Isolated Brain Stem-spinal Cord Preparation Research Articles

Glycine and GABAA receptors suppressively regulate the inspiratory-related calcium rise in the thoracic inspiratory cells of the neonatal rat

We previously demonstrated that in an isolated brainstem–spinal cord preparation from neonatal rats, a local bath application of strychnine (a broad antagonist of glycine and GABAA receptors) to the spinal cord enhances thoracic inspiratory motor activity. Herein, to investigate the involvement of the inspiratory spinal interneurons that provide excitatory input to the motoneuron, we conducted calcium imaging using this preparation. Oregon Green 488 BAPTA-1 AM, a fluorescent calcium indicator, was injected into the ventromedial surface of the thoracic cord. In all cells that showed inspiratory-related fluorescence changes > 2% of the baseline fluorescence intensity, the inspiratory-related fluorescence change decreased when the focal depth was deepened. The application of strychnine to the spinal cord increased the inspiratory-related intracellular calcium rise in these cells. These results suggest that the enhancement of inspiratory interneuron activity could be involved in this enhancement of inspiratory motor activity.

ЗНАЧЕНИЕ ПРЕНАТАЛЬНОГО ПАССИВНОГО КУРЕНИЯ ДЛЯ РАЗВИТИЯ ЦЕНТРАЛЬНЫХ МЕХАНИЗМРОВ РЕГУЛЯЦИИ ДЫХАНИЯ У НОВОРОЖДЕННЫХ КРЫС IN VITRO

In the prenatal period, neural networks undergo the most significant morphological changes and subsequent functional transformations. The influence of a combination of factors of passive tobacco smoking to the development of the central mechanisms of respiratory control in the prenatal period remains unexplored. The present study was devoted to the study of the prenatal effect of passive smoking factors on the functioning of the central mechanisms of respiratory control in newborn rat. The study was carried out on 107 isolated brain stem-spinal cord preparations of the brain of newborn white outbred rats in vitro. Passive smoking was modeled by fumigating experimental animals with cigarette smoke. To simulate the hypercapnic effect, the bulbospinal preparations were perfused for 10 minutes with a solution of artificial cerebrospinal fluid with a saturated gas mixture, consisting of 10 % CO2 and 90 % O2; to simulate hypoxic exposure, the preparations were irrigated with a solution of artificial cerebrospinal fluid with a saturated gas mixture for 20 minutes 5 % CO2 and 95 % N2. As a result of the study, it was found that in newborn rats that underwent prenatal passive smoking, there is a high frequency of generation of inspiratory discharges, a shift in the spectral power density peaks of respiratory discharges in both ranges towards low frequencies, a decrease in their power, a decrease in the amplitude and a shorter duration of inspiratory discharges. in comparison with intact animals. A feature of the reaction to hypoxia in newborn rats who have undergone prenatal passive smoking is a late formation of the hypoxic depression of respiratory rhythm, reaction to hypercapnia characterized by no reduction in duration respiratory cycle. Thus performed by us research shows that prenatal secondhand smoke affects the mechanism of formation of the respiratory rhythm and pattern and also modifies the respiratory response to change in the partial tension of respiratory gases in cerebrospinal fluid in vitro.

Influence of Brain Stem on Axial and Hindlimb Spinal Locomotor Rhythm Generating Circuits of the Neonatal Mouse.

The trunk plays a pivotal role in limbed locomotion. Yet, little is known about how the brain stem controls trunk activity during walking. In this study, we assessed the spatiotemporal activity patterns of axial and hindlimb motoneurons (MNs) during drug-induced fictive locomotor-like activity (LLA) in an isolated brain stem-spinal cord preparation of the neonatal mouse. We also evaluated the extent to which these activity patterns are affected by removal of brain stem. Recordings were made in the segments T7, L2, and L5 using calcium imaging from individual axial MNs in the medial motor column (MMC) and hindlimb MNs in lateral motor column (LMC). The MN activities were analyzed during both the rhythmic and the tonic components of LLA, the tonic component being used as a readout of generalized increase in excitability in spinal locomotor networks. The most salient effect of brain stem removal was an increase in locomotor rhythm frequency and a concomitant reduction in burst durations in both MMC and LMC MNs. The lack of effect on the tonic component of LLA indicated specificity of action during the rhythmic component. Cooling-induced silencing of the brain stem reproduced the increase in rhythm frequency and accompanying decrease in burst durations in L2 MMC and LMC, suggesting a dependency on brain stem neuron activity. The work supports the idea that the brain stem locomotor circuits are operational already at birth and further suggests an important role in modulating trunk activity. The brain stem may influence the axial and hindlimb spinal locomotor rhythm generating circuits by extending their range of operation. This may represent a critical step of locomotor development when learning how to walk in different conditions and environments is a major endeavor.

A behaviorally related developmental switch in nitrergic modulation of locomotor rhythmogenesis in larval Xenopus tadpoles.

Locomotor control requires functional flexibility to support an animal's full behavioral repertoire. This flexibility is partly endowed by neuromodulators, allowing neural networks to generate a range of motor output configurations. In hatchling Xenopus tadpoles, before the onset of free-swimming behavior, the gaseous modulator nitric oxide (NO) inhibits locomotor output, shortening swim episodes and decreasing swim cycle frequency. While populations of nitrergic neurons are already present in the tadpole's brain stem at hatching, neurons positive for the NO-synthetic enzyme, NO synthase, subsequently appear in the spinal cord, suggesting additional as yet unidentified roles for NO during larval development. Here, we first describe the expression of locomotor behavior during the animal's change from an early sessile to a later free-swimming lifestyle and then compare the effects of NO throughout tadpole development. We identify a discrete switch in nitrergic modulation from net inhibition to overall excitation, coincident with the transition to free-swimming locomotion. Additionally, we show in isolated brain stem-spinal cord preparations of older larvae that NO's excitatory effects are manifested as an increase in the probability of spontaneous swim episode occurrence, as found previously for the neurotransmitter dopamine, but that these effects are mediated within the brain stem. Moreover, while the effects of NO and dopamine are similar, the two modulators act in parallel rather than NO operating serially by modulating dopaminergic signaling. Finally, NO's activation of neurons in the brain stem also leads to the release of NO in the spinal cord that subsequently contributes to NO's facilitation of swimming.

Organization of pontine reticulospinal inputs to motoneurons controlling axial and limb muscles in the neonatal mouse.

Using optical recording of synaptically mediated calcium transients and selective spinal lesions, we investigated the pattern of activation of spinal motoneurons (MNs) by the pontine reticulospinal projection in isolated brain stem-spinal cord preparations from the neonatal mouse. Stimulation sites throughout the region where the pontine reticulospinal neurons reside reliably activated MNs at cervical, thoracic, and lumbar levels. Activation was similar in MNs ipsi- and contralateral to the stimulation site, similar in medial and lateral motor columns that contain trunk and limb MNs, respectively, and similar in the L2 and L5 segments that predominantly contain flexor and extensor MNs, respectively. In nonlesioned preparations, responses in both ipsi- and contralateral MNs followed individual stimuli in stimulus trains nearly one-to-one (with few failures). After unilateral hemisection at C1 on the same side as the stimulation, responses had substantially smaller magnitudes and longer latencies and no longer followed individual stimuli. After unilateral hemisection at C1 on the side opposite to the stimulation, the responses were also smaller, but their latencies were not affected. Thus we distinguish two pontine reticulospinal pathways to spinal MNs, one uncrossed and the other crossed, of which the uncrossed pathway transmits more faithfully and appears to be more direct.

Spinal locomotor inputs to individually identified reticulospinal neurons in the lamprey

Locomotor feedback signals from the spinal cord to descending brain stem neurons were examined in the lamprey using the uniquely identifiable reticulospinal neurons, the Müller and Mauthner cells. The same identified reticulospinal neurons were recorded in several preparations, under reduced conditions, to address whether an identified reticulospinal neuron shows similar locomotor-related oscillation timing from animal to animal and whether these timing signals can differ significantly from other identified reticulospinal neurons. Intracellular recordings of membrane potential in identified neurons were made in an isolated brain stem-spinal cord preparation with a high-divalent cation solution on the brain stem to suppress indirect neural pathways and with D-glutamate perfusion to the spinal cord to induce fictive swimming. Under these conditions, the identified reticulospinal neurons show significant clustering of the timings of the peaks and troughs of their locomotor-related oscillations. Whereas most identified neurons oscillated in phase with locomotor bursting in ipsilateral ventral roots of the rostral spinal cord, the B1 Müller cell, which has an ipsilateral descending axon, and the Mauthner cell, which has a contralateral descending axon, both had oscillation peaks that were out of phase with the ipsilateral ventral roots. The differences in oscillation timing appear to be due to differences in synaptic input sources as shown by cross-correlations of fast synaptic activity in pairs of Müller cells. Since the main source of the locomotor input under these experimental conditions is ascending neurons in the spinal cord, these experiments suggest that individual reticulospinal neurons can receive locomotor signals from different subsets of these ascending neurons. This result may indicate that the locomotor feedback signals from the spinal locomotor networks are matched in some way to the motor output functions of the individual reticulospinal neurons, which include command signals for turning and for compensatory movements.

Transgenic Mice Lacking Serotonin Neurons Have Severe Apnea and High Mortality during Development

Central serotonin (5-HT) neurons modulate many vital brain functions, including respiratory control. Whether breathing depends critically on 5-HT neurons, or whether their influence is excitatory or inhibitory, remains controversial. Here we show that neonatal Lmx1b(flox/flox;ePet-Cre/+) mice (also called Lmx1b(f/f/p) mice), which selectively lack serotonin neurons, display frequent and severe apnea lasting as long as 55 s. This was associated with a marked decrease in ventilation to less than one-half of normal. These respiratory abnormalities were most severe during the postnatal period, markedly improving by the time the pups were 2-4 weeks old. Despite the severe breathing dysfunction, many of these mice survived, but there was a high perinatal mortality, and those that survived had a decrease in growth rate until the age at which the respiratory defects resolved. Consistent with these in vivo observations, respiratory output was markedly reduced in isolated brainstem-spinal cord preparations from neonatal Lmx1b(f/f/p) mice and completely blocked in perfused brain preparations from neonatal rats treated with selective antagonists of 5-HT(2A) and neurokinin 1 (NK-1) receptors. The ventilatory deficits in neonatal Lmx1b(f/f/p) mice were reversed in vitro and in vivo with agonists of 5-HT(2A) and/or NK-1 receptors. These results demonstrate that ventilatory output in the neonatal period is critically dependent on serotonin neurons, which provide excitatory drive to the respiratory network via 5-HT(2A) and NK-1 receptor activation. These findings provide insight into the mechanisms of sudden infant death syndrome, which has been associated with abnormalities of 5-HT neurons and of cardiorespiratory control.

Differential origin of reticulospinal drive to motoneurons innervating trunk and hindlimb muscles in the mouse revealed by optical recording

To better understand how the brainstem reticular formation controls and coordinates trunk and hindlimb muscle activity, we used optical recording to characterize the functional connections between medullary reticulospinal neurons and lumbar motoneurons of the L2 segment in the neonatal mouse. In an isolated brainstem-spinal cord preparation, synaptically induced calcium transients were visualized in individual MNs of the ipsilateral and contralateral medial and lateral motor columns (MMC, LMC) following focal electrical stimulation of the medullary reticular formation (MRF). Stimulation of the MRF elicited differential responses in MMC and LMC, according to a specific spatial organization. Stimulation of the medial MRF elicited responses predominantly in the LMC whereas stimulation of the lateral MRF elicited responses predominantly in the MMC. This reciprocal response pattern was observed on both the ipsilateral and contralateral sides of the spinal cord. To ascertain whether the regions stimulated contained reticulospinal neurons, we retrogradely labelled MRF neurons with axons coursing in different spinal funiculi, and compared the distributions of the labelled neurons to the stimulation sites. We found a large number of retrogradely labelled neurons within regions of the gigantocellularis reticular nucleus (including its pars ventralis and alpha) where most stimulation sites were located. The existence of a mediolateral organization within the MRF, whereby distinct populations of reticulospinal neurons predominantly influence medial or lateral motoneurons, provides an anatomical substrate for the differential control of trunk and hindlimb muscles. Such an organization introduces flexibility in the initiation and coordination of activity in the two sets of muscles that would satisfy many of the functional requirements that arise during postural and non-postural motor control in mammals.

Spatiotemporal organization of frog respiratory neurons visualized on the ventral medullary surface

We visualized the spatiotemporal activity of respiratory-related neurons in the frog using the isolated brainstem spinal cord preparation. We recorded optical signals from the ventral surface of the medulla using a voltage-sensitive dye, and calculated cross-correlations with the integrated respiratory activity of the trigeminal nerve. Lung burst-related depolarizing optical signals were observed bilaterally as longitudinal columns in the ventrolateral medulla between the levels of trigeminal and hypoglossal rootlets, mostly caudal to the vagal rootlet. However, we could not differentiate between neurons involved in rhythm generation and motoneurons. The dye weakened the buccal rhythm and slowed the lung rhythm, which might have influenced the results. Extracellular recording of respiratory neurons verified the optically identified area. Strychnine disrupted the spatiotemporal organization of optical signals, although trigeminal periodic bursts persisted. We conclude that the pattern generator but not the rhythm generator of lung burst in the frog involves glycinergic mechanisms and lies as longitudinal columns in the reticular formation of the ventrolateral medulla.

Development and pH sensitivity of the respiratory rhythm of fetal mice in vitro

In newborn and adult mammals, chemosensory drive exerted by CO2 and H+ provides an essential tonic input: without it the rhythm of respiration is abolished. It is not known, however, whether this chemosensory drive and the respiratory rhythm appear simultaneously during development. In isolated brainstem–spinal cord preparations from fetal mice, we determined at what stage of fetal life the respiratory rhythm appeared in third to fifth cervical ventral roots (phrenic motoneurons) and whether this fetal rhythm was sensitive to chemosensory inputs. A respiratory-like rhythm consisting of short duration bursts of discharges recurring at 2–16 min−1 was detected in two of nine embryonic day 13 fetuses; it was abolished by transection of the spinal cord between the first to second cervical segments and was phase-related to rhythmic activity from medullary units of the ventral respiratory group. At embryonic day 13, it coexisted with a slow rhythm (0.1–2.0 min−1) of long duration bursts of action potentials which was generated by the spinal cord. At later fetal stages, the respiratory-like rhythm became more robust and of higher frequency, while the spinal cord rhythm became less obvious. At all fetal stages, acidification of the superfusion medium from pH 7.5–7.2 or 7.4–7.3 or 7.4 to 7.2 increased the frequency of both the respiratory-like and the spinal cord rhythms. In addition, acidification reduced the amplitude of the integrated burst activity of the spinal cord rhythm of embryonic day 13–embryonic day 16 fetuses and the respiratory-like rhythm of embryonic day 17 and older fetuses. Our results indicate that the rhythms transmitted by phrenic motoneurons during fetal development are chemosensitive from early fetal stages. Through its effects on induction and patterning of the rhythm, chemosensory drive may play a role in activity-dependent formation of respiratory neural networks.

Membrane Potential Oscillations in Reticulospinal and Spinobulbar Neurons During Locomotor Activity

Feedback from the spinal locomotor networks provides rhythmic modulation of the membrane potential of reticulospinal (RS) neurons during locomotor activity. To further understand the origins of this rhythmic activity, the timings of the oscillations in spinobulbar (SB) neurons of the spinal cord and in RS neurons of the posterior and middle rhombencephalic reticular nuclei were measured using intracellular microelectrode recordings in the isolated brain stem-spinal cord preparation of the lamprey. A diffusion barrier constructed just caudal to the obex allowed induction of locomotor activity in the spinal cord by bath application of an excitatory amino acid to the spinal bath. All of the ipsilaterally projecting SB neurons recorded had oscillatory membrane potentials with peak depolarizations in phase with the ipsilateral ventral root bursts, whereas the contralaterally projecting SB neurons were about evenly divided between those in phase with the ipsilateral ventral root bursts and those in phase with the contralateral bursts. In the brain stem under these conditions, 75% of RS neurons had peak depolarizations in phase with the ipsilateral ventral root bursts while the remainder had peak depolarizations during the contralateral bursts. Addition of a high-Ca2+, Mg2+ solution to the brain stem bath to reduce polysynaptic activity had little or no effect on oscillation timing in RS neurons, suggesting that direct inputs from SB neurons make a major contribution to RS neuron oscillations under these conditions. Under normal conditions when the brain is participating in the generation of locomotor activity, these spinal inputs will be integrated with other inputs to RS neurons.

Significance of extracellular potassium in central respiratory control studied in the isolated brainstem–spinal cord preparation of the neonatal rat

The significance of extracellular potassium in central respiratory control was investigated using the isolated brainstem-spinal cord preparation of the neonatal rat. Depth profiles of extracellular potassium activity ([K+])ECF in the medulla were measured with ion-sensitive microelectrodes. Although [K+]ECF increased with depth in medullary tissue during control (4 mM) and low (1 mM) potassium concentration ([K+])CSF superfusion, this gradient disappeared with higher [K+]CSF. With low [K+]CSF (1 mM), respiratory CO2 responsiveness was abolished, and increased with high [K+]CSF (8 mM). Respiratory frequency (fR) was diminished at low [K+]CSF (1 mM), and increased with elevated [K+]CSF (8 and 16 mM); with yet higher [K+]CSF (32 mM) apnea occurred after a transient increase in fR. Perforated patch recording revealed that high [K+]ECF decreased membrane resistance, depolarized membrane potential, and increased firing frequency in most of the recorded medullary neurons. High [K+]ECF also increased excitatory and inhibitory post-synaptic potentials of medullary neurons and augmented the functional connectivity among neurons. It is concluded that [K+]ECF is of importance in the maintenance of respiratory rhythm and central chemosensitivity.

Respiratory responses to pH in the absence of pontine and dorsal medullary areas in the newborn mouse in vitro

The contribution of pons and dorsal medulla in establishing the pattern of fictive respiration and in mediating the respiratory response to acidification was studied using the isolated brainstem–spinal cord preparation from neonatal mouse. About 40% of ponto-medullary preparations (retaining pons) showed spontaneous, but irregular respiratory-like rhythm. In the other 60%, the elimination of the pons often was followed by the initiation of a respiratory-like rhythm. Medullary preparations, derived from either inactive or rhythmic ponto-medullary preparations, showed a regular respiratory-like rhythm, which was also of a higher frequency and a bigger amplitude than that observed in ponto-medullary preparations. In contrast, ventral medullary preparations, derived from medullary preparations by eliminating the dorsal medulla, showed an irregular rhythm with a reduced amplitude of the integrated inspiratory burst. In ponto-medullary and ventral medullary preparations, acidification of the superfusion medium increased the respiratory frequency, while in medullary preparations, it increased the frequency and reduced the amplitude of the inspiratory burst. Our results suggest that pontine structures influence negatively the rate and depth of the respiratory-like rhythm, while dorsal medullary structures influence positively the depth of the rhythm. They also suggest that the pattern of response to pH supported by the ventral medulla is modified by the input provided from pons and dorsal medulla.

In vitro approach to the chemical drive of breathing

Since its introduction two decades ago, the isolated brain stem-spinal cord preparation of neonatal rodents has been the preferred method used to reveal the mystery underlying the genesis of the respiratory rhythm. Little research using this in vitro approach has focused on the study of the central respiratory chemosensitivity. Some unexpected findings obtained with the brain stem-spinal cord preparation have added new questions that challenge our previous theoretic framework. Some of these findings are addressed here.

Mechanisms regulating hypoxic respiratory depression during fetal and postnatal life.

Selected topics in the respiratory response to acute hypoxia in the fetus and newborn are reviewed. Peripheral chemoreceptors acting through ionotrophic glutamate receptors play an important role in affecting the initial augmentation phase. Whether fall off in peripheral chemoreceptor activity contributes to the secondary depressive phase remains controversial. A number of approaches including permanent electrolytic and reversible cooling lesions, Fos protein activation, and double-labeling immunohistochemistry has converged to show that an area in and around the locus ceruleus in the rostral pons affects the central depression. There is evidence that this is mediated by catecholamines acting at alpha(2)-adrenergic receptors. Tonic activity in early expiratory (postinspiratory) neurons may contribute to hypoxia-induced apneic episodes in the fetus and newborn. Desensitization of alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptors has been demonstrated in respiratory-related neurons both in vivo and in vitro. The role that this process might play in the depressive phase of the hypoxic ventilatory response has not been established. In vitro experiments with isolated brain stem-spinal cord preparations or transverse brain stem slices usually involve anoxia, whereas whole animal experiments use 8-15% O(2). Therefore, caution must be exercised in attempting to construct a unifying framework from these two approaches.

Newborn rat brainstem preparation with the trigeminal nerve attached for pain study

Studies using a brainstem–spinal cord preparation isolated from newborn rats have provided substantial information on neuro-physiology, -pharmacology and -anatomy of the respiratory center, such as mechanisms of respiratory rhythm generation, development of a respiratory center or respiratory reflex [T. Murakoshi, T. Suzue, S. Tamai, A pharmacological study on respiratory rhythm in the isolated brainstem–spinal cord preparation from newborn rat, Br. J. Pharmac. 86 (1985) 95–104 [5]; H. Onimaru, A. Arata, I. Homma, Primary respiratory rhythm generator in the medulla of brainstem–spinal cord preparations from newborn rats, Brain Res. 445 (1988) 314–324 [6]; H. Onimaru, I. Homma, Whole cell recordings from respiratory neurons in the medulla of brainstem–spinal cord preparations isolated from newborn rats, Pflügers Arch. 420 (1992) 399–406 [7]; J.C. Smith, K. Ballanyi, D.W. Richter, Whole-cell patch clamp recordings from respiratory neurons in neonatal rat brainstem in vitro, Neurosci. Lett. 314 (1992) 153–156 [10]; T. Suzue, Respiratory rhythm generation in the in vitro brainstem–spinal cord preparation of the neonatal rat, J. Physiol. (London) 354 (1984) 173–183 [11], reviewed in H. Onimaru, A. Arata, I. Homma, Neuronal mechanisms of respiratory rhythm generation: An approach using in vitro preparation, Jpn. J. Physiol. 47 (1997) 385–403 [8]]. Recently, the dissecting method of the preparation was modified to introduce a brainstem preparation with the trigeminal primary afferents attached for pain studies [M. Hamba, Repetitive stimulation potentiated the stimulus-evoked firing in the trigeminal caudalis—in vitro study. Neurosci. Res. 20 (1996) s163 [2]; M. Hamba, Stimulation-induced responses of the trigeminal caudal neurons in the brainstem preparation isolated from newborn rats, Brain Res. 785 (1998) 66–74 [3]]. As reported previously [3], the activity-dependent change in the excitability of pain-processing neurons, wind-up phenomenon, was studied in the trigeminal caudalis by stimulating the mandibular nerve, using a modified brainstem preparation isolated from newborn rats. The caudalis, the medulla dorsal horn, is known as the center for processing pain and sensory information from the cranio-facial area. The results indicated that the brainstem preparation is applicable for studies on the neuroplasticity at the pain-processing synapses. Here, we describe the method for isolation of a brainstem preparation with the trigeminal mandibular nerve attached and for recording the synaptic response evoked in the caudal neurons, using a whole-cell patch clamp technique. In the present study, we demonstrated repetitive stimulation-induced responses of caudal neurons at postnatal day 1 as an example showing the feasibility of the preparation for pain studies. Themes: Sensory systems Topics: Pain modulation: anatomy and physiology

K+ channels of expiratory neurones in the brain stem preparation of the newborn rat.

Single channel activity of expiratory neurones was studied in outside-out recordings. Expiratory neurones were identified in the ventrolateral region of the in vitro isolated brain stem-spinal cord preparation of newborn rats in cell-attached and whole-cell configurations by their pattern of firing related to phrenic motor output. Three potassium (K+) channels of 10, 30 and 70 pS exhibited steady-state activity during long voltage commands (up to 5 min) and could be found associated together in the same patches. The 30pS channel showed voltage dependency, being most active at small depolarizations. The 70 pS channel showed little activity with < 1% of openings per sample time and 1 mM tetraethylammonium (TEA) sensitivity. At similar concentrations, the discharge of the phrenic nerve was also altered, as shown by the increase of the respiratory frequency and a tonic discharge. The association of these K+ channel types on the same patches may be specific of respiratory neurones and could contribute to their bursting activity.

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.

Descending Inhibition in Neonatal Rat Spinal Cord: Actions of Pentobarbital and Morphine

Descending inhibition plays an important role in modulating spinal nociceptive neurotransmission. Barbiturates have been suggested to be poor analgesics or anti-analgesic because they block descending inhibition from supraspinal centers to the spinal cord. Opiate analgesics, on the other hand, are postulated to increase descending inhibition. We tested this hypothesis in an isolated brain stem-spinal cord preparation from neonatal rats, using as the test response a nociceptive-related slow ventral root potential (sVRP) recorded in the lumbar region. Brain stem and spinal cord were separately perfused. Transecting the spinal cord, applying the local anesthetic lidocaine to the brain stem, or cooling the brain stem increased the area of the sVRP, thus demonstrating that tonic descending inhibition is present in this preparation. Pentobarbital (Pb) (1–10 μM) applied to the spinal cord depressed the sVRP in a dose-dependent fashion. Spinal cord transection did not significantly change Pb potency. Pb (5–10 μM) applied to the brain stem alone did not significantly increase sVRP amplitude. Morphine (15–35 nM) applied to the spinal cord also depressed the sVRP but had no effect when applied to the brain stem. The results show that there are functional synaptic connections mediating tonic descending inhibition in the neonatal rat. They do not support interaction with tonic descending inhibition as an explanation for morphine analgesia or as a reason for lack of analgesic properties in the barbiturates.

Thyrotropin-releasing hormone stimulates perinatal rat respiration in vitro.

In the present study, we test whether thyrotropin-releasing hormone (TRH) stimulates respiratory frequency in perinatal rats by acting at regions of the medulla responsible for respiratory rhythmogenesis, the pre-Bötzinger complex. We also test whether TRH stimulates respiration in the fetal rat at a time shortly after the inception of respiratory rhythmogenesis [embryonic days (E) 17-18]. Two in vitro experimental models were utilized: the isolated brain stem-spinal cord preparation from fetal (E17-E18) and neonatal [postnatal days (P) 0-2] rats and the medullary slice preparation isolated from neonatal rats (P1-P2). Bath application of TRH caused a dose-dependent, reversible increase (maximum increase approximately 60%) in the frequency of respiratory rhythmic neural discharge generated by brain stem-spinal cord [half-maximal effective concentration (EC50) approximately 9 nM] and medullary slice (EC50 approximately 2.5 nM) neonatal rat preparations. Pressure injection of TRH unilaterally into the region of the pre-Bötzinger complex of the neonatal medullary slice caused an approximately 28% increase in the frequency of respiratory discharge. Application of TRH to the medium bathing fetal rat brain stem-spinal cord preparations caused an approximately threefold increase in respiratory discharge frequency. We conclude that TRH stimulates respiratory discharge frequency from the time near inception of respiratory motor discharge and acts directly at the pre-Bötzinger complex.