INTRACEREBROVENTRICULAR DELIVERY OF ADENO-ASSOCIATED VIRUSES INTO NEONATAL MOUSE BRAIN FOR GENE THERAPY PRECLINICAL STUDIES FOR GNAO1 ENCEPHALOPATHY

In the present work we validated neonatal intracerebroventricular injections for AAV-mediated transgene delivery into GNAO1-rich regions of the murine brain. This administration route can be used for testing efficacy of AAV- based gene therapy for GNAO1 encephalopathy in the mouse models.

L-Lysine as the Molecule Influencing Selective Brain Activity in Pain-Induced Behavior of Rats

Lysine-rich proteins are some of the most important proteins of neurons and it has become necessary to investigate the possible role of L-lysine as a brain functioning regulator. The purpose of our study is to identify the characteristics and the mechanisms of L-lysine effects on the different types of pain-induced behavior in the stimulation of tail and foot-shock models in 210 adult male Wistar rats. L-lysine was administered in intraperitoneal or intracerebroventricular injections in doses of 0.15–50.0 µg/kg. When a tail is irritated, L-lysine was found to enhance pain sensitivity and affective defense after both intraperitoneal and intracerebroventricular administration. In the case of unavoidable painful irritation of a pair of rats with both types of L-lysine administration, there was no direct correlation of the severity of pain with defensive reactions and outbursts of aggression. This indicates a more complex integration of the activity of brain structures in this situation of animal interaction, which was confirmed by the results of the direct amino acid action on the periventricular brain structures. Our findings show that L-lysine influences the selective brain activity in dependence on the biological significance of pain-induced behavior.

NPY2 receptor activation in the dorsal vagal complex increases food intake and attenuates CCK-induced satiation in male rats

Neuropeptide Y (NPY), peptide YY (PYY), and their cognate receptors (YR) are expressed by subpopulations of central and peripheral nervous system neurons. Intracerebroventricular injections of NPY or PYY increase food intake, and intrahypothalamic NPY1 or NPY5 receptor agonist injections also increase food intake. In contrast, injection of PYY in the periphery reduces food intake, apparently by activating peripheral Y2R. The dorsal vagal complex (DVC) of the hindbrain is the site where vagal afferents relay gut satiation signals to the brain. While contributions of the DVC are increasingly investigated, a role for DVC YR in control of food intake has not been examined systematically. We used in situ hybridization to confirm expression of Y1R and Y2R, but not Y5R, in the DVC and vagal afferent neurons. We found that nanoinjections of a Y2R agonist, PYY-(3–36), into the DVC significantly increased food intake over a 4-h period in satiated male rats. PYY-(3–36)-evoked food intake was prevented by injection of a selective Y2R antagonist. Injection of a Y1R/Y5R-preferring agonist into the DVC failed to increase food intake at doses reported to increase food intake following hypothalamic injection. Finally, injection of PYY-(3–36) into the DVC prevented reduction of 30-min food intake following intraperitoneal injection of cholecystokinin (CCK). Our results indicate that activation of DVC Y2R, unlike hypothalamic or peripheral Y2R, increases food intake. Furthermore, in the context of available electrophysiological observations, our results are consistent with the hypothesis that DVC Y2R control food intake by dampening vagally mediated satiation signals in the DVC.

Lipopolysaccharide reduces gonadotrophin-releasing hormone (GnRH) gene expression: role of RFamide-related peptide-3 and kisspeptin

RFamide-related peptide (RFRP)-3 reduces luteinising hormone (LH) secretion in rodents. Stress has been shown to upregulate the expression of the RFRP gene (Rfrp) with a concomitant reduction in LH secretion, but an effect on expression of the gonadotrophin-releasing hormone (GnRH) gene (Gnrh1) has not been shown. We hypothesised that lipopolysaccharide (LPS)-induced stress affects expression of Rfrp, the gene for kisspeptin (Kiss1) and/or Gnrh1, leading to suppression of LH levels in rats. Intracerebroventricular injections of RFRP-3 (0.1, 1, 5 nmol) or i.v. LPS (15μgkg−1) reduced LH levels. Doses of 1 and 5 nmol RFRP-3 were then administered to analyse gene expression by in situ hybridisation. RFRP-3 (5 nmol) had no effect on Gnrh1 or Kiss1 expression. LPS stress reduced GnRH and Kiss1 expression, without affecting Rfrp1 expression. These data indicate that LPS stress directly or indirectly reduces Gnrh1 expression, but this is unlikely to be due to a change in Rfrp1 expression.

Effect of salsolinol on ACTH and cortisol response to handling stress in early anestrous sheep

Inhibition of hypothalamic–pituitary–adrenal (HPA) axis activity by salsolinol was demonstrated in lactating sheep. We assessed whether salsolinol regulates, besides lactation, also adrenocorticotropic hormone (ACTH) and cortisol release, and if its action is prolactin-dependent. We examined two groups of early anestrous sheep, which received for three days salsolinol or vehicle-only intracerebroventricular injections, and a group of lactating sheep injected with the vehicle only. On day 3, blood samples were collected for over six hours and the anterior pituitary was dissected. Plasma ACTH, cortisol, and prolactin concentrations, and proopiomelanocortin (POMC) and prolactin (PRL) mRNA expression within the anterior pituitary were assayed. In all groups, ACTH and cortisol concentrations were higher (P < 0.05 and P < 0.001) during the first half of sampling than in the second half; there were no differences in prolactin concentration. Lactating sheep had lower (P < 0.05 and P < 0.001) plasma ACTH and cortisol concentrations and higher (P < 0.001) plasma prolactin concentration than both groups of anestrous sheep during the first half of sampling. In the second half, there were no differences in ACTH and cortisol concentrations between all groups, but prolactin concentration was still higher (P < 0.001) in lactating animals. Salsolinol treatment decreased ACTH and cortisol concentrations during the first half of sampling (P < 0.05 and P < 0.001) compared to the anestrous controls, but had no effect on prolactin concentration. POMC mRNA expression was lower (P < 0.05) and PRL mRNA expression was higher (P < 0.05) in lactating sheep than in anestrous sheep. Salsolinol did not affect POMC and PRL mRNA expression. In conclusion, increased ACTH and cortisol concentrations during the first half of sampling occurred in response to handling stress. Salsolinol inhibited the HPA axis response to stress in early anestrous sheep, and it was unrelated to prolactin secretion.