Long lasting activity of nociceptive muscular afferents facilitates bilateral flexion reflex pattern in the feline spinal cord.

Chronic muscular limb pain requires the adoption of motor patterns distinct from the classic ipsilateral flexion, crossed extension and corresponding reciprocal inhibitions to acute exteroceptive stimulation. Using selective chemical activation of group III/IV afferents in gastrocnemius-soleus (GS) muscles we investigated bilaterally their reflex responses conditioned by (a) acute 'myositis' induced by intramuscular carrageenan; and (b) sub-acute 'myositis' induced by infusion of complete Freund's adjuvant (CFA). Reflex transmission was detected by monosynaptic testing and c-fos staining used to identify increased neuronal activity. In all control experiments with chemical stimulation of group III/IV afferents, ipsilateral responses conformed to the flexor reflex pattern. However, the expected contralateral facilitation of GS motoneurones occurred in fewer than 50% trials while only 9% of trials induced contralateral inhibition of flexor posterior-biceps-semitendinosus (PBSt) motoneurones. During carrageenan acute myositis contralateral PBSt was transiently facilitated by selective activation of group III/IV afferents. During CFA-induced myositis, contralateral only inhibition of GS motoneurones occurred instead of any facilitation, while bidirectionally a crossed facilitation of PBST dominated. These reflex changes were mirrored in an enhanced number of neurones with enhanced c-fos expression. Muscle pain, particularly if chronically persistent, requires another behavioural response pattern than acute exteroceptive pain.

My first 20 years in neuroscience.

Intriguing facts were obtained in the first electrophysiological investigations (1964) that the action potentials (AP) produced by direct depolarization of the cell membrane in different species of mollusks showed specific relations to changes in external ionic composition. In Helix neurons, the generation of AP was well maintained in sodium-free solutions with high calcium or barium content. The amplitude of the spike overshoot in the case was linearly related to the logarithm of calcium concentration. It is interesting that increase in external calcium ions decreased the ionic conductance of the resting membrane (R0) also in linear relation to the logarithm of Ca2+ or Ba2+ concentration. It was found for the first time (1965) that addition of Ba2+ to the external solution produced in the neurons well-developed prolonged (protracted) APs in all cases. However, the excitability of Planorbis and Limnea neurons was rapidly (during 3 min) reversibly depressed in sodium-free solutions. We found that, after injections of horse radish peroxidase (HRP) in the spinal cord of cats, the enzyme was transported retrogradely to brain stem neurons in the bulbar medial reticular formation, the vestibular complex, and the red nucleus. We obtained (1975) intriguing facts in our investigations: we recorded the labeled neurons in the locus coeruleus and subcoeruleus, as well as in the paraventricular hypothalamic nucleus. The existence of straight pathways from hypothalamus to the spinal cord has not been demonstrated earlier. The next step of our study was to determine the corresponding spinal funiculi where descending fibers (from various brain stem cell groups) are located. Indeed, in our pioneer studies (1977), we found that the fibers from the hypothalamus, which descend throughout the spinal cord, are located mainly in the lateral funiculus, ipsilaterally.

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.

Distinctive pattern of c-fos expression in the feline cervico-lumbar spinal cord after stimulation of vanilloid receptors in dorsal neck muscles.

In the present study, c-fos expression in the spinal cord has been used as a marker of neuronal activation induced by capsaicin-sensitive sensory afferents from the dorsal neck muscles in cats (n = 6). The number of Fos-immunoreactive neurons, which were revealed using the avidin-biotin-peroxidase method, was significantly increased in the cervical and lumbar spinal cord. In contrast to the control group (n = 3), 2 h after intramuscular capsaicin injection, c-fos expression was more extensive ipsilaterally to the injected side in the C3-C6 segments, and bilaterally in the L4-L6 segments. Most labeled neurons in the cervical spinal cord were small and giant cells, predominantly located in the middle and lateral parts of lamina I and, additionally, at the neck of the dorsal horn (lamina V), i.e., within the zones of termination of high-threshold muscle afferents. The widespread distribution of labeled cells throughout the cervical cord within the intermediate zone (lamina VII) coincided with the sites of last-order premotor interneurons and cells of origin of long crossed and uncrossed descending propriospinal pathways to the lumbar spinal cord. These findings suggest possible mechanisms for spreading of nociceptive signals between cervical and lumbar regions.

Fos and nitric oxide synthase in rat brain with chronic mesostriatal dopamine deficiency: effects of nitroglycerin and hypoxia.

We found sustained proto-oncogene c-fos expression in neurons of the lateral and medial neostriatum and suppression of this expression in nitric oxide synthase (NOS)-containing cells within the islands of Calleja after lesions of the dopaminergic mesostriatal system induced by 6-hydroxydopamine. Systemic administration of nitroglycerin (NTG) or mild hypoxia resulted in a decreased of c-fos expression in the dorsolateral part of the denervated neostriatum. However, in other brain structures NTG or mild hypoxia evoked sustained c-fos expression in NOS-containing neurons and in the sources catecholaminergic projections involved in the control of cardiovascular function. We propose that the administration of NTG, an NO donor, or hypoxia partially attenuate the consequences of an excessively increased glutamate level in the denervated neostriatum which are manifest in high level of c-fos expression.

c-fos Expression and NADPH-d reactivity in spinal neurons after fatiguing stimulation of hindlimb muscles in the rat.

The distribution of Fos-immunoreactive (Fos-ir) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-reactive neurons in the rat lumbar spinal cord was examined following muscle fatigue caused by intermittent high-rate (100 s(-1)) electrical stimulation of the triceps surae muscle or the ventral root L5 (VRL5) for 30 min. Following both types of stimulation, the fatigue-related c-fos gene expression was more extensive in the L2-L5 segments on the stimulated side, and the majority of Fos-ir neurons were concentrated in the dorsal horn. After direct muscle stimulation, the highest number of Fos-ir neurons were detected in two regions: layer 5, and superficial layers (1 and 2(o)), although many labeled cells were also found in layers 3, 4, 6, and 7. In response to VRL5 stimulation, the maximal density of Fos-ir neurons was detected in the middle and lateral parts of layers 1 and 2(o), the zone of termination of high-threshold muscle afferents(.) Statistically significant prevalence of Fos-ir cell number was also found in layers 5 and 7 on the stimulated side. A few Fos-ir neurons were detected in the ventral horn (layer 8 and area 10) on both sides. The lamellar distribution of NADPH-d-reactive neurons was similar over all experimental groups of animals. In the L3-L6 segments, such reactive cells were arranged in two distinct regions: dorsal horn (layers 2(i), 3, and 5) and area 10; in the L1 and L2 segments, an additional cluster of NADPH-d positive cells was found in the intermediolateral cell column (IML). Double-labeled cells were not detected. We suggest that c-fos expression in response to muscle fatigue reveals activity of functionally different types of spinal neurons which could operate together with NOS-containing cells in pre-motoneuronal networks to modulate the motoneuron output.

Persistent c-fos expression and NADPH-d reactivity in the medulla and the lumbar spinal cord in rat with short-term peripheral anosmia.

Here we examine hypothesis that short-term peripheral ZnSO(4)-induced anosmia can produce effects on c-fos expression within spinal cord and caudal medulla in male Wistar rats (n=4). Fos-like-immunoreactive cells revealed by avidin-biotin-peroxidase method show a significant bilateral increase in the nucleus proprius (layers 3 and 4) and medial part of layers 5 and 6. In substantia gelatinosa (layer 2(i)) and area 10 Fos-positive neurons were intermixed together with nicotin-amide adenine dineucleotide phosphate-diaphorase (NADPH-d)-reactive cells. Short-term anosmia enhanced c-fos expression in ventral horn (layers 7 and 8), ventrolateral segment and dorsal part of the spinal trigeminal nuclei. In anosmic rats varicose fibres and numerous NADPH-d-stained neurons were present in the gelatinous layer of the spinal trigeminal nucleus caudalis, and a separate population of Fos-positive cells was detected within this layer. Nucleus tractus solitaris also contained a few NADPH-d-reactive, medium sized neurons intermixed with Fos-immunoreactive cells.

Topographical organization of the sources of discrete cortical projections within the striatum as determined by a retrograde fluorescence tracing technique in the cat.

The projections from the neostriatum and the paleostriatum to the cerebral cortex in the cat were examined by means of retrogradely transported fluorescent tracers primuline, Fast Blue, Nuclear Yellow and Evans Blue injected into different neocortical fields. In all cases after dye injections only large labelled cells of sources of striatocortical ipsilateral projections were observed. The main projections from the caudate nucleus and the putamen are directed to the auditory and neighbouring "associative" cortex, and more numerous projections from the globus pallidus are addressed to the motor cortex. No sources of cortical projections within the entopeduncular nucleus were found. Simultaneous injections of Fast Blue and primuline into even closely located and tightly bound functional regions of parietal or temporal cortex failed to reveal double-labelled neurons in the caudate nucleus, internal capsule, putamen and globus pallidus. Thus, our findings on cats are consistent with recent studies on rats and monkeys that suggest that striatal neurons innervate relatively small, restricted fields of the neocortex. Again, the results show evidence for a significant contribution to cholinergic cortical innervation not only of magnocellular neurons of the basal forebrain but also of large neo- and paleostriatal cells.

Sources of cortical, hypothalamic and spinal serotonergic projections: topical organization within the nucleus raphe dorsalis.

Retrograde axonal transport of fluorescent tracers (primuline, FluoroGold and Nuclear Yellow) from the spinal cord, frontal cortex, lateral hypothalamus to various neuronal groups of the midbrain periventricular gray substance (periaqueductal gray matter) and to the dorsolateral pontine tegmentum in the rat has been studied. Two large groups of serotonin-containing neurons have been found to be localized in the dorsomedial region of the nucleus raphe dorsalis. They are sources of projections into the thoracic segments of the spinal cord. A part of these neurons gives divergent axon collaterals to the frontal cortex and to the spinal cord. Non-collateral projections of the dorsolateral pontine tegmental catecholaminergic neurons to the spinal cord and the frontal cortex have been revealed. The data obtained give support to the fact that antinociceptive effect of stimulation of the "pure analgesic zone" [Fardin et al. (1984) Brain Res. 306, 105-123.] of the midbrain periaqueductal gray matter may be due to direct involvement of the nucleus raphe dorsalis into a descending control over transmitting nociceptive stimuli at the spinal cord level. Neurotransmissive and neuroregulatory roles of separate cortical, hypothalamic and spinal serotonergic projections of the nucleus raphe dorsalis neurons are discussed.

A method for bevelling of microelectrodes by means of vibration.

A technique for the rapid bevelling of glass and metallic microelectrodes is described. The method utilizes end friction of the electrode tips on the fine-grained surface of a quartz plate vibrating at the mechanical resonance frequency of the system. A electromagnetic vibrator is supplied with alternating current at a frequency equal to the mechanical resonance frequency of the system (200-300 s-1).

Catecholamine projections to the spinal cord in the rat and their relationship to central cardiovascular neurons.

The organization of the neuroanatomical substrate which provides the supraspinal catecholaminergic innervation of the upper thoracic spinal cord in the rat was studied by means of retrograde labelling of neurons with primuline and other dyes, combined with simultaneous catecholamine fluorescence (FAGLU-method). It was shown that spinally projecting catecholaminergic neurons lie predominantly in the A6 and A11 groups, while a minority of these neurons are also located in the A1/C1 and A5 groups. The number of catecholamine-containing neurons in the A11 group in the rat was estimated to be 173 +/- 4. Most of them (up to 86%) were retrogradely labelled with primuline. The possible functional role of dopaminergic diencephalospinal, noradrenergic pontospinal, and adrenergic bulbospinal neuronal systems in cardiovascular control is discussed.