It has recently been proposed that extracellular signalregulated kinases 1 and 2 (ERK1/2) are one of the factors mediating seizure development. We hypothesized that inhibition of ERK1/2 activity could prevent audiogenic seizures by altering GABA and glutamate release mechanisms. Krushinsky-Molodkina rats, genetically prone to audiogenic seizure, were recruited in the experiments. Animals were i.p. injected with an inhibitor of ERK1/2 SL 327 at different doses 60 min before audio stimulation. We demonstrated for the first time that inhibition of ERK1/2 activity by SL 327 injections prevented seizure behavior and this effect was dose-dependent and correlated with ERK1/2 activity. The obtained data also demonstrated unchanged levels of GABA production, and an increase in the level of vesicular glutamate transporter 2. The study of exocytosis protein expression showed that SL 327 treatment leads to downregulation of vesicle-associated membrane protein 2 and synapsin I, and accumulation of synaptosomal-associated protein 25 (SNAP-25). The obtained data indicate that the inhibition of ERK1/2 blocks seizure behavior presumably by altering the exocytosis machinery, and identifies ERK1/2 as a potential target for the development of new strategies for seizure treatment. Temporal lobe epilepsy is one the most common chronic neurological diseases, and there is no available treatment capable of fully preventing the development of epilepsy or ensuring complete recovery.Recently, it has been proposed that extracellular signalregulated kinases 1 and 2 (ERK1/2), members of the MAPK family, are one of the factors mediating seizure development (Nateri et al. 2007). ERK1/2 are activated by dual-specificity MAP/ERK kinases (MEK1/2), which use ERK1/2 kinases as the only known substrates (Shaul and Seger 2007). Mice with over-expression of constitutively active brain-specific form of MEK1 demonstrate spontaneous seizures (Nateri et al. 2007). It has been reported that ERK1/2 activity is also upregulated following pilocarpine treatment (Berkeley et al. 2002) or by electric stimulation of seizures (Baraban et al. 1993;Kang et al. 1994;Bhat et al. 1998). However, little is known about the role of ERK1/2 in epileptiform seizure progression in animals genetically prone to seizures. Address correspondence and reprint requests to Dr Margarita Glazova, Group of Molecular mechanisms of neuronal functioning, Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 44 Thorez pr., 194223 Saint-Petersburg, Russia. E-mail: marglazova@gmail.com Abbreviations used: ERK1/2, extracellular signal-regulated kinases 1 and 2; GAD65 and GAD67, glutamic acid decarboxylases 65 and 67; KM, Krushinsky-Molodkina rats; MEK1/2, dual-specificity MAP/ERK kinases; SNAP-25, synaptosomal-associated protein 25; SNARE, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor; VAMP2, vesicle-associated membrane protein 2; VGLUT2, vesicular glutamate transporter 2.

Obtained results demonstrated a decrease activity in synapsin I, and accumulation of VGLUT2 in the striatum after blockade of audiogenic seizure (AGS) by SL 327 that could lead to inhibition of glutamate release. While in the striatum GAD65/67 level was diminished, in the substantia nigra GAD65/67 was increased showing enhanced inhibitory output to the compact part of the substantia nigra. Analysis of dopaminergic system showed a significant reduction of tyrosine hydroxylase activity and expression in the substantia nigra, and decreased D1 and D2 receptor expression in the striatum. In summary, we propose that changes in the nigrostriatal system could be mediated by inhibitory effect of SL 327 on AGS expression.

The present study analysed the effects of audiogenic kindling on the functional state of the vasopressinergic system of Krushinsky-Molodkina (KM) rats. KM rats represent a genetic model of audiogenic reflex epilepsy. Multiple audiogenic seizures in KM rats lead to the involvement of the limbic structures and neocortex in the epileptic network. The phenomenon of epileptic activity that overspreads from the brain stem to the forebrain is called audiogenic kindling and represents a model of limbic epilepsy. In the present study, audiogenic kindling was induced by 25 repetitive audiogenic seizures (AGS) with 1 AGS per day. A proportion of KM rats did not express AGS to sound stimuli, and these rats were characterised as the AGS-resistant group.The data demonstrated that audiogenic kindling did not change activity of extracellular signal-regulated kinase 1/2 or cAMP response element-binding protein, although it led to an increase in vasopressin (VP) expression in the supraoptic nucleus (SON) and in the magnocellular division of the paraventricular nucleus (PVN). Additionally, we observed a decrease in GABAergic innervation of the hypothalamic neuroendocrine neurones after audiogenic kindling, whereas glutamatergic innervation of the SON and PVN was not altered. By contrast, analysis of AGS-resistant KM rats did not reveal any changes in the activity of the VP-ergic system, confirming that the activation of VP expression was caused by repetitive AGS expression, rather than by repetitive acoustic stress. Thus, we suggest that overspread of epileptiform activity in the brain is the main factor that affects VP expression in the hypothalamic magnocellular neurones. K E Y W O R D S audiogenic kindling, GABA, Krushinsky-Molodkina rats, limbic epilepsy, vasopressin S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section. How to cite this article: Harbachova EL, Chernigovskaya EV, V.Glazova M, Nikitina LS. Audiogenic kindling activates expression of vasopressin in the hypothalamus of Krushinsky-Molodkina rats genetically prone to reflex epilepsy. J Neuroendocrinol. 2020;32:e12846. https://doi.

Dehydration activates the vasopressinergic system of the hypothalamus. We analyzed the effects of dehydration induced by water deprivation for 3 days on the activities of ERK1/2 and transcription factors, Elk1 and cAMP response element-binding protein (CREB) in vasopressinergic neurons, as well as the distribution and level of the motor protein, kinesin, in the hypothalamo-neurohypophyseal system. We showed that dehydration resulted in enhanced vasopressin (VP) expression and activation of CREB, and increased the activity of the MEK/ERK/Elk1 pathway in magnocellular neurons of the supraoptic nucleus. The activation of VP secretion was associated also with accumulation of phospho-ERK1/2 in the VP-ergic terminals of the posterior lobe of the pituitary. Analysis of the amount and distribution of kinesin and SNAP25, the proteins associated with transport and secretion, demonstrated that dehydration enhanced kinesin content in the perikarya of magnocellular neurons in the supraoptic nucleus and decreased kinesin and SNAP25 levels in the posterior pituitary. ERK1/2 and ERK1/2-dependent transcription factors, Elk1 and CREB, participate in the regulation of dehydration-evoked VP expression. We propose that ERK1/2 and kinesin participate in regulation of anterograde transport of VP dense core vesicles.