Modulation of glycinergic synaptic transmission in the trigeminal and hypoglossal motor nuclei by the nitric oxide-cyclicGMP signaling pathway.

In a previous work we found that nitric oxide (NO) and cyclicGMP (cGMP) inhibit glutamatergic synaptic transmission in trigeminal motoneurons (MnV). Here we study the actions of the NO/cGMP signaling pathway on glycinergic synaptic transmission in trigeminal and hypoglossal motoneurons (MnXII) in brain stem slices of neonatal rats. Glycinergic inhibitory postsynaptic currents (IPSCs) were recorded in MnV by stimulation of the supratrigeminal nucleus (SuV) and in MnXII by stimulation of the nucleus of Roller. The NO donor DETA/NONOate (DETA/NO) reduced the amplitude of the IPSC to 58.1±4.2% of control values in MnV. In the presence of YC-1, a modulator of guanylate cyclase that acts as a NO sensitizer, lower and otherwise ineffective concentrations of DETA/NO induced a reduction of the IPSC to 47.2±15.6%. NO effects were mimicked by 8 bromo cyclicGMP (8BrcGMP). They were accompanied by an increase in the paired pulse facilitation (PPF) and in the failure rate of evoked IPSCs. 8BrcGMP did not modify the glycinergic currents elicited by exogenous glycine. In MnXII the IPSCs were also reduced by NO donors and 8BrcGMP to 52.9±6.3% and 45.9±4% of control values, respectively. In these neurons, but not in MnV, we also observed excitatory postsynaptic actions of NO donors. We propose that the differences between the two motor pools may be due to a differential development of the nitrergic system in the two nuclei. Our data show that NO, through its second messenger cGMP, reduces inhibitory glycinergic synaptic transmission in both MnV and MnXII. For MnV, evidence in favor of presynaptic inhibition of glycine release is presented. Given our previous data together with the current results, we propose that the NO/cGMP signaling pathway participates pre- and postsynaptically in the combined regulation of MnV and MnXII activities in motor acts in which they participate.

Nitrergic ventro-medial medullary neurons activated during cholinergically induced active (rapid eye movement) sleep in the cat.

The rostral ventro-medial medullary reticular formation is a complex structure that is involved with a variety of motor functions. It contains glycinergic neurons that are activated during active (rapid eye movement (REM)) sleep (AS); these neurons appear to be responsible for the postsynaptic inhibition of motoneurons that occurs during this state. We have reported that neurons in this same region contain nitric oxide (NO) synthase and that they innervate brainstem motor pools. In the present study we examined the c-fos expression of these neurons after carbachol-induced active sleep (C-AS). Three control and four experimental cats were employed to identify c-fos expressing nitrergic neurons using immunocytochemical techniques to detect the Fos protein together with neuronal nitric oxide synthase (nNOS) or nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity. The classical neurotransmitter content of the nitrergic cells in this region was examined through the combination of immunocytochemical techniques for the detection of glutamate, glycine, choline acetyltransferase (ChAT), tyrosine hydroxilase (TH) or GABA together with nNOS. During C-AS, there was a 1074% increase in the number of nitrergic neurons that expressed c-fos. These neurons did not contain glycine, ChAT, TH or GABA, but a subpopulation (15%) of them displayed glutamate-like immunoreactivity. Therefore, some of these neurons contain both an excitatory neurotransmitter (glutamate) and an excitatory neuromodulator (NO); the neurotransmitter content of the rest of them remains to be determined. Because some of the nitrergic neurons innervate brainstem motoneurons it is possible that they participate in the generation of tonic and excitatory phasic motor events that occur during AS. We also suggest that these nitrergic neurons may be involved in autonomic regulation during this state. In addition, because NO has trophic effects on target neurons, the present findings represent the first, albeit indirect, evidence for a possible trophic function of this nature during AS.

The possible additional role of the cystic fibrosis transmembrane regulator to motoneuron inhibition produced by glycine effects.

In the present work we study the contribution of the chloride channel of the Cystic Fibrosis Transmembrane Regulator (CFTR) in the postsynaptic inhibition of somatic motoneurons during rapid-eye-movement (REM) sleep atonia. Postsynaptic inhibition of motoneurons is partially responsible for the atonia that occurs during REM sleep. Disfacilitation is an additional mechanism that lowers motoneuron excitability in this state. Postsynaptic inhibition is mediated by the release of glycine from synaptic terminals on motoneurons, and by GABA that plays a complementary role to that of glycine. In this work we look in brain stem motoneurons of neonatal rats at a mechanism unrelated to the actions of glycine, GABA or to disfacilitation which depends on the chloride channel of the CFTR. We studied the presence of CFTR by immunocytochemistry. In electrophysiological experiments utilizing whole cell recordings in in vitro slices we examined the consequences of blocking this chloride channel. The effects on motoneurons of the application of glycine, of the application of glibenclamide (a CFTR blocker) and again of glycine during the effects of glibenclamide were studied. Glycine produced an hyperpolarization, a decrease in motoneuron excitability and a decrease in input resistance, all characteristic changes of the postsynaptic inhibition produced by this neurotransmitter. Glibenclamide produced an increase in input resistance and in motoneurons' repetitive discharge as well as a shift in the equilibrium potential for chloride ions as indicated by the displacement of the reversal potential for glycinergic actions. In motoneurons treated with glibenclamide, glycine produced postsynaptic inhibition but this effect was smaller when compared to that elicited by glycine in control conditions. The fact that blocking of the CFTR-chloride channel in brain stem motoneurons influences glycinergic inhibition suggests that this channel may play a complementary role in the glycinergic inhibition that occurs during REM sleep.

Direct hypothalamic innervation of the trigeminal motor nucleus: a retrograde tracer study.

It is currently thought that the hypothalamus influences motor output through connections with premotor structures which in turn project to motor nuclei. However, hypocretinergic/orexinergic projections to different motor pools have recently been demonstrated. The present study was undertaken to examine whether hypocretinergic/orexinergic neurons are the only source of projections from the hypothalamus to the trigeminal motor nucleus in the guinea-pig. Cholera toxin subunit b was injected into the trigeminal motor nucleus in order to retrogradely label premotor neurons. Two anatomically separated populations of labeled neurons were observed in the hypothalamus: one group was distributed along the dorsal zone of the lateral hypothalamic area, the lateral portion of the dorsomedial hypothalamic nucleus and the perifornical nucleus; the other was located within the periventricular portion of the dorsomedial hypothalamic nucleus. Numerous cholera toxin subunit b+ neurons in both populations displayed glutamate-like immunoreactivity. In addition, premotor neurons containing hypocretin/orexin were distributed throughout the lateral dorsomedial hypothalamic nucleus, perifornical nucleus and lateral hypothalamic area. Other premotor neurons were immunostained for melanin concentrating hormone; these cells, which were located within the lateral hypothalamic area and the perifornical nucleus, were intermingled with glutamatergic and hypocretinergic/orexinergic neurons. Nitrergic premotor neurons were located only in the periventricular zone of the dorsomedial hypothalamic nucleus. None of the hypothalamic premotor neurons were GABAergic, cholinergic or monoaminergic. The existence of diverse neurotransmitter systems projecting from the hypothalamus to the trigeminal motor pool indicates that this diencephalic structure may influence the numerous functions that are subserved by the trigeminal motor system.

Cuneiform neurons activated during cholinergically induced active sleep in the cat.

In the present study, we report that the cuneiform (Cun) nucleus, a brainstem structure that before now has not been implicated in sleep processes, exhibits a large number of neurons that express c-fos during carbachol-induced active sleep (AS-carbachol). Compared with control (awake) cats, during AS-carbachol, there was a 671% increase in the number of neurons that expressed c-fos in this structure. Within the Cun nucleus, three immunocytochemically distinct populations of neurons were observed. One group consisted of GABAergic neurons, which predominantly did not express c-fos during AS-carbachol. Two other different populations expressed c-fos during this state. One of the Fos-positive (Fos(+)) populations consisted of a distinct group of nitric oxide synthase (NOS)-NADPH-diaphorase (NADPH-d)-containing neurons; the neurotransmitter of the other Fos(+) population remains unknown. The Cun nucleus did not contain cholinergic, catecholaminergic, serotonergic, or glycinergic neurons. On the basis of neuronal activation during AS-carbachol, as indicated by c-fos expression, we suggest that the Cun nucleus is involved, in an as yet unknown manner, in the physiological expression of active sleep. The finding of a population of NOS-NADPH-d containing neurons, which were activated during AS-carbachol, suggests that nitrergic modulation of their target cell groups is likely to play a role in active sleep-related physiological processes.

Neurotrophin-induced rapid enhancement of membrane potential oscillations in mesencephalic trigeminal neurons.

We have proposed that neurotrophins, in addition to their trophic actions, act as neuromodulators in the adult central nervous system. As a first step to test this hypothesis, we examined in the adult rat slice preparation whether nerve growth factor and neurotrophin-3 are capable of altering the excitability of neurons of the mesencencephalic trigeminal nucleus. In contrast to vehicle pressure microapplication, which did not evoke changes in the electrophysiological properties of these neurons, neurotrophin application produced a significant increase in amplitude of the membrane potential oscillatory activity that is observed in these cells and a significant decrease in their threshold current. The latency of these effects ranged from 2 to 80 s and the duration ranged from 2 to 11 min. Neurotrophin-3 induced a decrease in input resistance and resting membrane potential in 58% of the cells; nerve growth factor induced a decrease in input resistance and resting membrane potential in 35% of the neurons. The spike configuration and action potential afterhyperpolarization potential remained unchanged following neurotrophin application. Tetrodotoxin blocked the membrane potential oscillatory activity of trigeminal mesencephalic neurons. Neurotrophin-induced effects were not blocked by the tyrosine kinase inhibitor K-252a, whereas IgG-192, an antibody directed to the neurotrophin low-affinity receptor, enhanced excitability, as did neurotrophins. These results demonstrate that neurotrophins are capable of producing a rapid increase in the excitability of trigeminal mesencephalic neurons and suggest that their effects may be mediated by low-affinity neurotrophin receptors.

Oscillatory membrane potential activity in the soma of a primary afferent neuron.

In the present report, we provide evidence that mesencephalic trigeminal (Mes-V) sensory neurons, a peculiar type of primary afferent cell with its cell body located within the CNS, may operate in different functional modes depending on the degree of their membrane polarization. Using intracellular recording techniques in the slice preparation of the adult rat brain stem, we demonstrate that when these neurons are depolarized, they exhibit sustained, high-frequency, amplitude-modulated membrane potential oscillations. Under these conditions, the cells discharge high-frequency trains of spikes. Oscillations occur at membrane potential levels more depolarized than -53 +/- 2.3 mV (mean +/- SD). The amplitude of these oscillations increases with increasing levels of membrane depolarization. The peak-to-peak amplitude of these waves is approximately 3 mV when the cells are depolarized to levels near threshold for repetitive firing. The frequency of oscillations is similar in different neurons (108.9 +/- 15.5 Hz) and was not modified, in any individual neuron, by changes in the membrane potential level. These oscillations are abolished by hyperpolarization and by TTX, whereas blockers of voltage-dependent K+ currents slow the frequency of oscillations but do not abolish the activity. These data indicate that the oscillations are generated by the activation of inward Na+ current/s and shaped by voltage-dependent K+ outward currents. The oscillatory activity is not modified by perfusion with low-calcium, high-magnesium, or cobalt-containing solutions nor is it modified in the presence of cadmium or Apamin. These results indicate that a calcium-dependent K+ current does not play a significant role in this activity. We postulate that the membrane oscillatory activity in Mes-V neurons is synchronized in adjoining electrotonically coupled cells and that this activity may be modulated in the behaving animal by synaptic influences.

Experimental analysis of the method of 'peeling' exponentials for measuring passive electrical properties of mammalian motoneurons.

Trigeminal motoneurons of the guinea pig brain stem slice preparation were studied using intracellular recording techniques. The voltage response to a 100-ms constant-current pulse was studied and a population of cells was found which did not exhibit sag or overshoot of their voltage response to a pulse of hyperpolarizing current of < 1 nA but did exhibit both phenomena when a current pulse of > 1 nA was used. The sag and overshoot observed with large-current pulses were reduced or blocked when 4 mM CsCl was added to the bathing solution. This observation supports the hypothesis that these phenomena were due to the voltage- and time-dependent activation of the Q-current. The method of peeling exponentials was then used to correct raw voltage data from cells in which the Q-current was present. The mean membrane time constant was within 1% and the mean input resistance was within 2% of the means for these parameters when measured in these same cells under conditions in which the Q-current was absent. We conclude from these experiments that the method of peeling exponentials is valid for obtaining estimates of the membrane time constant and input resistance from cells that exhibit sag and overshoot due to voltage- and time-dependent changes in the magnitude of the Q-current.

Velocity of ultrasound in active and passive cat medial gastrocnemius muscle.

The lengths and pinnation angles of muscle fibers in the medial gastrocnemius (MG) muscle have recently been measured in freely moving cats [Hoffer et al., Progr. Brain Res. 80, 75-85 (1989); Muscle Afferents and Spinal Control of Movement (1992)] using an ultrasound transit-time (USTT) technique. This method assumed that the velocity of ultrasound through intact muscles was constant, independent of fiber orientation, muscle activity, load, belly shape, or fiber movement. However, the velocity of ultrasound along and across the fibers has been reported to depend on the state of muscle activation in frog muscle experiments in vitro [Hatta et al., J. Physiol. 403, 193-209 (1988)]. In the present study, the assumption of constant velocity of ultrasound in the cat MG muscle was evaluated. In acute experiments, done in situ with intact blood supply, the USTT was measured along and across cat MG muscle fibers in the passive, reflexly activated and tetanically activated states, with and without changes in muscle fiber length, for situations that reproduced the length and force ranges normally used by cats during locomotion. The velocity of ultrasound was found to be independent of the state of activation or motion of the muscle, and independent of the direction of the measurement with respect to the fiber orientation, within a measurement uncertainty less than or equal to 0.2%. These results validate the use of the USTT technique for the measurement of intramuscular dimensions in freely moving animals.

Roles of muscle activity and load on the relationship between muscle spindle length and whole muscle length in the freely walking cat.

The objective of this research was to compare the length of muscle spindles to the length of the whole muscle, during normal movements. Pairs of piezoelectric crystals were implanted near the origin and insertion of muscle fibres in the medial gastrocnemius (MG) muscle of cats. The distance between crystals was measured with pulsed ultrasound, the origin-to-insertion length of the MG muscle was measured with a transducer made of saline-filled silicone tubing, MG force was measured with a tendon force transducer and EMG activity was selectively recorded in the vicinity of implanted crystals. These signals were simultaneously recorded during posture or locomotion on a motorized treadmill. Three periods were identified in the step cycle, during which the relation between muscle length and spindle length changed dramatically. In period I (roughly corresponding to the late F and E1 phases of swing), the MG muscle and spindles followed similar length changes: both were stretched and then shortened by about 6 mm. In period II (corresponding to the stance phase, E2-E3) the MG muscle yielded under the weight of the body and was stretched by 1-3 mm, whereas the MG spindles typically continued shortening. In period III, the MG muscle shortened rapidly by 6-8 mm after the foot left the ground and then stretched again by about the same amount, whereas the spindles could remain nearly isometric. We attribute these large discrepancies in muscle and spindle length to the architecture of the MG muscle and the compliance of long tendinous elements in series with the spindles. We conclude that the length changes imposed on muscle spindles during voluntary movements are not simply related to the parent muscle length changes and cannot be estimated without taking into account the muscle architecture, the location of the spindle within the muscle, the level of muscle activation and the external load.

Effect of xipamide and furosemide on guinea pig cochlear recorded potentials.

The effects of furosemide and xipamide on guinea pig cochlear potentials were studied under acute conditions. Auditory nerve action potentials (AP) and cochlear microphonics (CM) were depressed by both diuretics in a dose-related manner. Furosemide was more effective on AP than on CM. In contrast, the xipamide-induced reductions of AP and CM were similar. Our results suggest that the depressive effects of furosemide or xipamide may be related to a direct action on cochlear mechanisms.