Daily acute intermittent hypoxia enhances serotonergic innervation of hypoglossal motor nuclei in rats with and without cervical spinal injury.

Intermittent hypoxia elicits protocol-dependent effects on hypoglossal (XII) motor plasticity. Whereas low-dose, acute intermittent hypoxia (AIH) elicits serotonin-dependent plasticity in XII motor neurons, high-dose, chronic intermittent hypoxia (CIH) elicits neuroinflammation that undermines AIH-induced plasticity. Preconditioning with repeated AIH and mild CIH enhance AIH-induced XII motor plasticity. Since intermittent hypoxia pre-conditioning could enhance serotonin-dependent XII motor plasticity by increasing serotonergic innervation density of the XII motor nuclei, we tested the hypothesis that 3 distinct intermittent hypoxia protocols commonly studied to elicit plasticity (AIH) or simulate aspects of sleep apnea (CIH) differentially affect XII serotonergic innervation. Sleep apnea and associated CIH are common in people with cervical spinal injuries and, since repetitive AIH is emerging as a promising therapeutic strategy to improve respiratory and non-respiratory motor function after spinal injury, we also tested the hypotheses that XII serotonergic innervation is increased by repetitive AIH and/or CIH in rats with cervical C2 hemisections (C2Hx). Serotonergic innervation was assessed via immunofluorescence in male Sprague Dawley rats, with and without C2Hx (beginning 8 weeks post-injury) exposed to 28 days of: 1) normoxia; 2) daily AIH (10, 5-min 10.5% O2 episodes per day; 5-min normoxic intervals); 3) mild CIH (5-min 10.5% O2 episodes; 5-min intervals; 8 h/day); and 4) moderate CIH (2-min 10.5% O2 episodes; 2-min intervals; 8 h/day). Daily AIH, but neither CIH protocol, increased the area of serotonergic immunolabeling in the XII motor nuclei in both intact and injured rats. C2Hx per se had no effect on XII serotonergic innervation density. Thus, daily AIH may increases XII serotonergic innervation and function, enhancing the capacity for serotonin-dependent, AIH-induced plasticity in upper airway motor neurons. Such effects may preserve upper airway patency and/or swallowing ability in people with cervical spinal cord injuries and other clinical disorders that compromise breathing and airway defense.

Microglia support ATF3-positive neurons following hypoglossal nerve axotomy.

Microglia are essential in developmental processes and maintenance of neuronal homeostasis. Experimental axotomy of motor neurons results in neurodegeneration, and microglia in motor nuclei become activated and migrate towards injured neurons. However, whether these activated microglia are protective or destructive to neurons remains controversial. In the present study, we transected the hypoglossal nerve in BALB/c mice, causing activating transcription factor 3 (ATF3) and growth associated protein 43 (GAP43) induction, and partial neuronal death. Inhibition of microglial accumulation by minocycline administration impaired microglial accumulation, decreased GAP43 mRNA expression, and reduced motor neuron survival. Expression of ATF3 contributed to nerve regeneration, and increased within 6 h after axotomy, prior to microglial migration. Further, microglial contact with neuronal cell bodies was associated with neuronal ATF3 expression. Colchicine administration blocked lesion-induced ATF3 transcription in axotomized neurons and microglial accumulation. In addition, perineuronal microglia-derived ciliary neurotrophic factor (CNTF) increased, indicating that perineuronal microglia in the hypoglossal nucleus protect axotomized motor neurons by releasing trophic factors. We also observed that microglia secrete CNTF and that neurons have CNTFRα and can respond to it in vitro. CNTF promote neurite elongation and neuronal survival of primary cultured neurons. Microglia make contact through unknown neuronal signals that are possibly regulated by ATF3 in hypoglossal nucleus. Moreover, they play important roles in regenerating motor neurons and are potential new therapeutic targets for motor neuron diseases.

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.

Developmental dissociation of presynaptic inhibitory neurotransmitter and postsynaptic receptor clustering in the hypoglossal nucleus.

At postsynaptic densities of mouse hypoglossal motoneurons, the proportion of glycine receptors co-clustered with GABAA receptors increases from neonatal to adult animals, suggesting that mixed synapses might play a greater role in adult synaptic inhibition. We visualized the presynaptic correlates of these developmental changes using immunocytochemistry. At P5, presynaptic terminals contained glycine and GlyT2 and/or GABA and GAD65, but at P15, the majority of inhibitory terminals contained glycine and GlyT2 only. The GABAergic component of evoked inhibitory postsynaptic currents in HMs decreased strongly between P5 and P15. Similarly, miniature inhibitory postsynaptic currents evolved from mainly glycinergic and mixed glycinergic/GABAergic events at P3-5 to predominantly glycinergic currents at P15. These results indicate that the decrease in the proportion of functional mixed inhibitory synapses with maturation results from a loss of the ability of presynaptic terminals to release both neurotransmitters during development while co-aggregation of GlyRs + GABAARs at postsynaptic loci remained.

Glycine receptors and GABA receptor alpha 1 and gamma 2 subunits during the development of mouse hypoglossal nucleus.

In the hypoglossal nucleus, GABA and glycine mediate inhibition at separate or mixed synapses containing glycine receptors (GlyRs) and/or GABA(A) receptors (GABA(A)Rs). The functional development of mixed inhibitory synapses depends on the brain area studied, but their relative proportion to total synapses generally decreases with time. We have determined the sequential process of inhibitory synapse maturation in the hypoglossal nucleus in vivo. Immunocytochemistry and confocal microscopy were used for codetection of VIAAT, the common presynaptic vesicular transporter of glycine and GABA, GlyRs, GABA(A)R alpha1 and gamma2 subunits, and gephyrin, the scaffold protein implicated in the synaptic localization of inhibitory receptors. In E17 embryos, GlyRs were already clustered while GABA(A)R alpha1 and gamma2 subunit immunoreactivity (IR) displayed both diffuse and clustered patterns. Quantitative analysis at this stage revealed that the majority of GlyR clusters were apposed to VIAAT-IR accumulation and that 30% of them colocalized with gamma2GABA(A)R clusters. This proportion increased with age to 50% at P30. GlyR clusters that did not colocalize with gamma2GABA(A)R clusters were associated with GABA(A)R gamma2 diffuse IR. Interestingly, the percentage of GlyR clusters surrounded by GABA(A)R gamma2 diffuse IR decreased with age, while GlyR clusters colocalized with gamma2GABA(A)R clusters increased. The developmental coclustered pattern of gephyrin and GABA(A)R alpha1 and gamma2 subunits paralleled the coclustered pattern of GlyRs and GABA(A)R alpha1 and gamma2 subunits. Our results indicate that the proportion of GlyR-GABA(A)R coclusters increases until adulthood. A developmental sequence of the postsynaptic events is proposed in which diffuse extrasynaptic GABA(A)Rs accumulate at inhibitory synapses to form postsynaptic clusters, most of them being colocalized with GlyR clusters in the adult.

Early onset of NMDA receptor GluR epsilon 1 (NR2A) expression and its abundant postsynaptic localization in developing motoneurons of the mouse hypoglossal nucleus.

Oro-facial sensorimotor function conducted by the brainstem is vital to newborn mammals, and N-methyl-D-aspartate (NMDA) receptors play an important role in the regulation. Here we examined the expression of NMDA receptor subunits in the mouse hypoglossal nucleus from embryonic day 13 (E13) through postnatal day 21 (P21). Compared with other brainstem regions, early onset of GluRepsilon1 (NR2A) mRNA expression was conspicuous to the embryonic hypoglossal nucleus. The expression peaked at P1-P7, when other brainstem regions just started to express it. At P1, GluRepsilon1 subunit was localized to asymmetrical synapses on motoneuron dendrites, particularly, on the postsynaptic junction membrane. In developing motoneurons, expressions of GluRepsilon2 (NR2B), GluRepsilon4 (NR2D), and GluRzeta1 (NR1) mRNAs were accompanied. Until P21, however, all of these subunits were down-regulated with particular reduction for GluRepsilon2 and GluRepsilon4 mRNAs. Similar patterns of temporal expressions were observed in motoneurons of other brainstem motor nuclei. Taking that high levels of GluRepsilon1, GluRepsilon2, and GluRzeta1 subunits are also found in the adult hippocampus and cerebral cortex, it can be assumed that NMDA receptors in developing motoneurons are highly potent and potentially involved in structural and functional development of the brainstem motor system.

Single cell laser dissection with molecular beacon polymerase chain reaction identifies 2A as the predominant serotonin receptor subtype in hypoglossal motoneurons.

We hypothesize that sleep state-dependent withdrawal of serotonin (5-hydroxytryptamine, 5-HT) at upper airway (UAW) dilator motoneurons contributes significantly to sleep-related suppression of dilator muscle activity in obstructive sleep apnea. Identification of 5-HT receptor subtypes involved in postsynaptic facilitation of UAW motoneuron activity may provide pharmacotherapies for this prevalent disorder. We have adapted two assays to provide semi-quantitative measurements of mRNA copy numbers for 5-HT receptor subtypes in single UAW motoneurons. Specifically, soma of 111 hypoglossal (XII) motoneurons in 10 adult male rats were captured using a laser dissection microscope, and then used individually in single round molecular beacon polymerase chain reaction (PCR) for real-time quantitation of 5-HT(2A), 5-HT(2C), 5-HT(3), 5-HT(4), 5-HT(5A), 5-HT(5B), 5-HT(6) or 5-HT(7) receptor. Receptor mRNA copy numbers from single XII motoneurons were compared to control samples from within the XII nucleus and lateral medulla. All 20 motoneuronal soma assayed for the 5-HT(2A) receptor had measurable copy numbers (7028+/-2656 copies/cell). In contrast, copy numbers for the 5-HT(2A) receptor in XII non-motoneuronal (n=17) and lateral medulla (n=15) samples were 81+/-51 copies and 83+/-35 copies, respectively, P<0.05. Seven of 13 XII motoneurons assayed had measurable 5-HT(2C) receptor copy numbers of mRNA (287+/-112 copies/cell). XII soma had minimal 5-HT(3), 5-HT(4), 5-HT(5A), 5-HT(5B), 5-HT(6) or 5-HT(7) receptor mRNA. 5-HT(2A) receptor mRNA presence within XII motoneurons was confirmed with digoxigenin-labeled in situ hybridization. In summary, combined use of laser dissection and molecular beacon PCR revealed 5-HT(2A) receptor as the predominant 5-HT receptor mRNA in XII motoneurons, and identified small quantities of 5-HT(2C) receptor. This information will allow a more complete understanding of serotonergic control of respiratory activity.

Location of renin-angiotensin system components in the hypoglossal nucleus of the rat.

The renin-angiotensin system (RAS) in the hypoglossal nuclei of the rat was studied by immunohistochemistry. Antibodies to angiotensin AT(1) receptor (AT1), angiotensinogen (ANG), renin (REN), angiotensin converting enzyme (ACE) and angiotensin II (AII) were used. All the components of the RAS with the exception of renin were detected. Light and electron microscopy revealed the following results: ANG was predominantly found in astrocytes, with small amounts in neuronal dendrites; ACE was found in the cytoplasm of neurons, dendrites and astrocyte processes; AT1 was found in the cytoplasm of neurons and dendrites, but not on the membrane; and AII was found mainly in astrocytes with some located in the dendrites and cytoplasm. Right hypoglossal nerve lesion caused an increase in expression of AT1 in neurons as early as 2 days post-lesion. An increase in expression of ANG in astrocytes was also seen, but at a much later time of 3 weeks post-lesion. For AII, staining occurred in both the neurons and astrocytes in the undamaged hypoglossal nucleus. Nerve lesion caused a disappearance of neuronal stains and an increase in astrocyte stains. There were no changes in ACE staining after nerve lesion. We speculate that ANG and AII are made within the astrocytes, whereas ACE could either be uptaken from blood or de novo synthesized. AT1 may potentially be internal soluble receptors. As to the function of AII in the hypoglossal nucleus, the data do not support AII as a neurotransmitter in the hypoglossal nucleus. It may function as a neuromodulator and also be involved in basic cellular activities, e.g. regulation of transcription factors.

Modulation of hypoglossal motoneuron excitability by NK1 receptor activation in neonatal mice in vitro.

1. The effects of substance P (SP), acting at NK1 receptors, on the excitability and inspiratory activity of hypoglossal (XII) motoneurons (MNs) were investigated using rhythmically active medullary-slice preparations from neonatal mice (postnatal day 0-3). 2. Local application of the NK1 agonist [SAR(9),Met (O(2))(11)]-SP (SP(NK1)) produced a dose-dependent, spantide- (a non-specific NK receptor antagonist) and GR82334-(an NK1 antagonist) sensitive increase in inspiratory burst amplitude recorded from XII nerves. 3. Under current clamp, SP(NK1) significantly depolarized XII MNs, potentiated repetitive firing responses to injected currents and produced a leftward shift in the firing frequency-current relationships without affecting slope. 4. Under voltage clamp, SP(NK1) evoked an inward current and increased input resistance, but had no effect on inspiratory synaptic currents. SP(NK1) currents persisted in the presence of TTX, were GR82334 sensitive, were reduced with hyperpolarization and reversed near the expected E(K). 5. Effects of the alpha(1)-noradrenergic receptor agonist phenylephrine (PE) on repetitive firing behaviour were virtually identical to those of SP(NK1). Moreover, SP(NK1) currents were completely occluded by PE, suggesting that common intracellular pathways mediate the actions of NK1 and alpha(1)-noradrenergic receptors. In spite of the similar actions of SP(NK1) and PE on XII MN responses to somally injected current, alpha(1)-noradrenergic receptor activation potentiated inspiratory synaptic currents and was more than twice as effective in potentiating XII nerve inspiratory burst amplitude. 6. GR82334 reduced XII nerve inspiratory burst amplitude and generated a small outward current in XII MNs. These observations, together with the first immunohistochemical evidence in the newborn for SP immunopositive terminals in the vicinity of SP(NK1)-sensitive inspiratory XII MNs, support the endogenous modulation of XII MN excitability by SP. 7. In contrast to phrenic MNs (Ptak et al. 2000), blocking NMDA receptors with AP5 had no effect on the modulation of XII nerve activity by SP(NK1). 8. In conclusion, SP(NK1) modulates XII motoneuron responses to inspiratory drive primarily through inhibition of a resting, postsynaptic K+ leak conductance. The results establish the functional significance of SP in controlling upper airway tone during early postnatal life and indicate differential modulation of motoneurons controlling airway and pump muscles by SP.

Hypocretin (orexin) input to trigeminal and hypoglossal motoneurons in the cat: a double-labeling immunohistochemical study.

In trigeminal and hypoglossal motor nuclei of adult cats, hypocretin immunoreactive fiber varicosities were observed in apposition to retrogradely labeled motoneuron somata and dendrites. Among those lateral hypothalamus neurons that project to the hypoglossal nucleus some were determined to be hypocretin immunoreactive and were located amongst the single-labeled hypocretinergic neurons. These data suggest that hypocretin may play a role in the synaptic control of these motoneurons.

Expression of hypoglossal long-term facilitation differs between substrains of Sprague-Dawley rat.

Long-term facilitation (LTF) is a prolonged, serotonin-dependent augmentation of respiratory motor output following episodic hypoxia. Previous observations lead us to hypothesize that LTF is subject to genetic influences and, as a result, differs between Sprague-Dawley (SD) rats from two vendors, Harlan (H) and Charles River Laboratories/Sasco (CRL/S). Using a blinded experimental design, we recorded integrated phrenic (integralPhr) and hypoglossal neurograms in anesthetized, vagotomized, paralyzed, and ventilated rats. At 60 min following three 5-min hypoxic episodes (Pa(O(2)) = 40 +/- 1 Torr; 5-min hyperoxic intervals), integralPhr was elevated from baseline in both SD substrains (i.e., LTF; P < 0.05). Conversely, hypoglossal LTF was present in CRL/S but not H rats (P < 0.05 between substrains). Serotonin immunoreactivity within the hypoglossal nucleus was not different between H and CRL/S rats. We conclude that the expression of hypoglossal LTF differs between SD rat substrains, indicating a difference in their genetic predisposition to neural plasticity.

Morphological changes and expression of protein kinase CK2 beta subunit in the microglia after hypoglossal nerve transection.

Following hypoglossal nerve transection, the microglia of the rat hypoglossal nucleus expressed protein kinase CK2 beta subunit immunoreactivity. CK2 beta immunostaining occurred on the operated side from postoperative day 3; on day 5 we observed strong immunoreactivity and the immunopositive microglial cell processes surrounded the injured neurones. Thereafter, the immunoreactivity decreased gradually and on day 10 the immunopositive cells surrounded only a few injured neurones. Electron microscopic observations on the hypoglossal nucleus revealed microglia-neuronal contact within 3 hours of nerve injury, and by day 3 all the injured neurones were in contact with microglial cells. These observations indicated that microglia-neuronal contact occurred earlier than the CK2 beta subunit immunoreactivity. CK2 may not be implicated during the initial migration of the microglia to the injured neurones; however, it may enhance the growth and elongation of the microglial cell processes around the injured neurones.

Fos induction in selective hypothalamic neuroendocrine and medullary nuclei by intravenous injection of urocortin and corticotropin-releasing factor in rats.

CRF and urocortin, administrated systemically, exert peripheral biological actions which may be mediated by brain pathways. We identified brain neuronal activation induced by intravenous (i.v.) injection of CRF and urocortin in conscious rats by monitoring Fos expression 60 min later. Both peptides (850 pmol/kg, i.v.) increased the number of Fos immunoreactive cells in the paraventricular nucleus of the hypothalamus, supraoptic nucleus, central amygdala, nucleus tractus solitarius and area postrema compared with vehicle injection. Urocortin induced a 4-fold increase in the number of Fos-positive cells in the supraoptic nucleus and a 3.4-fold increase in the lateral magnocellular part of the paraventricular nucleus compared with CRF. Urocortin also elicited Fos expression in the accessory hypothalamic neurosecretory nuclei, ependyma lining the ventricles and choroid plexus which was not observed after CRF. The intensity and pattern of the Fos response were dose-related (85, 255 and 850 pmol/kg, i.v.) and urocortin was more potent than CRF. Neither CRF nor urocortin induced Fos expression in the lateral septal nucleus, Edinger-Westphal nucleus, dorsal raphe nucleus, locus coeruleus, or hypoglossal nucleus. These results show that urocortin, and less potently CRF, injected into the circulation at picomolar doses activate selective brain nuclei involved in the modulation of autonomic/endocrine function; in addition, urocortin induces a distinct activation of hypothalamic neuroendocrine neurons.

Contribution of single-channel properties to the time course and amplitude variance of quantal glycine currents recorded in rat motoneurons.

The amplitude of spontaneous, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) recorded in hypoglossal motoneurons (HMs) in an in vitro brain stem slice preparation increased over the first 3 postnatal weeks, from 42 +/- 6 pA in neonate (P0-3) to 77 +/- 11 pA in juvenile (P11-18) HMs. Additionally, mIPSC amplitude distributions were highly variable: CV 0.68 +/- 0.05 (means +/- SE) for neonates and 0.83 +/- 0.06 for juveniles. We wished to ascertain the contribution of glycine receptor (GlyR)-channel properties to this change in quantal amplitude and to the amplitude variability and time course of mIPSCs. To determine whether a postnatal increase in GlyR-channel conductance accounted for the postnatal change in quantal amplitude, the conductance of synaptic GlyR channels was determined by nonstationary variance analysis of mIPSCs. It was 48 +/- 8 pS in neonate and 46 +/- 10 pS in juvenile HMs, suggesting that developmental changes in mIPSC amplitude do not result from a postnatal alteration of GlyR-channel conductance. Next we determined the open probability (Popen) of GlyR channels in outside-out patches excised from HMs to estimate the contribution of stochastic channel behavior to quantal amplitude variability. Brief (1 ms) pulses of glycine (1 mM) elicited patch currents that closely resembled mIPSCs. The GlyR channels' Popen, calculated by nonstationary variance analysis of these currents, was approximately 0.70 (0.66 +/- 0.09 in neonates and 0.72 +/- 0.05 in juveniles). The decay rate of patch currents elicited by brief application of saturating concentrations of glycine (10 mM) increased postnatally, mimicking previously documented changes in mIPSC time course. Paired pulses of glycine (10 mM) were used to determine if rapid GlyR-channel desensitization contributed to either patch current time course or quantal amplitude variability. Because we did not observe any fast desensitization of patch currents, we believe that fast desensitization of GlyRs underlies neither phenomenon. From our analysis of glycinergic patch currents and mIPSCs, we draw three conclusions. First, channel deactivation is the primary determinant of glycinergic mIPSC time course, and postnatal changes in channel deactivation rate account for observed developmental changes in mIPSC decay rate. Second, because GlyR-channel Popen is high, differences in receptor number between synapses rather than stochastic channel behavior are likely to underlie the majority of quantal variability seen at glycinergic synapses throughout postnatal development. We estimate the number of GlyRs available at a synapse to be on average 27 in neonate neurons and 39 in juvenile neurons. Third, this change in the calculated number of GlyRs at each synapse may account for the postnatal increase in mIPSC amplitude.

Ultrastructure of enkephalin- and substance P-immunoreactive axon terminals in the hypoglossal nucleus of the chicken.

The morphology and distribution of leucine-enkephalin (LENK)- and substance P (SP)-containing axon terminals in the hypoglossal nucleus of the chicken was investigated immunocytochemically at the electron microscopic level. The major findings of the present study were as follows: 1) Both LENK- and SP-immunoreactive (IR) profiles including unmyelinated axons, varicosities and axon terminals were found throughout the hypoglossal nucleus. 2) LENK-IR profiles without synapses (preterminal axons and varicosities) were observed more frequently (66%) than LENK-IR synaptic profiles (34%). In contrast, SP-IR synaptic terminals were seen more often (68%) than SP-IR terminals without synapses (32%). 3) Both LENK- and SP-IR synaptic terminals ending on dendrites were more numerous (83% and 94%, respectively) than those making synapses on somata (17% and 6%, respectively). 4) The average diameter of SP-IR synaptic terminals were significantly larger than that of LENK-IR ones. 5) Both LENK- and SP-IR synaptic terminals contained numerous small clear vesicles and several large dense-cored vesicles (LDV). Although both LENK- and SP-IR synaptic terminals contained LDV consistently, SP-IR synaptic terminals contained significantly more LDV than LENK-IR types. 6) Postsynaptic dense bodies (Taxi bodies) were seen more frequently in SP-IR synaptic terminals than in LENK-IR ones. These findings confirm both LENK and SP innervation in the hypoglossal nucleus and suggest a functional role as neuromodulator for the two neuropeptides in tongue control.

Kir2.4: a novel K+ inward rectifier channel associated with motoneurons of cranial nerve nuclei.

Members of the Kir2 subfamily of inwardly rectifying K+ channels characterized by their strong current rectification are widely expressed both in the periphery and in the CNS in mammals. We have cloned from rat brain a fourth subfamily member, designated Kir2.4 (IRK4), which shares 53-63% similarity to Kir2.1, Kir2.2, or Kir2.3 on the amino acid level. In situ hybridization analysis identifies Kir2.4 as the most restricted of all Kir subunits in the brain. Kir2. 4 transcripts are expressed predominantly in motoneurons of cranial nerve motor nuclei within the general somatic and special visceral motor cell column and thus are uniquely related to a functional system. Heterologous expression of Kir2.4 in Xenopus oocytes and mammalian cells gives rise to low-conductance channels (15 pS), with an affinity to the channel blockers Ba2+ (Ki = 390 microM) and Cs+ (Ki = 8.06 mM) 30-50-fold lower than in other Kir channels. Low Ba2+ sensitivity allows dissection of Kir2.4 currents from other Kir conductances in hypoglossal motoneurons (HMs) in rat brainstem slices. The finding that Ba2+-mediated block of Kir2.4 in HMs evokes tonic activity and increases the frequency of induced spike discharge indicates that Kir2.4 channels are of major importance in controlling excitability of motoneurons in situ.

Localization of parvalbumin, calretinin, and calbindin D-28k in identified extraocular motoneurons and internuclear neurons of the cat.

Calcium-binding proteins have been shown to be excellent markers of specific neuronal populations. We aimed to characterize the expression of calcium-binding proteins in identified populations of the cat extraocular motor nuclei by means of immunohistochemistry against parvalbumin, calretinin, and calbindin D-28k. Abducens, medial rectus, and trochlear motoneurons were retrogradely labeled with horseradish peroxidase from their corresponding muscles. Oculomotor and abducens internuclear neurons were retrogradely labeled after horseradish peroxidase injection into either the abducens or the oculomotor nucleus, respectively. Parvalbumin staining produced the highest density of immunoreactive terminals in all extraocular motor nuclei and was distributed uniformly. Around 15-20% of the motoneurons were moderately stained with antibody against parvalbumin, but their axons were heavily stained, indicating an intracellular segregation of parvalbumin. Colchicine administration increased the number of parvalbumin-immunoreactive motoneurons to approximately 85%. Except for a few calbindin-immunoreactive trochlear motoneurons (1%), parvalbumin was the only marker of extraocular motoneurons. Oculomotor internuclear neurons identified from the abducens nucleus constituted a nonuniform population, because low percentages of the three types of immunostaining were observed, calbindin being the most abundant (28.5%). Other interneurons located within the boundaries of the oculomotor nucleus were mainly calbindin-immunoreactive. The medial longitudinal fascicle contained numerous parvalbumin- and calretinin-immunoreactive but few calbindin-immunoreactive axons. The majority of abducens internuclear neurons projecting to the oculomotor nucleus (80.7%) contained calretinin. Moreover, the distribution of calretinin-immunoreactive terminals in the oculomotor nucleus overlapped that of the medial rectus motoneurons and matched the anterogradely labeled terminal field of the abducens internuclear neurons. Parvalbumin immunostained 42% of the abducens internuclear neurons. Colocalization of parvalbumin and calretinin was demonstrated in adjacent semithin sections, although single-labeled neurons were also observed. Therefore, calretinin is proven to be a good marker of abducens internuclear neurons. From all of these data, it is concluded that parvalbumin, calretinin, and calbindin D-28k selectively delineate certain neuronal populations in the oculomotor system and constitute valuable tools for further analysis of oculomotor function under normal and experimental conditions.

P2 receptor excitation of rodent hypoglossal motoneuron activity in vitro and in vivo: a molecular physiological analysis.

The role of P2 receptors in controlling hypoglossal motoneuron (XII MN) output was examined (1) electrophysiologically, via application of ATP to the hypoglossal nucleus of rhythmically active mouse medullary slices and anesthetized adult rats; (2) immunohistochemically, using an antiserum against the P2X2 receptor subunit; and (3) using PCR to identify expression of P2X2 receptor subunits in micropunches of tissue taken from the XII motor nucleus. Application of ATP to the hypoglossal nucleus of mouse medullary slices and anesthetized rats produced a suramin-sensitive excitation of hypoglossal nerve activity. Additional in vitro effects included potentiation of inspiratory hypoglossal nerve output via a suramin- and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS)-sensitive mechanism, XII MN depolarization via activation of a suramin-sensitive inward current, decreased neuronal input resistance, and a slow-onset theophylline-sensitive reduction of inspiratory output likely resulting from hydrolysis of extracellular ATP to adenosine and activation of P1 receptors. Immunohistochemically, P2X2 receptors were detected in inspiratory XII MNs that were labeled with Lucifer yellow. These data, combined with identification of mRNA for three P2X2 receptor subunit isoforms within the hypoglossal nucleus (two of which have not been localized previously in brain) and the previous demonstration that P2X receptors are ubiquitously expressed in cranial and spinal motoneuron pools, support not only a role of P2 receptors in modulating inspiratory hypoglossal activity but a general role of P2 receptors in modulating motor outflow from the CNS.

NMDA and non-NMDA receptors are co-localized at excitatory synapses of rat hypoglossal motoneurons.

We used whole-cell patch clamp recordings in a rat brainstem slice preparation to characterize the properties of miniature excitatory postsynaptic currents (mEPSCs) in hypoglossal motoneurons. The distinct kinetic characteristics of N-methyl-D-aspartate (NMDA) and non-NMDA receptor-mediated synaptic responses allowed us to study dual component mEPSCs mediated by the two receptor types. Using this approach, NMDA and non-NMDA receptors were found to be co-localized at the same synaptic locations. In addition, some sites contain only NMDA receptors since a large proportion of mEPSCs were apparently mediated by NMDA receptors only. Furthermore, the amplitudes of pharmacologically isolated NMDA receptor-mediated mEPSCs were highly variable in individual cells and their decay kinetics were modulated by membrane potential.

Distribution of neuropeptide-immunoreactive nerve fibres in the medulla oblongata of the chicken.

The anatomical distribution of nerve fibres containing galanin (GAL), leucine enkephalin (lENK), methionine enkephalin (mENK) and substance P (SP) was investigated in the medulla oblongata of the chicken by means of immunohistochemistry. In the medulla oblongata, dense networks of mENK-, lENK-, SP- and GAL-immunoreactive nerve fibres were mainly restricted within and around the dorsal vagal complex, i.e., the nucleus of the solitary tract and dorsal motor nucleus of the vagus nerve. The relative density of immunoreactive nerve fibres was usually decreased in the order of mENK-, SP-, lENK- and GAL-immunoreactive ones. Immunoreactive nerve fibres for each antiserum had a unique distribution among the subnuclear groups of the dorsal vagal complex. Sparser networks of immunolabelled nerve fibres were found in the nucleus of the hypoglossal nerve, nucleus ambiguus, nucleus of the descending trigeminal tract, raphe nucleus and caudal olivary complex. In the colchicine-pretreated chicken, some nerve cell bodies expressing the immunoreaction for ENK and GAL antisera were detected in the nucleus of the solitary tract and nucleus of the descending trigeminal tract. There were some differences in the distribution pattern of immunoreactive nerve elements among the four kinds of antisera. The selective distribution of neuropeptide-containing nerve elements within various nuclei of the medulla oblongata suggests that these neuropeptides may be differentially involved in central neural regulation.