Neonatal Rat Brainstem-spinal Cord Preparation Research Articles

PH sensitivity of spinal cord rhythm in fetal mice in vitro.

The spinal respiratory rhythm generator is a neural network localized at the segments C4-C6 able to produce synchronous long lasting rhythmic bursts of action potentials on both phrenic nerves (Dubayle and Viala, 1996). As other spinal cord rhythms, the spinal respiratory rhythm can be induced in curarized rabbits after cervical transection by administration of nialamide-DOPA (Viala and Freton, 1983). This spinal rhythm can also be induced in the in vitro brainstem-spinal cord preparation from newborn rat or mouse by deep diethyl ether anaesthesia, or bath administration of glutamic acid, N-methyl-D-aspartic acid, amphetamine, 5-hydroxytryptophan, or high potassium concentration (Dubayle and Viala, 1998). It has been shown that the spinal respiratory rhythm recorded from neonatal rat brainstem-spinal cord preparation is sensitive to H+ and CO2 (Dubayle and Viala, 1998). However, in most of the experiments the extent to what this chemosensitive response depends directly on the spinal respiratory generator was undefined, because chemical stimulation or the pharmacological inductors affect also the activity of the medullary respiratory pattern generator and both, the medullary and spinal respiratory oscillators, are coupled (Dubayle and Viala, 1996). Uncoupling of these generators by spinal transection at C2 segment caused the disappearance of the spinal respiratory rhythm in most of the neonatal preparations (Dubayle and Viala, 1998).KeywordsVentral RootFetal MouseRespiratory RhythmHigh Potassium ConcentrationSuperfusion MediumThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Hypothermia and recovery from respiratory arrest in a neonatal rat in-vitro brainstem preparation

In mammals, progressive hypothermia leads to loss of reflex responses, respiratory arrest, and finally, cardiac arrest and death. Under acute conditions in neonatal mammals, if progressive rewarming occurs soon enough, both the heart beat and breathing will resume spontaneously and they will recover fully but in adult rats, once respiratory arrest occurs, the animals must be artificially resuscitated. The mechanistic basis of the initial respiratory arrest in hypothermia as well as the ontogenetic changes in tolerance and in the ability of the system to autoresuscitate on rewarming are poorly understood. Onimura and Homma [1], demonstrated that when temperature was lowered from 26 to 24°C in an en bloc brainstem-spinal cord preparation from newborn rats, respiratory rate was depressed and bursts of activity in Pre-I neurons were not always followed by bursts of inspiratory activity in respiratory motor neurons. This suggested that either the number of active Pre-I neurons decreased and were no longer sufficient to activate the motor neuron pools, or that the threshold for the generation of inspiratory-modulated efferent activity in the motor neuron pools was raised, or both. This in turn suggested that respiratory arrest might occur at the level of pre-motor or motor neurons rather than at the level of the rhythm generator itself. This study was designed to examine whether respiratory arrest during hypothermia occurs at the level of pre motor or motor neurons rather than at the level of the central rhythm generator itself. Specifically we sought to determine the consequences of hypothermic cooling until respiratory arrest, and subsequent re warming, on neurons in the preBotzinger Complex (via field potential recordings), as an indication of the output of the entire rhythmo-genic network; and from cervical spinal (phrenic) ventral roots (via suction electrode recording), as an indication of motor neuron output, in an in vitro neonatal rat brainstem-spinal cord preparation. With this preparation, progressive cooling slowed the frequency of fictive breathing with the cycle period increasing exponentially until respiratory related rhythmic activity ceased. There was no decrease in the peak, integrated sum or duration of the field potential during this process; ie, while slowing was progressive, arrest was not. The same was not completely true of the phrenic motor output. During progressive cooling there was a 14% fall in peak amplitude and a 32% fall in the integrated sum of the activity associated with each fictive burst. There were also rare instances at low temperature during both cooling and recovery where bursts of activity in the field potential were not accompanied by any increase in activity in the phrenic motor output. These data suggest that there must have been some increase in the threshold for generation of activity in the phrenic motor neuron pool relative to the neuron pool from which the field potential was recorded. Ultimate arrest, however, appears to occur at the level of the central rhythm generating network giving rise to complete and abrupt cessation of activation of the motor neuron pool(s). On rewarming, the motor output often was fractionated suggesting that changes occurred in network synchronization either during cooling or during reactivation following hypothermic arrest.

Three types of putative presympathetic neurons in the rostral ventrolateral medulla studied with rat brainstem–spinal cord preparation

To study the electrophysiological properties of presympathetic neurons in the rostral ventrolateral medulla (RVLM), intracellular recordings were performed by the whole-cell patch-clamp technique. We utilized the neonatal rat brainstem–spinal cord preparation, in which the sympathetic neuronal network is thought to be preserved, unlike in slice preparation. In response to stimulation in the ipsilateral Th 2 spinal segment including intermediolateral cell column (IML), 33 of 151 non-respiratory RVLM neurons showed antidromic action potentials with a constant latency of 45 ms, and can be considered as presympathetic neurons. We classified and characterized the RVLM presympathetic neurons into three types: ‘regularly firing neurons ( n=7)’, which showed ramp depolarization and frequent action potentials (4.2±0.9 spikes/s) with rare excitatory postsynaptic potentials (EPSPs); ‘irregularly firing neurons ( n=21)’, which exhibited many EPSPs that modulated the firing rate; and ‘silent-type neurons ( n=5)’, which discharged action potentials only during current-induced depolarization. Lucifer-Yellow staining showed that the irregularly firing neurons were significantly larger and had more dendrites than the regularly firing neurons. All regularly firing neurons retained their discharges during low-Ca 2+–high-Mg 2+ superfusion that blocks synaptic input, whereas the discharges in 11 of 16 irregularly firing neurons were abolished, suggesting that the regularly firing neurons discharged independently of synaptic input. Seven of 31 RVLM neurons were hyperpolarized by stimulation of vagal afferent nerves. In summary, three types of RVLM presympathetic neurons were characterized by the patch-clamp technique in the brainstem–spinal cord preparation, in which the connection was preserved from vagal afferent to the Th 2 spinal segment through the RVLM. Since antidromic action potentials were demonstrated by stimulation in the Th 2 spinal segment in 33 neurons of all three types, all types of RVLM neurons constitute a part of the sympathetic neuronal network.

Concurrent Inhibition and Excitation of Phrenic Motoneurons during Inspiration: Phase-Specific Control of Excitability

The movements that define behavior are controlled by motoneuron output, which depends on the excitability of motoneurons and the synaptic inputs they receive. Modulation of motoneuron excitability takes place over many time scales. To determine whether motoneuron excitability is specifically modulated during the active versus the quiescent phase of rhythmic behavior, we compared the input-output properties of phrenic motoneurons (PMNs) during inspiratory and expiratory phases of respiration. In neonatal rat brainstem-spinal cord preparations that generate rhythmic respiratory motor outflow, we blocked excitatory inspiratory synaptic drive to PMNs and then examined their phase-dependent responses to superthreshold current pulses. Pulses during inspiration elicited fewer action potentials compared with identical pulses during expiration. This reduced excitability arose from an inspiratory-phase inhibitory input that hyperpolarized PMNs in the absence of excitatory inspiratory inputs. Local application of bicuculline blocked this inhibition as well as the difference between inspiratory and expiratory firing. Correspondingly, bicuculline locally applied to the midcervical spinal cord enhanced fourth cervical nerve (C4) inspiratory burst amplitude. Strychnine had no effect on C4 output. Nicotinic receptor antagonists neither potentiated C4 output nor blocked its potentiation by bicuculline, further indicating that the inhibition is not from recurrent inhibitory pathways. We conclude that it is bulbospinal in origin. These data demonstrate that rapid changes in motoneuron excitability occur during behavior and suggest that integration of overlapping, opposing synaptic inputs to motoneurons is important in controlling motor outflow. Modulation of phasic inhibition may represent a means for regulating the transfer function of PMNs to suit behavioral demands.

Age- and temperature-dependent effects of opioids on medulla oblongata respiratory activity: an in vitro study in newborn rat

Influences of post-natal age (P0–P4) and temperature (22.5°–31.5°C) on the action(s) of opioids on respiratory activities from neonatal rat brainstem–spinal cord preparations were examined in this study. A temperature-dependent μ-opioid receptor effect on respiration was found. In addition, the effect of morphine increased with postnatal age (P0–P4). Hence, age and temperature must be taken into account when performing studies on medullary respiration-related structures using the neonatal rat brainstem–spinal cord preparation.

Potential role for imidazole in the rhythmic respiratory activity of the in vitro neonatal rat brainstem

We used the imidazole-binding agent, diethylpyrocarbonate (DEPC), to test the hypothesis that rhythmic respiratory activity of the in vitro neonatal rat brainstem-spinal cord preparation was functionally dependent on imidazole. Neural activity was recorded from spinal nerves (C1-C4) during superfusion with 95%O 2/5%CO 2 buffer at pH 7.3 and T=26°C. Superfusate containing DEPC (40 mM) caused cessation of rhythmic activity within minutes. In eight of 33 preparations, microinjection of DEPC (32 nmol) onto the ventral medullary surface (VMS) reduced burst amplitude by at least 50% within 10 min, and in 12 of 33 preparations, microinjection of DEPC produced neural apnea. Therefore, we conclude that proteins containing imidazole near the VMS are critically important for the maintenance of rhythmic respiratory activity in vitro. Furthermore, alphastat regulation of respiration may be an essential trait of this preparation.

GABAergic inhibition of neonatal rat phrenic motoneurons

Phrenic motoneurons (PMNs) receive intermittently glutaminergic inspiratory drives and GABAergic inhibition in adult mammals. Since γ-amino-butyric acid (GABA) might act as an excitatory amino acid in early stages of development, we here investigated if GABA A receptors inhibit PMNs in neonates. Using in vitro neonatal rat brainstem–spinal cord preparation, local application of GABA and muscimol (a GABA A receptor agonist) to the vicinity of PMNs consistently reduced the inspiratory activity of C4 ventral roots. Under whole-cell patch-clamp conditions and in the presence of 0.5 μM TTX to block synaptic transmission, muscimol (10 μM) decreased whole-cell input resistance, and surprisingly, when PMNs were voltage-clamped at their resting membrane potential, muscimol induced depolarizing-inward, rather than hyperpolarizing-outward membrane current. Our findings indicate that GABA A receptors mediate a depolarizing blockade of the glutaminergic excitatory inputs to neonatal rat PMNs.

Serotonergic inhibition of phrenic motoneuron activity: an in vitro study in neonatal rat

In vitro experiments were conducted on neonatal rat brainstem-spinal cord preparations to test the hypothesis of an inhibitory modulation of phrenic activity by serotonin (5-HT) via non-5-HT 2A receptors [Lindsay, A.D. and Feldman, J.L., Modulation of respiratory activity of neonatal rat phrenic motoneurones by serotonin, J. Physiol., 461 (1993) 213–233]. The changes induced by 5-HT and related agents on phrenic root discharges and membrane currents in identified phrenic motoneurons were analysed after blockade of spinal 5-HT 2A receptors. Spinal application of 5-HT 1B (but not 5-HT 1A) receptor agonists depressed the phrenic activity and the effect was prevented by pretreatment with 5-HT 1B (but not 5-HT 1A, 5-HT 2A and 5-HT 3) receptor antagonists. Results from phrenic motoneuron whole cell recordings do not reject a presynaptic location of the 5-HT receptors responsible for this depression.

Effects of ethanol on respiratory activity in the neonatal rat brainstem-spinal cord preparation

Ethanol (1–12 mM) added to the superfusion medium of the isolated brainstem-spinal cords of newborn rats did not affect phrenic activity but significantly reduced hypoglossal activity by 54%, 67% and 55% at 3, 6 and 12 mM, respectively. Although the reasons for the suppression of hypoglossal activity remain unknown, this preparation may be a useful model for determining why cranial motoneurons are more vulnerable than phrenic motoneurons to various agents and, more generally, how ethanol impairs neural function.

Medullary‐evoked EPSPs in neonatal rat sympathetic preganglionic neurones in vitro.

1. Whole-cell patch clamp recordings were made from twenty-three sympathetic preganglionic neurones (SPNs) in the upper thoracic segments of a neonatal rat brainstem-spinal cord preparation to study their synaptic responses to stimulation of the rostral ventrolateral medulla (RVLM) and the receptors involved. 2. SPNs were identified by their antidromic activation following stimulation of a ventral root, their morphology and their location in the spinal cord. 3. Electrical stimulation within the RVLM elicited EPSPs in all SPNs tested (n = 23). These EPSPs consisted of one or more components that had different time courses, voltage relationships and pharmacological sensitivities. 4. All SPNs responded to RVLM stimulation with a constant-latency fast EPSP that increased in size as the membrane was hyperpolarized. This EPSP was reduced in amplitude by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10-20 microM). 5. In thirteen SPNs the response to RVLM stimulation was a complex EPSP consisting of a fast EPSP and a slow EPSP that either followed or summed with the fast EPSP. The amplitude of the slow EPSP was (i) either reduced in size or not affected as the membrane was hyperpolarized, and (ii) reduced by the NMDA receptor antagonist, D, L-2-amino-5-phosphonovaleric acid (50 microM). 6. Selective activation of neuronal cell bodies in the RVLM by chemical stimulation elicited slow depolarizations and increases in synaptic activity in SPNs. 7. These results provide evidence that an excitatory amino acid is involved in transmitting sympathoexcitatory drive from the RVLM, partly via a monosynaptic pathway. Both non-NMDA and NMDA receptors play a role in mediating this drive.

Opioid depression of respiration in neonatal rats.

1. The effects of opioid receptor agonists and antagonists on the breathing pattern of neonatal rats were studied. Three experimental approaches were taken. In the first approach, the effects of opioid agonists and antagonists on the spontaneous respiratory neural activity generated by brainstem-spinal cords isolated from neonatal rats aged 0-4 days postnatal (P0-4) maintained in vitro were studied. Secondly, similar studies were performed utilizing medullary slice preparations consisting of respiratory rhythm-generating regions (pre-Bötzinger complex). Thirdly, whole-body plethysmographic recordings were obtained from unanaesthetized neonatal (P0-18) rats before and after I.P. administration of opioid-receptor agonists and antagonists. 2. The mu-receptor agonists morphiceptin and DAGO (Tyr-D-Ala-Gly-[NMePhe]-Gly-ol), when added either to the solutions bathing the brainstems of neonatal rat brainstem-spinal cord preparations or bathing the medullary slice preparations, resulted in a naloxone-reversible, dose-dependent decrease in the frequency of respiratory rhythmic discharge. 3. The respiratory burst frequency and amplitude in vitro were unaffected by the addition of the delta-opioid receptor agonist DPDPE ([D-pen2,5]-enkephalin) and the kappa-opioid receptor agonist U50488 (trans-[+]-3,4-dichloro-N-methyl-N-(2-[1- pyrrolidinyl]cyclohexyl) benzene-acetamide) or the opioid receptor antagonist naloxone. 4. Intraperitoneal administration of the mu-opioid receptor agonist fentanyl resulted in a naloxone-reversible, dose-dependent decrease in the frequency and amplitude of breathing of unanaesthetized neonatal rats (P0-P10). I.P. administration of the delta-opioid receptor agonist DPDPE did not affect breathing of neonatal rats until the second week postnatally. 5. We conclude that opioids suppress the frequency of neonatal rat respiration by acting via mu-opioid receptors located within regions of the ventral medulla containing respiratory rhythm-generating centres (the pre-Bötzinger complex). delta-Opioid receptor activation does not affect breathing in neonatal rats until approximately the second week postnatally.

A study of sympathetic preganglionic neuronal activity in a neonatal rat brainstem-spinal cord preparation

Extracellular recordings were made from 46 sympathetic preganglionic neurones (SPNs) in a neonatal rat brainstem-spinal cord preparation. Neurones were identified as SPNs as they were: ( i) activated at constant latencies (2–10 ms) following stimulation of the ventral root, which indicated antidromic activation and ( ii) recorded at sites located either in the intermediolateral cell column or the intercalated nucleus of the thoracic spinal cord. Over one-third of the neurones ( n = 17) recorded displayed ongoing activity with firing frequencies of 0.3–5 Hz. Of the neurones analyzed only one showed a very obvious phasic firing pattern. Dorsal root stimulation evoked firing in 16 of 26 SPNs recorded from the same spinal segment (6 of 10 with ongoing activity). The types of responses observed varied between neurones. The excitation of all neurones was characterised by a response occurring at a latency of 6–50 ms. In addition, SPNs in ‘spinalised’ preparations ( n = 2) responded with latencies of 10–40 ms, similar to those observed in the intact preparation. The latencies of responses in SPNs were longer and more variable than those observed in ventral horn motor neurones. This indicates that a spinal polysynaptic pathway was involved in mediating these responses. In 7 SPNs dorsal root stimulation also elicited longer latency responses which were observed up to 1000 ms after stimulation. These responses may involve activation of bulbospinal and/or propriospinal pathways. These results show that the neonatal rat brainstem-spinal cord preparation is viable for studying SPNs and that dorsal root-SPN reflexes are intact.

Whole-cell patch-clamp recordings from respiratory neurons in neonatal rat brainstem in vitro

Whole-cell recordings were obtained from respiratory neurons by applying patch-clamp techniques in the en bloc medulla of in vitro neonatal rat brainstem-spinal cord preparations. Stable voltage-clamp recordings of excitatory or inhibitory synaptic drive currents and current-clamp recordings of spike discharge of inspiratory and expiratory neurons could be maintained for periods of 1–2 h. Parameters of whole-cell recording, including membrane seal resistances and series resistances, obtained in the en bloc medulla were similar to those obtained in corresponding regions of thin slices where neurons were directly visualized to optimize conditions for whole-cell patch clamp.