Endogenous Rho-kinase signaling maintains synaptic strength by stabilizing the size of the readily releasable pool of synaptic vesicles.

Rho-associated kinase (ROCK) regulates neural cell migration, proliferation and survival, dendritic spine morphology, and axon guidance and regeneration. There is, however, little information about whether ROCK modulates the electrical activity and information processing of neuronal circuits. At neonatal stage, ROCKα is expressed in hypoglossal motoneurons (HMNs) and in their afferent inputs, whereas ROCKß is found in synaptic terminals on HMNs, but not in their somata. Inhibition of endogenous ROCK activity in neonatal rat brainstem slices failed to modulate intrinsic excitability of HMNs, but strongly attenuated the strength of their glutamatergic and GABAergic synaptic inputs. The mechanism acts presynaptically to reduce evoked neurotransmitter release. ROCK inhibition increased myosin light chain (MLC) phosphorylation, which is known to trigger actomyosin contraction, and reduced the number of synaptic vesicles docked to active zones in excitatory boutons. Functional and ultrastructural changes induced by ROCK inhibition were fully prevented/reverted by MLC kinase (MLCK) inhibition. Furthermore, ROCK inhibition drastically reduced the phosphorylated form of p21-associated kinase (PAK), which directly inhibits MLCK. We conclude that endogenous ROCK activity is necessary for the normal performance of motor output commands, because it maintains afferent synaptic strength, by stabilizing the size of the readily releasable pool of synaptic vesicles. The mechanism of action involves a tonic inhibition of MLCK, presumably through PAK phosphorylation. This mechanism might be present in adults since unilateral microinjection of ROCK or MLCK inhibitors into the hypoglossal nucleus reduced or increased, respectively, whole XIIth nerve activity.

Protein kinase G-dependent mechanisms modulate hypoglossal motoneuronal excitability and long-term facilitation.

Since protein kinase-dependent modulation of motoneuronal excitability contributes to adaptive changes in breathing, we hypothesized that cGMP-dependent pathways activating protein kinase G (PKG) modulate motoneuronal inspiratory drive currents and long-term plasticity. In a medullary slice preparation from neonatal rat (postnatal days 0-4) generating spontaneous respiratory-related rhythm, hypoglossal (XII) motoneuronal inspiratory drive currents and respiratory-related XII nerve activity were recorded. Focal application of a PKG activator, 8-bromoguanosine-3',5'-cyclomonophosphate (8-Br-cGMP), to voltage-clamped XII motoneurones decreased inspiratory drive currents. In the presence of tetrodotoxin (TTX), 8-Br-cGMP decreased the exogenous postsynaptic inward currents induced by focal application of AMPA. Intracellular dialysis of XII motoneurones with an inhibitory peptide to PKG (PKGI) increased endogenous inspiratory-drive currents and exogenous AMPA-induced currents. Application of 8-Br-cGMP with PKGI had no further effect on spontaneous or evoked currents, confirming that the observed effects were induced by PKG. However, PKG differentially increased longer-term plasticity. Three 3 min applications (separated by 5 min) of the α(1)-adrenergic agonist phenylephrine (PE) in combination with 8-Br-cGMP yielded greater in vitro long-term facilitation than PE alone. These data indicate the presence of a cGMP/PKG-dependent signalling pathway in XII motoneurones that modulates inspiratory drive currents and plasticity of XII motoneurones, possibly contributing to their adaptation during physiological challenges, such as sleep and exercise.

Simultaneous expression of glutathione, thioredoxin-1, and their reductases in nerve transected hypoglossal motor neurons of rat.

Anti-oxidative stress responses are crucial for the survival of nerve-injured motor neurons. Herein, we examined changes in expression of glutathione reductase (GSHr), thioredoxins (TRX1 and TRX2), and thioredoxin reductases (TRXr1 and TRXr2), important constituents of anti-oxidative pathways, following rat hypoglossal nerve transection. RT-PCR and in situ hybridization demonstrated that GSHr, TRX1, and TRXr1 mRNAs were significantly up-regulated during the first few weeks in nerve-injured motor neurons, while TRX2 and TRXr2 mRNAs were unchanged throughout 8 weeks after nerve transection. The up-regulation of GSH, GSHr, TRX1, and TRXr1 proteins in injured neurons was confirmed by immunohistochemical analysis. Western blotting also demonstrated up-regulation of GSHr, TRX1, and TRXr1 in injured neurons. These data suggest that the two major redox systems, GSH/GSHr and TRX1/TRXr1, are simultaneously activated in injured neurons, and likely provide protection of injured neurons against oxidative stress.

Neurogenesis in an adult avian song nucleus is reduced by decreasing caspase-mediated apoptosis.

Neuron death and replacement are fundamental components of brain plasticity. Much remains unknown, however, about the mechanistic interaction between neuron death and neurogenesis in adult vertebrates. In seasonally breeding adult male white-crowned sparrows, the song system nucleus HVC loses approximately 26% of its neurons via caspase-dependent apoptosis within 4 d after a transition to nonbreeding physiological conditions. To determine whether neuronal death is necessary for the recruitment of new neurons, we infused caspase inhibitors into HVC in vivo and suppressed neurodegeneration for at least 20 d after the transition to nonbreeding conditions. The blockade of HVC neuron death reduced the number and density of new neurons recruited to the ipsilateral HVC by 48 and 29%, respectively, compared with contralateral HVC. Our results are the first to show that reducing neuronal death in the adult brain decreases the recruitment of new neurons.

Hypoglossal premotor neurons of the intermediate medullary reticular region express cholinergic markers.

The inspiratory drive to hypoglossal (XII) motoneurons originates in the caudal medullary intermediate reticular (IRt) region. This drive is mainly glutamatergic, but little is known about the neurochemical features of IRt XII premotor neurons. Prompted by the evidence that XII motoneuronal activity is controlled by both muscarinic (M) and nicotinic cholinergic inputs and that the IRt region contains cells that express choline acetyltransferase (ChAT), a marker of cholinergic neurons, we investigated whether some IRt XII premotor neurons are cholinergic. In seven rats, we applied single-cell reverse transcription-polymerase chain reaction to acutely dissociated IRt neurons retrogradely labeled from the XII nucleus. We found that over half (21/37) of such neurons expressed mRNA for ChAT and one-third (13/37) also had M2 receptor mRNA. In contrast, among the IRt neurons not retrogradely labeled, only 4 of 29 expressed ChAT mRNA (P < 0.0008) and only 3 of 29 expressed M2 receptor mRNA (P < 0.04). The distributions of other cholinergic receptor mRNAs (M1, M3, M4, M5, and nicotinic alpha4-subunit) did not differ between IRt XII premotor neurons and unlabeled IRt neurons. In an additional three rats with retrograde tracers injected into the XII nucleus and ChAT immunohistochemistry, 5-11% of IRt XII premotor neurons located at, and caudal to, the area postrema were ChAT positive, and 27-48% of ChAT-positive caudal IRt neurons were retrogradely labeled from the XII nucleus. Thus the pre- and postsynaptic cholinergic effects previously described in XII motoneurons may originate, at least in part, in medullary IRt neurons.

Melatonin preserves superoxide dismutase activity in hypoglossal motoneurons of adult rats following peripheral nerve injury.

Peripheral nerve injury (PNI) produces functional changes in lesioned neurons in which oxidative stress is considered to be the main cause of neuronal damage. As superoxide dismutase (SOD) is an important antioxidative enzyme involved in redox regulation of oxidative stress, the present study determined whether melatonin would exert its beneficial effects by preserving the SOD reactivity following PNI. Adult rats subjected to hypoglossal nerve transection were intraperitoneally injected with melatonin at ones for 3, 7, 14, 30 and 60 days successively. The potential neuroprotective effects of melatonin were quantitatively demonstrated by neuronal nitric oxide synthase (nNOS), mitochondrial manganese SOD (Mn-SOD), and cytosolic copper-zinc SOD (Cu/Zn-SOD) immunohistochemistry. The functional recovery of the lesioned neurons was evaluated by choline acetyltransferase (ChAT) immunohistochemistry along with the electromyographic (EMG) recordings of denervation-induced fibrillation activity. The results indicate that following PNI, the nNOS immunoreactivity was significantly increased in lesioned neurons peaking at 14 days. The up-regulation of nNOS temporally coincided with the reduction of ChAT and SOD in which the Cu/Zn-SOD showed a greater diminution than Mn-SOD. However, following melatonin administration, the nNOS augmentation was successfully suppressed and the activities of Mn-SOD, Cu/Zn-SOD, and ChAT were effectively preserved at all postaxotomy periods. EMG data also showed a decreased fibrillation in melatonin-treated groups, suggesting a potential effect of melatonin in promoting functional recovery. In association with its significant capacity in preserving SOD reactivity, melatonin is suggested to serve as a powerful therapeutic agent for treating PNI-relevant oxidative damage.

Mild hypoxic preconditioning attenuates injury-induced NADPH-d/nNOS expression in brainstem motor neurons of adult rats.

Excessive production of nitric oxide (NO) might have detrimental effects on the hypoxia-related neuropathology. This study aimed to test if mild hypoxic preconditioning (MHPC) would attenuate the pathological changes in the brainstem motoneurons having a different functional component after peripheral nerve crush injury (PNCI). Prior to PNCI treatment, young adult rats were caged in the mild hypoxic altitude chamber with 79Torr of the partial oxygen concentration ( pO(2)) (i.e., 0.5atm at 5500m in height) for 4 weeks to adapt the environmental changes. After that, all the animals having successfully crushed both the hypoglossal and vagus nerves (left-side) were allowed to survive for 3, 7, 14, 30 and 60 successive days in normoxic condition. Nicotinamine adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry revealed that MHPC reduces NADPH-d/nNOS expression in the hypoglossal nucleus (HN) and the dorsal motor nucleus of the vagus (DMN) at different time points after PNCI. The morphological findings were further ascertained by Western blot analysis of nNOS and nitrite assay for NO production. Both the morphological and quantitative results peaked at 7 days in HN, whereas for those in DMN were progressively increased up to 60 days following PNCI. The staining intensity of NADPH-d/nNOS(+) neurons, expression of nNOS protein, NO production levels as well as the neuronal loss in HN and DMN of MHPC rats following PNCI were attenuated, especially for those having a longer survival period over 14 days. The MHPC treatment might induce minute amounts of NO to alter the state of milieu of the experimental animals to protect against the PNCI.

Inhibition of resting potassium conductances by long-term activation of the NO/cGMP/protein kinase G pathway: a new mechanism regulating neuronal excitability.

Glutamate-induced excitotoxicity, the most common pathological mechanism leading to neuronal death, may occur even with normal levels of glutamate if it coincides with a persistent enhancement of neuronal excitability. Neurons expressing nitric oxide (NO) synthase (NOS-I), which is upregulated in many human chronic neurodegenerative diseases, are highly susceptible to neurodegeneration. We hypothesized that chronic production of NO in damaged neurons may increase their intrinsic excitability via modulation of resting or "leak" K+ currents. Peripheral XIIth nerve injury in adult rats induced de novo NOS-I expression and an increased incidence of low-threshold motor units, the latter being prevented by chronic inhibition of the neuronal NO/cGMP pathway. Accordingly, sustained synthesis of NO maintained an enhanced basal activity in injured motoneurons that was slowly reverted (over the course of 2-3 h) by NOS-I inhibitors. In slice preparations, persistent, but not acute, activation of the NO/cGMP pathway evoked a robust augment in motoneuron excitability independent of synaptic activity. Furthermore, chronic activation of the NO/cGMP pathway fully suppressed TWIK-related acid-sensitive K+ (TASK) currents through a protein kinase G (PKG)-dependent mechanism. Finally, we found evidence for the involvement of this long-term mechanism in regulating membrane excitability of motoneurons, because their pH-sensitive currents were drastically reduced by nerve injury. This NO/cGMP/PKG-mediated modulation of TASK conductances might represent a new pathological mechanism that leads to hyperexcitability and sensitizes neurons to excitotoxic damage. It could explain why de novo expression of NOS-I and/or its overexpression makes them susceptible to neurodegeneration under pathological conditions.

Nitrergic innervation of trigeminal and hypoglossal motoneurons in the cat.

The present study was undertaken to determine the location of trigeminal and hypoglossal premotor neurons that express neuronal nitric oxide synthase (nNOS) in the cat. Cholera toxin subunit b (CTb) was injected into the trigeminal (mV) or the hypoglossal (mXII) motor nuclei in order to label the corresponding premotor neurons. CTb immunocytochemistry was combined with NADPH-d histochemistry or nNOS immunocytochemistry to identify premotor nitrergic (NADPH-d(+)/CTb(+) or nNOS(+)/ CTb(+) double-labeled) neurons. Premotor trigeminal as well as premotor hypoglossal neurons were located in the ventro-medial medullary reticular formation in a region corresponding to the nucleus magnocellularis (Mc) and the ventral aspect of the nucleus reticularis gigantocellularis (NRGc). Following the injection of CTb into the mV, this region was found to contain a total of 60 +/- 15 double-labeled neurons on the ipsilateral side and 33 +/- 14 on the contralateral side. CTb injections into the mXII resulted in 40 +/- 17 double-labeled neurons in this region on the ipsilateral side and 16 +/- 5 on the contralateral side. Thus, we conclude that premotor trigeminal and premotor hypoglossal nitrergic cells coexist in the same medullary region. They are colocalized with a larger population of nitrergic cells (7200 +/- 23). Premotor neurons in other locations did not express nNOS. The present data demonstrate that a population of neurons within the Mc and the NRGc are the source of the nitrergic innervation of trigeminal and hypoglossal motoneurons. Based on the characteristics of nitric oxide actions and its diffusibility, we postulate that these neurons may serve to synchronize the activity of mV and mXII motoneurons.

Nitric-oxide-directed synaptic remodeling in the adult mammal CNS.

In adult mammals, learning, memory, and restoration of sensorimotor lost functions imply synaptic reorganization that requires diffusible messengers-mediated communication between presynaptic and postsynaptic structures. A candidate molecule to accomplish this function is the gaseous intercellular messenger nitric oxide (NO), which is involved in synaptogenesis and projection refinement during development; however, the role of NO in synaptic reorganization processes in adulthood remains to be established. In this work, we tested the hypothesis that this free radical is a mediator in the adult mammal CNS synaptic remodeling processes using a model of hypoglossal axonal injury recently developed by us. Axonal injury-induced disconnection of motoneurons from myocytes produces withdrawal of synaptic inputs to motoneurons and concomitant upregulation of the neuronal isoform of NO synthase (NOS-I). After recovery of the neuromuscular function, synaptic coverage is reestablished and NOS-I is downregulated. We also report, by using functional and morphological approaches, that chronic inhibition of the NO/cGMP pathway prevents synaptic withdrawal evoked by axon injury, despite the persistent muscle disconnection. After successful withdrawal of synaptic boutons, inhibition of NO synthesis, but not of cGMP, accelerated the recovery of synaptic coverage, although neuromuscular disconnection was maintained. Furthermore, protein S-nitrosylation was upregulated after nerve injury, and this effect was reversed by NOS-I inhibition. Our results suggest that during synaptic remodeling in the adult CNS, NO acts as a signal for synaptic detachment and inhibits synapse formation by cGMP-dependent and probably S-nitrosylation-mediated mechanisms, respectively. We also suggest a feasible role of NO in neurological disorders coursing with NOS-I upregulation.

Regulation of stearoyl-CoA desaturase-1 after central and peripheral nerve lesions.

BACKGROUND: Interruption of mature axons activates a cascade of events in neuronal cell bodies which leads to various outcomes from functional regeneration in the PNS to the failure of any significant regeneration in the CNS. One factor which seems to play an important role in the molecular programs after axotomy is the stearoyl Coenzyme A-desaturase-1 (SCD-1). This enzyme is needed for the conversion of stearate into oleate. Beside its role in membrane synthesis, oleate could act as a neurotrophic factor, involved in signal transduction pathways via activation of protein kinases C. RESULTS: In situ hybridization and immunohistochemistry demonstrated a strong up-regulation of SCD at mRNA and protein level in regenerating neurons of the rat facial nucleus whereas non-regenerating Clarke's and Red nucleus neurons did not show an induction of this gene. CONCLUSION: This differential expression points to a functionally significant role for the SCD-1 in the process of regeneration.

Postnatal changes in cytochrome oxidase expressions in brain stem nuclei of rats: implications for sensitive periods.

Previously, we reported that cytochrome oxidase (CO) activity in the rat pre-Bötzinger complex (PBC) exhibited a plateau on postnatal days (P) 3-4 and a prominent decrease on P12 (Liu and Wong-Riley, J Appl Physiol 92: 923-934, 2002). These changes were correlated with a concomitant reduction in the expression of glutamate and N-methyl-d-aspartate receptor subunit 1 and an increase in GABA, GABAB, glycine receptor, and glutamate receptor 2. To determine whether changes were limited to the PBC, the present study aimed at examining the expression of CO in a number of brain stem nuclei, with or without known respiratory functions from P0 to P21 in rats: the ventrolateral subnucleus of the solitary tract nucleus, nucleus ambiguus, hypoglossal nucleus, nucleus raphe obscurus, dorsal motor nucleus of the vagus nerve, medial accessory olivary nucleus, spinal nucleus of the trigeminal nerve, and medial vestibular nucleus (MVe). Results indicated that, in all of the brain stem nuclei examined, CO activity exhibited a general increase with age from P0 to P21, with MVe having the slowest rise. Notably, in all of the nuclei examined except for MVe, there was a plateau or decrease at P3-P4 and a prominent rise-fall-rise pattern at P11-P13, similar to that observed in the PBC. In addition, there was a fall-rise-fall pattern at P15-P17 in these nuclei, instead of a plateau pattern in the PBC. Our data suggest that the two postnatal periods with reduced CO activity, P3-P4 and especially P12, may represent common sensitive periods for most of the brain stem nuclei with known or suspected respiratory control functions.

IPP isomerase, an enzyme of mevalonate pathway, is preferentially expressed in postnatal cortical neurons and induced after nerve transection.

To understand the molecular mechanisms underlying the developmental processes of the cerebral cortex, we screened genes whose mRNA expression was up-regulated in neonatal in the rat cortex to a greater extent than in adult by differential display and obtained five genes. Among these genes, we focused on pyrophosphate (isopentenyl diphosphate, dimethylallyl diphosphate: IPP) isomerase gene, the product of which is known as an enzyme of the mevalonate pathway. Rat IPP isomerase was recently cloned and the gene expression was shown to be dependent on the activation of the mevalonate pathway. Its expression and roles in the brain, however, have not been investigated hitherto. In the present study, Northern blots and in situ hybridization analysis showed that at embryonic stage weak signals for IPP mRNA were diffusely detected in the CNS, and the signal in the cortex became intense at postnatal day 1 and maximized in almost all neurons of all layers at postnatal day 7 with a subsequent reduction. At 8 weeks, the expression of IPP isomerase mRNA in neurons decreased, while it was detected in the oligodendrocytes in the regions containing abundant nerve fibers. These findings suggested that IPP isomerase contributes to postnatal neuronal maturation and myelination. We also demonstrated that IPP isomerase mRNA is induced after nerve axotomy, which suggests a relationship between neuronal regeneration and IPP isomerase. Taken together, these results suggest that elevation of IPP isomerase mRNA levels in neurons contributes to construction of nerve fibers both during the postnatal period in the cortex and their regeneration.

CEP-1347/KT7515 prevents motor neuronal programmed cell death and injury-induced dedifferentiation in vivo.

CEP-1347, also known as KT7515, a derivative of a natural product indolocarbazole, inhibited motor neuronal death in vitro, inhibited activation of the stress-activated kinase JNK1 (c-jun NH terminal kinase) in cultured spinal motor neurons, but had no effect on the mitogen-activated protein kinase ERK1 in these cells. Results reported here profile the functional activity of CEP-1347/KT7515 in vivo in models of motor neuronal death or dedifferentiation. Application of CEP-1347/KT7515 to the chorioallantoic membrane of embryonic chicks rescued 40% of the lumbar motor neurons that normally die during the developmental period assessed. Peripheral administration of low doses (0.5 and 1 mg/kg daily) of CEP-1347/KT7515 reduced death of motor neurons of the spinal nucleus of the bulbocavernosus in postnatal female rats, with efficacy comparable to testosterone. Strikingly, daily administration of CEP-1347/KT7515 during the 4-day postnatal window of motor neuronal death resulted in persistent long-term motor neuronal survival in adult animals that received no additional CEP-1347/KT7515. In a model of adult motor neuronal dedifferentiation following axotomy, local application of CEP-1347/KT7515 to the transected hypoglossal nerve substantially reduced the loss of choline acetyl transferase immunoreactivity observed 7 days postaxotomy compared to untreated animals. Results from these experiments demonstrate that a small organic molecule that inhibits a signaling pathway associated with stress and injury also reduces neuronal death and degeneration in vivo.

Evidence for reduced nitric oxide synthase (NOS) activity in the ipsilateral medial vestibular nucleus and bilateral prepositus hypoglossi following unilateral vestibular deafferentation in the guinea pig.

The aim of the present study was to examine, using a radioenzymatic assay technique, nitric oxide synthase (NOS) activity in the bilateral medial vestibular nuclei (MVN) and prepositus hypoglossi (PH), during the development of vestibular compensation for unilateral vestibular deafferentation (UVD) in the guinea pig. In the MVN ipsilateral to the UVD, and bilaterally in PH, NOS activity decreased following UVD compared to sham controls and did not recover significantly up to 50 h later, when a substantial degree of behavioural vestibular compensation had occurred. These results suggest that UVD causes a decrease in NOS activity in the ipsilateral MVN and the bilateral PH, and that a consequent decrease in NO may be responsible for some of the ocular motor and postural symptoms of UVD.

Expression of glutamate receptor subunit 1 and nitric oxide synthase in the hypoglossal nucleus and dorsal vagal nucleus in the rat after neurectomy.

Using nitric oxide synthase (NOS) and glutamate receptor subunit 1 (GluR1) immunohistochemistry, the present study demonstrated changes in the expression of NOS and GluR1 in the hypoglossal (HN) and dorsal vagal nucleus (DVN) after neurectomy. Two and 7 days after sectioning the left hypoglossal nerve, NOS expression was seen in a few neurons but GluR1 immunoreactivity was drastically reduced in the ipsilateral HN. The upregulation of NOS immunoreactivity in the HN appeared to peak at 14 days postoperation (dpo). At this period, however, the GluR1 immunoreactivity almost completely disappeared. Twenty-one, 35 and 56 days after neurectomy, NOS immunoreactivity was still expressed in the ipsilateral HN; at the same time, GluR1 immunoreactivity reappeared in a few neurons of the nucleus. Ninety days after operation, NOS immunoreactivity completely disappeared on the operated side of the nucleus, but GluR1 immunoreactivity was re-expressed in many hypoglossal neurons. The number of such neurons was obviously less than that on the unoperated side. After sectioning the left vagus nerve in the same animals, the expression of NOS immunoreactivity in the ipsilateral DVN resembled that in the HN. On the unoperated side, NOS immunoreactivity was demonstrated in some neurons in the DVN, like that in the normal. In both normal and operated rats, only a few neurons expressed GluR1 immunoreactive products on both the operated and unoperated sides of the DVN. Combining with previous results on protein synthesis observed at 14 dpo, the present investigation suggested that in the early stages after neurectomy, the expression of NOS immunoreactivity and loss of GluR1 expression in the HN may indicate the organism's double protective mechanism. Lastly, the reappearance of GluR1 in the same nucleus from 21 to 90 days after operation may reflect functional recovery of the hypoglossal neurons.

Regulation of nitric oxide synthase expression in motoneurons following nerve injury.

This study investigated the influence of postsynaptic targets and axonal ensheathing cells on the expression of nitric oxide synthase (NOS) in axotomized neurons. The hypoglossal and vagus nerves of adult rats were lesioned unilaterally, and axotomized neurons expressing NOS, identified by NADPH-diaphorase histochemistry and NOS immunocytochemistry, were counted as a function of time between 1 and 70 days postaxotomy. In the dorsal motor nucleus of the vagus (DMV), the number of NOS-positive neurons increased steadily with a time course which was not significantly different between the nerve crushed and transected groups. In the hypoglossal nucleus, each of four axotomy groups, namely, crushed, transected, ligated/transected, and avulsed, exhibited a distinct time course and extent of NOS expression, suggesting that postsynaptic targets and axonal ensheathing cells regulated NOS expression. The disparity between hypoglossal and DMV neurons in NOS expression may be due to the latters' unresponsiveness to, or inability to obtain the proper regulatory signals. Since cell loss was most severe in the hypoglossal nucleus following nerve avulsion, it appears that prolonged expression of NOS at high levels may be neurotoxic.

Pre- and postnatal development of the catecholamine innervation of the hypoglossal nucleus in the rat: an immunocytochemical study.

The pre- and postnatal development of the catecholamine (CA) innervation to the hypoglossal nucleus (nXII) in the rat was investigated immunocytochemically with antisera to tyrosine hydroxylase (TH). Immunoreactive profiles positive for TH were first identified in nXII on gestational day (GD) 16. By GD 18, the adult-like distribution pattern was evident, characterized by the preferential targeting of the ventromedial region of nXII, but this pattern was not consistently found in all fetuses until GD 19. From GD 19 to postnatal day (PD) 180, the overall density of TH immunoreactivity, particularly in the ventromedial region, increased with further growth and maturation of nXII. These results establish the early prenatal CA innervation of nXII and support the hypothesis that CA are important in regulating motor tongue behavior in the newborn. Moreover, because the ventral compartment of nXII contains motoneurons that innervate protrusor muscles of the tongue, and tongue protrusor mechanisms play an essential role in suckling, deglutition, and respiratory (maintaining a patent upper airway) behaviors, it is further proposed that the CA innervation of nXII is critical to the survival of the newborn.

Axotomy induces intranuclear immunolocalization of neuron-specific enolase in facial and hypoglossal neurons of the rat.

Neuron-specific enolase as an enzyme of the glycolytic pathway is localized in the cytoplasm of nerve cells, but not in the cell nucleus. We have applied immunocytochemistry with 1:64,000 polyclonal anti-rat neuron-specific enolase to the brainstem of male and female adult Wistar rats following: (a) transection of the facial nerve with immediate microsurgical nerve suture (facial-facial anastomosis), (b) transection of the hypoglossal nerve with immediate suture (hypoglossal-hypoglossal anastomosis) and (c) transection of the facial and hypoglossal nerve with immediate suture of the proximal hypoglossal to the distal facial nerve stump (hypoglossal-facial anastomosis). Studying the intracellular immunolocalization of neuron-specific enolase in neurons of the facial and hypoglossal nucleus we detected that (1) in normal rats about 20% of all facial and hypoglossal neurons display not only cytoplasmic, but also intranuclear neuron-specific enolase-like immunoreactivity and (2) following any axotomy of the facial or hypoglossal peripheral nerve, the perikarya of all injured motoneurons react by an outstanding increase of neuron-specific enolase-like immunoreactivity in the karyoplasm. Similar findings were obtained in experiments on non-fixed cultured Neuro-2a cells that had been lesioned with hydrogen peroxide. Counting the absolute numbers of normal and reactive neurons at 1-365 days post axotomy revealed that the increase of neuron-specific enolase in neuronal cell nuclei is temporary and reversible. It is first detected at 2 days post axotomy, reaches its maximum at 10-18 days post axotomy and is no longer evident 56 days following surgery.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific expression of type II protein kinase c after axotomy in the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus.

Protein kinase C (PKC) and growth-associated protein-43 (GAP-43) were investigated immunohistochemically in the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus after axotomy using monoclonal antibodies against type I, II and III PKC and GAP-43. In the control side of both nuclei, anti-type I and II PKC weakly stained neuronal cell bodies, while anti-type III PKC did not show any reaction with neurons. In the axotomized side of both nuclei, anti-type II PKC antibody intensely stained affected nerve cell bodies as well as plasma membrane. Some of the severed neurons showed intensified reactions for both anti-type II PKC and anti-GAP-43 antibodies in the serial sections. These findings suggest that axotomy increases the type II PKC of the severed neurons, and type II PKC seems to phosphorylate some protein, such as GAP-43, and plays some role in the retrograde neuronal reaction.