Genetic Deletion of Somatostatin Receptor 1 Alters Somatostatinergic
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Neuropharmacology 45 (2003) 1080–1092 www.elsevier.com/locate/neuropharm Genetic deletion of somatostatin receptor 1 alters somatostatinergic transmission in the mouse retina Massimo Dal Monte a, Cristina Petrucci a, Anna Vasilaki b, Davide Cervia a, Dominique Grouselle c, Jacques Epelbaum c, Hans-Jurgen Kreienkamp d, Dietmar Richter d, Daniel Hoyer b, Paola Bagnoli a,∗ a Dipartimento di Fisiologia e Biochimica, Universita` di Pisa, via San Zeno 31, 56127 Pisa, Italy b Nervous System Research, Novartis Pharma AG, WSJ-386/745, 4002 Basel, Switzerland c INSERUM, U-549, IFR Broca-Sainte Anne, Centre Paul Broca, 2ter rue d’Alesia, 75014 Paris, France d Institut fu¨r Zellbiochemie und Klinische Neurobiologie, Universita¨ts Kronkenhaus Eppendorf, 20246, Hamburg, Germany Received 30 April 2003; received in revised form 8 July 2003; accepted 16 July 2003 Abstract In the mammalian retina, sparse amacrine cells contain somatostatin-14 (SRIF) which acts at multiple levels of neuronal circuitry through distinct SRIF receptors (sst1–5). Among them, the sst1 receptor has been localised to SRIF-containing amacrine cells in the rat and rabbit retina. Little is known about sst1 receptor localisation and function in the mouse retina. We have addressed this question in the retina of mice with deletion of sst1 receptors (sst1 KO mice). In the retina of wild type (WT) mice, sst1 receptors are localised to SRIF-containing amacrine cells, whereas in the retina of sst1 KO mice, sst1 receptors are absent. sst1 receptor loss causes a significant increase in retinal levels of SRIF, whereas it does not affect SRIF messenger RNA indicating that sst1 receptors play a role in limiting retinal SRIF at the post-transcriptional level. As another consequence of sst1 receptor loss, levels of expression of sst2 receptors are significantly higher than in control retinas. Together, these findings provide the first demonstration of prominent compensatory regulation in the mouse retina as a conse- quence of a distinct SRIF receptor deletion. The fact that in the absence of the sst1 receptor, retinal SRIF increases in concomitance with an increase in sst2 receptors suggests that SRIF may regulate sst2 receptor expression and that this regulatory process is controlled upstream by the sst1 receptor. This finding can be important in the design of drugs affecting SRIF function, not only in the retina, but also elsewhere in the brain. 2003 Elsevier Ltd. All rights reserved. Keywords: Somatostatin receptor 1 null mutation; Retinal cells; Somatostatin levels; Somatostatin receptor 2 expression; Compensatory events 1. Introduction are present in the innermost inner nuclear layer (INL) and in the ganglion cell layer (GCL) of the mammalian Somatostatin-14 (somatotropin release-inhibiting fac- retina (Bagnoli et al., 2003, for review). Those in the tor, SRIF) is a regulatory peptide produced throughout INL have been identified as amacrine cells, whereas the central nervous system and in most major peripheral those in the GCL are either displaced amacrine or gang- organs. It exerts its functions through the activation of lion cells. In the mouse retina, SRIF immunoreactivity distinct G-protein-coupled SRIF subtype receptors desig- (IR) has been recently localised to sparse-occurring nated sst1–5 (Barnett, 2003, for review). amacrine cells present both in the proximal INL of the SRIF and its receptors are found in the retina. In parti- entire retina and in the GCL of the ventral retina cular, sparse-occurring SRIF-immunoreactive neurons (Cristiani et al., 2002). In spite of the low density of SRIF-containing somata, dense SRIF-positive processes are widely distributed to ∗ Corresponding author. Tel.: +39-050-2213514; fax: +39-050- distinct laminae of the inner plexiform layer (IPL) of the 2313527. entire retina, thus suggesting that SRIF may act at mul- E-mail address: [email protected] (P. Bagnoli). tiple levels of retinal circuitry (Bagnoli et al., 2003, for 0028-3908/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0028-3908(03)00296-X M.D. Monte et al. / Neuropharmacology 45 (2003) 1080–1092 1081 review). Indeed, mRNAs for sst1–5 receptors have 2. Methods been reported in the mouse, rat, rabbit and human ret- ina (Cristiani et al., 2000, 2002; Klisovic et al., 2001; 2.1. Animals Mori et al., 1997) and SRIF receptor proteins have been visualised in retinal cells by immunocytochemis- Experiments were performed on retinas of 93 mice of try (Bagnoli et al., 2003, for review). Most of the stud- either WT or sst1 KO strains of both sexes at 6–8 weeks ies concur in reporting an extensive retinal distri- after birth (20–30 g body weight). sst1 KO mice were bution for sst2A receptors and particularly for its splice generated by recombination of a neomycin cassette with the entire sst coding region on a 129Sv/C57BL6 hybrid variant sst2A, and a somewhat more restricted distri- 1 background (Kreienkamp et al., 1999). WT mice were bution for sst1 and sst4 receptors (Cristianietal.,2000, 2002; Fontanesi et al., 2000; Helboe and Moller, 1999, derived by interbreeding heterozygous 129Sv/C57BL6 2000; Johnson et al., 2000; Vasilaki et al., 2001). In mice. Animals were kept in a regulated environment (23 ± 1 °C, 50 ± 5% humidity) with a 12 h light/dark particular, sst2 receptor expression by rod bipolar cells has been consistently found in all species examined, cycle (lights on at 8 a.m.) with food and water ad lib. although its localisation to distinct amacrine cells may No major effects of sst1 deletion were found on gross vary between species (Bagnoli et al., 2003,for visual ability as evaluated by feeding and exploratory behaviour. Eye blink and pupillary reflex were also nor- review). In the human retina, the sst2 receptor is expressed in all retinal layers with the most intensive mal. In all experiments, mice were anaesthetised by i.p. injection of Avertin (1.2% tribromoethanol and 2.4% immunoreactivity in the INL and the IPL (Klisovic et amylene hydrate in distilled water, 0.02 ml/g body al., 2001; Van Hagen et al., 2000). weight; Sigma, St. Louis, MO). Experiments were per- There is some evidence suggesting that SRIF modu- formed in compliance with the Italian law on animal care lates the physiology of retinal cells through the acti- No. 116/1992 and in accordance with the European vation of its distinct receptors (Akopian et al., 2000; Community Council Directive (EEC/609/86). All efforts Johnson et al., 2001; Petrucci et al., 2001). Recent results were made to reduce both animal suffering and the num- in the rabbit retina suggest that SRIF mediates its actions ber of animals used. by interacting mainly with sst2 receptors that couple to All experiments were performed at the same time of α Go (Vasilaki et al., 2003). However, little is known the day (between 11.00 and 13.00 h) in order to exclude about SRIF receptor function in retinal physiology. For possible circadian influences. The data were collected instance, a role of sst2 receptors in the modulation of from male and female control WT and KO animals, and 2+ + Ca and K currents has been demonstrated (Akopian the results combined since there was no apparent gen- et al., 2000; Petrucci et al., 2001) and the sst2 receptor der difference. appears to mediate SRIF inhibition of glutamate trans- mitter release in the mouse retina (Dal Monte et al., 2.2. Optic nerve transection 2003). In addition, recent evidence demonstrates that sst2 receptors mediate SRIF regulation of nitric oxide (NO) The optic nerve was transected intracranially after a production in the retina (Vasilaki et al., 2002). procedure described by Cristiani et al. (2002). Briefly, At present, no information is available on sst1 receptor in three anaesthetised WT mice, the cerebral cortex over- function in retinal physiology. Based on its localisation lying the optic nerve was removed and the optic nerve to somatostatinergic cells, as shown in the rat and rabbit was transected approximately 2 mm behind the sclera. retina (Helboe and Moller, 1999; Cristiani et al., 2000) After 120-day survival, retinas were removed immedi- and in the rat hypothalamus (Helboe et al., 1998), the ately after euthanasia, and the completeness of the optic sst1 receptor may play a role as an autoreceptor. nerve transection was assessed. Retinas were then pro- In the present study, we have investigated sst1 receptor cessed for immunocytochemistry. role(s) in retinal physiology by using gene-knockout technology. We have first determined the cellular local- 2.3. Semiquantitative RT-PCR isation of sst1 receptors in the retina of wild type mice (WT mice) and assessed the identity of retinal neurons The methods for RT-PCR analysis were similar to expressing these receptors with double-label immunocy- those used in a previous study of the mouse retina tochemistry. Then, in the retina of WT mice and in that (Cristiani et al., 2002). Fifteen WT or sst1 KO mice were deeply anaesthetised and killed by decapitation. For each of mice with no functional copy of the sst1 gene (sst1 KO mice), we determined the relative levels of SRIF and sample, total RNAs were extracted from six retinas that its distinct receptors in order to establish possible role(s) were dissected out for each strain in guanidine hydroch- loride using Simple Nucleic Acid Preparation kit of sst1 receptors in the control of SRIF and its recep- tor expression. (Invitrogen, Paisley, PA). Total RNAs were also extracted from fronto-parietal cortical samples used for 1082 M.D. Monte et al. / Neuropharmacology 45 (2003) 1080–1092 comparison. Six samples were dissected out for each plateau. Curves of cyclophilin B and mRNA amplifi- strain. Cyclophilin B mRNA, a presumably stable cation were parallel. All the conditions were chosen in mRNA, was used as an internal standard.