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,#,1
* Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan;
Graduate School of Science and Engineering, University of Toyama, Toyama, Japan;
Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, Toyama, Japan;
Department of Translational Medical Sciences, Course of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan;
|| National Kyushu Cancer Center, Fukuoka, Japan;
¶ Department of Translational Medical Sciences, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan; and
# Advanced Institute for Biological Science, Waseda University, Tokyo, Japan
1Correspondence: Department of Biology, Faculty of Science, University of Toyama, Rm. B214, 3190 Gofuku, Toyama-city, Toyama 930-8555, Japan. E-mail: msikeda{at}sci.u-toyama.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: cholecystokinin-A receptor mutant mice • glycinergic amacrine cells • melanopsin retinal ganglion cells • suprachiasmatic nucleus neurons
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Although these rhythms persist under time cue-free conditions, they are usually synchronized to the environmental light-dark cycles, a process called photoentrainment. The classic photoreceptor cells, rods and cones, are not the only cells involved in this process, however, because photoentrainment still occurs in mutant (rdta/cl) mice lacking all rods and cones (3)
. It has been shown that intrinsically photosensitive retinal ganglion cells, which contain the pigment melanopsin (4)
, respond directly to light and send monosynaptic projections to the SCN and the olivary pretectal nuclei, as part of nonimage-forming visual functions including photoentrainment and the pupillary light reflex (5
, 6)
. Photoentrainment is still possible, however, in melanopsin-deficient (Opn4–/–) mice and only becomes absent with the subtraction of both the photosensitivity of retinal ganglion cells and classic photoreceptors in double mutant melanopsin-deficient and retinal degeneration (Opn4–/–; rd/rd) mice (7)
. These results suggest that classic photoreceptors and intrinsically photosensitive retinal ganglion cells represent compensatory photoreception mechanisms for circadian photoentrainment. In mice, intrinsically photosensitive retinal ganglion cell dendrites in the inner ON region of the inner plexiform layer receive both bipolar and amacrine cell terminals, whereas intrinsically photosensitive retinal ganglion cell dendrites stratifying in the outer OFF region of the inner plexiform layer receive only amacrine cell terminals (8)
. The circuitry between photoreceptor pathways, retinal ganglion cells, and amacrine cells underlying photoentrainment, however, is not fully understood.
In SCN neurons, some subcellular responses have been identified that may be involved in photoentrainment. After photic stimulation at night, retino-recipient SCN neurons respond to glutamate release from retinohypothalamic tract terminals with an activation of N-methyl-D-aspartate glutamate receptors, an increase in cytosolic Ca2+ concentration, and immediate expression of clock genes, such as mPer1 and mPer2 (9
10
11)
. Several other signaling mechanisms also appear to be involved. For example, pituitary adenylate cyclase-activating polypeptide is released from retinohypothalamic tract terminals in addition to glutamate (12)
, and serotonin-1B receptors (13)
and GABA-B receptors (14)
on retinohypothalamic tract terminals may synergistically control neurotransmitter release on SCN pacemaker neurons. The interconnections of these elements remain unclear, however.
Cholecystokinin (CCK) is one of the most abundant neuropeptides in the central nervous system, but its function in many pathways has not been identified. CCK immunoreactive cell bodies and axons have been shown to be sparsely dispersed throughout the SCN of rats, mice, and hamsters (6
, 15
16
17)
. In addition, CCK peptide has been shown to be present in a variety of vertebrate retinas (18
19
20
21)
. In rat retinas, it is located predominantly in amacrine cells (22)
and may function for local signal transduction pathways, as expression of CCK-A and CCK-B receptor mRNAs has been reported for retinal homogenates (23)
. The function of CCK within this circadian clock circuitry has not been elucidated, but it is a reasonable hypothesis that CCK peptides have a role in the SCN clock work itself and/or photic input pathways to the SCN. Indeed, we have reported that photoentrainment of locomotor rhythms and immediate early gene (c-fos) expression in SCN neurons in response to light are significantly reduced in Otsuka Long Evans Tokushima Fatty (OLETF) rats, a strain of obese mutant rats, that lack CCK-A receptor genes (24
25
26
27)
. Although these data are highly suggestive, multiple other genes are also lacking in OLETF rats; thus, further experiments are needed to identify the specific gene responsible for the circadian photoentrainment (28
, 29)
.
To examine the specific function of CCK-A receptors in the circadian clock system, in the present study we used mutant mice lacking CCK-A receptors (CCKAR–/–) that carry the lacZ reporter gene within a gene-knockout cassette (30)
, which allowed precise study of the localization of CCK-A receptors. We also examined the function of these receptors at the gene expression, intracellular signaling, and behavioral levels. Based on these data, we propose that CCK-A receptors on a subpopulation of retinal amacrine cells may have an important role in nonimage-forming visual functions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In short, the targeting vector was designed to replace the SalI-BglII 1.9-kb genomic fragment of the mouse CCK-A receptor gene with a NLS-lacZ and pGK-neo cassette. The homologous recombination deleted the first 122 amino acids, including the first membrane-spanning region of the CCK-A receptor. J1 embryonic stem (ES) cells were electroporated with the targeting vector and selected with G418 on embryonic fibroblast feeder cells. After Southern blot analysis screening, the successful ES clones were microinjected into blastocysts of C57BL/6J females. Finally, two independent ES clones generated germline chimeras. The chimeras were bred with C57BL6J mice to generate CCKAR+/– mutant F1 mice. CCKAR–/– mice were finally generated by mating CCKAR+/– mice followed by sufficient backcross to C57BL/6J wild-type mice. The experiments below were approved by the Committee of Animal Care of Kyusyu University and University of Toyama.
Locomotor activity recording
Wild-type and CCKAR–/– mice were housed in temperature-controlled (23±2°C) rooms, with a 12:12-h light-dark cycle. Food and water were provided ad libitum. To observe locomotor activity rhythms, mice were transferred to transparent plastic cages (30x35x17 cm), and their locomotor activity was measured using an infrared area sensor (F5B; Omron, Kyoto, Japan) located 30 cm above the surface of the cage. Locomotor activity was continuously recorded in 6-min epochs via a 48-channel parallel interface installed in a personal computer. To observe the free-running locomotor activity rhythm in constant darkness, wild-type (n=8) and CCKAR–/– (n=8) mice were housed in constant darkness for 2–3 wk. The free-running period during the first 2 wk was calculated using a 
2 periodogram. For animals housed in constant darkness, circadian time 12 was defined as the onset of locomotor activities. To evaluate the response to photic stimuli, mice were maintained in constant darkness for 9 days and then exposed to a full-spectrum light pulse (15 min at 10–500 lux) at circadian time 16. The activity-onset delays in free-running rhythms were calculated on the basis of the distance between the two regression lines drawn from daily onset of locomotor activity for 7 days before and after the light pulse.
Pupillometry
Consensual pupillary constriction was measured in response to an adirectional light stimulus. Light was exposed for 60 s by a flexible-arm 100-W halogen lamp house (LA100USW; Hayashi Watch-Works Co. LTD., Tokyo, Japan), by which intensity was adjusted (20 or 100 lux at the animal levels) or maximized (>1000 lux). UV wavelength (<400 nm) was eliminated by a UV cut-filter installed in the lamp house. Adult male wild-type mice (n=5) and CCKAR–/– mice (n=4) were dark adapted for 1–2 h and placed on a custom-built stereotactic apparatus, by which animal movement was restricted by a 27 mm
polyethylene tube. Pupillary constriction was monitored with an infrared video system (BBCAM130 Night Vision-II; Timely Computer, Inc., Tokyo, Japan), which is composed of a digital charge-coupled device (CCD) camera centering on eight coaxial infrared (>850 nm) light-emitting diode arrays. At the end of the experiment, 1% atropine sulfate (Wako Pure Chemicals, Tokyo, Japan) dissolved in saline was dropped on the eye to estimate maximal pupil dimensions. Pupil dimensions were measured from the video images using Video Studio version 6.0 software (Ulead System, Inc., Tokyo, Japan). All experiments were conducted during the light period of 12:12-hour light-dark cycles.
5-Bromo-4-chloro-3-indoyl-β-D-galactopyranoside (X-gal) staining
Adult wild-type and CCKAR+/– mice (6 wk old) were used for the X-gal staining experiments. These animals were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight) and transcardially perfused with PBS for 5 min and then with ice-cold 2% paraformaldehyde in 0.1 M phosphate buffer for 15 min. Eyes and the whole brain were removed and further fixed in the same fixative (4°C, 2 h). Then the vitreous and connective tissues were carefully removed from the eye, and the cerebellum and olfactory bulb were cut off from the brain in ice-cold PBS. The eyecups and brain tissue were immersed in 20% sucrose PBS and stored overnight. Frozen sections of 20 µm thickness were cut using a cryostat microtome and mounted on glass slides. These samples were then stained for 3 h at 37°C with a β-galactosidase (β-gal) staining kit (K1465–01; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. As a negative control, samples from wild-type mice were also stained, but no staining was observed with a 3-h reaction period. These samples were imaged using a color CCD camera (Ds-5mc; Nikon, Tokyo, Japan) mounted on an inverted microscope (Axiovert 135TV with a Plan-Neofluar x10 objective; Carl Zeiss, Thornwood, NY, USA).
Immunohistochemistry
Retinal frozen sections prepared as above were also used for immunofluorescence double-labeling studies. After three PBS rinses, the fixed samples were incubated in 10% donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) dissolved in 0.01% Triton X-100 (Sigma-Aldrich Corp., St. Louis, MO, USA) in PBS for 2 h at room temperature (22–26°C). As a first step, samples were incubated with 1:2000 mouse anti-β-galactosidase (Sigma-Aldrich Corp.) dissolved in 10% donkey serum in PBS for 24 h at 4°C. After three 20-min PBS rinses, samples were incubated in 1:200 Cy3-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories). As an additional staining step, 1:50 goat anti-Chx10 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1:2000 goat anti-glycine transporter 1 (Chemicon International Inc., Temecula, CA, USA), 1:1000 goat anti-calretinin (Chemicon International Inc.), or 1:1000 rabbit anti-calbindin D28k (Chemicon International Inc.) was used as a primary antibody, and 1:200 fluorescein isothiocyanate (FITC) -conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories) or 1:200 FITC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories) was used as a secondary antibody. Double-labeled samples were embedded with Vectashield containing 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). The fluorescent images were acquired using a confocal laser-scanning unit (LSM510; Carl Zeiss) mounted on an inverted microscope (Axiovert 200M with an oil immersion objective lens, Plan-Apochromat x63/1.40; Carl Zeiss).
Organotypic cultures
Wild-type and CCKAR–/– mouse pups (2–3 days old) were used to make organotypic cultures of retina and SCN. For retinal cultures, eyeballs were removed from the body after deep pentobarbital anesthesia and immediately immersed in ice-cold PBS. Under a stereomicroscope, the sclera and cornea were carefully torn and peeled off using fine forceps, and the neural retina was isolated. After removal of pigment epithelium and lens, the neural retina was trimmed using microscissors and placed in a 0.40-µm filter cup (Millicell-CM; Millipore, Bedford, MA, USA) with the ganglion cell layer being on the surface. The neural retinas on filter cups were transferred to standard six-well culture plates and cultured with 1 ml of low-glucose Dulbecco’s modified Eagle’s medium supplemented with 1:50 B-27 (Invitrogen), 5 µM forskolin (Sigma-Aldrich Corp.), 20 ng/ml brain-derived neurotrophic factor (Invitrogen), and 50 µg/ml gentamicin (Invitrogen). The medium was changed every other day until recording (7–14 days in culture). This procedure enables the survival of the outer and inner nuclear layers and the ganglion cell layer in the in vitro slice cultures (31)
.
SCN cultures were prepared as described previously (32)
. Briefly, coronal hypothalamic slices containing the SCN were cut using a vibrating-blade microtome in artificial cerebrospinal fluid containing 138.6 mM NaCl, 3.35 mM KCl, 21 mM NaHCO3, 0.6 mM NaH2PO4, 9.9 mM D-glucose, 0.5 mM CaCl2, and 3 mM MgCl2 and bubbled with 95% O2 and 5% CO2. These slices were trimmed to an 
4-mm square containing the ventral end of the hypothalamus centered on the third ventricle. The slices were placed in Millicell-CM filter cups and cultured with 1 ml of medium consisting of 50% Eagle’s basal medium, 25% Earle’s balanced salt solution, and 25% heat-inactivated horse serum, supplemented with glucose and Glutamax (Invitrogen). The slice containing the rostrocaudal center of the SCN was used for further experiments. The SCN and retinal cultures were maintained in a CO2 incubator at 35.5 ± 0.5°C and 5% CO2.
Ca2+ imaging in retinal cultures
The retinal cultures on a membrane filter were incubated for 30 min in culture medium containing 10 µM Fura-2 AM (Molecular Probes, Eugene, OR, USA) in a CO2 incubator at 35.5 ± 0.5°C and 5% CO2 and rinsed 3x with buffered salt solution (BSS) consisting of 128 mM NaCl, 5 mM KCl, 2.7 mM CaCl2, 1.2 mM MgCl2, 1 mM Na2HPO4, 10 mM glucose, and 10 mM HEPES/NaOH (pH 7.3). Then the retinal cultures were gently removed from membrane filters using a fine brush and transferred to a recording chamber on the microscope. Fluorescent images were obtained with an upright microscope (Axioskop FS; Carl Zeiss) with a water immersion objective (Achroplan x40 NA0.75; Carl Zeiss). The wavelength of the excitation UV light (340 or 380 nm pulse; 100 ms) was switched using a filter wheel (Lambda 10-2; Sutter Instruments, Novato, CA, USA). The UV light was generated by a full-spectrum 175-W xenon bulb (Lambda LS; Sutter) conducted to the microscope through a liquid light guide and reflected using a dichroic mirror (FT 395 nm; Carl Zeiss). The pair of fluorescent images were processed using a band-pass filter (BP 485–515 nm; Carl Zeiss) and exposed to a multiple-format cooled CCD camera (CoolSnap-FS; Photometrics, Tokyo, Japan) at 6-s intervals. The filter wheel and the CCD camera were controlled using digital imaging software (MetaFluor version 6.0; Japan Molecular Devices, Tokyo, Japan). The background fluorescence was also subtracted using the software. During recording, slices were placed in a 0.5-ml bath chamber and perfused with BSS supplemented with tetrodotoxin (0.5 µM) at a flow rate of 2.5 ml/min. CCK-8 sulfate (CCK-8s), lorglumide, thapsigargin (all purchased from Sigma-Aldrich Corp.), Ca2+-free BSS, and 60 mM potassium (high K+) BSS were applied by switching the perfusate.
Ca2+ imaging in SCN cultures
Isolation of neuronal and glial images is difficult in SCN slices using conventional Fura-2 AM staining methods; therefore, neuron-specific Ca2+ imaging was performed using a yellow cameleon 2.1 sensor linked to a neuron-specific enolase promoter (32)
. The cameleon-expressing SCN slice was cut from a filter cup and transferred into the microscope chamber together with the membrane filter. The Ca2+ response was observed as above using an upright microscope with an x40 water-immersion objective. The SCN neurons were exposed to 440 ± 5 nm light using a liquid light guide lamp house (Lambda LS) with a band-pass filter (440NBD10; Omega Optical Inc., Brattleboro, VT, USA). The resultant fluorescence image was separated using a dichroic mirror (455DRLP; Omega Optical Inc.) and fed into double-view optics (A4313; Hamamatsu Corporation, Bridgewater, NJ, USA), in which one image was split into bilateral images via internal reflection mirrors and processed using two dichroic mirrors (515 DRLPXR; Omega Optical Inc,) and band-pass filters (480DF30 and 535DF25 filters). The lamp house, shutter, and CCD camera were controlled as above using digital imaging software.
Quantitative reverse transcriptase-polymerase chain reaction (PCR)
Total RNA was extracted from mouse tissue using RNAzolB reagent (Tel-Test Inc., Friendswood, TX, USA). Mice were deeply anesthetized with ether, and their brains were quickly removed. Coronal brain slices (1 mm) were prepared using a rodent brain matrix (RBM-2000C; ASI Instruments, Warren, MI, USA), and the SCN was punched out bilaterally from the brain slices. The cDNAs of mPer1 (GenBank accession number AF022992; sense, 5'-CCAGGCCCGGAGAACCTTTTT-3'; antisense, 5'-CGAAGTTTGAGCTCCCGAAGTG-3'), mPer2 (GenBank accession number AF035830; sense, 5'-ACACCACCCCTTACAAGCTTC-3'; antisense, 5'-CGCTGGATGATGTCTGGCTC-3'), CCK-A receptor (GenBank accession number AK004730; sense, 5'-ACAGGAGTGAGCCATTCACCAGC-3'; antisense, 5'-GATGTTGGTGACAGTCCGCATCC-3'), and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GenBank accession number M32599; sense, 5'-GGGAAGCTTGTCATCAA-3'; antisense, 5'-TGCTTCA-CCACCTTCTTG-3') were amplified using a program temperature control system PC-700 (ASTEC, Fukuoka, Japan). The reaction solution consisted of 2.5 µl of 10x PCR buffer (Boehringer Mannheim, Mannheim, Germany), 2.5 µl of 2.5 mM dNTP, 1.25 µl of 10 µM sense, 1.25 µl of 10 µM antisense, 17.0 µl of distilled water, 0.25 µl of Taq polymerase (Boehringer Mannheim), and 0.2 µl of 20 µCi [32P]dCTP (Amersham Pharmacia Biotech, Ltd., Little Chalfont, Buckinghamshire, UK). The PCR product was run on a 5% acrylamide gel. Images were visualized using an imaging plate (BAS-IP MS 2040; Fujifilm, Tokyo, Japan) and analyzed using Image Gauge (Fujifilm). The exponential phase of GAPDH amplification in all experimental conditions was located between cycles 22 and 24, and the exponential phase of mPer1 and mPer2 was located between cycles 29 and 31. The amplified efficiency of GAPDH and mPer1 or mPer2 was quantified at cycle 30. The ratio of the amplified target to the amplified internal control was compared.
In situ hybridization
Mice were deeply anesthetized with ether and intracardially perfused with chilled saline (25 ml) followed by 0.1 M phosphate buffer (pH=7.4) containing 4% paraformaldehyde (25 ml). Brains were removed, postfixed in 0.1 M phosphate buffer containing 4% paraformaldehyde for 24 h at 4°C, and transferred into 20% sucrose in 0.1 M phosphate buffer for 72 h at 4°C. Slices (40 µm thick) that included the SCN were cut using a cryostat and divided into three equal groups from rostral to caudal regions for the measurement of mPer1 and mPer2 mRNAs. The mPer1 and mPer2 cRNA probes were a gift from Dr. Hitoshi Okamura at Kobe University (nucleotide positions: mPer1, 538–1752; mPer2, 1–638) for use in these in situ hybridization studies. Slices were placed in 2x standard sodium citrate (SSC) and were treated with 1 µg/ml proteinase K in 10 mM Tris-HCl buffer (pH=7.5) containing 10 mM EDTA for 10 min at 37°C, followed by treatment with 0.25% acetic anhydride in 0.1 M triethanolamine and 0.9% NaCl for 10 min. The slices were then incubated in hybridization buffer [60% formamide, 10% dextran sulfate, 10 mM Tris-HCl (pH=7.4), 1 mM EDTA, 0.6 M NaCl, Denhardt’s solution (0.02% Ficoll, 0.02% polyvinyl pyrrolidone, and 0.02% bovine serum albumin), 0.2 mg/ml tRNA, and 0.25% sodium dodecyl sulfate] containing 33P-labeled cRNA probes for 16 h at 60°C. Radioisotope (
[-33P]UTP; PerkinElmer Life and Analytical Sciences, Boston, MA, USA) -labeled antisense cRNA probes were made from restriction enzyme-linearized cDNA templates. After high-stringency posthybridization washes with 2x SSC/50% formamide, slices were treated with RNaseA (10 µg/ml) for 30 min at 37°C. Images were prepared as autoradiograms using BioMax MR film (Eastman Kodak, Rochester, NY, USA). After conversion into optical density by 14C-autoradiographic microscales (Amersham Biosciences Corp., Piscataway, NJ, USA), they were analyzed using an image analysis system (MCID; Imaging Research, St. Catharines, ON, Canada). Further, mounted slices after exposure to X-ray film were dipped into emulsion (NTB2, diluted 1:1 with distilled water; Eastman-Kodak), air-dried for 3 h, and stored in light-tight slide boxes at 4°C for 3 wk. The slides were developed with a D19 developer (Eastman Kodak), fixed with Fujifix (Fujifilm), and counterstained with cresyl violet (Sigma-Aldrich Corp.).
Statistical analysis
All data are presented as means with SE. Unless otherwise noted, one-way ANOVA followed by Duncan’s multiple-range tests was used for the statistical comparisons across multiple means. Two-tailed Student’s t test was used for the pairwise comparisons. A 95% confidence level was considered to indicate statistical significance. The irradiance dependency for the pupillary light reflex was analyzed using two-way ANOVA. The irradiance response curve for circadian phase shifts was analyzed using a four-parameter Hill function using SigmaPlot software (version 7.1; SPSS Inc., Chicago, IL, USA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). Within the SCN, only scattered staining was observed in the core region, whereas the shell boundary zone (peri-SCN) and periventricular zone contained a relatively larger number of stained cells (Fig. 1B
). The number of stained cells in the SCN was less than that in control hypothalamic regions, such as in the arcuate nuclei (number of X-gal stained cells=23.8±6.6/SCN slice and 137.6±19.4/rostral arcuate nucleus slice, number of slices=5, P<0.01 by Student’s t test; Fig. 1C
).
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To determine the cell types in the retina that express CCK-A receptors, we examined double immunofluorescent staining of retina with antibodies against β-gal and several different neural markers (Fig. 2
). Staining with the amacrine cell marker, glycine transporter-1, was strongly colocalized with β-gal staining (74.6±3.5%, number of slices=5). Neither calretinin-positive amacrine cells nor calbindin-D28k-positive amacrine cells exhibited β-gal staining (number of slices=5). Also, neither horizontal cells visualized using calbindin-D28k staining nor bipolar cells visualized using Chx10 staining exhibited β-gal staining (number of slices=5). These results clearly indicate that CCK-A receptors were expressed specifically on amacrine cells, most of which also expressed glycine transporters.
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Stimulation of CCK-A receptors mobilized intracellular Ca2+ in retinal amacrine cells but not in SCN neurons
We used Ca2+ imaging of organotypic cultures of retina and SCN to analyze the functional expression of CCK-A receptors within the photic input pathways to the circadian clock. First, Fura-2 AM-stained retinal cultures of wild-type (n=8), CCKAR+/– (n=8), and CCKAR–/– (n=8) mice were stimulated using the CCK-A receptor agonist, CCK-8s (300 nM, 1 min). CCK-8s evoked Ca2+ transients in 7.1 ± 1.8% of the Fura-2 AM-stained cells (total number of cells=234) in wild-type retina and 5.7 ± 1.9% of the cells (total number of cells=228) in CCKAR+/– retina (Fig. 3
A, C). These responding cells were identified as amacrine cells by the postexperimental X-gal staining of CCKAR+/– retina (Fig. 3C
). The CCK-8s-induced Ca2+ transient but not the high K+-induced Ca2+ flux was absent in CCKAR–/– retina (total number of cells=230) (Fig. 3A, C
). The CCK-8s-induced Ca2+ transient observed in wild-type retina was significantly inhibited using the CCK-A antagonist, lorglumide (300 nM, application 5 min before the CCK-8s application; F5,42=7.08, P<0.01 by one-way ANOVA followed by Duncan’s multiple-range tests) (Fig. 3C
). The CCK-8s-induced Ca2+ transients observed in wild-type retinas were resistant to the replacement of normal extracellular buffer with Ca2+-free buffer (responding cell population=7.8±2.2%, total number of cells=215). CCK-8s-induced Ca2+ transients were completely eliminated, however, after thapsigargin-depletion of internal Ca2+ stores (F5,42=7.08, P<0.01 by one-way ANOVA followed by Duncan’s multiple-range tests) (Fig. 3B, C
).
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Second, organotypic slice cultures of SCN expressing a yellow cameleon 2.1 sensor linked to a neuron-specific enolase promoter were used to examine the effects of exposure to either CCK-8s or glutamate (Fig. 4
). Within the core region, 75% of cultured SCN neurons (55 of 73 neurons, number of slices=11) exhibited a significant increase in cytosolic Ca2+ in response to exposure to glutamate (300 µM, 1 min), but exhibited no response to CCK-8s (300 nM, 1 min) (Fig. 4A
). A similar response to glutamate was observed in slice cultures of CCKAR–/– mouse SCN (14 of 20 neurons, number of slices=3). Of a total of 73 neurons analyzed in the wild-type slice, one neuron in the dorsomedial periventricular region exhibited a sustained increase in cytosolic Ca2+ in response to CCK-8s, and this effect was completely inhibited in the presence of lorglumide (300 nM) (Fig. 4B
). The amplitude of the glutamate response after exposure to CCK-8s was similar to that in the presence of lorglumide in this particular neuron.
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Light pulse-induced mPer1 and mPer2 gene expression was reduced in the SCN of CCKAR–/– mice
To examine the effects of CCKAR–/– on light-induced responses of SCN neurons, we examined quantitative PCR for mPer1 and mPer2 mRNAs in SCN grafts. Under constant darkness conditions, the levels of mPer1 and mPer2 mRNAs oscillated in a circadian fashion both in the wild-type and CCKAR–/– SCN (Fig. 5
A, B). The approximate peaks of mPer1 and mPer2 occurred at identical times in the wild-type and CCKAR–/– SCN: at circadian time 6 for mPer1 and at circadian time 9 for mPer2 (Fig. 5B
). Brief light pulse exposure (70 lux, 15 min) applied 4 h after the activity-onset time (circadian time 16), significantly increased the expression of mPer1 (3.2-fold of unexposed control; P<0.01 by Student’s t test) and mPer2 (3.6-fold of unexposed control; P<0.01 by Student’s t test) in the wild-type SCN (Fig. 5C
). The light pulse-induced mPer1 expression (1.6-fold of unexposed control; P=0.16 by Student’s t test) and mPer2 expression (1.7-fold of unexposed control; P=0.09 by Student’s t test) were less evident in the SCN grafts of CCKAR–/– mice (Fig. 5C
). The light pulse-induced increase in mPer1 and mPer2 mRNAs in wild-type SCN and the reduction in the responsiveness in the CCKAR–/– SCN were also visualized using in situ hybridization in SCN sections (Fig. 5D
).
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Light pulse-induced behavioral phase shifts and pupillary reflex were reduced in CCKAR–/– mice
Infrared sensor detection of activity/rest patterns across 24-h light-dark cycles (data not shown) and free-running periods of behavioral rhythms in constant darkness (Fig. 6
A) were almost identical between wild-type mice (
=23.8±0.06 h, n=8) and CCKAR–/– mice (=23.9±0.04 h, n=8). The magnitude of behavioral phase delays caused by a light pulse exposure (10–500 lux for 15 min) at circadian time 16 was analyzed in wild-type and CCKAR–/– mice. Both types exhibited activity-onset delays depending on the intensity of light exposure (Fig. 6B
). The magnitude of the phase delays caused by 50 lux or larger, however, was significantly smaller in CCKAR–/– mice than in wild-type mice. The maximal reduction in responsiveness was observed at 50 lux, at which CCKAR–/– mice exhibited a 42% smaller magnitude in phase shift than that of wild-type mice (P<0.05 by Student’s t test, n=8 for both types). The maximal response at 500 lux was still 35% smaller in CCKAR–/– mice (P<0.01 by Student’s t test, n=8 for both types) (Fig. 6B
).
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To examine the effects of CCKAR–/– on nonimage-forming visual functions, we further analyzed the consensual pupillary reflex using infrared video systems. The dark-adapted aperture areas were not significantly different between the wild-type mice (2.43±0.07 mm, n=5) and CCKAR–/– mice (2.48±0.03 mm, n=4). Also, atropine-induced aperture areas observed at the end of experiments were not significantly different between the wild-type mice (2.60±0.08 mm, n=5) and CCKAR–/– mice (2.67±0.09 mm, n=4). The irradiance-dependent pupillary constrictions caused by 20, 100, or >1000 lux light were also observed both for the wild-type mice (F2,92=440.2, P<0.01 by two-way ANOVA) and CCKAR–/– mice (F2,92=183.9, P<0.01 by two-way ANOVA for the wild-type group) (Fig. 7
A). However, the minimal pupillary dimensions followed by exposures of 100-lux light (+64.7%; P<0.05 by Student’s t test) or >1000-lux light (+154.3%; P<0.05 by Student’s t test) were significantly larger in CCKAR–/– mice (Fig. 7B, C
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Little CCK function in retino-recipient SCN neurons
CCK peptides have previously been reported to be expressed to some degree in SCN neurons (6
, 15
16
17)
, so we carefully analyzed the possible functionality of CCK-A receptors in SCN neurons. X-gal stained only sparsely scattered cells in the SCN, with a rather denser distribution in the peri-SCN and periventricular zones. Consistent with this result, our Ca2+ imaging studies in organotypic SCN cultures demonstrated that core SCN neurons did not respond to the CCK-A receptor agonist, CCK-8s. An increase in cytosolic Ca2+ evoked by either CCK-8s or glutamate was observed in only one neuron located in the periventricular region, where retino-recipient neurons are absent or rare (16)
. Although lack of CCK-8s effects on glutamate receptive SCN neurons may be due to limitations of samples in our Ca2+ imaging studies, this result is consistent with the reported distribution of CCK peptides in the mouse, because dorsal efferent fibers from the SCN but not retinohypothalamic tracts are CCK-immunoreactive in mice (16)
. Therefore, CCK peptides may have a role in the efferent transmission from SCN neurons and/or transmission between intra-SCN neurons but not for transmission to retino-recipient SCN neurons.
Because CCK is one of the most abundant neuropeptides in the brain, and several brain loci responsible for visual input, such as the dorsal lateral geniculate, express CCK-A receptors (33)
, careful discussion is needed to identify the localization of CCK-A receptors responsible for the modulation of nonimage-forming visual functions. The SCN receive geniculohypothalamic tracts (GHT) from the intergeniculate leaflet (IGL) of the thalamus (6)
. It has been shown that the GHT uses neuropeptide-Y and GABA as neurotransmitters to the SCN, and this pathway is involved in the behavioral phase shifts via nonphotic inputs, such as that via activity elevations and triazolam administrations (6)
. Also, it has been shown that a free running period during constant light is reduced by IGL lesions, and thus the GHT pathway may be involved in the tonic effects of light (6)
. However, to our knowledge, there have been no reports showing that light pulse-induced phase shifts are significantly modulated by IGL lesions in mice. Whether CCK-A receptors are expressed on any of the GHT pathways to the SCN is currently not known; therefore, it is reasonable to consider that significant reduction of photoentrainment in CCKAR–/– mice is due to the modulation of reticulohypothalamic (RHT) pathways not of GHT pathways. Within the RHT pathways, the results of the lacZ reporter assays and Ca2+ imaging in the present study suggest that the principal site of CCK-A receptor actions for circadian photoentrainment is confined within the retina not in SCN neurons.
Possible function of CCK-A receptors in mouse amacrine cells
Approximately 30 different types of amacrine cells have been identified in the mammalian retina (34)
. About half of the amacrine cells contain GABA and the other half contain glycine as inhibitory neurotransmitters, and both of these types of amacrine cells express ionotropic glutamate receptors to receive glutamatergic input from bipolar cells (35)
. Despite the abundant knowledge about the morphological diversity of amacrine cells, their physiological functions are poorly understood, especially at a systems level. One of the best characterized functions is inhibitory neurotransmission on the synapse between bipolar cells and retinal ganglion cells (36)
. This amacrine cell function contributes to the transient firing pattern of retinal ganglion cells, which is required for background control for motion perception (37
, 38)
. The present results suggest that amacrine cells have a novel role in circadian photoentrainment, because 1) CCK-A receptors were concentrated on glycine transporter-positive amacrine cells but not on retino-recipient SCN neurons, 2) a CCK-A receptor agonist evoked calcium responses in amacrine cells of wild-type but not of CCKAR–/– mice nor in the SCN of wild-type mice, 3) CCKAR–/– mice exhibited reduced light pulse-induced mPer1/mPer2 expression in the SCN, and 4) CCKAR–/– mice exhibited reduced light pulse-induced behavioral phase shifts. Notably, these results suggest that CCK-A receptor activation results in an excitatory effect on retinal ganglion cells that project to the SCN, because the photic response of the SCN was significantly reduced in CCKAR–/– mice.
The present results showed that the cytosolic Ca2+ concentration was increased by CCK-8s in cultured amacrine cells. Activation of phospholipase C with generation of inositol 1,4,5-trisphosphate and diacylglycerol, subsequent Ca2+ release from internal Ca2+ stores, and activation of protein kinase C is the proposed intracellular signaling common to a wide variety of cells after CCK-A receptor activation (39
40
41)
. Accordingly, the CCK-8s-induced Ca2+ response in cultured amacrine cells remained in Ca2+-free extracellular medium but was abolished by the depletion of internal Ca2+ stores by thapsigargin. In chick retinal cultures, protein kinase C
colocalizes with -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) -type glutamate receptors, and phosphorylation of AMPA receptors by protein kinase C causes translocation of AMPA receptors to the plasma membrane and increases excitatory synaptic transmission (42)
. Axons from glycinergic amacrine cells have been reported to terminate on neighboring GABAergic amacrine cells, which terminate on the synapse between bipolar cells and retinal ganglion cells (38)
. Therefore, one possible explanation of the excitatory action of CCK-A receptor activation on the ON bipolar-ganglion cell synapse is that activation of CCK-A receptors excites glycinergic amacrine cells, which inhibit GABAergic amacrine cells. Inhibition of GABAergic amacrine cells would then disinhibit the bipolar ganglion cell synapse. Retinal ganglion cells, which project to the SCN, could then be activated by ON bipolar cell activity (Fig. 8
). It is also possible that the excitation of glycinergic amacrine cells by activation of CCK-A receptors may inhibit OFF bipolar terminals, which may also allow activation of retinal ganglion cells. It has been suggested, however, that intrinsically photosensitive retinal ganglion cells, which are the predominant type (75%) of SCN-projecting retinal ganglion cells (43)
only receive ON bipolar terminals and amacrine cell terminals (8)
.
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Rhythmic mPer1 expression has been reported to occur in the majority of GABAergic amacrine cells but not glycinergic amacrine cells (44)
. Also, the AII amacrine cells exhibit circadian rhythms in parvalbumin expression in constant darkness (45)
. Therefore, a subpopulation of amacrine cells may contain an intrinsic molecular clock mechanism. It has not been shown previously, however, that amacrine cell-mediated pathways have a critical role in circadian photoentrainment. The present results showed that CCKAR–/– mice exhibited significantly reduced bright light-induced behavioral phase shifts; thus, we propose that the CCK-A receptor-mediated amacrine cell pathway has an important role in circadian photoentrainment via cone photoreceptor pathways, although we cannot exclude the possible involvement of rods in this signaling pathway only with the results of behavioral phase shifts. In specific photoreceptor-deficient mutants, such as midwavelength coneless mice (46)
, exposure to full-spectrum light or saturating bright light (>100 lux) masks their deficiency in circadian photoentrainment. In the present study, however, CCKAR–/– mice exhibited a significantly reduced saturating bright light response, suggesting that multiple cone-mediated pathways may be processed via amacrine cells that express CCK-A receptors. This hypothesis is consistent with the dense lateral distribution of CCK-A receptors within the inner nuclear layer that we observed in the present study using X-gal staining.
CCK-A receptors may control outputs and inputs of the circadian clock
The present data show that CCK-A receptors are involved in the photic input to the SCN for photoentrainment. This finding raises the possibility that CCK receptors in related structures may have additional functions in other aspects of circadian control of behaviors. For example, the CCK peptide is known as an important regulator for feeding behaviors, and CCK-A receptors may participate in control of satiety both via central and gastrointestinal systems (47)
. As described above, efferent fibers from the SCN contain CCK peptides (16)
, and they may terminate on satiety-controlling hypothalamic nuclei such as the paraventricular nucleus and dorsal medial hypothalamus that express CCK-A receptors (47)
, observable also in our results (Fig. 1)
. This suggestion raises the possibility that CCK outputs from the SCN contribute to the circadian rhythms in feeding behaviors. If so, the CCK-A receptor-mediated pathway could not be the sole output from the SCN because CCKAR–/– mice maintain normal nocturnal feeding rhythms (48)
. Consistent with this hypothesis, our locomotor activity recordings also showed strong circadian rhythms in CCKAR–/– mice.
We have previously observed that light pulse-induced phase shifts and c-fos expression in the SCN were reduced in obese mutant OLETF rats (26
, 27)
, which lack multiple genes, including genes encoding CCK-A receptors (28
, 29)
. The present study further demonstrated that CCKAR–/– mice exhibited impaired circadian photoentrainability similar to that observed in OLETF rats. The CCKAR–/– mice are not obese, presumably due to the basal energy balance of mice, although food intake activity is up-regulated in mutant mice (30
, 48
, 49)
. Therefore, a lack of CCK-A receptors, but not an obese phenotype, underlies impaired circadian photoentrainability. The present study demonstrated that in CCKAR–/– mice a key cause of impaired photoentrainability is a deficiency in CCK-A receptor expression in amacrine cells.
As in cerebral CCK-receptive neurons, amacrine cells may receive CCK peptides from neighboring retinal neurons, because CCK peptides have been found in retina (18
19
20
21
22)
, and blood-retinal barriers may prevent peptide transport from the gastrointestinal system. It has recently been suggested, however, that there may be direct regulation of satiety control centers by peripheral nutritional signals, such as CCK, ghrelin, and leptin, via "leaky" portions of the blood-brain barrier and circumventricular organs (47
, 50)
. This suggestion raises the possibility that peripheral CCK harmonistically activates cerebral and retinal CCK-A receptors, adding an interesting aspect to possible circadian clock mechanisms, especially at a systems level and in relation to metabolic control.
In conclusion, our data suggest a novel function for retinal CCK in nonimage-forming visual functions, including circadian photoentrainment and pupillary light reflex. CCK-A receptors on glycinergic amacrine cells may have a key role in the process of photoentrainment, probably modulating retinal ganglion cell activation of the SCN.
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ACKNOWLEDGMENTS |
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Received for publication August 2, 2007. Accepted for publication November 15, 2007.
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REFERENCES |
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associates directly with the GluR4 -amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit: effect on receptor phosphorylation. J. Biol. Chem. 278,6307-6313
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