Neuroscience Letters, 153 (1993) 197-201 197 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00 NSL 09458 Somatostatin mRNA in the hippocampal formation following electroconvulsive shock in the rat Francesca Passarelli and Francesco Orzi Department of Neuroscienee, University of Roma 'La Sapienza', 1 Clinica Neurologiea, Rome ( ltalv ) (Received 3 February 1992; Revised version received 11 January 1993: Accepted 19 January 1993) Key words." Electroshock; Somatostatin mRNA; In situ hybridization; Hippocampus The effect of electroshock on the brain levels of somatostatin mRNA were evaluated by in situ hybridization using a selective oligonucleotide probe. Rats were submitted to single or repeated (7 days, one session for each day) sessions of electroshock. There was a marked increase of the expression of somatostatin mRNA in the hippocampal formation, mostly in the multiform layer of the hilus of the dentate gyrus, following repeated but not single electroshock. Our findings show that repeated ECS is associated with increase in the synthesis of somatostatin. The results also support previous data indicating that the hippocampal formation is selectively affected by the treatment. The growth-hormone-release inhibiting factor soma- tostatin (SS), a 14-amino-acid peptide [2], plays a role as a neurotransmitter [4, 5]. Alterations of SS tissue con- tents occur in different models of experimental epilepsy, in the hippocampal formation [14], and other selected brain areas [6]. We previously showed [10] that repeated electroshock (ECS), a procedure effective in the treat- ment of major depression [1], induces an increase of SS immunoreactivity in the rat hippocampal formation, and distinctively in the dentate gyrus. In this study we evalu- ated the brain tissue levels of SS mRNA, by means of in situ hybridization technique, in animals submitted to sin- gle or repeated ECS. Male adult Wistar rats weighing 250-300 g were used. The animals were divided into 3 groups one group (n=7) received ECS once daily for 7 days; a second group (n=6) received a single ECS; a third group (n=8) of unshocked rats served as control. ECS was applied through ear-clip electrodes from an Electroshock Seizure Apparatus (Ugo Basile, Italy), giving a current of 80 mA (100 Hz) per 0.8 s. All animals treated with ECS showed a full tonic clonic attack, followed by hindlimb extension and stuporous phase (30-40 s). One day after the single ECS session, or 1 day after the last of the 7 daily ECS sessions, the animals were killed. Brains were immediately frozen. Cryostat sections (10/lm) were thaw-mounted onto poly- Correspondence: F. Passarelli, Department of Neuroscience, University of Rome 'La Sapienza', 1 Clinica Neurologica, V. le dell'Universitfi 30, 1-00185 Rome, Italy. L-lysine-coated slides, fixed for 5 min in 4% paraformal- dehyde, and processed for in situ hybridization [9]. The oligonucleotide (45 mers) was constructed complemen- tary to the sequence encoding for amino acids 79-92 of the rat prosomatostatin gene [8] using an Applied Bio- system DNA synthesizer. The oligonucleotide was la- belled using terminal deoxynucleotidyl transferase (Pharmacia) with [35S]dATP (1,300 Ci/mmol New Eng- land Nuclear) to a specific activity of l09 dpm//ag. After incubation the sections were exposed to X-ray film (XAR 5, Kodak), dipped in liquid emulsion (Ilford K5 diluted l:l with 1 x SSC), air-dried, and stored desiccated in light-tight boxes. Following an exposure time of 3 weeks the sections were developed in Kodak D-19 for 4 min. at 15°C. The sections were then stained with thionin to per- mit identification of nuclei. Control and experimental brain slices were comounted in the same coverslip to minimize differences in handling of tissue sections, in- cluding freeze-thaw cycles and section thickness. A mor- phometric analysis of the autoradiographies was carried out by using a computerized image processing system (MCID Imaging Research, Brock University, St. Catharines, Ontario, Canada). For each brain section (2-3 sections per rat), computerized overlay of both his- tology and the corresponding autoradiographic image was performed to outline the hippocampal formation as a whole or the dentate gyrus (Fig. 1) on the autoradi- ograms. The extent of the outlined areas was then meas- ured (mm2). An optical density threshold was used to objectively discriminate the grains in the autoradiograms