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ISSN 0022-0930, Journal of Evolutionary Biochemistry and Physiology, 2011, Vol. 47, No. 6, pp. 603—606. © Pleiades Publishing, Ltd., 2011. Original Russian Text © N. G. Lopatina, T. G. Zachepilo, E. G. Chesnokova, 2011, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2011, Vol. 47, No. 6, pp. 513—515.

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Again about Central Metabotropic Glutamate Receptors of the Group I in the Honeybee Apis mellifera N. G. Lopatina, T. G. Zachepilo, and E. G. Chesnokova† Pavlov Institute of Physiology, Russian Academy of Sciences, St. Petersburg, Russia E-mail: [email protected] Received November 24, 2010

DOI: 10.1134/S0022093011060123

The study of metabotropic L-glutamate receptors (mGluR) was begun in the mid-1980s [1]. The first publications about the presence of mGluR in the invertebrate head ganglion and their role in learning appeared at the mid-1990s [2]. Our studies allowed us to conclude [3] about the presence in the honeybee head ganglion of I, II и III groups of mGluR responsible for the long-term preservation of the individually acquired experience, with the receptors similar by the pharmacological profile to those in mammals. Our data differed from those obtained in studies of the foreign authors showing the presence in the honeybee head ganglion only of mGluR of the II group [4, 5]. It was necessary to obtain additional proofs in favor of the function/absence in the honeybee head ganglion of the heterogeneous mGluR group (I, II, III). Considering the presence of homology between the sequences of the I and II groups of mGluR, we needed the method that would allow identification particularly of the mGluR taking part in the honeybee associative learning. As such method, we used the knock-down method of ImGluR1 with aid of antisense oligonucleotide (asON). The goal of this work was the study of the behavioral consequences of intracerebral asON injections. The ImGluR1 state in the honeybee

head ganglion after the injections was evaluated by using an immunohistochemical method. The object of the study served honeybees of the krain race Apis mellifera carnica Polm at the age of 10–15 days. According to the posed tasks of the work, we used the behavioral and immunocytochemical methods of the study. Behavioral test. The elaboration of conditioned reflexes (CR) started from that 3 h before the beginning of experiment the honeybees were deprived of food to achieve the standard level of the conditioned-unconditioned reflex backgrounds. Prior to the learning performance the sensory and alimentary excitabilities were evaluated. The conditioned reflex of the proboscis pulling was elaborated in the honeybees immobilized by wings at the single combination of the conditioned stimulus (carnation smell) with the unconditioned alimentary reinforcement (the 50% sucrose solution). Immunohistochemical method. The insects were submitted to the cold narcosis and the brain was removed and fixed in paraformaldehyde for 4 h. The tissue was dehydrated in ethanols (40, 70, 96, and 100%) and in a methylbenzoate:paraffin, then immersed into a mixture methylbenzoate–paraf†

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genes of metabotropic glutamate receptors of the II group—IImGluR2, IImGluR3 (the genes of the I, II, and III groups mGluR have the common origin and high homology in conservative fields). Then the chosen sequences were compared with the honeybee genome. The following sequences (the database www.ncbi.nih.gov, program Vector NTI 9.1) were chosen: asON: 5'-TGGACGTAGTATATACCGACGG-3', sON: 5'-ACCTGCATCATATGGCTGGCGTTCGT-3'. Fig. 1. The amount of honeybees (%) retaining in the long-term memory the conditioned reflex after intracerebral injections: (1)—saline, (2)—sON, (3)— asON. *—The differences with control are statistically significant ( р ≤ 0.05).

fin (50:50). The preparations were embedded into paraffin for 3 h. The paraffin blocks of samples were prepared, the 7-μm thick sections were cut from them, cleared of paraffin (with xylene and ethanols (100, 96, 70, and 40%), heated for 5 min in the citrate buffer in a microwave oven at power of 450 W, and treated for 30 min in 0.3% Н2О2. The sections were incubated with normal serum (Vectastain ABC-elite Kit, Vector) for 2 h at 25°С. Then they were incubated with primary antibodies to rabbit ImGluR (solution 1:100; Chemicon, USA) overnight at 4°С, then with the secondary biotined antibodies (Vectastain ABC-elite Kit, Vector, Great Britain) and with avidin–biotin complex (Vectastain ABC-elite Kit, Vector, Great Britain) for 1 h at 37°С. The staining was performed by using diaminobenzidine. The preparations were dehydrated in ethanols and xylene, embedded in D-Pex (Sigma, Germany), and analyzed by light microscopy and an installation containing a digital CCD-camera and computer with the software Videotest-FISH (Russia). Then the densitometry was performed by the inserted method of Videotest-FISH. The obtained data were compared by using the non-parametric Mann– Whitney criterion. In total, 25 individuals were used in experiments with honeybees (3 groups: asON—12 individuals, sON—7, saline—6). Antisense knock-down. At selection of the nucleotide sequences used in the antisense knock-down procedure, we chose fragments of the rat gene ImGluR1 differing from the nucleotide sequences for

The chosen nucleotide sequences of the honeybee genome have a high degree of homology with the ImGluR1 gene of rat (~95%), and are not similar with the nucleotide sequences for genes of metabotropic glutamate receptors of the II group of the rat mGluR. The protocol of the experiment was as follows: the cooled honeybees of three groups were administrated intracerebrally into the field of medial calices of mushroom bodies by means of a microinjector with the solution of oligonucleotides complementary to mRNA of rat mGluR1—asON (200 nl, 43 pmol/μl), which produced the temporary suppression of mRNA translation of mGluR1 (experiment); the solution of sense nucleotides was administrated as a control—sON (control 1) and saline (control 2). The honeybees were kept for 6–7 h after the injections in a thermostat with constant temperature of 26°С in net test tubes with food that was removed 3 h prior to learning. The learning procedure was performed once. The interval (6–7 h) was chosen with taking into account the «mean lifespan» of the receptor protein molecule. The retention of the conditioned reflex was tested 3 h after learning (the long-term memory). In total, 94 honeybees participated in the experiment (asON—39 honeybees, sON—42, saline—13). The data were processed statistically by using Wilcoxon paired criterion. Then the honeybees were used for the immunohistochemical analysis. The results of 6 repeated experiments showed the significant ( р ≤ 0.05) suppression of long-term memory in honeybees injected with asON (Fig. 1). The level of retention of conditioned reflex was similar in honeybees after injections of sON and saline (66% and 67%, respectively) and exceeded 6 times that after asON injections (11%). None of

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Fig. 2. Comparison of expression of the I group metabotropic receptors in the honeybee brain in control. (a) Injections of sense oligonucleotide, (b) injections of saline, (c) after antisense-knock-down. After injections of the specially chosen antisense oligonucleotides the expression of ImGluR in calices (shown in the frame) of mushroom bodies is decreased.

these injections affected the sensory and alimentary excitability [6]. Results of the immunohistochemical staining of the honeybee brain sections are presented in Fig. 2 ((a)—sON), (b)—saline, (c)—asON. In control (Figs. 2a, 2b) the entire brain is stained, the high-

est receptor concentration being observed in calices of mushroom bodies (0.35–0.36 optical units). In the case of asON administration the picture was different (Fig. 2c): the brain was stained weakly and uniformly (0.14 optical units, р ≤ 0.05). Earlier, we [6], with aid of the complex ap-


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proach with use of immunocytochemical, electrophoretic, and behavioral methods, studied localization of metabotropic glutamate receptors (ImGluR1) in the honeybee Apis mellifera brain ganglion and the mechanisms responsible for participation of activated mGluR in processes of the CNS plasticity. However, the presence of homology between sequences of the I and II mGluR groups required use of the method allowing identification of the presence of the particular ImGluR1. As such method, in this work the method of the ImGluR1 gene antisense-knockdown was used. The obtained data clearly indicate that the used sequence of asON inhibits synthesis particularly of the ImGluR1 protein in the honeybee brain at the time interval of 6–10 h; this produces an inhibitory effect on development of the long-term memory traces. The obtained facts confirm the correctness of our conclusion about the presence in the honeybee head ganglion of the ImGluR1 similar with mammalian metabotropic receptors of the I group participating in the long-term preservation of the individually acquired experience. REFERENCES 1. Sladeczek, F., Pin, J., Recasens, M., Bockaert, J.,

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and Weiss, S., Glutamate Stimulates Inositol Phosphate Formation in Striatal Neurons, Nature, 1985, vol. 317, pp. 717–719. Parmentier, M., Pin, J., Bockaert, J., and Grau, Y., Cloning and Functional Expression of a Drosophila Metabotropic Glutamate Receptor Expressed in the Embryonic CNS, J. Neurosci., 1996, vol. 16, no. 21, pp. 6687–6694. Lopatina, N.G., Ryzhova, I.V., Zachepilo, T.G., Smirnov, V.B., and Chesnokova, E.G., L-Glutamate in Forming of Long-Term Memory in the Honeybee Apis mellifera, Zh. Evol. Biokhim. Fiziol., 2004, vol. 40, no. 6, pp. 539–545. Funada, M., Yasuo, S., Yoshimura, T., Ebihara, S., Sasagawa, H., Kitagawa, Y., and Kadowaki, T., Characterisation of the Two Distinct Subtypes of Metabotropic Glutamate Receptors from Honeybee, Apis mellifera, Neurosci. Lett., 2004, vol. 359, pp. 190–194. Kucharski, R., Mitri, C., Grau, Y., and Maleszka, R., Characterisation of a Metabotropic Glutamate Receptor in the Honeybee (Apis mellifera): Implication for Memory Formation, Invert. Neurosci., 2007, vol.7, pp. 99–108. Ryzhova, I.V., Zachepilo, T.G., Chesnokova, E.G., and Lopatina, N.G., Metabotropic Gutamate Receptors in Mechanisms of Plasticity of the Central Nervous System of the Honeybee Apis mellifera, Zh. Evol. Biokhim. Fiziol., 2010, vol. 46, pp. 211–217.
