Transgenic approach reveals expression of the VPAC<sub>2</sub> receptor in phenotypically defined neurons in the mouse suprachiasmatic nucleus and in its efferent target sites.
Circadian rhythms in mammals depend on the properties of cells in the suprachiasmatic nucleus (SCN). The retino‐recipient core of the mouse SCN is characterized by vasoactive intestinal peptide (VIP) neurons. Expression within the SCN of VPAC2, a VIP receptor, is required for circadian rhythmicity. Using transgenic mice with β‐galactosidase as a marker for VPAC2, we have phenotyped VPAC2‐expressing cells within the SCN and investigated expression of the VPAC2 marker at sites previously shown to receive VIP‐containing SCN efferents. In situ hybridization and immunohistochemistry demonstrated identical distributions for VPAC2 mRNA and β‐galactosidase and coexpression of the two signals in the SCN. Double‐label confocal immunofluorescence identified β‐galactosidase in 32% of the VIP and 31% of the calretinin neurons in the SCN core. Of the arginine‐vasopressin neurons that characterize the SCN shell, 45% expressed β‐galactosidase. In contrast, this marker was not apparent in astrocytes within the SCN core or shell. Cell bodies containing β‐galactosidase were detected at sites reportedly receiving VIP‐containing SCN efferents, including the subparaventricular zone and lateral septum and the anteroventral periventricular, preoptic suprachiasmatic, medial preoptic and paraventricular hypothalamic nuclei. The detection of a marker for VPAC2 expression in the SCN in almost one‐third of the VIP and calretinin core neurons and nearly half of the arginine‐vasopressin shell neurons and also in cell bodies at sites receiving VIP‐immunoreactive projections from the SCN indicates that VPAC2 may contribute to autoregulation and/or coupling within the SCN core and to the control of the SCN shell and sites distal to this nucleus.
Keywords: calretinin; circadian; glial fibrillary acidic protein; vasoactive intestinal peptide; vasopressin
The suprachiasmatic nucleus (SCN) of the hypothalamus contains the predominant circadian pacemaker, capable of driving a plethora of physiological and behavioural rhythms ([56]). Components within the SCN also enable the entrainment of circadian rhythms to photic or nonphotic Zeitgebers ([25]; [22]; [75]). Increasing evidence indicates that circadian rhythmicity in mammals depends not only on intracellular clock mechanisms, autonomously ticking within SCN cells ([53]), but also on interactions between distinct SCN neuronal populations ([20]; [39]; [49]). Recent studies have begun to elucidate the mechanisms underlying SCN cellular interactions and their significance in the integration of local cellular activity for coherent output ([11]) or response to photic input ([50]; [75]; [49]).
Several studies indicate that, within the SCN, peptidergic signalling involving vasoactive intestinal peptide (VIP) is critical for the generation and entrainment of circadian rhythms. Mice overexpressing the VIP receptor VPAC2 exhibit circadian rhythms of reduced period and faster phase shifts in response to advances in the light–dark schedule ([57]). Conversely, mice lacking VIP ([14]) or VPAC2 ([23]; [15]) display pronounced deficits in circadian rhythmicity and sensitivity to photic stimuli. Furthermore, chronic application of the selective VPAC2 receptor antagonist PG 99‐465 to the wild‐type SCN slice disrupts electrophysiological rhythms ([15]).
The VIP‐synthesizing cells in the mouse SCN constitute 10% of the neurons in the nucleus and are located in the core subdivision, an area further characterized by a slightly smaller population expressing the calcium‐binding protein calretinin ([64]; [44]; [1]). The core SCN represents a nodal site as it receives projections mediating the photic Zeitgeber directly via the retinohypothalamic tract ([26]) and indirectly via the geniculohypothalamic tract ([1]). As a corollary, exposure of mice to light induces c‐fos and Per1 gene expression in these VIP neurons ([10]; [36]). The VIP neurons within the SCN core establish a dense immunoreactive network of fibres that extends into the shell ([64]; [1]), a subdivision characterized by arginine‐vasopressin (AVP)‐synthesizing neurons.
This study was designed to establish the phenotype of cells expressing VPAC2 receptors within the mouse SCN; such cells would be potential mediators of the effects of locally released VIP. Using in situ hybridization histochemistry and/or immunohistochemistry, we examined the SCN of transgenic mice that express β‐galactosidase as a marker for the human VPAC2 receptor ([57]). Core VIP‐ and calretinin‐immunoreactive neurons, shell AVP‐immunoreactive neurons and glial fibrillary acidic protein (GFAP)‐ immunoreactive astrocytes throughout the SCN were analysed. Sites outside the SCN that might be responsive to the VIP projections from the SCN ([1]) were also examined for the marker for the VPAC2 receptor.
Materials and methods
Animals and tissue collection
Adult male mice, wild type (n = 12) and transgenic which over‐express the human VPAC2 receptor gene (n = 12), were bred on a mixed CBA × C57Bl6J background in the Division of Neuroscience, University of Edinburgh ([57]). Animals were housed under a 12 : 12 light/dark cycle (lights on at 07:00 h) for at least 2 weeks and food and water were provided ad libitum. Mice were perfused transcardially (between 14:00 and 18:00 h) with 4% paraformaldehyde under pentobarbital anaesthesia. Brains were removed from the cranium, postfixed in 4% paraformaldehyde overnight and stored in phosphate‐buffered saline (PBS) until cut into 30‐µm thick sections with a vibratome. Sections were then stored in antifreeze until further processing for immunohistochemical and/or in situ hybridization procedures. Every third section was committed to a specific histological study, i.e. immunohistochemistry, in situ hybridization or immunohistochemistry in combination with in situ hybridization.
Immunohistochemistry
Procedures for immunohistochemistry were performed on free‐floating sections, according to [32]). Sections from transgenic and wild‐type mice were processed concurrently in the same containers (the use of micropunched codes in the tissue allowed for subsequent attribution of the sections to specific brains and correct orientation of the mounted sections). Immunohistochemical controls included preabsorption of the primary antibody with excess (5 µg/mL) Escherichia coliβ‐galactosidase (Sigma‐Aldrich, Gillingham, Dorset, UK) and/or omission of primary or secondary antibodies. Such procedures resulted in a complete absence of immunoreactive signals.
Single‐ and double‐label immunofluorescence
The following steps took place at room temperature and sections were thoroughly rinsed in 0.1 m PBS (3 × 20 min) between each step unless otherwise stated. After transfer of the sections from antifreeze, they were pretreated consecutively with 1% sodium borohydride (15 min), 0.5% Triton X‐100 (30 min), 0.5% hydrogen peroxide (10 min) and 2% normal donkey serum (30 min), each dissolved in 0.1 m PBS. To reveal β‐galactosidase immunoreactivity, antibodies raised in two different species (rabbit antiβ‐galactosidase, 1 : 10 000, 5′‐3′, Inc., Boulder, CO, USA; goat antiβ‐galactosidase, 1 : 500 000, Paesel + Lorei, Hanau, Germany) were used for 2 days at 4 °C. Sections were incubated in biotinylated secondary antibodies raised in donkey (antirabbit or antigoat, 1 : 1000; Jackson Immunoresearch, West Grove, PA, USA) for 2 h and then in avidin‐biotin‐peroxidase complex (ABC Elite, 1 : 1000; Vector Laboratories, Burlingame, CA, USA) for 60 min. To enhance the signal detection, biotinylated tyramide (1 : 200; Dupont‐NEN, Stevenage, Herts, UK) was applied in Tris buffer for 20 min. The final incubation was in fluorescein‐streptavidin (1 : 500; Jackson Immunoresearch) overnight at 4 °C.
Sections were further processed for double‐label immunofluorescence studies. The use of β‐galactosidase antibodies raised in two different species allowed us to combine the second primary antibodies as follows. Mouse antivasopressin‐neurophysin (PS41, 1 : 1000; from Dr H. Gainer, NIH/NINDS, Bethesda, MD, USA) was used after rabbit antiβ‐galactosidase and, after staining with goat antiβ‐galactosidase, we used rabbit antiVIP (1 : 5000; Diasorin, Stillwater, MN, USA), rabbit anticalretinin (1 : 5000; from Dr J.H. Rogers, University of Cambridge, UK) or rabbit antiGFAP (1 : 500; Sigma‐Aldrich) antisera. Subsequently, rhodamine red‐X‐donkey antimouse IgG or rhodamine red‐X‐donkey antirabbit IgG (1 : 500; Jackson Immunoresearch) was used for overnight incubation at 4 °C. After a final rinse in PBS, sections were mounted on slides, air‐dried and coverslipped with Prolong Antifade medium (Prolong Antifade Kit; Cambridge BioScience, Cambridge, UK).
Nickel‐enhanced diaminobenzidine immunoperoxidase staining
Pretreatment steps and incubation of sections with primary (rabbit antiβ‐galactosidase) and secondary (biotinylated donkey antirabbit IgG) antibodies took place as described above. Immunoreactivity was revealed by nickel‐enhanced diaminobenzidine and the reacting solution for the peroxidase enzyme contained diaminobenzidine tetrahydrochloride (0.5 mg/mL; Sigma‐Aldrich), 0.005% H2O2 (Sigma‐Aldrich) and 6 mg/mL ammonium nickel sulphate (Sigma‐Aldrich) in 0.1 m Tris buffer, pH 7.6. Sections were mounted onto glass slides and allowed to air dry. Following a brief transfer in 100% ethanol and xylene clearing, sections were coverslipped with DEPEX (BDH, Poole, UK).
In situ hybridization histochemistry
Probe synthesis
Radiolabelled VPAC2 cRNA probes were produced using the RPR4/Pbluescript construct, containing bases 711–1192 of the published sequence of the rat receptor ([42]), sharing 97 and 82% homology with mouse and human ([43]; [57]) VPAC2, respectively. Antisense cRNA VPAC2 probes were generated following linearization of the construct with EcoRI and transcription with T7 RNA polymerase. Sense strand probes were synthesized following linearization with BamHI and transcription with T3 RNA polymerase. Riboprobes were transcribed in the presence of 35S‐rUTP (Dupont‐NEN) and radiolabelled probes were purified using G‐50 Sephadex columns (Roche, Lewes, UK).
Pretreatment and hybridization
Vibratome sections from transgenic and wild‐type animals, incorporating the rostro‐caudal SCN, were thaw‐mounted onto gelatin (Sigma‐Aldrich)‐treated slides. Sections were then fixed in 4% paraformaldehyde in 0.1 m PBS, acetylated in 0.1 m triethanolamine and 0.25% acetic anhydride in 0.9% NaCl, treated with proteinase K (10 µg/mL; Sigma‐Aldrich), dehydrated through increasing concentrations of ethanol and delipidated in chloroform. Sections were stored at −70 °C until used for in situ hybridization histochemistry. Sections were hybridized for 16 h at 55 °C with buffer (60 µL) containing 2 × 106 cpm of labelled VPAC2 cRNA probe. The concentration of probe used here provided optimal labelling without a significant background signal as demonstrated in preliminary studies. Following RNAse A treatment (40 µg/mL; Sigma‐Aldrich) at 37 °C and stringency washing (0.1 × SSC, 60 °C), sections were dehydrated and exposed to MR1 film (Kodak, Hemel Hempstead, Herts, UK). Following film development, sections were dipped in K‐9 nuclear track emulsion (Ilford Imaging, Mobberley, Knutsford, UK). After 6–9 weeks of exposure, the emulsion‐coated sections were developed and coverslipped. To ensure specificity of results, several sections were subjected to concurrent control treatments. These included hybridization with cRNA probes following RNAse pretreatment, competition with 100‐fold excess unlabelled probe and hybridization with sense strand probes. The signals were not above background after such procedures.
Combined immunohistochemistry and in situ hybridization
Immunohistochemistry preceded in situ hybridization steps. Blocking of nonspecific antibody binding was carried out by incubation of sections in diethylpirocarbonate‐treated PBS containing heparin (500 µg/mL; Sigma‐Aldrich) and 2% bovine serum albumin (2% bovine serum albumin/PBS; Sigma‐Aldrich) for 30 min at room temperature. Primary antibody incubations were carried out with rabbit antiβ‐galactosidase (1 : 5000; 5′‐3′, Inc.) diluted in heparinized 2% bovine serum albumin/PBS for 16–24 h at 4 °C. After washing, sections were incubated in biotinylated donkey antirabbit IgG (1 : 500; Jackson Immunoresearch) in heparinized 2% bovine serum albumin/PBS (14–16 h, 4 °C) and the avidin‐biotin‐peroxidase complex (ABC Elite, 1 : 1000; Vector Laboratories; 60 min). Sections were processed for biotin tyramide amplification of the peroxidase signal ([2]) by sequential incubations in biotin tyramide (30 min) ([2]) and ABC Elite (1 h; Vector Laboratories) working solutions and the final peroxidase reaction was developed in 0.1 m Tris buffer, pH 7.6, containing diaminobenzidine tetrahydrochloride (0.5 mg/mL; Sigma‐Aldrich) and 0.005% H2O2 (Sigma‐Aldrich). Sections were then mounted onto gelatinized slides and allowed to air dry. In situ hybridization for detection of VPAC2 on the immunostained sections took place as described for nonimmunostained sections.
Cell counting, photomicrography and statistical analysis
To determine the incidence of β‐galactosidase expression in phenotypically defined cell populations in the SCN, a confocal microscope with the MRC‐600 laser scanning confocal imaging system (Bio‐Rad) and COMOS software was used. For each double‐labelling combination, we examined one to three sections from levels belonging to the rostral (approx. −0.3 to −0.4 mm from Bregma; Franklin & Paxinos, 1997), mid‐rostral (−0.4 to −0.55 mm), mid‐caudal (−0.55 to −0.70 mm) and caudal (−0.70 to −0.82 mm) SCN in three animals. Cells within the SCN were analysed unilaterally to ensure rostro‐caudal precision for the representative levels. To avoid signal 'bleed‐through', sections were excited with the argon–krypton laser using the standard excitation wavelengths sequentially for rhodamine red‐X and fluorescein. At each rostro‐caudal level confocal images, covering the total area of the SCN containing cells immunoreactive for AVP, VIP, calretinin or GFAP were obtained. For each section, 15–20 confocal images were collected from 1.0‐µm thick optical slices and imported into Image J (version 1.30; Wayne Rasband, NIH, USA) for conversion into a dual colour (red–green) stack. Immunolabelled profiles for AVP, VIP, calretinin and GFAP were identified, assessed for coexpression with β‐galactosidase and labelled across the Z‐axis using the Image J software. Cells exclusively displaying punctate signals for β‐galactosidase were considered double‐labelled only if this signal was enveloped by the second fluorescent signal. Collage images of the SCN were generated by importing confocal images to Adobe PhotoShop (version 5.0.2; Adobe Systems, Mountain View, CA, USA).
The percentages of double‐labelled cells at each rostro‐caudal level in the nucleus or for the whole SCN were analysed using anova. Posthoc comparisons were made using the Fisher LSD test. Statistical significance was defined as P < 0.05.
Results
Distribution of β‐galactosidase‐immunoreactive and VPAC 2 receptor mRNA‐expressing cells in t...
Numerous cells immunopositive for β‐galactosidase, a marker for the VPAC2 receptor in these mice, were detected by biotinylated tyramide‐amplified immunofluorescence histochemistry across the rostro‐caudal extent of the SCN (Fig. 1A–H). At the rostral tip of the SCN (Fig. 1A) two clusters of β‐galactosidase‐positive cells were detected, one located medially below the third ventricle, spanning the midline, and the other located more laterally (also see Fig. 1O). Similarly, at mid‐rostro‐caudal levels (Fig. 1D and E) two distinct populations of β‐galactosidase cells were observed, a large population in the dorsomedial SCN and a smaller cluster in the ventrolateral margin of the SCN. At this rostro‐caudal level, a lower incidence of β‐galactosidase cells was found in the central region between the two positive cell clusters (Fig. 1D and E). At the caudal pole of the SCN in the retrochiasmatic area (Fig. 1H), a single cluster of immunopositive perikarya was observed medially, below the third ventricle, within a dense plexus of immunoreactive fibres (Fig. 1H). β‐galactosidase‐immunoreactive perikarya were observed embedded within the optic chasm. Immunopositive perikarya and fibres located lateral or dorsal to the SCN within the subparaventricular zone (SPVZ) were found along the rostro‐caudal extent of this nucleus (Fig. 1A–H). VPAC2 receptor mRNA, as revealed by isotopic in situ hybridization, was abundantly expressed in the SCN of transgenic (Fig. 1I–L) and wild‐type (inset in Fig. 1I) mice. The distribution of VPAC2 receptor mRNA‐positive cells in the SCN of transgenic and wild‐type mice (Fig. 1I–L) corresponded to that of β‐galactosidase at equivalent rostro‐caudal levels. Furthermore, the combination of immunohistochemistry, using diaminobenzidine as the chromogen, and in situ hybridization revealed colocalization of β‐galactosidase and VPAC2 mRNA signals in cells of the SCN (Fig. 1M and N). Within individual SCN cells, immunoreactivity for β‐galactosidase was detected as diffuse or punctate signals (Fig. 1O–Q); the latter type of signal had a diameter of 1–2 µm and has been previously described as a 'doughnut‐like' ring in LacZ transgenics ([18]; [66]).
Graph: 1 Photomicrographs of the suprachiasmatic nucleus (SCN) in transgenic mice with LacZ as a reporter for the human VPAC2 receptor gene. Rostro‐caudal distribution of β‐galactosidase‐immunoreactive (A–H) and VPAC2 receptor mRNA‐expressing (I–L) cells. At equivalent levels in these transgenic mice, correspondence is apparent between the immunoreactivity revealed by fluorescein (A, C, E and G) and the in situ hybridization histochemistry (ISHH) signals visualized by dark‐field microscopy (I–L). The inset (in I) shows a bright field image of the hybridization signal for VPAC2 mRNA in the SCN (arrowhead) and subnuclei of the thalamus in wild‐type mice. Immunoreactive or mRNA‐positive cells are abundant at all levels of the SCN; relatively few are visible in the central portion of the mid‐SCN (D, E and K). At rostral (A) and caudal (H) levels immunoreactive cells span the midline. Combination of ISHH for VPAC2 mRNA and immunohistochemistry for β‐galactosidase indicates colocalization of the two signals (silver grains and diaminobenzidine reaction product, respectively), as shown in the medium power (M) and high power (N, area enclosed by a box in M) photomicrographs of the rostral SCN. (O) β‐galactosidase immunoreactivity revealed by nickel‐diaminobenzidine‐enhanced immunohistochemistry at the level of the rostral SCN; at higher magnification (P and Q, areas enclosed by boxes in O) this immunoreactivity appears as diffuse signals (arrows) and punctate signals (arrowheads). 3V, third ventricle. oc, optic chiasm.
Phenotypic characterization of cells expressing β‐galactosidase across the rostro‐caudal exte...
Double‐label immunofluorescence and confocal microscopy were employed to identify cells expressing AVP, VIP, calretinin or GFAP, together with β‐galactosidase, across the rostro‐caudal extent of the SCN. The distribution of peptide‐immunoreactive or calcium‐binding protein‐immunoreactive cells within the SCN was similar in the transgenic and wild‐type mice and consistent with that previously reported for the wild‐type SCN ([64]; [1]). β‐galactosidase was found to be differentially expressed in the cell populations examined, the highest incidence (44.6 ± 1.7%) being displayed by the AVP population within the SCN shell (Table 1). Expression of the β‐galactosidase marker in AVP neurons was detected across the rostro‐caudal extent of the SCN in all subcompartments of the nucleus containing AVP‐immunoreactive neurons (Fig. 2A–J). Of the VIP‐immunoreactive cells that characterize the ventral core of the SCN, 31.6 ± 2.3% showed β‐galactosidase expression (Table 1; Fig. 3A and B). Within the SCN core, coexpression of β‐galactosidase was detected in 30.8 ± 2.6% of calretinin‐immunoreactive cells (Table 1; Fig. 3C).
1 Percentages of neurons with AVP, VIP or calretinin that also express the VPAC 2 receptor marker along the rostro‐caudal axis of the mouse SCN
| Rostral SCN | Mid‐rostral SCN | Mid‐caudal SCN | Caudal SCN | Rostral to caudal SCN |
|---|
| AVP + β‐gal | 47.7 ± 11.2 | 36.1 ± 9.7 | 49.2 ± 7.5 | 50.6 ± 7.1 | 44.6 ± 1.7a |
| (63) | (98) | (276) | (92) | (530) |
| VIP + β‐gal | NA | NA | 36.7 ± 2.3
(133) | 22.4 ± 5
(76) | 31.6 ± 2.3b
(210) |
| Calretinin + β‐gal | 40.9 ± 8.7 | 28.6 ± 5.9 | 35.5 ± 6.9 | 17.5 ± 5.9 | 30.8 ± 2.6c |
| (43) | (50) | (145) | (69) | (308) |
1 Data are presented as percentages ± SEM. Numbers in parentheses represent the mean number of peptidergic cells counted per animal (n = 3). One‐way anova and the Fisher LSD were used to establish significant (P < 0.01) differences (with a > b, c). NA, not applicable due to the very low numbers of VIP neurons; β‐gal, β‐galactosidase.
Graph: 2 Confocal photomicrographs demonstrating immunoreactivity for arginine vasopressin (AVP, red) and β‐galactosidase (β‐gal, green), a marker for the VPAC2 receptor within the transgenic mouse suprachiasmatic nucleus. The boxed areas in A, C, F and I (rostral to caudal sequence) are shown in higher magnification in B, D, E, G, H and J. Arrows indicate AVP cells with diffuse β‐galactosidase immunoreactivity. An arrowhead in B points to punctate β‐galactosidase immunoreactivity within a double‐labelled cell. 3V, third ventricle.
Graph: 3 Confocal photomicrographs of double‐labelled sections showing immunoreactivity for β‐galactosidase (β‐gal; green), as a marker for the VPAC2 receptor, and (A and B) vasoactive intestinal peptide (VIP, red), (C) calretinin (CR, red) or (D) glial fibrillary acidic protein (GFAP, red) within the transgenic mouse suprachiasmatic nucleus (SCN). The boxed area in A is shown in higher magnification in B. Arrows indicate double‐labelled cells displaying diffuse β‐galactosidase immunoreactivity. The β‐galactosidase marker for VPAC2 receptor is not apparent in astrocytes of the core or shell regions of the SCN (to the left and right, respectively, of the vertical dotted line). 3V, third ventricle; oc, optic chiasm.
There were no statistically significant differences across the rostro‐caudal levels of the SCN for the incidence of β‐galactosidase expression within each of the neuronal populations analysed (Table 1). Examination of sections throughout the SCN failed to detect β‐galactosidase in GFAP‐immunoreactive astrocytes (Fig. 3D). At mid‐rostro‐caudal levels, an increased incidence of large immunoreactive astrocytes was observed in the central region of the SCN (Fig. 3D), an area characterized by a lower incidence of β‐galactosidase‐immunoreactive cells (also see Fig. 1D and E). The distribution of GFAP‐positive astrocytes was similar in the wild‐type and transgenic animals and corresponded to that previously reported ([10]).
β‐galactosidase expression at sites putatively innervated by efferent projections from the su...
When we examined sites previously reported ([1]) to be recipients of VIP‐containing projections from the mouse SCN, β‐galactosidase‐immunoreactive perikarya were observed in the SPVZ (Figs 1 and 4A), ventral and dorsal parvocellular subdivisions of the paraventricular hypothalamic nucleus (PVN; Fig. 4A–C), ventral subdivision of the lateral septal nucleus (Fig. 5A), anteroventral periventricular nucleus (AVPV) (Fig. 5B), medial preoptic nucleus (MPN) and suprachiasmatic preoptic nucleus (Fig. 5C). Sparse β‐galactosidase‐immunoreactive perikarya were also observed in the nucleus of the vertical limb of the diagonal band and in the dorsomedial hypothalamic nucleus.
Graph: 4 Photomicrographs demonstrating immunoreactivity for β‐galactosidase (β‐gal; green), as a marker for the VPAC2 receptor, together with arginine vasopressin (AVP, red) in the transgenic mouse brain. In the caudal suprachiasmatic nucleus (SCN) (A) a high degree of correspondence between the distribution of AVP‐ and β‐galactosidase‐immunoreactive profiles is evident (yellow). Cell bodies immunoreactive for β‐galactosidase (arrowheads) are apparent dorsal to the SCN within the subparaventricular zone (SPVZ) and sparsely within the paraventricular hypothalamic nucleus (PVN) at mid (A), rostral (B) and caudal (C) levels. β‐galactosidase‐immunoreactive cells are also present in the zona incerta (ZI, A). 3V, third ventricle; oc, optic chiasm.
Graph: 5 Photomicrographs demonstrating immunoreactivity for β‐galactosidase, as a marker for the VPAC2 receptor, in the transgenic mouse brain at sites reported to be targets for vasoactive intestinal peptide‐containing projections from the suprachiasmatic nucleus (SCN). β‐Galactosidase‐immunoreactive perikarya within the (A) ventral subdivision of the lateral septal nucleus (vLS), (B) anteroventral periventricular nucleus (AVPV) and (C) medial preoptic nucleus (MPN) and suprachiasmatic preoptic nucleus (PSCH). Abundant β‐galactosidase‐immunoreactive fibres are present in the periventricular nucleus (Pe). The inset (in C) shows the boxed area enlarged. LV, lateral ventricle; ac, anterior commissure; oc, optic chiasm.
The biotinylated tyramide amplification of the immunofluorescence enabled detection of β‐galactosidase immunoreactivity in fibres. Dense immunoreactive fibres were detected in the retrochiasmatic area (Fig. 1H), SPVZ, ventral and dorsal parvocellular subdivisions of the PVN, suprachiasmatic preoptic nucleus and periventricular nucleus (Figs 1, 4 and 5). More diffuse β‐galactosidase‐immunoreactive fibres were observed in the lateral septal nucleus, MPN, AVPV (Fig. 5), the vertical and horizontal limbs of the diagonal band, the dorsomedial hypothalamic nucleus and the anterior thalamic paraventricular and paratenial nuclei. As these sites are recipients of AVP‐ and VIP‐containing projections from the mouse SCN ([1]), the observed distribution may reflect diffusion of the VPAC2 marker along processes from the SCN.
Discussion
Previous studies have indicated that VIP and its receptor VPAC2 are components of the SCN that contribute to the production and entrainment of circadian rhythms ([3]; [67]; [57]; [8]; [23]; [14]; [15]) and the regulation of period length ([47]; [41]; [27]; [57]). The SCN VIP cells lacking afferent inputs, as in the case of dispersed SCN cells or SCN slice preparations, have been reported to release VIP in a circadian manner ([28]; [63]). Nevertheless, the dependence on the light/dark cycle for daily rhythmicity of VIP mRNA ([4]; [76]; [62]) and VIP peptide ([69]; [60], [61]) within the adult SCN in situ is well established. Despite such evidence about the regulation of VIP itself, the presence of circadian rhythmicity within SCN cells containing either VIP or presumptive VPAC2 is apparent from their circadian rhythms of PER1 and PER2 expression ([34]).
The present study examined the distribution of VPAC2 across the rostro‐caudal extent of the mouse SCN using β‐galactosidase as a marker; the results are consistent with and extend previous findings ([34]). Identical distributions for VPAC2 mRNA and β‐galactosidase and coexpression of the two signals are described here. These findings, together with evidence for VPAC2 binding within the mouse SCN ([23]), indicate that the β‐galactosidase marker maps the sites of VPAC2 expression within the SCN. In contrast to AVP‐, VIP‐ or calretinin‐immunoreactive neurons, GFAP‐positive astrocytes within the SCN do not show expression of the VPAC2 marker which suggests neuron‐specific expression of VPAC2 in the SCN. The presence of neurons with this marker in the shell and core subdivisions of the SCN is consistent with evidence for VIP‐induced electrophysiological responses in each of these subdivisions in SCN slices from both mice ([31]) and rats ([52]).
Possible significance of VPAC 2 in the suprachiasmatic nucleus shell
Nearly half of the AVP‐synthesizing neurons of the SCN shell expressed the marker for VPAC2. The evidence for VPAC2 expression by AVP neurons is consistent with data indicating regulation of SCN AVP by VIP. The VIP‐immunoreactive synapses on AVP neurons are relatively common in the rat SCN ([29]). Although comparable ultrastructural data are not currently available for the mouse, there is a dense network of VIP‐immunoreactive fibres in the SCN shell (Fig. 3A) ([64]; [1]). The release of AVP is elicited by VIP applied to the SCN slice ([30]) or to dispersed SCN cells ([72]) and this can be inhibited by VIP antibodies ([30]). As signalling pathways downstream of the VPAC2 receptor primarily involve the cAMP cascade ([68]), it may be significant that the cAMP response element is present in the AVP gene ([33]) and that activation of adenylate cyclase in the SCN slice leads to increased AVP gene expression ([6]).
A phase‐shifting action of VIP on circadian AVP release has been demonstrated in dissociated SCN cells ([73]). Phase‐shifts in electrophysiological or behavioural circadian rhythms can also be induced by VIP or a VPAC2 receptor agonist ([48]; [51]). It has been postulated that VIP mediates light‐evoked mPER2 induction in AVP‐expressing cells in the SCN shell ([34]). This is supported by evidence that VIP induces Per2 mRNA in the SCN in vitro ([46]) and that photic stimulation in vivo or N‐methyl‐d‐aspartate in vitro evokes VIP release in the SCN ([58]; [61]), thereby inducing phase‐shifts ([58]). The possibility that behavioural phase‐shifts occur only when light‐induced Per gene expression spreads from the retino‐recipient core to the shell has been proposed by [75]). The discovery of the VPAC2 marker in shell AVP neurons indicates that VIP signals from the predominantly retino‐recipient SCN to the shell are mediated by VPAC2. In this context it may be significant that mice overexpressing the VPAC2 receptor adjust their locomotor activity rhythms to an advance in the light/dark cycle faster than wild‐types ([57]).
It should be noted that the loss of circadian expression for AVP and for clock genes (Per1, Per2 and Cry1) in the SCN of mice lacking VPAC2 has led to the proposition ([23]) that VIP signalling between SCN neurons is required for sustained rhythm generation.
Possible significance of VPAC 2 in the suprachiasmatic nucleus core
Almost one‐third of the VIP and calretinin neurons in the SCN core were found to contain the VPAC2 marker. Although it remains to be established whether VIP and calretinin are coexpressed, our findings suggest that VPAC2 contributes to coupling between these cells or autoregulation; such effects may be enhanced by the cAMP response element sequence in the VIP gene ([19]). In the context of putative autoregulatory actions, it should be noted that VIP has been shown to augment GABAergic neurotransmission within the mouse SCN via presynaptic activation of VPAC2 receptors ([31]). VPAC2‐expressing VIP neurons may also participate in reciprocal interactions between the ventral region of each SCN; bilateral connections between these retino‐recipient regions have been identified in the mouse ([1]) and rat ([38]). As pituitary adenylate cyclase‐activating peptide (PACAP) is a major component in retinal projections to the SCN ([21], [22]; [26]) and the VPAC2 receptor has an affinity for PACAP equal to that for VIP ([42]), this receptor may contribute to signalling from the retina to the SCN, a possibility which remains to be explored. Nevertheless, the mRNA for PAC1, a receptor with a 1000‐fold higher affinity for PACAP than VPAC2 ([13]), is also present in the mouse SCN ([21]).
Possible significance of VPAC 2 at sites distal to the suprachiasmatic nucleus
Perikarya containing the VPAC2 marker were detected in the SPVZ, suprachiasmatic preoptic nucleus, MPN, ventral subdivision of the lateral septal nucleus, AVPV and parvocellular subdivisions of the PVN. Each of these sites receives VIP‐immunoreactive projections from the SCN in mice ([1]). Lesions of the SPVZ lead to varying degrees of disruption of locomotor activity, body temperature or sleep rhythms in rats ([40]; [45]). Mutant mice with reduced VIP‐immunoreactive fibres extending into the SPVZ show a prolonged activity rhythm period ([59]). The mouse SPVZ receives direct retinal input ([1]). Such projections may mediate the suppression of locomotor activity by light independent of the circadian oscillator ([35]). As PACAP binds VPAC2 ([42]) and is present in retinohypothalamic projections ([22]), our evidence for VPAC2 in the SPVZ suggests that retinohypothalamic PACAP may also act downstream of the SCN.
Numerous VPAC2‐expressing neurons are present in the MPN. Efferent projections to the MPN derive from the core and shell SCN in the mouse ([1]). The MPN has been implicated in circadian regulation of sleep and arousal as it forms a relay between the SCN and the ventrolateral preoptic nucleus and locus coeruleus ([7]; [16]). Efferents to the sleep‐active ventrolateral preoptic nucleus also derive from the ventral subdivision of the lateral septal nucleus ([12]), a site expressing the VPAC2 marker. A role for SCN VIP signalling via VPAC2 in reproductive neuroendocrine processes is suggested by the presence of the VPAC2 marker in the AVPV. The oestrogen‐responsive AVPV ([65]; [32]) is implicated in initiating the luteinizing hormone surge in rats ([74]; [37]). Furthermore, inhibition of VIP synthesis in the rat SCN reduces the amplitude and delays the onset of the oestrogen‐induced luteinizing hormone surge ([24]).
The marker for VPAC2 was also found in parvocellular subdivisions of the PVN, particularly in ventral and dorsal regions, a distribution that corresponds to that reported for VIP‐immunoreactive processes ([1]). This region contains neuroendocrine corticotrophin‐releasing hormone‐synthesizing neurons ([54]). A role for VIP signalling in regulating the circadian rhythm of corticosterone has been demonstrated in rats and antisense oligonucleotides for VIP mRNA in the vicinity of the SCN disrupt this rhythm ([55]). Furthermore, stimulation of corticosterone secretion by VIP in the PVN is blocked by a corticotrophin‐releasing hormone antagonist ([5]). The possibility that SCN VIP neurons projecting to the PVN form part of a multisynaptic pathway regulating various autonomic functions is supported by transneuronal retrograde tracing studies in the rat. Thus, pseudorabies virus injected into the adrenal ([9]) or pineal ([70]) gland is detected in SCN VIP cells following its appearance in parvocellular PVN cells.
Conclusions
The approach adopted in this study has resulted in the detection of the VPAC2 marker in nearly half of the AVP neurons in the SCN shell and in one‐third of the VIP and calretinin neurons in the SCN core. This suggests that the VPAC2 receptor participates in autoregulation and/or coupling of neurons within the predominantly retino‐recipient part of the nucleus and in the control of the SCN shell. In this context it should be noted that signalling pathways downstream of the VPAC2 receptor primarily involve the cAMP cascade ([42]; [68]) and that the cAMP response element sequence is present in Per1 and Per2 genes ([71]). Detection of the VPAC2 marker in perikarya in regions receiving VIP‐immunoreactive efferents from the SCN also implicates VPAC2 signalling in the circadian control of autonomic functions at multiple sites beyond the SCN.
Acknowledgements
The authors thank Dr H. Gainer for mouse antivasopressin‐neurophysin (PS41) and Dr J.H. Rogers for rabbit anticalretinin. This study was supported by funds provided by the Wellcome Trust and the BBSRC.
- Abbreviations
- AVP arginine‐vasopressin
- AVPV anteroventral periventricular nucleus
- GFAP glial fibrillary acidic protein
- MPN medial preoptic nucleus
- PACAP pituitary adenylate cyclase‐activating peptide
- PBS phosphate‐buffered saline
- PVN paraventricular nucleus of the hypothalamus
- SCN suprachiasmatic nucleus
- SPVZ subparaventricular zone
- VIP vasoactive intestinal peptide.
[
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By Imre Kalló; Theodosis Kalamatianos; Nzinga Wiltshire; Sanbing Shen; W. John Sheward; Anthony J. Harmar and Clive W. Coen
Reported by Author; Author; Author; Author; Author; Author; Author
