Substance P-like immunoreactivity in the suprachiasmatic nucleus of the rat

271

Brain Research, 619 (1993) 271-277 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 19107

Substance P-like immunoreactivity in the suprachiasmatic nucleus of the rat Yasumasa Otori a,b, Keiko Tominaga a,c, Chiaki Fukuhara a,d, Jing Yang a, Shin Yamazaki a, Felino Ramon A. Cagampang a, Hitoshi Okamura e and Shin-Ichi T. Inouye a a Laboratory of Integrative Brain Function, Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo (Japan), b Department of Ophthalmology, Osaka University Medical School, Osaka (Japan), c Department of Pharmaceutical Science, Kyushu University, Fukuoka (Japan), d Life Science Institute, Sophia University, Kioicho, Tokyo (Japan) and e Department of Anatomy, Kyoto Prefectural University of Medicine, Kyoto (Japan) (Accepted 16 March 1993)

Key words: Suprachiasmatic nucleus; Substance P; Enzyme immunoassay; Rat; Circadian rhythm; Retinohypothalamic tract

The content of substance P (SP)-like immunoreactivity (LI) within the suprachiasmatic nucleus (SCN) of rats was determined by enzyme immunoassay to evaluate the effect of light on SP-LI in the rat SCN. Male rats were kept under various lighting conditions: light-dark cycles, constant darkness, continuous light exposure for 24 h or light pulse interrupting constant darkness. Animals were also subjected to ocular enucleation. The present study showed that SP-LI in the SCN was unaffected by environmental lighting conditions or by bilateral ocular enucleation. Immunohistochemical studies also confirmed that SP immunoreactivity, which was found in the ventrolateral (VL) subdivision of the SCN, was not reduced significantly even after ocular enucleation. These results suggest that, in contrast to other neurotransmitters in the VL portion of the SCN such as vasoactive intestinal polypeptide (VIP), gastrin releasing peptide (GRP) and neuropeptide Y (NPY), SP level in the SCN is quite stable to light and arises from an area other than the retina.

INTRODUCTION

The presence of a circadian pacemaker within the suprachiasmatic nucleus (SCN) of mammalian brain has been well documented 19. Anatomical studies 2°'34 have shown that neurons in the SCN are divided into two groups, the dorsomedial (DM) and ventrolateral (VL) portions. Neurons within the DM and VL subdivisions produce different peptides. Within the DM portion of the SCN arginine-vasopressin (AVP) and somatostatin (SS) neurons a r e f o u n d 6'34. A V P 33,36 and SS 1°'29 levels in the SCN show circadian rhythms independent of environmental lighting cues with a peak during the early subjective day and a trough during the subjective night. On the other hand, the VL part of the SCN contains cells that produce vasoactive intestinal polypeptide (VIP), gastrin releasing peptide (GRP) or peptide histidine isoleucine amide (PHI) 34. The cellular levels of VIP and GRP in the SCN were regulated by photic stimuli and these peptides were implicated in

the entrainment of the circadian pacemaker to light 3°. A number of other substances have also been localized within cell bodies of the SCN, i.e. corticotropin-releasing factor, neurotensin, thyrotropin-releasing hormone, angiotensin II, ~/-aminobutyric acid (GABA). Serotonin and neuropeptide Y (NPY) are neurotransmitters that are synthesized outside the SCN and are present in high density within the SCN. Serotonin is released by afferent fibers from the raphe nucleus, and NPY is released from terminals of fibers projected from the intergeniculate leaflet and the ventral lateral geniculate nuclei 19. Both serotonin (Cagampang et al., unpublished data) and NPY levels in the SCN were found to be drastically modified by environmental lighting conditions 3°. Responsiveness to light appeared a common feature of neurotransmitters located in the VL subdivision of the SCN, either intrinsic (VIP and GRP) or extrinsic (serotonin and NPY). Another peptide, substance P (SP) has also been localized in terminals in the VL portion of the S C N 32. In the present study, we

Correspondence: Y. Otori, Laboratory of Integrative Brain Function, Mitsubishi Kasei Institute of Life Sciences, 11, MinamiOoya, Machida Tokyo 194, Japan. Fax: (81)(427)291252.

272 examined whether the similar responsiveness to light was also found in SP-like immunoreactivity (LI) in the SCN in order to see if the rule could be generalized that levels of neurotransmitters in the VL subdivision are regulated by light. Moreover, the effects of light on SP-LI level in the SCN are also interesting from a different point of view. Anatomical studies have defined two afferent pathways from the retina to the SCN, a direct projection from the retina called the retinohypothalamic tract (RHT) 21 and a secondary photic projection from the intergeniculate leaflet called the geniculohypothalamic tract (GHT) ~2. A principal neurotransmitter in the GHT, neuropeptide Y, has been reported to be involved in the circadian pacemaking activity It. The involvement of neurotransmitters in the RHT in entrainment of the rhythm in the SCN has not been clearly defined. A recent report has shown that in the rat retinal ganglion ceils which contain SP immunoreactivity projected to the ventral part of the SCN where retinal fibers terminate 3~. In fact, immunohistochemical studies demonstrated that SP immunoreactive ganglion cells were present in the frog ~5, chick 9, rat 7 and rabbit 4 retina, and SP immunoreactive terminals in selected central retino-recipient nuclei. In addition, a recent report has shown that exogenous SP application shifted the phase of circadian rhythms of neural activity in rat hypothalamic slice preparations 28. Hence, in the present study we evaluated the effect of light on SP-LI in the SCN by determining SP-LI contents in the SCN of animals subjected to various lighting conditions. MATERIALS

AND METHODS

Animals Mate Wistar rats (SLC Co. Shizuoka, Japan), 5 weeks postpartum at purchase, were maintained under 12:12 h light-dark cycles (lights on at 09.00 h, off at 21.00 h) for 2 weeks and had free access to food and water. All experiments were performed in accordance with the guide for care and use of laboratory animals (DHEW publication, NIH 80-23).

Experimental protocols Lighting protocols in this experiment are illustrated in Fig. 1. One group of rats (Fig. lb) was sacrificed under light-dark (LD) conditions at 4 h intervals. Another group of rats (Fig. la) was transferred from this light-dark regimen to constant darkness 48 h prior to the experiments. The animals were sacrificed at 4 h intervals on the 3rd day of free-running under constant dark (DD) conditions. To determine the effect of continuous light (Fig. lc) or light pulse (Fig ld), other groups of rats were exposed to either continuous light (lights were turned on at CT 0) or 1 h light pulse falling at circadian time (CT) 15 (the time of largest phase delay) after being kept in constant darkness for 2.5 days or 2 days, respectively. Light intensity was approximately 500 lux at the center of the cage. In free-running condition, 09.00 h and 21.00 h were designated as CT 0 and CT 12, respectively, based on locomotor activity measurements which showed that 2.5 days of free-running did not shift the phase of the animal's rhythm longer than 1 h.

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Fig. 1. Time schedule of lightings and samplings. All rats were entrained to 12 h light-dark (LD) cycle for 2 weeks. In the constant dark (DD) experiments (a), rats were transferred from light-dark regimen to constant darkness 48 h prior to the experiments. The animals were sacrificed at 4 h intervals on the 3rd day of free-running under DD conditions. In the LD experiments (b), rats were kept in LD cycles and sacrificed during the day. In the continuous light exposure experiments (c), animals were transferred to DD conditions for 2.5 days and then were exposed to light at circadian time (CT) 0. They were sacrificed at 0, 1, 4, 8, 12, 16, 20 and 24 h after light onset. In the light pulse experiments (d), they were transferred to DD conditions for 2 days and were exposed to 1 h light pulse at CT 15. They were sacrificed at CT 15, 16, 17, 18, 20 and 24. Sampling points are indicated by arrows.

Tissue preparation Rats were sacrificed by decapitation in the dark under safe red light. The brains were rapidly removed and placed on a Rodent Brain Matrix (Activational Systems Inc., MI, USA). One mm thick sections, 0.8-1.8 mm posterior to the bregma, were obtained using razor blades. These sections were immediately frozen on a rubber plate over a cold stage. The bilateral SCNs were punched out from the frozen sections under a stereomicroscope with a microdissecting needle having an inside diameter of 600/zm. This size of the needle was used so as to punch out consistently from the base of the optic chiasma to the dorsal edge of the SCN. Samples were stored at - 8 0 ° C until subjected to enzyme immunoassay.

Tissue extraction The samples were boiled in 1 N acetic acid and 0.02 N HCI to inactivate endogenous peptidases. After 10 min boiling, samples were chilled on ice, homogenized by sonication for 30 s and centrifuged at 10,000 g for 15 rain. Aliquots (750 ~1) of the supernatant were lyophilized and reconstituted in 150 p.1 of an assay buffer (0.14 M phosphate buffer pH 7.4 containing 25 mM EDTA, 0.5% bovine serum albumin and 0.05% Tween 20) for enzyme immunoassay (EIA),

EIA procedure The content of immunoreactive SP was determined by the double-antibody solid-phase method of Arai et al. 3 with minor modifications. Briefly, the microplate (Ninety-six Multi-Well Plates for ELISA, Sumitomo Bakelite Co. Ltd., Japan) were coated with the second antibody (anti-rabbit IgG, 1 : 800 diluted with carbonate-bicarbonate buffer, pH 9.6) and incubated overnight at 4°C. The wells were washed 3 times with washing buffer (0.02 M phosphate buffer saline, pH 7.4). Subsequently, 100 ~1 assay buffer, 50 #.1 of the sample or the standard and the diluted antiserum (1:25,000)were added into each well. The microplate was then incubated overnight at 4°C. Fifty /zl of HRP-SP conjugate prepared by the periodate oxidation method (1 : 1,600) was added into each well and incubation was continued for another 3 h. The microplate was then washed and incubated at room temperature with 250 /zl of the substrate (18.4 mg o-pbenylenedi-

273 amine dihydrochloride and 25 t~l of 3% H202 in 25 ml of 0.2 M citrate phosphate buffer, pH 5.2) for 20 rain. The reaction was stopped by adding 50 p,l of 5 N H2SO 4 to each well. The absorbance of the resulting reaction products was measured by an EIA reader (Model 2550, Bio-Rad, USA) at 492 rim. Each plate contained standards (B), enzyme blanks (non-specific binding (NSB)) and wells without free antigen (B0). Fifty% inhibitory concentration (ICs0) was 15.7-1-0.78 fmol/well. The sensitivity of our assay system was 2.14+ 0.22 fmol/well at 90% B / B 0 level. Samples were diluted serially and in parallel with synthetic SP standard. Protein content was determined using a protein assay kit (Bio-Rad, USA), with bovine serum albumin as the standard. Student's t-test was used for statistical analysis.

Eye enucleation Bilateral eye enucleation was carefully performed under anesthesia. This was done by cutting the conjunctiva and the extra ocular muscles before cutting the optic nerve under a stereomicroscope. After the eyes were removed, an antibiotic was applied to the orbit and the eye lids were sutured. Enucleated rats were killed at 7 days after surgery under DD conditions. Control rats were sacrificed at CT 15 on the second day of free-running under DD conditions. Immunohistochemistry Control and eye enucleated animals were anesthetized with pentobarbital and perfused with 100 ml of 0.1 M phosphate buffered saline (PBS, pH 7.4), followed by 200 ml of 0.1 M phosphate buffer (PB) containing 4% paraformaldehyde and 0.2% picric acid. The brains were removed and further fixed with the same fixative at 4°C for 24 h. Coronal sections (25 v.m in thickness) were made with a

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cryostat after immersion of the brain in 0.1 M PB with 20% sucrose for 24 h. Sections from the control and the enucleated animals were incubated in the same reaction glass bottle with the rabbit SP antiserum (dilution 1:5,000) for 4 days at 4°C. The sections were incubated with biotinylated goat anti-rabbit IgG (Vector, dilution 1:200) for 2 h, followed by avidin-biotinylated peroxidase complex (Vector, dilution 1:200) for 2 h at room temperature. All the sera were diluted with 0.1 M PBS containing 0.1% Triton X-100. Between each step of the reaction, sections were washed with 0.1 M PBS. Bound peroxidase was visualized in 50 mM Tris-HCl buffer (pH 7.5) containing 0.025% 3,Y-diaminobenzidine tetrahydrochloride, and 0.6% nickel ammonium sulfate in 0.002% H 2 0 2. Cross-reactivity of the SP-antiserum was checked for some other neuroactive substances (adrenocorticotropic hormone, /3-endorphin, bombesin, calcitonin, cholecystokinin, corticotropin-releasing factor, dynorphin, Leu- and Met-enkephalins, neuropeptide Y, neurotensin, somatostatin, vasoactive intestinal polypeptide, calcitonin gene-related peptide, y-aminobutyric acid, glutamate and aspartate) by dot immuno-blotting method 17 on nitrocellulose paper as described elsewhere 23'27. The specificity of the SP-antiserum was also confirmed by immunocytochemicat preabsorption test. No positive immunoreactivity was detected in sections reacted with the serum preabsorbed with synthetic SP (1-20/zg/ml).

Chemicals and other materials Chemicals were obtained from the following sources: synthetic SP (Peptide Research Foundation, Japan); rabbit antiserum against SP (first antibody; raised by Dr. T. Amano, Tokai University, Japan; the polyclonal SP antiserum was directed against the C-terminus.); goat antiserum against rabbit IgG (second antibody; Shibayagi Co. Ltd.,

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Fig. 2. Circadian profiles of SP-LI in the SeN under various lighting conditions. A: circadian profile of SP-LI in the SeN on the third day under constant darkness (n = 8), B: circadian profile of SP-LI in the SeN under LD conditions (n = 8). C: time course of the effect of continuous light exposure on SP-LI in the SeN. Following various durations (1 to 24 h) of light exposure at CT 0, SP-LI levels were measured (n = 8-4). d: time course of the effect of light pulse on SP-LI in the SeN. Following various circadian time (CT 15 to CT 24) of 1 h light pulse at CT 15, SP-LI levels were measured (n = 8-6). Values are means +_S.E.M. (fmol/mg protein).

274 Japan). Other chemicals were purchased from Wako Pure Chemical Industries, Ltd., Japan. RESULTS

The SP-LI content in the SCN under different lighting conditions The pattern of the SP-LI in the SCN on the third day under D D conditions is shown in Fig. 2a. No

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significant change in SP-LI content in the SCN was found at any time during the sample period. The average SP co,ltent in the SCN was 430 + 27.2 f m o l / m g protein. The average protein content of a SCN tissue was 114 + 3 p~g/SCN. Under LD conditions, SP-LI in the SCN also did not exhibit any significant changes Fig. 2b. The average SP content was 476_+28,7 f m o l / m g protein. In the third experiment, rats that

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Fig. 3. SP immunoreactivityin the suprachiasmatic nucleus (SCN). a: an intact rat. b: a bilateral enucleated rat. OC, optic chiasma. Bar = 200 /~m.

275 had been kept under DD condition for 60 h were exposed to continuous light stimuli for various durations from CT 0. The SP-LI content did not change during continuous light exposure for 24 h Fig. 2c. Rats that had been kept under DD conditions for 48 h were exposed to light pulse for 1 h starting at CT 15 in order to determine whether SP-LI content was affected by light pulse. SP-LI level did not show any significant changes for up to 8 h after 1 h light pulse Fig. 2d. Overall results showed that SP-LI contents were unaffected by environmental lighting conditions. In the final experiment, bilateral eye enucleation did not alter SP-LI content in the SCN even up to 7 days after surgery (450 + 33.5 fmol/mg protein, n = 7). These values were also not significantly different from SP-LI content in sighted control rats under DD condition (438 + 22.9 fmol/mg protein, n - 7).

Immunohistochemical study Although the SP immunoreactive fibers were densely detected in the surrounding anterior hypothalamic area, SCN contains very few SP immunoreactive fibers in sighted and eye enucleated rats. In the SCN of both groups of animals, the SP immunoreactive fibers were restricted to the ventral part. No alteration of SP immunoreactivity was observed between bilateral eyeenucleated rats and the intact controls Fig. 3. DISCUSSION The present study demonstrated the absence of circadian or diurnal rhythmicity in the content of SP-LI in the SCN. In particular, SP-LI content within the SCN was unaffected by environmental lighting conditions. SP-LI content of the rat with the bilateral eyes enucleated was not different from that in intact rat. Moreover, immunohistochemical study showed no appreciable difference in SP immunoreactivity in the SCN between sighted and enucleated rats. The areas surrounding the SCN have stronger immunoreactivity than the SCN except the optic chiasma. This is clearly shown in Fig. 3, and by the SP-LI value for the anterior hypothalamus (AH) tissues in the same sections (1525 + 61.8 fmol/mg protein; n = 32). Hence, a small contamination of the surrounding areas may increase apparent SP-LI contents drastically. In the present experiment, we placed one edge of the punching needle right at the ventral boundary of the optic chiasma so as to exclude the areas dorsal to the SCN in our punched SCN tissues. The area dorsal to the SCN contains higher level of SP-LI while the chiasmatic region does not appear to contain significant level of SP-LI in comparison to the other brain areas. We

believe that this punching technique is more or less accurate judging from the facts that our A H / S C N ratio (--3.3) of SP-LI levels was high and consistent with a previous report 25. Even with maximum precautions, it was impossible to exclude any of the structure outside the SCN in our punched tissues and we have to be very cautious in interpreting data on SP levels in the SCN obtained by micropunching method. With this reservation in mind, we have performed immunohistochemical analysis of SP immunoreactivity in the SCN and the results of these experiments were found to be consistent with the conclusion reached with the micropunching experiments. It has been previously reported that the lighting condition did not significantly alter the content of SP-LI in the SCN 2. However, their study had only determined SP-LI contents at two time points during the day under LD conditions and at one point under constant conditions. In the present study, we tried to reduce the possibility of missing the changes that might occur during the period between two times of the day by measuring SP-LI contents at 6 time points. We did not find any significant changes in SP-LI levels in the SCN under LD or constant dark conditions. Our results, therefore, support the view that lighting conditions do not have any effect on SP-LI contents in the SCN. Peptides which are anatomically located in the ventrolateral (VL) or dorsomedial (DM) region of the SCN behave differently under DD conditions or in response to light. Circadian rhythms of the contents of peptides in the VL SCN were rapidly altered by environmental lighting conditions3° and exogenous transient manipulation in peptide concentrations induced a shift in the phase of the circadian pacemaker ~. VIP and GRP, whose cell bodies were found in the VL portion of the SCN, belong to this group of peptides 34. In sharp contrast to these peptides in the VL portion of the SCN, the present experiments revealed that SP-LI level in the SCN did not change under all lighting conditions used in this study despite the fact that SP immunoreactivity is mainly confined in the ventrolateral subdivision of the SCN. These results may indicate that SP in the SCN belongs to a new category of substances in the circadian rhythm system. A recent report has indicated the presence of the SP innervation from the retina to the SCN in the rat 32. However, the present results could not support this study. SP-LI levels in the SCN did not change in response to environmental light or after enucleation of the eyes. By double-labeling experiments employing retrograde labeling from the superior colliculus or dorsal optic tectum, 25-35%, 3% or 3% of all retinal

276 ganglion cells were reported to contain SP immunoreactivity in the rabbit 4, the chick 9 or the rat 7, respectively. It is established that retinal ganglion cell axons form the optic nerve and project to several different sites in the central nervous system, including the SCN, the accessory optic nuclei, the pretectum, the superior colliculus (SC), ventral lateral geniculate nuclei (vLGN), dorsal lateral geniculate nuclei (dLGN) 26 and the intergeniculate leaflet (IGL) 12. SP-LI in the rat retina has been attributed to amacrine ceils whose SP containing cell bodies are situated in the inner nuclear layer (INL) and the ganglion cell layer (GCL) 37. However, it remains to be elucidated whether SP containing cells in the GCL are intrinsic to the GCL or are displaced cells from the INL. So far preprotachykinin (PPT) genes (SP/Neurokinin A (NKA) or Neurokinin B (NKB) mRNA) containing cells were suggested to displace amacrine cells in the GCL, although the presence of ganglion cells could not be discounted with in situ hybridization histochemistry 5. However, reports of SP-LI in rat optic nerve extracts TM and SP immunoreactive fibers in the optic tract and supraoptic decussation 32 indicate the possibility that some ganglion cells of the rat retina express either or both of the PPT genes. Although previous immunohistochemical studies have reported various degrees of SP immunoreactivity in the S C N 8'13'16'22'31'32'34'35, all were in agreement that a majority of the SP immunoreactive fibers are found in the ventral part of the SCN. Recently, Takatsuji et al. 32 reported that more SP immunoreactive fibers existed in the ventral part of the SCN than those previously reported. In the present study, we found fewer SP immunoreactive fibers in our immunohistochemical specimens than those of Takatsuji. The reason for this discrepancy is not clear at present. One possible reason may be that the specificity of antisera was different. While Takatsuji et al. 32 employed SP antiserum against the segment other than C-terminal of the peptide, most SP antisera used in other studies including ours are C-terminal directed polyclonals. Compared to the unavoidable crossreactivity in immunohistochemical studies, EIA studies are not significantly compromised by crossreactivity of SP-LI with the mammalian tachykinins NKA and NKB, because the levels of these peptides were generally lower than those of SP TM. Hence, we believe the antisera that we used were specific enough to recognize SP-LI in the SCN. In the present study, we confirmed the presence of that SP-LI in the SCN by using both EIA and immunohistochemical techniques and demonstrated that SP-LI in the SCN was unaffected by environmental lighting

conditions or by eye enucleation. These results suggest that, unlike the other neurotransmitters in the VL portion of the SCN, SP in the SCN is quite stable to light and arises from an area other than the retina. Acknowledgements. The authors are grateful to Dr. Takehiko Amano from Tokai University for supplying antiserum raised against substance P. The authors wish to thank Miss Ako Tokumasu for her technical assistance. These experiments were supported by Mitsubishi Kasei Institute of Life Sciences Grant 'Project 10'.

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