Phrenic motor neuron survival below cervical spinal cord hemisection.

Cervical spinal cord injury (cSCI) severs bulbospinal projections to respiratory motor neurons, paralyzing respiratory muscles below the injury. C2 spinal hemisection (C2Hx) is a model of cSCI often used to study spontaneous and induced plasticity and breathing recovery post-injury. One key assumption is that C2Hx dennervates motor neurons below the injury, but does not affect their survival. However, a recent study reported substantial bilateral motor neuron death caudal to C2Hx. Since phrenic motor neuron (PMN) death following C2Hx would have profound implications for therapeutic strategies designed to target spared neural circuits, we tested the hypothesis that C2Hx minimally impacts PMN survival. Using improved retrograde tracing methods, we observed no loss of PMNs at 2- or 8-weeks post-C2Hx. We also observed no injury-related differences in ChAT or NeuN immunolabeling within labelled PMNs. Although we found no evidence of PMN loss following C2Hx, we cannot rule out neuronal loss in other motor pools. These findings address an essential prerequisite for studies that utilize C2Hx as a model to explore strategies for inducing plasticity and/or regeneration within the phrenic motor system, as they provide important insights into the viability of phrenic motor neurons as therapeutic targets after high cervical injury.

WGA-Alexa transsynaptic labeling in the phrenic motor system of adult rats: Intrapleural injection versus intradiaphragmatic injection.

BACKGROUND: Intrapleural injection of CTB-Alexa 488, a retrograde tracer, provides an alternative labeling technique to the surgically invasive laparotomy required for intradiaphragmatic injection. However, CTB-Alexa 488 is incapable of crossing synapses restricting the tracer to the phrenic nuclei and the intercostal motor nuclei in the spinal cord. NEW METHOD: Intrapleural injection of WGA-Alexa 488, a transsynaptic tracer, provides a method to label the respiratory motor pathway in both the spinal cord and medulla. Intradiaphragmatic injection of WGA-Alexa 594 and vagal nerve injections of True blue were used to confirm the phrenic nuclei and to differentiate between the rVRG and the NA in the medulla. RESULTS: Following intrapleural injection, WGA-Alexa 488 was retrogradely transported to the phrenic nuclei and to the intercostal motor nuclei. Subsequently WGA-Alexa 488 was transsynaptically transported from the phrenic motoneurons to the pre-motor neurons in the rVRG that provide the descending drive to the phrenic neurons during inspiration. In addition WGA-Alexa 488 was identified in select cells of the NA confirmed by a dual label of both WGA-Alexa 488 and True blue. COMPARISON WITH EXISTING METHOD: WGA-Alexa 488 demonstrates retrograde transsynaptic labeling following intrapleural injection whereas the previous method of injecting CTB-Alexa 488 only demonstrates retrograde labeling. CONCLUSIONS: Intrapleural injection of WGA-Alexa fluor conjugates is an effective method to transsynaptically label the phrenic motor system providing an alternative for the invasive laparotomy required for intradiaphragmatic injections. Furthermore, the study provides the first anatomical evidence of a direct synaptic relationship between rVRG and select NA cells.

Okadaic acid-sensitive protein phosphatases constrain phrenic long-term facilitation after sustained hypoxia.

Phrenic long-term facilitation (pLTF) is a serotonin-dependent form of pattern-sensitive respiratory plasticity induced by intermittent hypoxia (IH), but not sustained hypoxia (SH). The mechanism(s) underlying pLTF pattern sensitivity are unknown. SH and IH may differentially regulate serine/threonine protein phosphatase activity, thereby inhibiting relevant protein phosphatases uniquely during IH and conferring pattern sensitivity to pLTF. We hypothesized that spinal protein phosphatase inhibition would relieve this braking action of protein phosphatases, thereby revealing pLTF after SH. Anesthetized rats received intrathecal (C4) okadaic acid (25 nm) before SH (25 min, 11% O(2)). Unlike (vehicle) control rats, SH induced a significant pLTF in okadaic acid-treated rats that was indistinguishable from rats exposed to IH (three 5 min episodes, 11% O(2)). IH and SH with okadaic acid may elicit pLTF by similar, serotonin-dependent mechanisms, because intravenous methysergide blocks pLTF in rats receiving IH or okadaic acid plus SH. Okadaic acid did not alter IH-induced pLTF. In summary, pattern sensitivity in pLTF may reflect differential regulation of okadaic acid-sensitive serine/threonine phosphatases; presumably, these phosphatases are less active during/after IH versus SH. The specific okadaic acid-sensitive phosphatase(s) constraining pLTF and their spatiotemporal dynamics during and/or after IH and SH remain to be determined.

Prions in the peripheral nerves of bovine spongiform encephalopathy-affected cattle.

With the use of increasingly sensitive methods for detection of the abnormal isoform of prion protein (PrP(Sc)) and infectivity in prion diseases, it has recently been shown that parts of the peripheral nervous system (PNS) of bovine spongiform encephalopathy (BSE)-affected cattle may become infected. It has been reported that prions spread to the central nervous system (CNS) via the PNS in sheep scrapie, but the pathogenesis of BSE in cattle is less well understood. To determine whether parts of the PNS other than those implicated directly in the hypothetical pathogenetic spread of agent from the intestine to the CNS become involved before or after the CNS is affected, PrP(Sc) distribution was investigated by a highly sensitive Western blotting technique in dorsal root ganglia, stellate ganglion, phrenic, radial and sciatic nerves, adrenal gland and CNS of cattle that were inoculated orally with BSE-affected brain and culled sequentially. In experimentally BSE-affected cattle, PrP(Sc) was first detected in the CNS and dorsal root ganglia; subsequently, PrP(Sc) accumulation was detected in the peripheral nerve trunks. PrP(Sc) was also detected in the adrenal glands of cattle that showed clinical signs. No PrP(Sc) was detected in the PNS of BSE-negative cattle. This study shows that, with respect to dorsal root ganglia, a paravertebral sympathetic ganglion and the somatic nerves examined, PrP(Sc) is detected in the PNS during the disease course at the same time as, or after, it accumulates in the CNS.

The case for the bulbospinal respiratory nitric oxide synthase-immunoreactive pathway in the dog.

Previous investigations from our laboratory have documented that the neuropil of the phrenic nucleus contains a dense accumulation of punctate nicotinamide adenine dinucleotide phosphate diaphorase staining. In this study we investigated the occurrence and origin of punctate nitric oxide synthase immunoreactivity in the neuropil of the phrenic nucleus in C3-C5 segments, supposed to be the terminal field of the premotor bulbospinal respiratory nitric oxide synthase-immunoreactive pathway in the dog. As the first step, nitric oxide synthase immunohistochemistry was used to characterize nitric oxide synthase-immunoreactive staining of the phrenic nucleus and nitric oxide synthase-containing neurons in the dorsal and rostral ventral respiratory group and in the Bötzinger complex of the medulla. Dense punctate nitric oxide synthase immunoreactivity was found on control sections in the neuropil of the phrenic nucleus. Several thin bundles of nitric oxide synthase-immunoreactive fibers were found to enter the phrenic nucleus from the lateral and ventral column. Nitric oxide synthase-containing neurons were revealed in the dorsal respiratory group of medulla corresponding to the ventrolateral nucleus of the solitary tract and in the rostral ventral respiratory group beginning approximately 1 mm caudal to the obex and reaching to 650 microm rostral to the obex. Axotomy-induced retrograde changes, consisting in a strong upregulation of nitric oxide synthase-containing neurons, were found in the dorsal and rostral ventral respiratory group contralateral to the hemisection performed at the C2-C3 level. Concurrently, a strong depletion of the punctate nitric oxide synthase immunopositivity in the neuropil of the phrenic nucleus ipsilaterally with the hemisection was detected, thus revealing that a crossed premotor bulbospinal respiratory pathway contains a fairly high number of nitric oxide synthase-immunopositive fibers terminating in the phrenic nucleus. The use of the retrograde fluorescent tracer Fluorogold injected into the phrenic nucleus and an analysis of sections cut through the dorsal and rostral ventral respiratory group and Bötzinger complex of medulla and processed for nitric oxide synthase immunocytochemistry revealed that approximately 73.8% of crossed premotor bulbospinal respiratory nitric oxide synthase-immunoreactive axons originate in the rostral ventral respiratory group and 26.2% is given by nitric oxide synthase-containing neurons of the dorsal respiratory group. A few premotor nitric oxide synthase-immunoreactive axons originating from the Bötzinger complex were found. In summary, the present study provides evidence for a hitherto unknown premotor bulbospinal respiratory nitric oxide synthase-immunoreactive pathway connecting the bulbar respiratory centers with the motor neurons of the phrenic nucleus in the dog.

Scorpion alpha and alpha-like toxins differentially interact with sodium channels in mammalian CNS and periphery.

Scorpion alpha-toxins from Leiurus quinquestriatus hebraeus, LqhII and LqhIII, are similarly toxic to mice when administered by a subcutaneous route, but in mouse brain LqhII is 25-fold more toxic. Examination of the two toxins effects in central nervous system (CNS), peripheral preparations and expressed sodium channels revealed the basis for their differential toxicity. In rat brain synaptosomes, LqhII binds with high affinity, whereas LqhIII competes only at high concentration for LqhII-binding sites in a voltage-dependent manner. LqhII strongly inhibits sodium current inactivation of brain rBII subtype expressed in HEK293 cells, whereas LqhIII is weakly active at 2 microM, suggesting that LqhIII affects sodium channel subtypes other than rBII in the brain. In the periphery, both toxins inhibit tetrodotoxin-sensitive sodium current inactivation in dorsal root ganglion neurons, and are strongly active directly on the muscle and on expressed muI channels. Only LqhII, however, induced repetitive end-plate potentials in mouse phrenic nerve-hemidiaphragm muscle preparation by direct effect on the motor nerve. Thus, rBII and sodium channel subtypes expressed in peripheral nervous system (PNS) serve as the main targets for LqhII but are mostly not sensitive to LqhIII. Toxicity of both toxins in periphery may be attributed to the direct effect on muscle. Our data elucidate, for the first time, how different toxins affect mammalian central and peripheral excitable cells, and reveal unexpected subtype specificity of toxins that interact with receptor site 3.

Cysteine-rich domain isoforms of the neuregulin-1 gene are required for maintenance of peripheral synapses.

Neuregulin-1 (NRG-1) signaling has been implicated in inductive interactions between pre- and postsynaptic partners during synaptogenesis. We used gene targeting to selectively disrupt cysteine-rich domain-(CRD-) containing NRG-1 isoforms. In CRD-NRG-1-/-mice, peripheral projections defasciculated and displayed aberrant branching patterns within their targets. Motor nerve terminals were transiently associated with broad bands of postsynaptic ACh receptor (AChR) clusters. Initially, Schwann cell precursors accompanied peripheral projections, but later, Schwann cells were absent from axons in the periphery. Following initial stages of synapse formation, sensory and motor nerves withdrew and degenerated. Our data demonstrate the essential role of CRD-NRG-1-mediated signaling for coordinating nerve, target, and Schwann cell interactions in the normal maintenance of peripheral synapses, and ultimately in the survival of CRD-NRG-1-expressing neurons.

Functional characterisation of tetanus and botulinum neurotoxins binding domains.

Tetanus and botulinum neurotoxins constitute a family of bacterial protein toxins responsible for two deadly syndromes in humans (tetanus and botulism, respectively). They bind with high affinity to neurons wherein they cause a complete inhibition of evoked neurotransmitter release. Here we report on the cloning, expression and use of the recombinant fragments of the heavy chains of tetanus neurotoxin and botulinum neurotoxin serotypes A, B and E as tools to study the neurospecific binding of the holotoxins. We found that the recombinant 50 kDa carboxy-terminal domains of tetanus and botulinum neurotoxins alone are responsible for the specific binding and internalisation into spinal cord cells in culture. Moreover, we provide evidence that the recombinant fragments block the internalization of the parental holotoxins in a dose-dependent manner, as determined by following the neurotoxin-dependent cleavage of their targets VAMP/synaptobrevin and SNAP-25. In addition, the recombinant binding fragments cause a significant delay in the paralysis induced by the corresponding holotoxin on the mouse phrenic nerve-hemidiaphragm preparation. Taken together, these results show that the carboxy-terminal domain of tetanus and botulinum neurotoxins is necessary and sufficient for the binding and internalisation of these proteins in neurons and open the possibility to use them as tools for the functional characterisation of the intracellular transport of clostridial neurotoxins.

Pre-embedding staining for GAD67 versus postembedding staining for GABA as markers for central GABAergic terminals.

Pre-embedding immunocytochemistry for the active form of glutamate decarboxylase (GAD67) and postembedding staining for gamma-aminobutyric acid (GABA) were compared as markers for central GABAergic terminals in the phrenic motor nucleus, in which phrenic motor neurons had been retrogradely labeled with cholera toxin B-horseradish peroxidase. Nerve terminals with or without GAD67 immunoreactivity were identified in one ultrathin section. GABA was localized with immunogold in an adjacent section after etching and bleaching. GABA labeling density was assessed over 519 GAD67-positive and GAD67-negative nerve terminals in the phrenic motor nucleus. Frequency histograms showed that statistically higher densities of gold particles occurred over most GAD67-positive terminals. However, some GAD67-negative terminals also showed high densities of gold particles, and some GAD67-positive terminals showed low densities. Preabsorption of the anti-GABA antibody with a GABA-protein conjugate, but not with other amino acid-protein conjugates, significantly reduced gold labeling over both GAD67-positive and GAD67-negative terminals. These results show that the presence of GAD67 immunoreactivity correlates strongly with high densities of immunogold labeling for GABA in nerve terminals in the phrenic motor nucleus. Preabsorption controls indicate that authentic GABA was localized in the postembedding labeling procedure. Only a small proportion of intensely GABA-immunoreactive terminals lack GAD67, suggesting that both GAD67 and GABA are reliable markers of GABAergic nerve terminals.

Cervical dorsal rhizotomy enhances serotonergic innervation of phrenic motoneurons and serotonin-dependent long-term facilitation of respiratory motor output in rats.

We tested the hypothesis that spinal plasticity elicited by chronic bilateral cervical dorsal rhizotomy (C3-C5; CDR) has functional implications for respiratory motor control. Surgery was performed on rats (CDR or sham-operated) 26 d before phrenic motoneurons were retrogradely labeled with cholera toxin. Rats were killed 2 d later, and their spinal cords were harvested and processed to reveal the cholera toxin-labeled phrenic motoneurons and serotonin-immunoreactive terminals. The number of serotonin-immunoreactive terminals within 5 micrometer of labeled phrenic motoneuron soma and primary dendrites increased 2.1-fold after CDR versus sham-operation. Time-dependent phrenic motor responses to hypoxia were compared among CDR, sham-operated, and control rats. Anesthetized, paralyzed, vagotomized, and artificially ventilated rats were exposed to three, 5 min episodes of isocapnic hypoxia (FiO2 = 0.11), separated by 5 min hyperoxic intervals (FiO2 = 0.5). One hour after hypoxia, a long-lasting, serotonin-dependent enhancement of phrenic motor output (long-term facilitation) was observed in both sham and control rats. After CDR, long-term facilitation was 108 and 163% greater than control and sham responses, respectively. Pretreatment of CDR rats with a 5-HT2 receptor antagonist (ketanserin tartrate, 2 mg/kg, i.v.) before episodic hypoxia prevented long-term facilitation and revealed a modest (-28 +/- 13%; p < 0.05) long-lasting depression of phrenic motor output. The results indicate that CDR: (1) increases serotonergic innervation of the phrenic motor nucleus; and (2) augments serotonin-dependent long-term facilitation of phrenic motor output. These results further suggest a form of plasticity based on changes in the capacity for neuromodulation.

Temperature-sensitive neuromuscular transmission in Kv1.1 null mice: role of potassium channels under the myelin sheath in young nerves.

In mammalian myelinated nerves, the internodal axon that is normally concealed by the myelin sheath expresses a rich repertoire of K channel subtypes thought to be important in modulating action potential propagation. The function of myelin-covered K channels at transition zones, however, has remained unexplored. Here we show that deleting the voltage-sensitive potassium channel Kv1.1 from mice confers a marked temperature-sensitivity to neuromuscular transmission in postnatal day 14 (P14)-P21 mice. Using immunofluorescence and electrophysiology, we examined contributions of four regions of the peripheral nervous system to the mutant phenotype: the nerve trunk, the myelinated segment preceding the terminal, the presynaptic terminal membrane itself, and the muscle. We conclude that the temperature-sensitive neuromuscular transmission is accounted for solely by a deficiency in Kv1.1 normally concealed in the myelinated segments just preceding the terminal. This paper demonstrates that under certain situations of physiological stress, the functional role of myelin-covered K channels is dramatically enhanced as the transition zone at the neuromuscular junction is approached.

Synaptic plasticity of 5-hydroxytryptamine-immunoreactive terminals in the phrenic nucleus following spinal cord injury: a quantitative electron microscopic analysis.

The present study was conducted to examine the plasticity of 5-hydroxytryptamine (5-HT)-immunoreactive terminals in the rat phrenic nucleus following an ipsilateral C2 spinal cord hemisection and 30-day survival period. A retrograde horseradish peroxidase (HRP) labeling technique was used to identify the phrenic motoneurons at the electron microscopic (EM) level. After employing a pre-embedding immunocytochemical technique, the ultrastructural characteristics of 5-HT-immunoreactive terminals were qualitatively and then quantitatively analyzed with a computerized morphometric system before and after injury in separate groups of rats. The results indicated that the majority of the 5-HT-labeled terminals formed axodendritic contacts, but some 5-HT-labeled terminals made axosomatic contacts. 5-HT terminals were associated with either asymmetrical or symmetrical synapses, and some displayed postsynaptic dense bodies. Approximately 2% of the 5-HT terminals had dense-core vesicles. Although the total number of labeled and unlabeled terminals in the phrenic nucleus was reduced after hemisection, the number of 5-HT terminals in the hemisected group was greater than that of the control group. Moreover, the total number and length of asymmetrical and symmetrical synaptic active zones per 5-HT terminal were significantly greater after injury. Finally, the total number of 5-HT terminals with multiple synapses was significantly greater in the hemisected group as compared to controls. It is possible that 5-HT synaptic plasticity may be part of the morphological substrate for the unmasking of the latent crossed phrenic pathway which mediates recovery of the ipsilateral hemidiaphragm paralyzed by C2 spinal cord hemisection.

Vesicle shape and amino acids in synaptic inputs to phrenic motoneurons: do all inputs contain either glutamate or GABA?

Varicosities that made synapses or direct contacts with retrogradely labelled rat phrenic motoneurons were examined for their content of immunoreactivity for either glutamate or glutamate decarboxylase, the enzyme involved in synthesis of gamma-aminobutyric acid (GABA). Phrenic motoneurons were identified by retrograde tracing from the diaphragm with cholera toxin B subunit conjugated to horseradish peroxidase. Cell bodies and medium-sized to large dendrites were labelled. Preembedding immunocytochemistry identified glutamate decarboxylase-immunoreactive nerve fibres; glutamate-immunoreactive nerve terminals were identified using postembedding immunogold labelling of ultrathin sections. The presence of glutamate- or glutamate decarboxylase immunoreactivity in nerve terminals was correlated with the morphology of the synaptic vesicles. Two major classes of nerve terminals were identified. Nerve terminals with round (presumably spherical) synaptic vesicles (S terminals) comprised 55% of synapses and contacts on phrenic motoneuron somata and 58% of synapses and direct contacts with dendrites. Nerve terminals with flattened synaptic vesicles (F terminals) comprised 42% of synapses direct contacts with somata and 41% of synapses and direct contacts with dendrites. Analysis of immunogold-labelled sections showed that S terminals contained statistically higher levels of glutamate immunoreactivity than F terminals. At the light microscope level, many glutamate decarboxylase-immunoreactive nerve terminals surrounded retrogradely labelled motoneurons. Varicosities with glutamate decarboxylase immunoreactivity made 33% of all synapses and direct contacts on somata, and 33% of synapses and direct contacts with dendrites of the retrogradely labelled phrenic motoneurons. Flattened synaptic vesicles were present in those glutamate decarboxylase-immunoreactive nerve terminals in which synaptic vesicle morphology could be judged. An additional 10% of all nerve terminals were of the F type, but were not glutamate decarboxylase-immunoreactive. Three percent of terminals on somata and 1% of nerve terminals on dendrites could not be classified as S or F types. These findings suggest that more than 90% of all inputs to phrenic motoneuron cell bodies and proximal dendrites could contain either GABA or glutamate. Some of these glutamatergic and GABAergic nerve fibres undoubtedly represent the source of inspiratory drive to, or expiratory inhibition of, phrenic motoneurons.

Mechanical ventilation increases substance P concentration in the vagus, sympathetic, and phrenic nerves.

Substance P (SP), a neurotransmitter localized to primary sensory neurons, is found in the vagus nerve, nodose ganglion, sympathetic chain, and phrenic nerve in various animal species. However, the changes in endogeneous SP concentration under various circumstances that involve the participation of cardiorespiratory afferent nerves are still unexplored. In the present study, attention was focused on the variations in SP content measured by radioimmunoassay (RIA) in respiratory afferent nerves (vagus nerve, cervical sympathetic chain, phrenic nerve) and respiratory muscles (diaphragm, intercostal muscles) during positive inspiratory pressure (PIP) breathing alone or PIP with an expiratory threshold load (ETL) in rabbits. SP was found in all sampled structures in spontaneously breathing control animals, prevailing in the nodose ganglion. Left-versus right-sided differences were noticed in nerves. As compared with that in control animals, the SP concentration was markedly higher in vagal and sympathetic nervous structures during PIP or PIP with ETL, and also in the phrenic nerve during ETL breathing. The SP content did not vary in respiratory muscles. These observations suggest that two very common circumstances of mechanical ventilation are associated with an increased SP concentration in nervous structures participating in the control of breathing.

Afferent innervation of the gallbladder in the cat, studied by the horseradish peroxidase method.

Following injection of horseradish peroxidase (HRP) into the wall of the gallbladder of cats, HRP-positive cells were found bilaterally in dorsal root ganglia T2-L3 (T2-L2, and T3-L2/L3 also observed in a few cats) and nodose ganglia. In about 33% of animals labelled cells were also distributed in cervical dorsal root ganglia C5-C7. Labelled cells were more frequently localized on the right side than the left. There was no apparent change in numbers of labelled cells in the nodose ganglion (NG) on either side following greater and lesser splanchnicotomy or section of the right phrenic nerve or removal of the celiac ganglion. After severing both the greater and lesser splanchnic nerves unilaterally, numbers of labelled afferent cells from the gallbladder in dorsal root ganglia (DRGs) significantly decreased on the ipsilateral side but there was no change in the pattern of distribution contralaterally. After section of the right phrenic nerve, labelled cells were not found in ipsilateral cervical ganglia. That some afferent fibers from the gallbladder travel via the phrenic nerves, particularly on the right side, may be a supplementary mechanism in the generation of referred pain in gallbladder disease. The splanchnic nerves are the main, but not the only pathway for afferent fibers from the gallbladder.