Rat Brainstem-spinal Cord Preparation Research Articles

Prenatal nicotine exposure alters glycinergic and GABAergic control of respiratory frequency in the neonatal rat brainstem-spinal cord preparation

Bath application of GABA-A receptor agonists in neonatal rat brainstem-spinal cord preparations (BSSC) reduces respiratory frequency, an effect that is enhanced by prenatal nicotine exposure. Here we test the hypothesis that these effects can be reproduced by microinjection of GABAergic and glycinergic agonists into the pre-Botzinger complex region (PBC). We recorded the activity of phrenic motor axons from the fourth cervical ventral root in 1-3 days old BSSC that were exposed to either nicotine (6 mg/(kg day)) or saline prenatally. Microinjection of glycine or muscimol into the PBC caused abrupt, reversible apnea in all experiments. Apnea duration with glycine averaged 50.3+/-5 s in saline-exposed (N=12), and 95.7+/-9.9 s in nicotine-exposed (N=12) neonates (P<0.001). Apnea duration with muscimol averaged 51+/-5.1 s in saline-exposed (N=10), and 86+/-10.6 s in nicotine-exposed (N=12) neonates (P<0.05). These data show that prenatal nicotine exposure alters development of central ventilatory control, and that neurons in the PBC region are involved.

Developmental changes in pontine influence on respiratory rhythm generation in neonatal rats: effect of age versus tissue oxygenation

Pontine ablation depressed respiratory frequency in in vitro rat brainstem spinal cord preparations from postnatal rat pups on the day of birth (P0) but increased burst frequency in 2 and 4 day old (P2, P4) preparations. To examine the extent to which the response to pontine removal and the effects of age on this response represented developmental changes versus changes in tissue mass and oxygenation of the preparation, we first examined the effect of chemical rather than physical lesion of the pons with tetrodotoxin (0.5μM) in a split bath preparation to inactivate the pons without reducing tissue mass. We found chemical lesions had a similar effect as physical ablation. Next, using an O2 sensing microelectrode, we found that pontine ablation did not affect PO2 in the medulla at the level of the PreBötzinger Complex. PO2 reached zero around 1mm from the ventral surface in the absence and presence of the pons, suggesting adequate oxygenation of the ventral respiratory group. These data confirm that the role of the pons and the changes seen in pontine influence on respiratory rhythm over early development are not an artifact of changes in tissue mass or oxygenation. Supported by the NSERC of Canada.

Early postnatal changes in respiratory activity in rat in vitro and modulatory effects of substance P

Developmental changes in the respiratory activity and its modulation by substance P (SP) were studied in the neonatal rat brainstem-spinal cord preparation from the day of birth to day 3 (P0-P3). The respiratory network activity in the ventrolateral medulla was represented by two types of bursts: basic regular bursts with typical decrementing shape and biphasic bursts appearing after augmented biphasic discharges in inspiratory neurons. With advancing postnatal age the respiratory output was considerably modified; the basic rhythm became faster by 20%, whereas the biphasic burst rate, which was originally 15 times slower, declined further by 180% and the C4 burst duration significantly decreased by 20% due to reduced decay time without preceding changes in the central inspiratory drive. SP had an age-dependent excitatory effect on respiratory activity. In the basic rhythm, SP could induce transient rhythm cessations on P0-P2 but not on P3. For the biphasic burst frequency, the sensitivity to SP significantly decreased from P0 to P3, whereas the range of SP-induced changes increased. In both types of bursts, SP prolonged C4 burst duration due to increasing decay time. This effect was three times greater on P3 and did not depend on the central inspiratory drive. Our results suggest that the potency of SP to regulate the respiratory activity elevates during the early postnatal period. The developmental changes in the respiratory activity appear to represent the transient stage in the maturation of rhythm and pattern generation mechanisms facilitating adaptive behavior of a quickly growing organism.

Point:Counterpoint: The parafacial respiratory group (pFRG)/pre-Bötzinger complex (preBötC) is the primary site of respiratory rhythm generation in the mammal

Point:Counterpoint: The parafacial respiratory group (pFRG)/pre-Bötzinger complex (preBötC) is the primary site of respiratory rhythm generation in the mammal

Propriospinal neurons contribute to bulbospinal transmission of the locomotor command signal in the neonatal rat spinal cord.

This study examines whether propriospinal transmission contributes to descending propagation of the brainstem locomotor command signal in the in vitro neonatal rat spinal cord. Using double bath partitions, synaptic transmission was suppressed in the cervicothoracic region while monitoring locomotor-like activity on lumbar ventral roots evoked by either chemical or electrical stimulation of the brainstem. Locomotor-like activity induced by electrical stimulation was more stable (cycle period coefficient of variation (CV) 11.7 +/- 6.1%) than the rhythm induced by chemical stimulation (CV 31.3 +/- 6.4%). Ca(2+)-free bath solution, elevated Mg(2+) ion concentration, excitatory amino acid receptor antagonists (AP5 and/or CNQX), and the muscarinic receptor antagonist, atropine, were used in attempts to block synaptic transmission. Each of these manipulations, except muscarinic receptor blockade, was capable of blocking locomotor-like activity induced by brainstem stimulation. However, locomotor-like activity induced by higher intensity electrical stimulation of the brainstem (1.2-5 times threshold) was relatively refractory to synaptic suppression using AP5 and CNQX, and Ca(2+)-free solution was more effective if combined with high Mg(2+) (15 mm) or EGTA. Enhancement of neuronal excitation in the cervicothoracic region, using Mg(2+)-free bath solution, facilitated brainstem activation of locomotor-like activity in the lumbar cord, consistent with a propriospinal mechanism of locomotor signal propagation. Blockade of brainstem-induced locomotor-like activity was related to the number of cervicothoracic segments exposed to synaptic suppression, being most effective if five or more segments were included. These results provide direct evidence that propriospinal pathways contribute to bulbospinal activation of the locomotor network in the in vitro neonatal rat brainstem-spinal cord preparation, and suggest that a propriospinal system is recruited in parallel with long direct projections that activate the locomotor network.

2P392 Two modes of respiratory rhythm generation in the newborn rat brainstem-spinal cord preparation in vitro(44. Neuro-biophysics,Poster Session,Abstract,Meeting Program of EABS &amp; BSJ 2006)

2P392 Two modes of respiratory rhythm generation in the newborn rat brainstem-spinal cord preparation in vitro(44. Neuro-biophysics,Poster Session,Abstract,Meeting Program of EABS &amp; BSJ 2006)

Dopaminergic modulation on respiratory rhythm in rat brainstem-spinal cord preparation.

Dopamine (DA) is known to regulate both higher order and autonomic brain functions. To elucidate the effects of DA on respiratory rhythm generation, isolated brainstem-spinal cord was treated with DA and each DA receptor agonist individually. DA application facilitated respiratory rhythm in pons-medulla-spinal cord preparations, but depressed it in medulla-spinal cord preparations. Furthermore, we found that DA application depressed Pre-Inspiratory (Pre-I) neurons but not Inspiratory or Expiratory neurons and that among the various DA receptor agonists, only a D4 agonist, PD168077, depressed respiratory rhythm. Therefore, the primary target of DA action in modulating respiratory rhythm generation is the Pre-I neurons in the medulla.

The excitability of lumbar motoneurones in the neonatal rat is increased by a hyperpolarization of their voltage threshold for activation by descending serotonergic fibres

Previous work has shown there is an increase in motoneurone excitability produced by hyperpolarization of the threshold potential at which an action potential is elicited (Vth) at the onset, and throughout brainstem-induced fictive locomotion in the decerebrate cat. This represents a transient facilitation in the membrane potential for activation dependent on the presence of fictive locomotion. The present study tests the hypothesis that a similar neuromodulatory mechanism facilitating neuronal recruitment also exists in the neonatal rat, and the endogenous pathway mediating the Vth hyperpolarization can be activated by electrical stimulation of the neonatal brainstem. Isolated brainstem-spinal cord preparations from 1- to 5-day-old neonatal rats, and whole-cell recording techniques were used to examine the patterns of ventral root (VR) activity produced, and the effect of electrical stimulation of the ventromedial medulla on lumbar spinal neurones. Hyperpolarization of Vth was seen in 10/11 (range -2 to -18 mV) neurones recorded during locomotor-like VR activity, and appeared analogous to the locomotor-dependent Vth hyperpolarization previously described in the cat. However, in the present study, Vth hyperpolarization was also seen during electrical brainstem stimulation that evoked alternating, rhythmic, or tonic VR activity, or failed to evoke VR activity. Thirty-six of 71 neurones were antidromically identified as lumbar motoneurones and 33/36 showed a hyperpolarization of Vth (-2 to -14 mV) during electrical brainstem stimulation. Of the unidentified lumbar ventral horn neurones, 31/35 also showed hyperpolarization of Vth (-2 to -20 mV) during brainstem stimulation. The hyperpolarization of Vth and VR activity induced by brainstem stimulation was reversibly blocked by cooling of the cervical cord, indicating it is mediated by descending fibres, and application of the serotonergic antagonist ketanserin to the spinal cord was effectively able to block the brainstem-evoked hyperpolarization of Vth. These results demonstrate a previously unknown action of the endogenous descending serotonergic system to facilitate spinal motoneuronal recruitment and firing by inducing a hyperpolarization of Vth. This modulatory process can be examined in the neonatal rat brainstem-spinal cord preparation without the requirement for ongoing locomotor activity.

Two types of rhythm in the respiratory network output in the isolated ventrolateral medulla in the neonatal rats

Effects of substance P and extracellular [K +] o on respiratory motor activity in the ventrolateral medulla in neonatal rat (0–4 days old) brainstem-spinal cord preparation were studied. In addition to fictive eupneic rhythm (8–13 bursts/minute), the respiratory motor output was composed of biphasic bursts which might underlie the sigh pattern in vivo. These bursts had considerably lower frequency (0.15–0.86 bursts/minute) and appeared when inspiratory neurons generated augmented biphasic discharges. The two rhythms were differently affected when the respiratory network excitability was increased by substance P or decreased by lowering external [K +] o, the effects on biphasic burst frequency being considerably greater. The augmented bursts could suppress inspiratory, but not pre-inspiratory neuron discharge, suggesting that pre-inspiratory neurons formed a supplementary rhythmic network which was not directly affected by biphasic burst generation.

Salicylate action on medullary inspiratory neuron activity in a brainstem-spinal cord preparation from newborn rats.

Salicylate affects central respiratory control. The inspiratory neurons are the most important component of the medullary respiratory control center because they modulate the final motor output via the phrenic nerve. We investigated changes in burst rate, intraburst firing frequency, and membrane properties of inspiratory neurons in the isolated brainstem after the administration of salicylate. Newborn rat brainstem-spinal cord preparations were superfused with salicylate. Whole-cell recordings were performed from inspiratory neurons. Application of 1 mM salicylate caused an increase in the inspiratory neuronal burst rate from 6.9 +/- 1.6 bursts/min to 8.2 +/- 1.9 bursts/min (P < 0.05). The inspiratory neuron burst rate decreased from 8.3 +/- 0.7 bursts/min to 4.5 +/- 1.1 bursts/min after the application of 10 mM salicylate (P < 0.01). The depressant effect of 10 mM salicylate was antagonized by the gamma-aminobutyric acid (GABA) receptor antagonist bicuculline (1 microM). Resting membrane potential and intraburst firing frequency did not change with the application of salicylate and bicuculline even when the burst rate did change. We conclude that the effects of salicylate on the medullary inspiratory neurons are mainly due to a presynaptic action. GABAergic mechanisms are probably involved in the salicylate-induced central respiratory depression.

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 brain stem preparation.

This study was designed to examine the possibility that respiratory arrest during hypothermia occurs at the level of premotor 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 rewarming, on neurons in the pre-Bötzinger Complex, as an indication of the output of the entire rhythmogenic network; and from cervical spinal (phrenic) ventral roots, as an indication of motor neuron output, in an in vitro neonatal rat brain stem-spinal cord preparation. We found that hypothermia led to a slowing of the respiratory rhythm with little or no decrease in the magnitude of phrenic motor output or the field potential of pre-Bötzinger Complex neurons. Ultimate arrest occurred abruptly and simultaneously in recordings from both sites, indicating that the arrest was due to failure of the central rhythm-generating network, primarily due to removal of a conditional excitation. On being rewarmed, the motor output recorded at both sites was usually fractionated, initially suggesting that changes occurred in network synchronization either during cooling or during reactivation following hypothermic arrest.

Opioid action on respiratory neuron activity of the isolated respiratory network in newborn rats.

Underlying mechanisms behind opioid-induced respiratory depression are not fully understood. The authors investigated changes in burst rate, intraburst firing frequency, membrane properties, as well as presynaptic and postsynaptic events of respiratory neurons in the isolated brainstem after administration of opioid receptor agonists. Newborn rat brainstem-spinal cord preparations were used and superfused with mu-, kappa-, and delta-opioid receptor agonists. Whole cell recordings were performed from three major classes of respiratory neurons (inspiratory, preinspiratory, and expiratory). Mu- and kappa-opioid receptor agonists reduced the spontaneous burst activity of inspiratory neurons and the C4 nerve activity. Forty-two percent of the inspiratory neurons were hyperpolarized and decreased in membrane resistance during opioid-induced respiratory depression. Furthermore, under synaptic block by tetrodotoxin perfusion, similar changes of inspiratory neuronal membrane properties occurred after application of mu- and kappa-opioid receptor agonists. In contrast, resting membrane potential and membrane resistance of preinspiratory and majority of expiratory neurons were unchanged by opioid receptor agonists, even during tetrodotoxin perfusion. Simultaneous recordings of inspiratory and preinspiratory neuronal activities confirmed the selective inhibition of inspiratory neurons caused by mu- and kappa-opioid receptor agonists. Application of opioids reduced the slope of rising of excitatory postsynaptic potentials evoked by contralateral medulla stimulation, resulting in a prolongation of the latency of successive first action potential responses. Mu- and kappa-opioid receptor agonists caused reduction of final motor outputs by mainly inhibiting medullary inspiratory neuron network. This inhibition of inspiratory neurons seems to be a result of both a presynaptic and postsynaptic inhibition. The central respiratory rhythm as reflected by the preinspiratory neuron burst rate was essentially unaltered by the agonists.

5-HT acting on 5-HT(1/2) receptors does not participate in the in vitro hypoxic respiratory depression.

The involvement of serotoninergic mechanisms in the central respiratory depression produced by hypoxia was studied in the newborn rat brainstem-spinal cord preparation. The respiratory frequency measured by the C4 ventral root activity was recorded. 5-HT (30 microM) superfusion elicited a rapid increase in respiratory frequency, prevented by a treatment with methysergide (a 5-HT(1/2) receptor antagonist) (40 microM). To investigate the possible participation of 5-HT in hypoxic respiratory depression, this concentration of methysergide was added to the bathing medium during hypoxia. Methysergide did not modify the decrease in respiratory frequency produced by hypoxia. In order to ensure that other 5-HT subtype receptors were not involved in hypoxic respiratory depression, 5-HT was added to the bath during hypoxic-methysergide tests; no effect on respiratory frequency was observed. These results suggest that in the newborn rat brainstem-spinal cord preparation, serotoninergic mechanisms are not involved in the elaboration of the in vitro respiratory response to hypoxia.

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.

Dorsal column nuclei neurons recorded in a brain stem-spinal cord preparation: characteristics and their responses to dorsal root stimulation.

Recordings were obtained from dorsal column nucleus (DCN) neurons in a neonatal rat brain stem-spinal cord preparation to study their basic electrophysiological properties and responses to stimulation of a dorsal root. Whole-cell patch-clamp recordings were made from 21 neurons that responded to dorsal root stimulation with a fast excitatory postsynaptic potential (EPSP). These neurons were located lateral to, but at the level of, the area postrema at depths of 100-268 microm below the dorsal surface of the brain. The neurons could be divided into groups according to the shape of their action potentials or voltage responses to hyperpolarizing current steps; however, the response profiles of the groups of neurons to dorsal root stimulation were not significantly different and all neurons were considered together. Dorsal root stimulation elicited excitatory postsynaptic potentials (EPSPs) in all neurons with a very low variability in onset latency and an ability to follow 100-Hz stimulation, indicating that they were mediated by activation of a monosynaptic pathway. The peak amplitude of the EPSP increased with membrane hyperpolarization, and applications of the non-NMDA receptor antagonists 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2, 3-dione (NBQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) decreased the amplitude of the EPSP to 14.2% of the control response (n = 6). The descending phase of the EPSP decreased with membrane hyperpolarization and was reduced by the N-methyl-D-aspartate (NMDA) receptor antagonist AP-5 (n = 2). The EPSPs were also reduced in amplitude by applications of the gamma-aminobutyric acid-B (GABA(B)) receptor agonist baclofen, which had no effect on membrane potential or input resistance. These results show that fast EPSPs in DCN neurons elicited by dorsal root stimulation are mediated by an excitatory amino acid acting at both non-NMDA and, to a lesser extent, NMDA receptors. In addition, GABA acting at presynaptic GABA(B) receptors can inhibit these responses.

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.

The adrenergic modulation of firings of respiratory rhythm-generating neurons in medulla-spinal cord preparation from newborn rat.

We analysed the modulation of respiratory neurons by adrenaline or noradrenaline (NA) in a newborn rat brainstem-spinal cord preparation. Adrenaline or NA caused a dose-dependent depression of the respiratory rhythm and induced C4 spinal tonic discharges. The inhibitory effect of adrenaline (ED50=0.5 microM) on the respiratory rhythm was stronger than NA (ED50=5 microM). The adrenaline respiratory rhythm depression was partially blocked by the alpha1-antagonist prazosin or by the alpha2-antagonist yohimbine. The C4 tonic discharge elicited by adrenaline was blocked by the alpha1-antagonist prazosin. The direct effects of adrenaline on pre-inspiratory (Pre-I) neurons were examined in a synaptic blockade solution (low Ca), and fifty-six percent of Pre-I neurons were found to continue firing. In low-Ca solution, Pre-I neurons were excited (n=29 of 39) or depressed (n=5 of 39) by adrenaline, and excited by alpha1-agonist phenylephrine or depressed by alpha2-agonist clonidine. These results suggest that the respiratory rhythm depression under intact network conditions is mediated by some other inhibitory system. The inhibitory effect of adrenaline on the respiratory rhythm was partially blocked by the GABA(A)-antagonists bicuculline or picrotoxin, but not by the GABA(B)-antagonist phaclofen. The present results suggest that: (1) respiratory rhythm generation is more sensitive to adrenaline than NA through alpha-adrenergic action of adrenaline; (2) the activity of Pre-I neurons could be directly regulated by excitation via alpha1-receptors and inhibition via alpha2-receptors; and (3) the depression of the respiratory rhythm by adrenaline is partly mediated by GABA(A)ergic neurons.

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.

Stimulation within the rostral ventrolateral medulla can evoke monosynaptic GABAergic IPSPs in sympathetic preganglionic neurons in vitro.

The inhibitory responses of identified sympathetic preganglionic neurons (SPNs) to stimulation within the rostral ventrolateral medulla (RVLM) were studied to determine their nature and pharmacology. Whole cell patch-clamp recordings were made from 36 SPNs in the upper thoracic segments of the spinal cord in a neonatal rat brain stem-spinal cord preparation. Neurons were identified as SPNs on the basis of their antidromic activation after stimulation of the ipsilateral segmental ventral root and their morphology and location in the intermediolateral cell column and intercalated nucleus. In all SPNs, electrical stimulation of the RVLM evoked fast excitatory postsynaptic potentials (EPSPs) that were mediated by non-N-methyl-D-aspartate (NMDA) and NMDA receptors. These excitatory responses were the most prominent response in control artificial cerebrospinal fluid and have been studied previously. In 22 of the SPNs, RVLM stimulation also elicited fast inhibitory postsynaptic potentials (IPSPs), which increased in amplitude as the membrane was depolarized. Five of these neurons were not studied further as they responded occasionally with IPSPs that had highly variable onset latencies indicating the involvement of a polysynaptic pathway. In the remaining SPNs (n = 17), the evoked IPSPs persisted in the presence of the excitatory amino acid antagonists 6-cyano-7-nitroquinoxaline-2,3,-dione and D,L-2-amino-5-phosphonopentanoic acid. In eight of these SPNs, it was necessary to block the EPSPs to reveal the IPSPs. In the 7 SPNs tested, the onset latencies of the IPSPs were not significantly different from the onset latencies of the fast EPSPs. The low sweep-to-sweep fluctuations in onset latency of individual IPSPs (absolute average deviation: 0.4 ms) indicated that the IPSPs were elicited by activation of a monosynaptic pathway. The amplitudes of the IPSPs decreased in amplitude as the membrane was hyperpolarized and reversed in polarity at -70.3 +/- 1.7 mV (mean +/- SD), which was close to the equilibrium potential for chloride ions. In addition, in seven SPNs, bath applications of 5 microM bicuculline, a gamma-aminobuturic acid-A (GABAA) antagonist, abolished or reduced the evoked IPSPs. Five SPNs also were studied that displayed ongoing IPSPs. The amplitudes of these IPSPs increased with membrane depolarization and were blocked by bath applications of 5 microM bicuculline, suggesting that they also were mediated by activation of GABAA receptors. These results demonstrate the existence of a bulbospinal GABAergic pathway impinging directly onto SPNs. This pathway may be tonically active in the neonatal rat brain stem-spinal cord preparation.