MODULATION OF THE MONOSYNAPTIC REFLEX POTENTIALSIN THE DECEREBRATED RATS UNDER THE INFLUENCE OF HYDROXYTRYPTOPHAN.

We studied the serotonin effect on monosynaptic reflex potentials (MSR) of spinal motorneurons in the decerebrated rats in control and after intraperitoneal administration of serotonin precursor ­ 5-Hydroxytryptophan (5-HTP). MSR of motorneurons in the lumbar spinal cord were registered using electrical stimulation of dorsal root of the 5th lumbar section. During stimulation physiological saline or 5-hydroxytryptophan was injected intraperitoneally. In comparison with average amplitude of the control MSR there were registered significant increase in amplitudes of the MSR (169% and +172%, P <0,001) in animals with injection 5-HTP. These data suggest that serotonin release after 5-HTP administration leads to activation of motorneurons in the lumbar spinal cord. The mechanism of this activation may be related to the weakening of the inhibitory control of interneurons in the transmission pathways of the excitatory influences from muscle afferent to motorneurons and to the postural (antigravity) reflex reactions which necessary for the initiation of locomotion.

[NADPH-DIAPHORASE REACTIVITY IN THE VENTRAL HORN OF THE FELINE SPINAL CORD DURING ACUTE MUSCLE INFLAMMATION].

The aim of this research was to reveal the changes in the NADPH-d reactivity in the lumbal spinal cord (L6/L7) of cats with unilateral acute myositis of the mm. gastrocnemius-soleus after intramuscular injections of carrageenan. The effect of unilateral muscle inflammation was expressed in a significant increase in the number of NADPH-d-reactive neurons in ipsilateral and contralateral intermediate (lamina VII; 17.62 ± 2.7 and 20.67 ± 13.3) and medial (lamina VIII; 7.3 ± 1.9 and 6.0 ± 2.1 respectively) zones of the ventral horns. However, a clear decline of the reactive cells was recorded on the ipsilateral side within the area around the central canal (lamina X). An increase in the NADPH-d reactivity within the ventral horns on both sides on the spinal cord and the induction of such reactivity (contralaterally) in large multipolar neurons localized in the dorsal part of the intermediate zone were revealed in cats with unilateral acute muscle inflammation. It is hypothesized, that during acute myositis, plastic changes in different layers of the dorsal and ventral horns activate the processes of disinhibition due to an increase in the number of NOS-containing/NADPH-d-reactive neurons in the spinal gray matter.

[Food-procuring stereotype movements is accompanied by changes of c-Fos gene expression in the amygdala and modulation of heart rate in rats].

The distribution of Fos-immunoreactive (Fos-ir) and NADPH Diaphorase reactive (NADPH-dr-) neurons in the different subnuclei of amygdala and insular cortex (on the level -2,12 to -3,14 mm from bregma), and the associated changes of heart rate (HR) in intact, food-deprivated and executed food-procuring movements of rats were studied. In comparison with other groups of animals, the mean number of the Fos-ir neurons in the central nucleus of amygdala (Ce) and the insular cortex (GI/DI) at all studied levels was significantly greater in the executed food-procuring movements in rats. The main focus of localization of the Fos-ir neurons was found in lateral part of the Ce (58.5 +/- 1.9 units in 40-microm-thick section) at the level -2.56 mm. The mean number of Fos-ir neurons was significantly greater also in the lateral and capsular parts of the Ce. The mean number of Fos-ir neurons in the GI/DI was 165.5 +/- 3.2 cells in section. The number and density of NADPH-d reactive neurons was not significantly different in the brain structures of all animal groups studied. The double stained neurons (Fos-ir + NADPH-dr) were registered in medial, basolateral, anterior cortical amygdaloid nuclei and substantia innominata (SI) in rats after realization food-procuring movements. It was found that realization of food-procuring movements by the forelimb during repeated sessions was accompanied with the gradual decline of mean values of the HR (from 5% to 12% of control level) with subsequent renewal of them to the initial values (tonic component). The analysis of dynamics of the HR changes during realization of separate purposeful motion has shown the transient period of the HR suppression (500 ms), which coincided with the terminal phase of grasping of food pellet (phasic component). We suggest that the revealed focuses of localization of Fos-ir neurons in the lateral and medial subregions of amigdaloid Ce and also GI/DI, and SI testified that these structures of brain are involved in generation of the goal-directed motions. Direct projections of these subnuclei (and hypothalamus) to the cardiovascular centers of the medulla determine the associated regulation of the cardiovascular system function in the period of realization of the goal-directed motions in animals.

[Laminar distribution of the active spinal cord neurons during the feeding-related stereotyped movements in the rat].

The comparative study of expression of early c-fos-gene (marker of neuronal activation) and NADPH-diaphorase reactivity (NADPH-dr) was performed in the cervical spinal cord of rats in the control (intact) animal, 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 comparison with control rats; in the starving rats or rats showed forelimb movement to reach-to-grasp the food, the number of Fos-immunoreactive (Fos-ir) cells in the dorsal and ventral horns of a 40-microm-thick slice was significantly greater (P < 0.05). The number of Fosir neurons in the starving state clearly exceeded that in the most layers after realization of movements. Increase of Fos immunoreactivity in the superficial (2i, 3) and deeper (4, 5) layers of the dorsal horn was initiated, evidently, by signals from peripheral and supraspinal structures. We also found labelled cells within layers 6-8, and 9 demonstrating the activity of interneurons and motoneurons directly involved into generation of operant forelimb movements. According to our data, high density of NADPH-dr/NO-generating neurons in the C6/C7 segments are observed in the substance gelatinosa (layer 2i) and layers 7 and 10. NADPH-dr cells and Fos-ir neurons were intermixed within the layers but did not demonstrate double-labelling. It is possible to suggest that NADPH-dr/NO-generating cells of the spinal cord did not operate under realization of the studied operant reflexes, which did not include nociceptive component.

[Intensification of c-fos expression in the spinal cord initiated by the signals from fatigue skeletal muscles].

Immunohistochemical studies of c-fos expression in the lumbar and neck segments of spinal cord after fatiguing stimulation of the mm. gastrocnemius-soleus or m. trapezius and m. splenius, were carried out on the anaesthetized (chloral-hydrate 400 mg/kg, i.p.) rats. The patterns of the Fos-immunoreactive neurons distribution in the grey matter of spinal cord in the L4 and L5 segments, as well as the C2 and C3 were similar. The highest number of marked cells was registered in the dorsal horn in the first, fourth and fifth layers of grey matter, i.e. within the areas of termination of high-threshold muscle afferents of group III and IV. It is assumed, that the types of afferents could be activated by muscle fatigue-induced metabolites. The signals incoming to the spinal cord could be involved into the presynaptic inhibition of muscle spindle volleys than resulted in the impairment of motor output performance. This mechanism is substantial for limitation of the force of muscles contraction and prevention the injury of the muscles under the excessive physical loading.

NADPH-diaphorase activity and neurovascular coupling in the rat cerebral cortex.

The distribution of NADPH-diaphorase-reactive (NADPH-dr) neurons and neuronal processes in the cerebral cortex and basal forebrain and their association with parenchymal vessels were studied in normal adult rats using NADPH-d histochemical protocol. The intensely stained cortical interneurons and reactive subcortically originating afferents, and stained microvessels were examined through a light microscope at law (x250) and high (x630) magnifications. NADPH-dr interneurons were concentrated in layers 2-6 of the M1 and M2 areas. However, clear predominance in their concentration (14 +/- 0.8 P < 0.05 per section) was found in layer 6. A mean number of labeled neurons in auditory (AuV), granular and agranular (GI, AIP) areas of the insular cortex was calculated to reach 12.3 +/- 0.7, 18.5 +/- 1.0 and 23.3 +/- 1.7 units per section, respectively (P < 0.05). The distinct apposition of labelled neurons to intracortical vessels was found in the M1, M2. The order of frequency of neurovascular coupling in different zones of the cerebral cortex was as following sequence: AuV (31.2%, n = 1040) > GI (18.0%, n = 640) > S1 (13.3%, n = 720) > M1 (6.3%, n = 1360). A large number of structural associations between labeled cells and vessels in the temporal and insular cortex indicate that NADPH-d-reactive interneurons can contribute to regulation of the cerebral regional blood flow in these areas.

[NADPH-diaphorase neurons and their interrelationship with microvascular vessels in the basal forebrain limbic structures and hypothalamus].

NADPH-diaphorase histochemistry was used to study the distribution and density of labeled neurons in the limbic structures and hypothalamus in intact rat. NADPH-diaphorase positive neurons were registered in the basal forebrain-medial septal nucleus (MS), the nuclei of the diagonal band of Broca (VDB, HDB), substancia innominata (SI) and the nucleus basalis of Meynert (B). These areas largely overlap with the cholinergic CH1-CH4 forebrain system of the rodent brain. The order of density of labeled neurons in different regions of the basal forebrain was as following sequence: HDB > VDB > SI > B. The highest densities of the reactive neurons (> 1000 labeled neurons per section 200x200 microm2) was found in the islands of Calleja (ICjs). In the supraoptic (SO) and paraventricular (Pa) nuclei of hypothalamus were recorded > 130 and > 100 labeled units, respectively. The lowest density of labeled neurons was recorded within the SI-B complex: < 10 reactive units. Reactive neurons, their dendrites and axon-like processes within the ICjs, SO, Pa, the lateral nucleus hypothalamus (LH) often surround arterioles which traverse the structures. We suggest that NADPH-diaphorase-reactive (NO-generating) neurons within the ICjs and hypothalamus are involved in regulation of the regional, blood flow (RBF) that is important to adapt the blood flow to changes in neuronal activity of the basal forebrain structures.

[Investigation of c-fos expression and NADPH-diaphorase activity in the spine cord and brain in the development of the neck muscle weakness in rats].

The distribution of c-fos expression as a marker of neuronal activation and NADPH-diaphorase reactivity were examined in the cervical spinal cord, limbic structures and hypothalamus in anaesthetized rats after fatigue induced by intermittent high-rate (100 s(-1)) electrical stimulation of the dorsal neck muscles (mm. trapezius and splenius). In comparison to the control or sham-stimulated animals fatiguing stimulation induced significant increase in ipsilaterally in the C -C4 spinal segments and the contralateral central (Ce), medial (Me) nuclei, paraventricular nucleus of hypothalamus (Pa) and ventrolateral periaqueductal gray (VLPAG). In spinal cord the highest mean number of Fos-immunoreactive (Fos-ir) neurons per section was found in layers 1,4 and 5 (5.8 +/- 0.9, 13.1 +/- 0.9 and 11.1 +/- 0.7, P < 0.05) of the dorsal horn. The order of intensity of c-fos expression in different regions of the brain was as following sequence: Pa > VLPAG > Ce (447.7 +/- 23.5, 196.3 +/- 12.7, 104.6 +/- 12.3, respectively). About 50 % of double-labeled (Fos-ir and NADPH-diaphorase reactive) cells were found in Pa nucleus. Received data show that limbic structures, hypothalamus and VLPAG are involved in activation after neck muscle fatigue and might contribute to nociceptive processing and generation of the autonomic and affective components of the muscle pain.

[Central neuronal pathways in the Bezold-Jarisch reflex in rats].

The distribution of the Fos-immunoreactive (Fos-ir) and NADPH-diaphorase-positive/NOS-containing neurons in the medulla and thoracic spinal cord in rats was studied after the veratrine injection into v. jugularis ext. followed by reproduction of the Bezold-Jarisch reflex. Fos-ir neurons were found in the nucleus tractus solitarius (Sol), coudoventrolateral (CVL) and rostroventrolateral (RVL) areas, intermediate reticular nucleus (IRt), and lateral paragiganrocellular nucleus (LPGi) of the medulla. The intensity of c-fos expression was as follows: Sol > CVL > RVL > LPGi. The intensity of NADPH-diaphorase reactivity was manifested in the following sequence: Sol > IRt > LPGi > RVL. In comparison to sham-operated animals, application of veratrine is associated with a statistically significant increase (P < 0.01) of the mean number of Fos-ir neurons in the Sol and CVL, but not in the RVL. Double-labelling (Fos + NA DPH) was found in a small number (about 2%) of neurones in the Sol, IRt and LPGi. In the thoracic spinal cord Fos-ir neurons were predominantly detected in the superficial layers of the dorsal horn, area around the central canal and the intermediate lateral or medial nuclei (IMl and IMM), where NOS-containing neurons in the thoracic spinal cord were accumulated. Received data show that signals arising from the veratrine-activated cardiopulmonary afferents activate neurons of the Sol and CVL that together with the RVL are incorporated into neuronal circuits for generation of the Bezold-Jarisch reflex. In the medulla NOS-containing neurons activated by input from cardiopulmonary receptors can contribute to the enhancement of cardioprotective depressor reflexes in the rats.

[Expression of c-fos in the spinal cord after electrical stimulation of low-threshold afferent fibres of the rat n. gastrocnemius-soleus]. / Ekspresiia c-fos u spynnomu mozku pislia elektrychnogo podraznennia nyz'koporohovykh aferentnykh volokon u skladi n. gastrocnemius-soleus shchura.

The specific pattern of c-fos gene expression was evaluated within the lumbo-sacral spinal cord during 30 min non-noxious stimulation n. gastrocnemius-soleus of male Wistar rats. A lot of small and large Fos-immunoreactive (Fos-ir) nuclei were located ipsilaterally, mainly in the middle of the neck and base of the dorsal horn (layers 5 and 6). Traditionally, these layers are considered to be the region of termination of the low-threshold afferent fibers. A few Fos-ir neurons were recorded in the intermediate zone (layer 7) and the area around central canal (layer 10). The scarce labeled cells were also revealed within the marginal zone and substantia gelatinosa of the dorsal horn, i. e. the zones of termination of the high-threshold muscle afferents. In conclusion, the revealed pattern of c-fos expression in the spinal cord following non-noxious stimulation differs from that induced by noxious stimulation. Thus, the pattern of c-fos expression in the spinal cord is dependent on the stimulus modality.

[Effect of muscle pain on c-fos expression and NADPH-diaphorase activity in the spinal cord]. / Vplyv M'iazovoho boliu na ekspresiiu c-fos i NADFN-diaforaznu aktyvnist' u spynnomu mozku.

Capsaicin (Sigma, 5 mg/l ml) was unilaterally injected into dorsal neck muscles (mm. trapezius, splenius) of the cat (n = 6) anesthetized with alpha-chloralose. The laminar distribution of Fos- immunoreactive (Fos-ir) and NADPH-d-reactive neurons was studied in the sections of the cervical and lumbar segments. In comparison to sham operated animals (n = 2), 2 h after capsaicin injection the significant c-fos expression was found throughout the cervical cord (C1 - C8) ipsilaterally and lumbar cord bilaterally (L1 - L7), while the predominant mean number of Fos-ir neurons was detected in the C1 - C4 and L3-L6 segments. In cervical spinal cord, c-fos expression was three times more than in lumbar spinal cord. Dorsal neck noxious stimulation produced widespread c-fos expression in the large and small labeled cells within lamina 1, at the neck of the dorsal horn (lamina V) and also in the medial part of the intermediate zone (lamina VII) where cells of origin of the long crossed and uncrossed descending propriospinal pathways to the lumbar cord were known to be localized. The main accumulations of NADPH-d-reactive cells were registered within the marginal zone (laminae I and II) and medial part of the intermediate zone (lamina VII). Fos-ir neurons were co-distributed with NADPH-d-reactive cells within the marginal zone, and the neck of the dorsal horn, however, double-staining spinal neurons were not registered. These results strongly suggest that the revealed c-fos expression in cervical and lumbar segments depends on the activity of vanilloid receptors of the cervical muscles and confirmed the anatomical coupling of both regions through long propriospinal pathways which may be underlying in the cervico-lumbar nociceptive processing. An influence of the spino-cerebro-spinal loop on modulation c-fos expression in these regions is not excluded also.