C5 Ventral Root Research Articles

D-serine regulation of the timing and architecture of the inspiratory burst in neonatal mice

d-serine, released from mouse medullary astrocytes in response to increased CO2 levels, boosts the respiratory frequency to adapt breathing to physiological demands. We analyzed in mouse neonates, the influence of d-serine upon inspiratory/expiratory durations and the architecture of the inspiratory burst, assessed by pwelch's power spectrum density (PSD) and continuous wavelet transform (CWT) analyses. Suction electrode recordings were performed in slices from the ventral respiratory column (VRC), site of generation of the respiratory rhythm, and in brainstem-spinal cord (en bloc) preparations, from the C5 ventral roots, containing phrenic fibers that in vivo innervate and drive the diaphragm, the main inspiratory muscle.In en bloc and slice preparations, d-serine (100 μM) reduced the expiratory, but not the inspiratory duration, and increased the frequency and the regularity of the respiratory rhythm. In en bloc preparations, d-serine (100 μM) also increased slightly the amplitude of the integrated inspiratory burst and the area under the curve of the integrated inspiratory burst, suggesting a change in the recruitment or the firing pattern of neurons within the burst. Time-frequency analyses revealed that d-serine changed the burst architecture of phrenic roots, widening their frequency spectrum and shifting the position of the core of firing frequencies towards the onset of the inspiratory burst. At the VRC, no clear d-serine induced changes in the frequency-time domain could be established. Our results show that d-serine not only regulates the timing of the respiratory cycle, but also the recruitment strategy of phrenic motoneurons within the inspiratory burst.

Spontaneous crossed phrenic activity in the neonatal respiratory network

Hemisection of the cervical spinal cord causes paralysis of the ipsilateral hemidiaphragm in adult rats. Activation of a latent crossed phrenic motor pathway can restore diaphragmatic function, although structural changes take place before the pathway can be activated. Since mechanisms are employed to eliminate non-functional projections during development, we predicted that this latent neural pathway might be active during development. Therefore, we examined the effect of spinal hemisection (C2) on respiratory-like activity bilaterally using the brainstem–spinal cord preparation from neonatal rats (0–4 days). Spontaneous crossed phrenic activity (respiratory-like activity recorded from the ipsilateral C4 or C5 ventral roots following C2 hemisection) was observed in an age-dependent manner; younger preparations exhibited more than older preparations. Increasing drive (increasing [K +] or superfusion of theophylline) either increased or induced crossed phrenic activity. Hemisection caused no change in the frequency, the burst area, duration or peak amplitude contralateral to hemisection. Unlike adult rats, this study shows that crossed phrenic activity is present in the in vitro respiratory network of neonatal rats suggesting that a crossed neural pathway may be functionally active in neonates.

Rescue of rat spinal motoneurons from avulsion-induced cell death by intrathecal administration of IGF-I and Cerebrolysin

Ventral root avulsion results in the loss of motoneurons in the corresponding spinal cord segment. In the present experiments we have tested effects of insulin-like growth factor-I (IGF-I) and Cerebrolysin on survival of avulsed motoneurons after their chronic intrathecal administration in the adult rats. We have found that avulsion of the C5 ventral roots results in significant loss of motoneurons in the same spinal cord segment due mainly to apoptosis. In comparison to the untreated control rats, the amount of motoneuron survival in avulsed ventral horn was significantly higher after 4 weeks intrathecal administration of IGF-I or Cerebrolysin. No significant differences were observed between effects of IGF-I and Cerebrolysin in our experimental model. The results suggest that both IGF-I and Cerebrolysin can reduce avulsion-induced loss of adult rat motoneurons.

Acellular nerve graft re-seeded by Schwann cells migrating from the nerve stump can stimulate spinal motoneurons for functional reinnervation of the rat muscle

The acellular nerve graft was utilised to restore a functional reinnervation of the biceps brachii muscle from the motoneuron pool of the cervical spinal cord. The musculocutaneous nerve stump was sutured to an acellular nerve graft, the opposite end of which was inserted into the cervical spinal cord cranial to the avulsed C5 ventral root. The acellular nerve graft was repopulated by Schwann cells heavily immunostained for NGFr within 90 days. The Schwann cells migrating from the nerve stump reached the spinal cord grey matter, where they stimulated the motoneurons to send axonal sprouts. The functional reinnervation of the biceps brachii muscle was assessed by means of the behavioural (grooming) test and EMG, the presence of myelinated and unmyelinated axons was demonstrated by light and electron microscopy. The axonal reconnection of the musculocutaneous nerve stump was verified by horseradish peroxidase retrograde labelling of the spinal motoneurons. Moreover, the motoneurons on the operated side of the C5 spinal segment displayed increased immunostaining for GAP-43 in comparison to the contralateral side, whereas the pattern of AChE histochemical reaction was similar on both the operated and contralateral side, of the C5 segment 150 days after acellular graft implantation. The regenerated axons bridged a 4-cm long originally acellular nerve graft to reach and reinnervate the biceps brachii muscle. The reinnervation of the neuromuscular junctions was morphologically determined by immunofluorescence for neurofilaments. The number of myelinated axons in the acellular nerve graft was significantly higher than those growing over the cellular graft, but their diameter was smaller. The results of experiments presented here demonstrate functional recovery of the biceps muscle reinnervation through the acellular nerve graft repopulated by migrating Schwann cells. The process of reinnervation by acellular nerve graft is however delayed and worse in comparison with the cellular graft.

Actions of opioids on respiratory activity via activation of brainstem μ-, δ- and κ-receptors; an in vitro study

Opioid-induced respiratory depression is well documented. However, exact sites of action and mechanisms for opioid-induced effects on respiration have not yet been elucidated. The present study was carried out on isolated brainstem–spinal cord preparations from newborn rats in order to explore the opioid activity on brainstem μ-, δ- and κ-receptors. The brainstem–spinal cord was isolated from 0- to 4-day-old Sprague-Dawley rats. The preparation was perfused with artificial cerebrospinal fluid (28.5°C) equilibrated with 95% O 2 and 5% CO 2 at a pH of 7.4. Neuronal respiratory activity was recorded from the ventrolateral part of the medulla oblongata and efferent impulses from C4 or C5 ventral roots. Effects of the μ-receptor agonist DAGO, the δ-receptor agonist DPDPE and the κ-receptor agonist U50,488 on respiratory frequency ( f R), inspiratory time ( T i) and peak integrated C4 amplitude ( Int C4) were measured. In addition, the effect of pre-treatment with the μ 1 receptor antagonist naloxanazine (35 mg/kg, subcutaneous injection) was evaluated. DAGO reduced f R and T i in a concentration-dependent manner and caused a reduction of Int C4 at high concentrations (10 μM). The μ 1 receptor antagonist naloxanazine shifted the f R concentration–response curve for DAGO to the right ( P<0.05). DPDPE had no effect on respiratory activities whereas U50,488, like DAGO, reduced f R and Int C4 in a concentration-dependent manner. It was concluded that μ-opioid receptors, including the μ 1 were involved in f R reduction whereas κ-opioid receptors were involved in reduction of both f R and respiratory amplitude. δ-Opioid receptors do not seem to participate in respiratory modulation at this age.

Adenosine modulates inspiratory neurons and the respiratory pattern in the brainstem of neonatal rats.

The role of adenosine in the modulation of respiration-related neurons was examined using an in vitro brainstem-spinal cord preparation from neonatal rats (0-4 d old). Respiratory activity was recorded from the C4 or C5 ventral roots by suction electrodes and from inspiratory related neurons (I neurons) in the rostral ventrolateral medulla by microelectrodes. The following substances were added to the preparation superfusate, and their effect was evaluated: the adenosine A1 receptor agonist N6-(2-phenylisopropyl)adenosine, R(-)isomer (R-PIA), the adenosine uptake blocker dipyridamole, the adenosine receptor antagonist theophylline, and the specific A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). R-PIA and dipyridamole decreased the activity of I neurons and the C4 respiratory burst rate. Furthermore, these compounds induced a significantly more irregular respiratory rate in three-quarters of preparations from the youngest animals (<48 h old) compared with that of controls. Theophylline or DPCPX reversed the effects of both R-PIA and dipyridamole on respiratory rate, regularity of respiratory rate, inspiratory time, amplitude, and intra-burst frequency of I neurons. Thus, adenosine depresses both the I neurons in the rostral ventrolateral medulla and the respiratory motor output. This depression of I neurons and respiratory rate can be abolished by theophylline primarily through a blockade of medullary adenosine A1 receptors. An age-dependent correlation of the effects of R-PIA and dipyridamole, with a more pronounced decrease in respiratory activity in preparations from younger animals, indicates that adenosinergic modulation of medullary respiration-related neurons changes during the first days of postnatal life.

Histometric effects of ciliary neurotrophic factor in wobbler mouse motor neuron disease

We investigated the histological effects of ciliary neurotrophic factor on degenerating motor neurons, their axons, and skeletal muscles in 68 wobbler mice with motor neuron disease. Treatment consisted of recombinant rat or human ciliary neurotrophic factor (or a vehicle solution), 1-mg/kg subcutaneous injection, three times per week for 4 weeks after the clinical diagnosis. The number of motor neurons immunoreactive for calcitonin gene-related peptide was higher in mice receiving rat ciliary neurotrophic factor (p < 0.03), although the number of choline acetyltransferase-reactive neurons was the same in both treated and untreated control groups. Treatment did not prevent vacuolar degeneration of motor neurons. In mice treated with human ciliary neurotrophic factor, the percentage of axons undergoing acute axonal degeneration (myelin ovoids) was smaller in the entire C5 ventral root (p < 0.02) and in the musculocutaneous nerve (p < 0.04), and the number of myelinated nerve fibers was 30% higher in both nerves (p < 0.01 and p < 0.04, respectively) than in controls. In ciliary neurotrophic factor-treated mice, the biceps muscle weight was 20% greater, the mean muscle fiber diameter was 30% larger, and the number of atrophied muscle fibers was 75% lower than that in the vehicle-treated wobbler mice (p < 0.001 for all three results). The number of terminal axonal branching points and the mean length of motor end-plates were also higher in the ciliary neurotrophic factor-treated mice (p < 0.001 and p < 0.02, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of CO2 on Vth nerve inhibition of respiration in isolated rat nervous system.

The role of hypercapnia in terminating trigeminal (Vth) nerve inhibition of respiration is not clear. Phrenic nerve responses to Vth nerve stimulation were examined during eucapnia and hypercapnia. We hypothesized that hypercapnic stimulation might not be sufficient to overcome the inhibitory effects of the Vth nerve, and thus would fail to restore respiratory center output to baseline values. Isolated rat brainstem-spinal cord preparations from newborn rats were placed in a tissue bath and superfused with modified Krebs solution at 27 degrees C. Respiratory related activity was recorded from cut C1 or C5 ventral roots. Tonic stimulation of the Vth nerve (1 Hz for 15 min) under eucapnic conditions initially inhibited the frequency of respiratory activity over the first 1-2 min, followed by partial recovery after 4 min to 50-60% of the control level. Stimulation of the Vth nerve at 0.5 Hz initially decreased the respiratory frequency to about 40% of baseline over the first 3 min, which then increased to control levels within 6 min despite continued Vth nerve stimulation. Exposure to hypercapnia (15 KPa PCO2) increased the baseline respiratory frequency. However, hypercapnic stimulation only partially reduced the degree of the Vth nerve inhibitory effect. We conclude that 15 KPa PCO2 only partially reverses the inhibitory effect of the Vth nerve stimulation at the level of the central respiratory controller.

Effects of NMDA and non-NMDA receptor antagonists on inspiratory neurons in the in vitro brainstem-spinal cord preparation of newborn rat

We examined the effects of N- methyl- d-aspartate (NMDA) antagonists, 2-amino-5-phosphonovaleric acid (APV) and non-NMDA antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on inspiratory neurons in the medulla oblongata and C4 and C5 ventral root activity in the in vitro brainstem-spinal cord preparation of newborn rats. Application of APV induced increased rate of inspiratory neuron bursts, and decreased burst duration. The C4 burst activity was almost abolished by application of CNQX, whereas inspiratory neurons continued to discharge rhythmically. The results suggest that non-NMDA receptors are not crucially involved in the medullary respiratory rhythmogenesis, and that NMDA-mediated neurotransmission could modify the respiratory pattern, in the in vitro model.

Changes in diaphragm motor unit EMG during fatigue

Fatigue-related changes in the waveform and root-mean-square (rms) values of evoked motor unit electromyographic (EMG) responses were studied in the right sternocostal region of the cat diaphragm. Motor units were isolated by microdissection and stimulation of C5 ventral root filaments and then classified as fast-twitch fatigable (FF), fast-twitch fatigue intermediate (FInt), fast-twitch fatigue resistant (FR), or slow-twitch (S) based on standard physiological criteria. The evoked EMG responses of S and FR units showed very little change during the fatigue test. The evoked EMG waveform and rms values of FF and FInt units displayed variable changes during the fatigue test. When changes were observed, they typically included a prolongation of the EMG waveform, a decrease in peak amplitude, and a decrease in rms value. The changes in EMG amplitude and rms values were not correlated. In more fatigable units, the decrease in force during the fatigue test generally exceeded the decrease in EMG rms values. Changes in the evoked force and EMG responses of multiple units innervated by C5 or C6 ventral roots were also examined during the fatigue test. The decrease in diaphragm force during the fatigue test closely matched the force decline predicted by the proportionate contribution of different motor unit types. However, the observed reduction in diaphragm EMG rms values during the fatigue test exceeded that predicted based on the aggregate contribution of different motor unit types. It was concluded that changes in EMG do not reflect the extent of diaphragm fatigue.

Oxidative capacity and capillary density of diaphragm motor units

Motor units in the cat diaphragm (DIA) were isolated in situ by microdissection and stimulation of C5 ventral root filaments. Motor units were classified based on their isometric contractile force responses and fatigue indexes (FI). The muscle fibers belonging to individual units (i.e., the muscle unit) were identified using the glycogen-depletion method. Fibers were classified as type I or II based on histochemical staining for myofibrillar adenosine triphosphatase (ATPase) after alkaline preincubation. The rate of succinate dehydrogenase (SDH) activity of each fiber was determined using a microphotometric procedure. The location of capillaries was determined from muscle cross sections stained for ATPase after acid (pH = 4.2) preincubation. The capillarity of muscle unit fibers was determined by counting the number of capillaries surrounding fibers and by calculating the number of capillaries per fiber area. A significant correlation was found between the fatigue resistance of DIA units and the mean SDH activity of muscle unit fibers. A significant correlation was also observed between DIA unit fatigue resistance and both indexes of muscle unit fiber capillarity. The mean SDH activity and mean capillary density of muscle unit fibers were also correlated. We conclude that DIA motor unit fatigue resistance depends, at least in part, on the oxidative capacity and capillary density of muscle unit fibers.

Mechanical properties of muscle units in the cat diaphragm.

1. Muscle units in the right sternocostal region of the cat diaphragm (DIA) were isolated in situ by dissecting filaments of the C5 ventral root. Isometric contractile and fatigue properties of DIA units were then measured. Contractile properties included: twitch contraction time (CT), peak twitch tension (Pt), maximum tetanic tension (P0), and the frequency dependence of tension production. Muscle-unit fatigue resistance was estimated using a 2-min fatigue test. 2. DIA muscle units were classified as fast (F) or slow (S) based on the presence or absence of sag in their unfused tetanic force responses. Muscle-unit fatigue indices (FI) were used to further classify DIA units as slow-twitch fatigue-resistant (S), fast-twitch fatigue-resistant (FR) fast-twitch fatigue-intermediate (FInt), or fast-twitch fatigable (FF) types. 3. Based on a total of 47 completely characterized DIA muscle units, 21% were classified as S, 4% as FR, 28% as FInt, and 47% as FF. In contrast to the distribution of unit types in other mixed appendicular muscles, the DIA was composed of a very low proportion of FR units and a relatively high proportion of FInt units. An interval of FIs between 0.50 and 0.75 separated units into fatigue-resistant and fatigable groups. The distribution of FIs for FF and most FInt units was continuous, indicating that they formed a single fatigable group. Relatively few FF units in the DIA had FIs less than 0.10. 4. A wide range of contractile properties was observed for DIA muscle units. Type S units had longer CTs and lower Pt and P0 values than type F units. The mean Pt and P0 of FF and FInt units were comparable, whereas the mean Pt and P0 of the two FR units were lower. Type S units produced a greater proportion of their P0 at lower frequencies of activation than type F units. The lower P0S produced by type F units in the DIA indicated that they were smaller than similar units in appendicular muscles. It was concluded that in meeting most normal ventilatory requirements, adequate force could be generated by the recruitment of only type S and FR units. The recruitment of the more fatigable FF and FInt units may occur only during more forceful respiratory and nonrespiratory behaviors of the DIA.