A comparative study of mmunoreactivity with respect to c-Fos protein in the motor (zones М1 and М2), medial prefrontal (PrL and IL), and cingular (Cg1 and Cg2) cortices allowed us to find significant differences between the intensities of expression of gene c-fos in these cortical regions in control rats (group 1) and animals trained to perform catching of food globules by the forelimb (i.e., realizing an operant food-procuring reflex, group 2). The density of distribution of Fos-immunoreactive (Fos-ir) neurons in rats of group 2 in motor and limbic cortical zones at +2.2 to +0.2 levels rostrally from the bregma were significantly lower than in control rats (Р < 0.05). In animals of group 2, we also found significantly greater numbers of Fos-ir neurons in the contralateral (with respect to the active extremity) zones of the cortex at all examined levels. These changes are probably related to functional changes in the cortex resulting from learning of motor habits in the course of training sessions for stabilization of the operant reflex. Histochemical estimation of the NADPH-diaphorase (NADPH-d) activity in the motor and limbic cortex showed that, in rats of both groups, the maximum number of labeled interneurons per slice in the М1 zone were observed in layers II/III, V, and VI (5.6 ± 0.4, 6.4 ± 0.5, and 14.0 ± 0.8, respectively, within 200 × 200 μm 2 areas). In the limbic cortex, NADPH-d-reactive (NADPH-d-r) interneurons were also met in layers II/III, V, and VI. Cortical NADPHd-r neurons with the Fos-ir nuclei were not found. The presence of spatial associations of the somata or processes of NADPH-d-r neurons with intraparenchimal arterioles and microvessels was a typical feature of the distribution of NADPH-d-reactivity in the М1 and М2 zones, as well as in Cg1, Cg2, PrL, and IL. The following succession of the density of neurovascular associations was observed: Cg1 > Cg2 > М1 > М2 > > PrL. As is supposed, NADPH-d-r neurons (i.e., cells generating NO) are involved in the control of regional blood flow in the studied cortical regions.
We estimated in rats the expression of early gene c-fos (marker of neuronal activation) and NADPH-diaphorase activity (NO-synthase marker) in the limbic structures of the basal forebrain and in the hypothalamus. Estimations were performed in the norm, in the state of starvation, and after realization of long-lasting (repeated 4 to 12 times per minute for 30 min) motivated stereotyped food-procuring forelimb movements. In food-deprived animals, a significantly greater (Р < 0.05), as compared with the control, number of Fosimmunoreactive (Fos-ir) and NADPH-diaphorase-reactive (NADPH-dr) neurons was observed in limbic structures, namely in the medial septum (MS), nuclei of the vertical and horizontal branches of the diagonal fascia (VDB and НDB), magnocellular preoptic nucleus (MCPO), complex of the substantia innominata−basal nucleus of Meynert of the pallidum, SI-GP(B), as well as in the laterodorsal tegmental nucleus (LDTg), medial part of the pallidum (MGP), paraventricular and lateral nuclei of the hypothalamus (Pa and LH), and islands of Calleja (ICj and ICjM). In the limbic structures and pontine nuclei of rats of the experimental group (that performed operant movements), greater mean densities of labeled neurons were found in the succession LDTg < SI < MCPO < GP(B) < MS < VDB < HDB. The maximum mean density of Fos-ir neurons (13.8 ± 0.9 labeled nuclei within 200 × 200 µm 2 area) was found in the HDB. In the hypothalamic nuclei of starving rats, c-fos expression was two times higher than that in the control. After realization of operant movements, the intensity of expression in the LH was somewhat smaller, while in the Ра it was higher. The maximum density of NADPH-dr neurons was observed in the Pa (303.4 ± 18.7 cells), in the ICj and ICjM (287 ± 11.6 and 260 ± ± 8.7 neurons, respectively), and in the MGP (93 ± 6.7 labeled cells). When analyzing the distribution of labeled neurons in experimental rats, we found high densities of double-labeled cells (Fos + NADPH-d positivity) in the Pa, MGP, ICj, and ICjM. Such specificity of changes in the c-fos expression and NADPH-d reactivity in the hypothalamus correlates, perhaps, with the formation of motivation signals related to a delay in food accessibility and supply of food. Modifications of neuronal activity in limbic structures reflect involvement of the latter in the formation of motor programs for food-procuring movements and their realization.
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