Ampakines stimulate phrenic motor output after cervical spinal cord injury.

Activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors increases phrenic motor output. Ampakines are a class of drugs that are positive allosteric modulators of AMPA receptors. We hypothesized that 1) ampakines can stimulate phrenic activity after incomplete cervical spinal cord injury (SCI), and 2) pairing ampakines with brief hypoxia could enable sustained facilitation of phrenic bursting. Phrenic activity was recorded ipsilateral (IL) and contralateral (CL) to C2 spinal cord hemisection (C2Hx) in anesthetized adult rats. Two weeks after C2Hx, ampakine CX717 (15 mg/kg, i.v.) increased IL (61 ± 46% baseline, BL) and CL burst amplitude (47 ± 26%BL) in 8 of 8 rats. After 90 min, IL and CL bursting remained above baseline (BL) in 7 of 8 rats. Pairing ampakine with a single bout of acute hypoxia (5-min, arterial partial pressure of O2 ~ 50 mmHg) had a variable impact on phrenic bursting, with some rats showing a large facilitation that exceeded the response of the ampakine alone group. At 8 weeks post-C2Hx, 7 of 8 rats increased IL (115 ± 117%BL) and CL burst amplitude (45 ± 27%BL) after ampakine. The IL burst amplitude remained above BL for 90-min in 7 of 8 rats; CL bursting remained elevated in 6 of 8 rats. The sustained impact of ampakine at 8 weeks was not enhanced by hypoxia exposure. Intravenous vehicle (10% 2-Hydroxypropyl-ß-cyclodextrin) did not increase phrenic bursting at either time point. We conclude that ampakines effectively stimulate neural drive to the diaphragm after cervical SCI. Pairing ampakines with a single hypoxic exposure did not consistently enhance phrenic motor facilitation.

Ampakine CX717 potentiates intermittent hypoxia-induced hypoglossal long-term facilitation.

Glutamatergic currents play a fundamental role in regulating respiratory motor output and are partially mediated by α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors throughout the premotor and motor respiratory circuitry. Ampakines are pharmacological compounds that enhance glutamatergic transmission by altering AMPA receptor channel kinetics. Here, we examined if ampakines alter the expression of respiratory long-term facilitation (LTF), a form of neuroplasticity manifested as a persistent increase in inspiratory activity following brief periods of reduced O2 [intermittent hypoxia (IH)]. Current synaptic models indicate enhanced effectiveness of glutamatergic synapses after IH, and we hypothesized that ampakine pretreatment would potentiate IH-induced LTF of respiratory activity. Inspiratory bursting was recorded from the hypoglossal nerve of anesthetized and mechanically ventilated mice. During baseline (BL) recording conditions, burst amplitude was stable for at least 90 min (98 ± 5% BL). Exposure to IH (3 × 1 min, 15% O2) resulted in a sustained increase in burst amplitude (218 ± 44% BL at 90 min following final bout of hypoxia). Mice given an intraperitoneal injection of ampakine CX717 (15 mg/kg) 10 min before IH showed enhanced LTF (500 ± 110% BL at 90 min). Post hoc analyses indicated that CX717 potentiated LTF only when initial baseline burst amplitude was low. We conclude that under appropriate conditions ampakine pretreatment can potentiate IH-induced respiratory LTF. These data suggest that ampakines may have therapeutic value in the context of hypoxia-based neurorehabilitation strategies, particularly in disorders with blunted respiratory motor output such as spinal cord injury.

Selective antagonism of opioid-induced ventilatory depression by an ampakine molecule in humans without loss of opioid analgesia.

Ventilatory depression is a significant risk associated with the use of opioids. We assessed whether opioid-induced ventilatory depression can be selectively antagonized by an ampakine without reduction of analgesia. In 16 healthy men, after a single oral dose of 1,500 mg of the ampakine CX717, a target concentration of 100 ng/ml alfentanil decreased the respiratory frequency by only 2.9 +/- 33.4% as compared with 25.6 +/- 27.9% during placebo coadministration (P < 0.01).Blood oxygenation and the ventilatory response to hypercapnic challenge also showed significantly smaller decreases with CX717 than with placebo. In contrast, CX717 did not affect alfentanil-induced analgesia in either electrical or heat-based experimental models of pain. Both ventilatory depression and analgesia were reversed with 1.6 mg of naloxone. These results support the use of ampakines as selective antidotes in humans to counter opioid-induced ventilatory depression without affecting opioid-mediated analgesia.

Distinct receptors underlie glutamatergic signalling in inspiratory rhythm-generating networks and motor output pathways in neonatal rat.

Despite the enormous diversity of glutamate (Glu) receptors and advances in understanding recombinant receptors, native Glu receptors underlying functionally identified inputs in active systems are poorly defined in comparison. In the present study we use UBP-302, which antagonizes GluR5 subunit-containing kainate (KA) receptors at < or = 10 microm, but other KA and AMPA receptors at > or = 100 microm, and rhythmically active in vitro preparations of neonatal rat to explore the contribution of non-NMDA receptor signalling in rhythm-generating and motor output compartments of the inspiratory network. At 10 microm, UBP-302 had no effect on inspiratory burst frequency or amplitude. At 100 microm, burst amplitude recorded from XII, C1 and C4 nerve roots was significantly reduced, but frequency was unaffected. The lack of a frequency effect was confirmed when local application of UBP-302 (100 microm) into the pre-Bötzinger complex (preBötC) did not affect frequency but substance P evoked a 2-fold increase. A UBP-302-sensitive (10 microm), ATPA-evoked frequency increase, however, established that preBötC networks are sensitive to GluR5 activation. Whole-cell recordings demonstrated that XII motoneurons also express functional GluR5-containing KA receptors that do not contribute to inspiratory drive, and confirmed the dose dependence of UBP-302 actions on KA and AMPA receptors. Our data provide the first evidence that the non-NMDA (most probably AMPA) receptors mediating glutamatergic transmission within preBötC inspiratory rhythm-generating networks are pharmacologically distinct from those transmitting drive to inspiratory motoneurons. This differential expression may ultimately be exploited pharmacologically to separately counteract depression of central respiratory rhythmogenesis or manipulate the drive to motoneurons controlling airway and pump musculature.

ATP sensitivity of preBötzinger complex neurones in neonatal rat in vitro: mechanism underlying a P2 receptor-mediated increase in inspiratory frequency.

P2 receptor (R) signalling plays an important role in the central ventilatory response to hypoxia. The frequency increase that results from activation of P2Y(1)Rs in the preBötzinger complex (preBötC; putative site of inspiratory rhythm generation) may contribute, but neither the cellular nor ionic mechanism(s) underlying these effects are known. We applied whole-cell recording to rhythmically-active medullary slices from neonatal rat to define, in preBötC neurones, the candidate cellular and ionic mechanisms through which ATP influences rhythm, and tested the hypothesis that putative rhythmogenic preBötC neurones are uniquely sensitive to ATP. ATP (1 mm) evoked inward currents in all non-respiratory neurones and the majority of respiratory neurons, which included inspiratory, expiratory and putative rhythmogenic inspiratory neurones identified by sensitivity to substance P (1 microM) and DAMGO (50 microM) or by voltage-dependent pacemaker-like activity. ATP current densities were similar in all classes of preBötC respiratory neurone. Reversal potentials and input resistance changes for ATP currents in respiratory neurones suggested they resulted from either inhibition of a K(+) channel or activation of a mixed cationic conductance. The P2YR agonist 2MeSADP (1 mm) evoked only the latter type of current in inspiratory and pacemaker-like neurones. In summary, putative rhythmogenic preBötC neurones were sensitive to ATP. However, this sensitivity was not unique; ATP evoked similar currents in all types of preBötC respiratory neurone. The P2Y(1)R-mediated frequency increase is therefore more likely to reflect activation of a mixed cationic conductance in multiple types of preBötC neurone than excitation of one, highly sensitive group.

Ultrasound measurements of fetal breathing movements in the rat.

The goal of this study was to determine when fetal breathing movements (FBMs) commence in the rat and to characterize age-dependent changes of FBMs in utero. These data provide a frame of reference for parallel in vitro studies of the cellular, synaptic, and network properties of the perinatal rat respiratory system. Ultrasound recordings were made from unanesthetized Sprague-Dawley rats from embryonic (E) day 15 (E15) to E20. Furthermore, the effects of respiratory stimulants (doxapram and aminophylline) and hypoxia on FBMs were studied. Single FBMs, occurring at a very low frequency (approximately 8 FBMs/h), commenced at E16. The incidence of single FBMs increased to approximately 80 FBMs/h by E20. Episodes of clustered rhythmic FBMs were first observed at E18 (approximately 40 FBMs/h). The incidence of episodic clustered FBMs increased to approximately 300 FMBs/h by E20, with the duration of each episode ranging from approximately 40 to 180 s. Doxapram, presumably acting to stimulate carotid body receptors, did not increase FBMs until E20, when the incidence of episodic clustered FBMs increased twofold. Aminophylline, a central-acting stimulant, caused an increase in episodic clustered FBMs after E17, reaching significance at E20 (3-fold increase). Exposing the dam to 10% O(2) caused a rapid, marked suppression of FBMs (5-fold decrease) that was readily reversed on exposure to room air.

Voltage-sensitive calcium currents and their role in regulating phrenic motoneuron electrical excitability during the perinatal period.

This study examined the ontogeny of voltage-sensitive calcium conductances in rat phrenic motoneurons (PMNs) and their role in regulating electrical excitability during the perinatal period. Specifically, we studied the period spanning from embryonic day (E)16 through postnatal day (P)1, when PMNs undergo fundamental transformation in their morphology, passive properties, ionic channel composition, synaptic inputs, and electrical excitability. Low voltage-activated (LVA) and high voltage-activated (HVA) conductances were measured using whole cell patch recordings utilizing a cervical slice-phrenic nerve preparation from perinatal rats. Changes between E16 and P0-1 included the following: an approximately 2-fold increase in the density of total calcium conductances, an approximately 2-fold decrease in the density of LVA calcium conductances, and an approximately 3-fold increase in the density of HVA conductances. The elevated expression of T-type calcium channels during the embryonic period lengthened the action potential and enhanced electrical excitability as evidenced by a hyperpolarization-evoked rebound depolarization. The reduction of LVA current density coupled to the presence of a hyperpolarizing outward A-type potassium current had a critical effect in diminishing the rebound depolarization in neonatal PMNs. The increase in HVA current density was concomitant with the emergence of a calcium-dependent "hump-like" afterdepolarization (ADP) and burst-like firing. Neonatal PMNs develop a prominent medium-duration afterhyperpolarization (mAHP) as the result of coupling between N-type calcium channels and small conductance, calcium-activated potassium channels. These data demonstrate that changes in calcium channel expression contribute to the maturation of PMN electrophysiological properties during the time from the commencement of fetal inspiratory drive to the onset of continuous breathing at birth.

Structure of the primordial diaphragm and defects associated with nitrofen-induced CDH.

The goals of this study were to further our understanding of diaphragm embryogenesis and the pathogenesis of congenital diaphragmatic hernia (CDH). Past work suggests that the pleuroperitoneal fold (PPF) is the primary source of diaphragmatic musculature. Furthermore, defects associated with an animal model of CDH can be traced back to the formation of the PPF. This study was designed to elucidate the anatomic structure of the PPF and to determine which regions of the PPF malform in the well-established nitrofen model of CDH. This was achieved by producing three-dimensional renderings constructed from serial transverse sections of control and nitrofen-exposed rats at embryonic day 13.5. Renderings of left- and right-sided defects demonstrated that the malformations were always limited to the dorsolateral portions of the caudal regions of the PPF. These data provide an explanation of why the holes in diaphragmatic musculature associated with CDH are characteristically located in dorsolateral regions. Moreover, these data provide further evidence against the widely stated hypothesis that a failure of pleuroperitoneal canal closure underlies the pathogenesis of nitrofen-induced CDH.

Development of potassium conductances in perinatal rat phrenic motoneurons.

Prior to the inception of inspiratory synaptic drive transmission from medullary respiratory centers, rat phrenic motoneurons (PMNs) have action potential and repetitive firing characteristics typical of immature embryonic motoneurons. During the period spanning from when respiratory bulbospinal and segmental afferent synaptic connections are formed at embryonic day 17 (E17) through to birth (gestational period is approximately 21 days), a pronounced transformation of PMN electrophysiological properties occurs. In this study, we test the hypothesis that the elaboration of action potential afterpotentials and the resulting changes in repetitive firing properties are due in large part to developmental changes in PMN potassium conductances. Ionic conductances were measured via whole cell patch recordings using a cervical slice-phrenic nerve preparation isolated from perinatal rats. Voltage- and current-clamp recordings revealed that PMNs expressed outward rectifier (I(KV)) and A-type potassium currents that regulated PMN action potential and repetitive firing properties throughout the perinatal period. There was an age-dependent leftward shift in the activation voltage and a decrease in the time-to-peak of I(KV) during the period from E16 through to birth. The most dramatic change during the perinatal period was the increase in calcium-activated potassium currents after the inception of inspiratory drive transmission at E17. Block of the maxi-type calcium-dependent potassium conductance caused a significant increase in action potential duration and a suppression of the fast afterhyperpolarizing potential. Block of the small conductance calcium-dependent potassium channels resulted in a marked suppression of the medium afterhyperpolarizing potential and an increase in the repetitive firing frequency. In conclusion, the increase in calcium-mediated potassium conductances are in large part responsible for the marked transformation in action potential shape and firing properties of PMNs from the time between the inception of fetal respiratory drive transmission and birth.

Recent advances in understanding the pathogenesis of nitrofen-induced congenital diaphragmatic hernia.

In this review, we discuss recent advances in the study of the pathogenesis of congenital diaphragmatic hernia (CDH). Much of the research has involved the use of an animal model of CDH in which diaphragmatic defects are produced in fetal rats by administering the herbicide nitrofen to dams during mid-gestation. The animal model is described and the relevance to the human condition is discussed. The data derived from the animal studies are critically assessed in the context of commonly cited hypotheses proposed for the pathogenesis of CDH. Finally, experimental strategies are proposed for systematically examining the normal and pathological formation of the pleuroperitoneal fold. We conclude that a malformation of the primordial diaphragm, the pleuroperitoneal fold, underlies the muscle defects associated with CDH.

Contractile and fatigue properties of the rat diaphragm musculature during the perinatal period.

The following two hypotheses regarding diaphragm contractile properties in the perinatal rat were tested. First, there is a major transformation of contractile and fatigue properties during the period between the inception of inspiratory drive transmission in utero and birth. Second, the diaphragm muscle properties develop to functionally match changes occurring in phrenic motoneuron electrophysiological properties. Muscle force recordings and intracellular recordings of end-plate potentials were measured by using phrenic nerve-diaphragm muscle in vitro preparations isolated from rats on embryonic day 18 and postnatal days 0-1. The following age-dependent changes occurred: 1) twitch contraction and half relaxation times decreased approximately two- and threefold, respectively; 2) the tetanic force levels increased approximately fivefold; 3) the ratio of peak twitch force to maximum tetanic force decreased 2.3-fold; 4) the range of forces generated by the diaphragm in response to graded nerve stimulation increased approximately twofold; 5) the force-frequency curve was shifted to the right; and 6) the propensity for neuromuscular transmission failure decreased. In conclusion, the diaphragm contractile and phrenic motoneuron repetitive firing properties develop in concert so that the full range of potential diaphragm force recruitment can be utilized and problems associated with diaphragm fatigue are minimized.

An overview of phrenic nerve and diaphragm muscle development in the perinatal rat.

In this overview, we outline what is known regarding the key developmental stages of phrenic nerve and diaphragm formation in perinatal rats. These developmental events include the following. Cervical axons emerge from the spinal cord during embryonic (E) day 11. At approximately E12.5, phrenic and brachial axons from the cervical segments merge at the brachial plexi. Subsequently, the two populations diverge as phrenic axons continue to grow ventrally toward the diaphragmatic primordium and brachial axons turn laterally to grow into the limb bud. A few pioneer axons extend ahead of the majority of the phrenic axonal population and migrate along a well-defined track toward the primordial diaphragm, which they reach by E13.5. The primordial diaphragmatic muscle arises from the pleuroperitoneal fold, a triangular protrusion of the body wall composed of the fusion of the primordial pleuroperitoneal and pleuropericardial tissues. The phrenic nerve initiates branching within the diaphragm at approximately E14, when myoblasts in the region of contact with the phrenic nerve begin to fuse and form distinct primary myotubes. As the nerve migrates through the various sectors of the diaphragm, myoblasts along the nerve's path begin to fuse and form additional myotubes. The phrenic nerve intramuscular branching and concomitant diaphragmatic myotube formation continue to progress up until E17, at which time the mature pattern of innervation and muscle architecture are approximated. E17 is also the time of the commencement of inspiratory drive transmission to phrenic motoneurons (PMNs) and the arrival of phrenic afferents to the motoneuron pool. During the period spanning from E17 to birth (gestation period of approximately 21 days), there is dramatic change in PMN morphology as the dendritic branching is rearranged into the rostrocaudal bundling characteristic of mature PMNs. This period is also a time of significant changes in PMN passive membrane properties, action-potential characteristics, and firing properties.

Electrophysiological properties of rat phrenic motoneurons during perinatal development.

Past studies determined that there is a critical period at approximately embryonic day (E)17 during which phrenic motoneurons (PMNs) undergo a number of pivotal developmental events, including the inception of functional recruitment via synaptic drive from medullary respiratory centers, contact with spinal afferent terminals, the completion of diaphragm innervation, and a major transformation of PMN morphology. The objective of this study was to test the hypothesis that there would be a marked maturation of motoneuron electrophysiological properties occurring in conjunction with these developmental processes. PMN properties were measured via whole cell patch recordings with a cervical slice-phrenic nerve preparation isolated from perinatal rats. From E16 to postnatal day 1, there was a considerable transformation in a number of motoneuron properties, including 1) 10-mV increase in the hyperpolarization of the resting membrane potential, 2) threefold reduction in the input resistance, 3) 12-mV increase in amplitude and 50% decrease duration of action potential, 4) major changes in the shapes of potassium- and calcium-mediated afterpotentials, 5) decline in the prominence of calcium-dependent rebound depolarizations, and 6) increases in rheobase current and steady-state firing rates. Electrical coupling among PMNs was detected in 15-25% of recordings at all ages studied. Collectively, these data and those from parallel studies of PMN-diaphragm ontogeny describe how a multitude of regulatory mechanisms operate in concert during the embryonic development of a single mammalian neuromuscular system.

Polysialylated NCAM expression during motor axon outgrowth and myogenesis in the fetal rat.

Polysialylation of the neural cell adhesion molecule (NCAM) converts it into an anti-adhesive molecule, attenuating intercellular adhesion and repelling apposed membranes. Previous studies have demonstrated that interaxonal repulsion, or defasciculation, induced by polysialylated NCAM (PSA-NCAM) expressed along outgrowing chick motor axons promotes intramuscular branching and facilitates differential guidance of segregating axonal populations. In the present study, we have examined the expression of PSA-NCAM in a developing mammalian motor system during axonal outgrowth, separation of distinct axonal populations, and intramuscular branching. Furthermore, we provide the first clear demonstration of the spatiotemporal modulation of PSA-NCAM expression on myotubes during each stage of myogenesis. Immunohistochemical labelling was used to compare the spatiotemporal pattern of PSA-NCAM expression with those of total-NCAM, the cell adhesion molecule L1, and growth associated protein (GAP-43) during development of the phrenic nerve and diaphragm of fetal rats (embryonic days, E11-E19). During segregation of phrenic and brachial axonal populations at the brachial plexus (E12.5-E13), PSA-NCAM expression was restricted to phrenics, being absent from brachial motoneurons. Both populations labelled equivalently for NCAM, L1, and GAP-43. We postulate that PSA-NCAM may be a component of the molecular machinery that specifically guides phrenic motoneuron growth at the brachial plexus. During diaphragmatic morphogenesis, PSA-NCAM expression: (i) remained high within the phrenic nerve throughout intramuscular branching; (ii) was transiently up-regulated on myotubes during myotube separation associated with primary and secondary myogenesis; (iii) was restricted to those regions of primary and secondary myotube membranes, which were juxtaposed and about to separate. These data suggest a role for PSA-NCAM in the guidance of specific subsets of mammalian motoneurons and in intramuscular branching, and demonstrate an intimate correlation between PSA-NCAM expression and myotube separation.

Pathogenesis of nitrofen-induced congenital diaphragmatic hernia in fetal rats.

Congenital diaphragmatic hernia (CDH) is a developmental anomaly characterized by the malformation of the diaphragm and impaired lung development. In the present study, we tested several hypotheses regarding the pathogenesis of CDH, including those suggesting that the primary defect is due to abnormal 1) lung development, 2) phrenic nerve formation, 3) developmental processes underlying diaphragmatic myotube formation, 4) pleuroperitoneal canal closure, or 5) formation of the primordial diaphragm within the pleuroperitoneal fold. The 2,4-dichloro-phenyl-p-nitrophenyl ether (nitrofen)-induced CDH rat model was used for this study. The following parameters were compared between normal and herniated fetal rats at various stages of development: 1) weight, protein, and DNA content of lungs; 2) phrenic nerve diameter, axonal number, and motoneuron distribution; 3) formation of the phrenic nerve intramuscular branching pattern and diaphragmatic myotube formation; and 4) formation of the precursor of the diaphragmatic musculature, the pleuroperitoneal fold. We demonstrated that previously proposed theories regarding the primary role of the lung, phrenic nerve, myotube formation, and the closure of pleuroperitoneal canal in the pathogenesis of CDH are incorrect. Rather, the primary defect associated with CDH, at least in the nitrofen rat model, occurs at the earliest stage of diaphragm development, the formation of the pleuroperitoneal fold.

Embryogenesis of the phrenic nerve and diaphragm in the fetal rat.

The embryogenesis of the mammalian phrenic nerve and diaphragm continues to be poorly understood. The purpose of this study was to reexamine this general issue and resolve some long-standing controversies. Specifically, we examined 1) the migratory path and the initial target for phrenic axons; 2) the relationship between the phrenic nerve and the primordial diaphragm during descent from the cervical to the thoracic spinal cord levels; and 3) the nature of the interaction between the progression of phrenic nerve intramuscular branching, myoblast and/or myogenic cell migration, and diaphragmatic myotube formation. We demonstrate that a leading group of "pioneering" phrenic axons migrate along a well-defined track of neural cell adhesion molecule (NCAM)-expressing and low-affinity nerve growth factor (p75) receptor-expressing cells to reach the primordial diaphragm, the pleuroperitoneal fold, at embryonic day (E) 13. During the next day of development, the phrenic nerve and the primordial diaphragm descend together toward the level of the thoracic spinal cord. By E14.5, intramuscular branching has commenced. There is a tight spatiotemporal correlation between the outgrowth of intramuscular phrenic nerve branches, the distribution of myoblasts and/or myogenic cells, and the formation of myotubes within the developing diaphragm, implicating intimate mutual regulation.

Development of phrenic motoneuron morphology in the fetal rat.

This study examined the morphological changes that a homogeneous mammalian spinal motoneuron population undergoes during foetal development. Retrograde labelling of the phrenic nerve with the carbocyanine dye, DiI, was used to visualise developmental changes in phrenic motoneuron morphology within the cervical spinal cord of perinatal rats from embryonic day (E) 13.5 to birth (ca. E21). Groups of intimately associated phrenic somata had migrated into the ventromedial region of cervical segments C3-C6 by E14. This migration was followed by their progressive compaction into a tightly aligned column by E18. During this period, close contact was maintained between phrenic somata throughout the motor pool, suggestive of the presence of gap junctions. From E15 to E18, extensive dendritic arborisations fanned out dorsolaterally and ventromedially into the white matter and the floor plate. By E19, however, dendritic fasciculation and retraction and the extension of newly formed rostrocaudally projecting dendrites had resulted in the approximation of the dendritic morphology observed at birth. These data demonstrate that morphological maturation of phrenic motoneurons occurs subsequently to the onset of functional recruitment and the arrival of central processes of dorsal root ganglion neurons within the ventral horn (ca. E17). By birth, a number of immature features remain, including a larger proportion of neurites that project into the white matter and into the floor plate, the presence of growth cones on a number of dendrites, and close contact between populations of contralaterally derived dendrites.

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.

Serotonergic modulation of the mudpuppy (Necturus maculatus) locomotor pattern in vitro.

The aims of the present study were to: (1) study the role of serotonin (5-HT) in modulating the central pattern generator (CPG) underlying locomotion in the mudpuppy (Necturus maculatus); (2) investigate whether there is an intrinsic spinal serotonergic system. These aims were achieved by the use of pharmacological and immunocytochemical methods. To study modulation of the locomotor pattern and rhythm, we applied 5-HT, its uptake blocker zimelidine, and a variety of 5-HT receptor agonists and antagonists to an in vitro brainstem-spinal cord preparation isolated from the mudpuppy. The preparation consisted of the first five segments of the spinal cord and the right forelimb attached by the brachial plexus. The spinal CPG for locomotion was activated chemically by adding NMDA to the superfusing solution. During locomotion, bipolar electromyographic (EMG) recordings were made unilaterally from flexor and extensor ulnae muscles. 5-HT on its own did not induce locomotion, but it did have a profound modulatory effect on NMDA-induced locomotion. 5-HT produced a dose-dependent increase in the overall cycle duration and enhanced the EMG burst duration. Use of zimelidine indicated that there is an endogenous release of 5-HT which modulated the locomotor rhythm. The endogenous release was antagonized by 5-HT1/5-HT2 receptor antagonist methiothepin. Immunocytochemical analysis, in which the entire spinal cord of the mudpuppy was used, revealed that there were more than one type of spinal serotonergic neuron. They were differentiated according to the cell diameter, shape, and arborization pattern of their processes. These neurons were located within the central gray matter ventrolateral to the central canal. Our results suggest that 5-HT plays an important role in modulating the locomotor CPG in the mudpuppy, by acting through a well-developed spinal serotonergic system. This is in contrast to what has been reported in higher vertebrates, where serotonergic innervation is derived from supraspinal structures.

Serotonergic and noradrenergic effects on respiratory neural discharge in the medullary slice preparation of neonatal rats.

Rhythmically active medullary slice preparations isolated from neonatal rats (postnatal days 0-3, P0-P3) were used to study the modulation of respiraory rhythmogenesis and hypoglossal (XII) nerve discharge by serotonin (5-hydroxytryptamine, 5-HT) and noradrenaline (NA). 5-HT, NA and their respective receptor agonists and antagonists were applied either to the bathing medium or focally via pressure injection into regions encompassing the pre-Botzinger complex or XII motoneurons. The effects of endogenously released 5-HT were also studied by chemical stimulation of neurons within the raphe obscurus. The frequency of respiratory burst discharge was increased when 5-HT was applied: (1) to the bathing medium (37+/-16%; 30 "mu"M; P < 0.05); (2) via pressure injection into the region of the pre-Botzinger complex (22 +/- 14%; < 25 pmol; P < 0. 05); or (3) endogenously released in response to activation of neurons within the raphe obscurus via pressure injection of (R,S)- "alpha"-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA, 34 +/- 15%; P < 0.05) or 5-HT (33 +/- 5%; P < 0. 05). All of these effects were antagonized by bath application of methysergide (30-40 "mu"M). NA caused a reduction of respiratory burst frequency when applied to the bathing medium (40 +/- 15%; 100 "mu"M; P < 0.05) or when pressure injected into the region of the pre-Botzinger complex (22 +/- 11%; < 25 pmol; P < 0.05). These effects were blocked by the bath application of the "alpha"2-receptor antagonist idazoxan (2 "mu"M). 5-HT and NA both caused an augmentation of tonic discharge of XII nerves when applied either to the bathing medium or via pressure injection into the XII motoneuron pool. The 5-HT-induced XII nerve tonic discharge was mimicked by the 5-HT2 receptor agonist R(-)2-(2, 5-dimethoxy-4-iodophenyl) (DOI.HCl, 5 "mu"M) and blocked by the 5-HT2 receptor antagonist ketanserine tartrate (30-40 "mu"M). The NA-induced XII nerve tonic discharge was mimicked by the "alpha"1-receptor agonist phenylephrine HCl (500 nM) and blocked by the "alpha"1-receptor antagonist prozasin HCl (1 "mu"M).