesthetized with tribromethanol i.p. (250 mg/kg b.wt.) 5-10 h after light onset. The animals were perfused through the heart first with 50 mM phosphate buffered saline (PBS; pH 7.4) to which 15.000 I U / l heparine was added and subsequently, they were fixed with 300 ml 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) equilibrated to room temperature for 15 min. The brain was rapidly removed, dissected in a diencephalic block and placed in the same fixative overnight. The tissue was placed in a solution of 30% sucrose in PBS for 24 h and 40/~m thick serial frontal sections were cut in a cryostat and placed in PBS until use. The sections were rinsed for 2 times 5 min in PBS with 0.02% KCl (KPBS; pH 7.4) to which 0.25% bovine serum albumin (BSA) and 0.1% Triton X-100 was added (KPBS-BT) and pretreated in 1% H 2 0 2 in KPBS for 10 min. The sections were then incubated for 20 min in a 4% swine serum solution in KPBS containing 0.3% Triton X-100 and 1% BSA, and incubated for 16-24 h at 4°C in the primary antiserum against I-1 (code G187) diluted 1:200 in KPBS-BT (pH 7.4). The antiserum raised against I-1 purified from rabbit skeletal muscle has been characterized previously I°,14. The antiserum recognizes a 29 kDa protein and shows no cross-reactivity with DARPP321°'14. The sections were then washed in KPBS-BT 3 times for 10 min followed by incubation with a biotinylated swine anti-rabbit IgG (no. E353, Dakopatts, Copenhagen) diluted 1:600 in KPBS-BT for 60 min at room temperature. They were next washed for 3 times 10

Fig. 2. Frontal sections of the hamster SCN showing the distribution of I-l-immunoreactive neurons in the middle SCN (A) and in the retrochiasmatic area (B). The figures demonstrate that a few I-l-positive neurons are found outside the borders of the SCN. l-l-immunoreactive cells are found in the lateroanferior hypothalamic area (arrows, A). The asteriks indicates a part of the SCN, where only few I-1 cell bodies are present. Single cell bodies are also found in the retrochiasmatic area (B). V, third ventricle. Bar = 200/~m.

150 rain in KPBS-BT, and finally incubated for 60 min at room temperature in an ABC-streptavidin-horseradish peroxidase complex (code #K377, Dakopatts, Copenhagen) diluted 1:125 in KPBS-BT. After

Washing in KPBS-BT for 10 rain, in KPBS alone for 10 min and in 50 mM Tris/HCl buffer (pH 7.6) for 10 min, the sections were reacted for peroxidase activity by incubation in a solution of 0.125% di-

4

Fig. 3. High-power photomicrographs demonstrating the morphology of single I-l-immunoreactive neurons in the hamster SCN. Fig. A demonstrates a few cell bodies embedded in the underlying optic chiasm (OCh) (arrows). Further caudal smoot h positive processes coursing in direction of the optic chiasm (arrows, B and C). In the retrochiasmatic area (D) the positive cell bodies are more elongated with processes directed either laterally or dorsally towards the SCN (arrows) or ventrally towards the optic tract. Bar ffi 50 ~m.

151 aminobenzidine (DAB) in 50 mM Tris/HCI buffer (pH 7.6) and 0.1% H 2 0 z for 20 rain. After washing 2 times for 5 min in distilled water, the sections were mounted on gelatinized glass slides, dried, dehydrated in a series of ethanols and embedded in Depex.

in the dorsomedial and the lateral hypothalamic areas. In the hamster forebrain, the highest number of I-l-ir cell bodies were observed in the caudate-putamen, hippocampal formation, and cerebral cortex (not shown). The I-l-Jr SCN neurons were present throughout the rostro-caudal axis of the nucleus (Figs. 1 and 4). In the prechiasmatic area, located in between the lamina terminalis and the SCN a few I-l-it cell bodies were observed, but exclusively in its most posterior part close to the SCN (Fig. 1A). Within the rostral pole of

RESULTS

The I-l-ir cell bodies in the SCN were filled with a homogenous cytoplasmic product and the nucleus stands out as the most immunoreactive structure in the entire hamster hypothalamus (Fig. 1). In other hypothalamic nuclei faintly stained cell bodies were found

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Fig. 4. Camera lucida drawings of frontal sections through the hamster SCN illustrating the distribution of I-l-ir cell bodies. Each circle represents an I-l-Jr cell body. OCh, optic chiasm; V, third ventricle.

152 the SCN, a modest number of I-l-ir cell bodies were distributed throughout the nucleus (Fig. 1B). In the middle part of the SCN, the number of I-l-ir cell bodies increased substantially, but mostly found in the ventral half of the nucleus (Fig. 1C,D). At this level, many I-l-ir cell bodies were embedded in the optic chiasm (Figs. 1C,D and 3A,B). T h e immunoreactive cell bodies found in the middle and caudal portion of the ventral SCN tended to be more concentrated in the medial than in the lateral part. The central and lateral portions of the SCN contained somewhat fewer I-l-ir cell bodies than the ventromedial part (Figs. 1D and 2A). In the dorsaloteral part a few I-l-ir cell bodies were seen, whereas in the dorsomedial part no I-l-ir cell bodies were identified. In the caudal part of the SCN, the labeled cell bodies were found predominantly in the ventromedial quadrant (Fig. 1E). A few labeled cell bodies were found outside the cytoarchitectural borders of the SCN (Figs. 2A, B, 3D and 4). These were found in the prechiasmatic area (Fig. 1A), the lateroanterior hypothalamic area (Fig. 2A), and in the retrochiasmatic area (Fig. 2B). However, the positive neurons in these areas were only found in that part of the given area closest to the SCN. The neurons were small and usually possessed relatively few processes, which were found to be oriented towards the SCN. These neurons appeared to be similar in size and morphology to those in the SCN. The I-l-ir neurons of the hamster SCN were round to oval with a maximum diameter of 9.86 + 1.58 /xm (n = 35). The neurons typically possessed one or two primary processes. The intensity of immunoreactivity in single SCN neurons differed. Some neurons contained a dark homogenous reaction product, whereas others displayed a weaker and more granular product. Usually, more intensely labeled neurons were found in the ventral part of the SCN, whereas less intensely labeled neurons were more often present in the dorsal part (Figs. 1C,D and 2A). The primary processes of the neurons were most often smooth and thick, and interpreted as dendrites. Due to the dense accumulation of immunoreactive neurons in the SCN the processes were discernable from one another only in areas of the SCN where the density of immunoreactive elements was low or moderate. In particular, in the caudal SCN dendrite-like processes could be seen to penetrate into the underlying optic chiasm (Fig. 3B,C). In the lateroanterior hypothalamic area and in the retrochiasmatic area, the neurons send often dendrite-like processes in direction of the SCN (Figs. 1 and 2). The immunoreactive fibers possessing boutons-enpassage were very fine and usually difficult to trace over long distances. However, I-l-ir axonal processses

were present within the SCN, and fibers coursed outside the borders of the SCN. I-l-ir nerve fibers were followed dorsally, most of them innervating the rostrodorsal (Fig. 1B) and the dorsomedial (Fig. 1C,D) part of the SCN, but some continued outside the SCN into the overlying subparaventricular area. A minor projection was followed into the prechiasmatic area. A prominent rostrodorsal I-l-containing projection coursed along the third ventricle in the medial preoptic area, but a definite relation to the positive cell bodies within the SCN could not be revealed. I-l-ir nerve fibers were found in several other forebrain and mesencephalic structures some of which are known to receive inputs directly from the SCN, e.g. the paraventricular thalamic nucleus and the periaquaductal grey, but any relation to the immunoreactive cell bodies in the SCN could not be demonstrated. DISCUSSION Examination of the sections reacted for I-l-immunoreactivity revealed that the hamster forebrain contained positive neurons in many areas, but many of the positive neurons in the SCN were more densely stained than those in any other area of the forebrain. Areas outside the SCN containing I-l-ir neurons included the lateral hypothalamic area, the dorsomedial hypothalamic area, the striatum, cortex, and hippocampus as reported by Gustafson et al. 1° in the rat, indicating that the antiserum, although raised against a rabbit protein, recognizes the same protein in the hamster. The apparent high concentration of I-1 in the SCN implies that the enzyme is involved in regulation of endogenous circadian rhythms. However, since I-l-ir cells have been detected in many other areas of the brain the enzyme plays distinct roles in many systems, and is not only related to circadian rhythmicity. It was notable that many positive neurons were found in the ventral part of the SCN, where the retinoand geniculohypothalamic pathways are known to terminate ml7'22'42. Both of these inputs are considered to be directly or indirectly responsible for the photic entrainment of the circadian pacemaker. The mechanism by which transmitters released by axons of these pathways induces shifts of neural activity is poorly understood. However, some indirect lines of evidence point to protein phosphorylation as one mechanism. It has been shown that the content of cAMP in the SCN shows a circadian rhythm z7. Furthermore, recent experiments have shown that application of cAMP to tissue slices containing the SCN, phase advances the rhythm of electrical activity 31,32. This effect is phase-dependent and can by mimicked by forskolin, which increases

153 intracellular c A M P 32. The anatomical and functional link between the afferents originating from the retina a n d / o r the intergeniculate leaflet of the thalamus and the I-1 containing cells remain to be established. It is well known that photic stimuli conveyed to the SCN are responsible for entrainment of the clock to the light-dark cycle, but the phase-response curves for light-stimuli in vivo and cAMP in vitro do not match. Recent studies have also shown that a variety of nonphotic stimuli can also shift free-running circadian rhythms 19'26. Interestingly, the phase response curve for non-photic stimuli, neuropeptide Y (NPY), serotonin, and cAMP are similar 19'31'33, indicating that I-1 is involved in intracellular mechanisms involved in nonphotic phase advances of the clock, could be mediated by serotoninergic or NPYergic afferents to the SCN. The phosphatase inhibitors belong to a family of enzymes, which after they are phosphorylated by a cAMP protein kinase, cGMP protein kinase, or Ca2+/calmodulin kinases exert a potent inhibition of protein phosphatase-1. The specific role of I-1 in the oscillation process is unknown, but the powerful effect of cAMP on the pacemaker may be mediated by activation of I-1. It is notable that this effect, whatever it may be, can not be attributed to all SCN neurons, because only a part of these were found to be I-l-immunoreacrive. As illustrated in Fig. 4, the I-l-ir neurons were found to be highest concentrated in the medial and ventral part of the SCN, but were in principal found throughout the SCN. It is known that vasopressinergic neurons are found in the dorsomedial part of the SCN, whereas vasoactive intestinal peptide (VIP)- a n d / o r gastrin releasing peptide (GRP)-immunoreactive neurons are found in the ventral part 4,23'38,39. The distribution of I-l-immunoreactive neurons overlapped with both populations of peptidergic neurons, although the overlap with VIP- and GRP-immunoreactive neurons was most dominant. The synthesis of these peptides, and expression of their respective mRNA's are differently synchronized to the external light-dark rhythms 37'43 and vasopressin is secreted in a circadian rhythm in vitro 6. The overlap of I-l-ir neurons with both populations of neurons indicates that the protein phosphorylation processes involving I-1 take place in many different neurons of the SCN. A number of densely labeled neurons were found outside the borders of the SCN as well. The boundaries of the SCN defined on the basis of uptake of 2-deoxyglucose during the subjective day 36 or on the distribution of c-fos-immunoreactive cells after a short light pulse in the early or late dark-phase 7,16 are not strictly similar to the architectural borders defined in !

Nissl-stained sections. The retinal and geniculate afferents also innervate areas outside the SCN, including those where the extrasuprachiasmatic I-l-ir cells were found 15'22'42. Thus, the I-l-ir cells located outside the SCN could functionally be considered to belong.to the SCN. A functionally similar protein, DARPP-32, has 15% amino acids in common with I-1, but those responsible for their inhibitory effects are more similar at. DARPP32 has been localized in D 1 dopamine-receptive neurons, and it is intriguing to speculate that a specific receptor is linked to phosphorylation of I-1 as well. DARPP-32 and I-1 are differently distributed in the brain TM,which excludes that I-1 is linked to a dopamine receptor. The transmitters which have been found in highest concentrations in axonal fibers of the SCN include N-acetylaspartylglutamate, NPY, and serotonin 4'5A1'21'24'39. It would be interesting to know whether any of these transmitters stimulate 1-1-activity in the SCN as well as in other systems, where I-1 has been shown to be present and thereby elucidate some of the cellular mechanisms linking synaptic transmission with a phase shift of the circadian clock. Acknowledgements.

We wish to thank Gitte G. Scrensen and Bianca Houlind for skillful technical and secretarial assitance. We acknowledge Drs. Angus Nairn, Jean-Antonie Girault and Paul Greengard for providing the I-1 antiserum. This study was supported by Psykiatrisk Forskningsfond, Landsforeningen til Bekaempelse af Ojensygdomme og Blindhed, Kong Chr X Fond, Nordisk Insulinfond, and The Danish Medical Research Council (12-0236). J.D.M. is recipient of a Hallas-M011er Research Fellowship financed by the NOVO Foundation. REFERENCES 1 Aitken A., Bilham, T. and Cohen, P., Complete primary structure of protein phosphatase inhibitor-1 from rabbit skeletal muscle, Eur J. Biochem., 126 (1982) 235-246. 2 Aswad, D. and Greengard, P., A specific substrate from rabbit cerebellum for guanosine 3':5'-monophosphate-dependent protein kinase. I. Purification and characterization, J. Biol. Chem., 256 (1981) 3487-3493. 3 Aswad, D. and Greengard, P., A specific substrate from rabbit cerebellum for guanosine 3':5'-monophosphate-dependent protein kinase. II. Kinetic studies on its phosphorylation by guanosine 3':5'-monophosphate-dependent and adenosine 3 ' : 5 ' monophosphate-dependent protein kinases, J. Biol. Chem., 256 (1981) 3494-3500. 4 Card, J.P. and Moore, R.Y., The suprachiasmatic nucleus of the golden hamster: immunohistochemical analysis of cell and fiber distribution, Neuroscience, 13 (1984) 415-431. 5 Card, J.P. and Moore, R.Y., Organization of lateral geniculatehypothalamic connections in the rat, J. Comp. Neurol., 284 (1989) 135-147. 6 Earnest, D.J. and Sladek, C.D., Circadian rhythms of vasopressin release from individual rat suprachiasmatic explants in vitro, Brain Res., 382 (1986) 129-133. 7 Ebling, F.J.P., Maywood, E.S., Staley, K., Humhy, T., Hancock, D.C., Waters, C.M., Evan, G.I. and Hastings, M.H., The role of N-Methyl-D-Aspartate-type glutamatergic neurotransmission in the photic induction of immediate-early gene expression in the

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