Evolution of vertebrate respiratory central rhythm generators.

Tracing the evolution of the central rhythm generators associated with ventilation in vertebrates is hindered by a lack of information surrounding key transitions. To begin with, central rhythm generation has been studied in detail in only a few species from four vertebrate groups, lamprey, anuran amphibians, turtles, and mammals (primarily rodents). Secondly, there is a lack of information regarding the transition from water breathing fish to air breathing amniotes (reptiles, birds, and mammals). Specifically, the respiratory rhythm generators of fish appear to be single oscillators capable of generating both phases of the respiratory cycle (expansion and compression) and projecting to motoneurons in cranial nerves innervating bucco-pharyngeal muscles. In the amniotes we find oscillators capable of independently generating separate phases of the respiratory cycle (expiration and inspiration) and projecting to pre-motoneurons in the ventrolateral medulla that in turn project to spinal motoneurons innervating thoracic and abdominal muscles (reptiles, birds, and mammals). Studies of the one group of amphibians that lie at this transition (the anurans), raise intriguing possibilities but, for a variety of reasons that we explore, also raise unanswered questions. In this review we summarize what is known about the rhythm generating circuits associated with breathing that arise from the different rhombomeric segments in each of the different vertebrate classes. Assuming oscillating circuits form in every pair of rhombomeres in every vertebrate during development, we trace what appears to be the evolutionary fate of each and highlight the questions that remain to be answered to properly understand the evolutionary transitions in vertebrate central respiratory rhythm generation.

Neonatal maternal separation opposes the facilitatory effect of castration on the respiratory response to hypercapnia of the adult male rat: Evidence for the involvement of the medial amygdala.

Respiratory manifestations of panic disorder (PD) include a greater respiratory instability and enhanced responsiveness to CO2 compared to normal individuals. Although the prevalence of PD is approximately three times greater in women compared to men, the origins of this sexual dimorphism remain poorly understood. Similar to PD patients, adult female rats previously subjected to neonatal maternal separation (NMS) show an increase in their ventilatory response to CO2 . Because this effect of NMS is not observed in males, we hypothesised that testosterone prevents NMS-induced hyper-responsiveness to CO2 . Pups subjected to NMS were placed in an incubator for 3 h d-1 from postnatal days 3-12. Control pups remained undisturbed. At adulthood (8-10 weeks of age), rats were then subjected either to sham surgery or castration. Fourteen days later, breathing was measured at rest (room air) and during acute exposure to hypercapnia (5 and 10% CO2 for 10 minutes each) using plethysmography. To gain insight into the mechanisms involved, c-fos expression was used as an indicator of neuronal activation. Brains were collected following air or CO2 exposure for quantification of c-fos positive cells by immunohistochemistry in selected regions, including the paraventricular nucleus of the hypothalamus, the dorsomedial hypothalamus and the amygdalar complex. Castration produced a 100% increase of hyperventilatory response to 10% CO2 in control rats. Unexpectedly, castration had no effect on the hyperventilatory response of NMS rats. The intensity of the hypercapnic response was inversely correlated with c-fos expression in the medial amygdala. We conclude that testosterone prevents the hyper-responsiveness to CO2 , whereas NMS attenuates sensitivity to hormone withdrawal. We propose that an inhibitory influence from the medial amygdala contributes to this effect.

The effects of sex and neonatal stress on pituitary adenylate cyclase-activating peptide expression.

NEW FINDINGS: What is the central question of this study? Does sex or neonatal stress affect the expression of pituitary adenylate cyclase-activating peptide or its receptors? What is the main finding and its importance? Neonatal-maternal separation stress has little long-lasting effect on the expression of pituitary adenylate cyclase-activating peptide or its receptors, but sex differences exist in these genes between males and females at baseline. Sex differences in classic stress hormones have been studied in depth, but pituitary adenylate cyclase-activating peptide (PACAP), recently identified as playing a critical role in the stress axes, has not. Here we studied whether baseline levels of PACAP differ between sexes in various stress-related tissues and whether neonatal-maternal separation stress has a sex-dependent effect on PACAP gene expression in stress pathways. Using quantitative RT-PCR, we found sex differences in PACAP and PACAP receptor gene expression in several respiratory and/or stress-related tissues, while neonatal-maternal separation stress did little to affect PACAP signalling in adult animals. We propose that sex differences in PACAP expression are likely to contribute to differences between males and females in responses to stress.

Role of pontine neurons in central O(2) chemoreflex during development in bullfrogs (Lithobates catesbeiana).

The present study used an in vitro brainstem preparation from pre-metamorphic tadpoles and adult bullfrogs (Lithobates catesbeiana) to understand the neural mechanisms associated with central O(2) chemosensitivity and its maturation. In this species, brainstem hypoxia increases fictive lung ventilation in tadpoles but decreases in adults. Previous studies have shown that alpha(1)-adrenoceptor inactivation prevents these responses, suggesting that noradrenergic neurons are involved. We first tested the hypothesis that the pons (which includes noradrenergic neurons from the locus coeruleus; LC) plays a role in the lung burst frequency response to central hypoxia by comparing the effects of brainstem transection at the LC level between pre-metamorphic tadpoles and adults. Data show that brainstem transection prevents the lung burst frequency response in both stage groups. During development, the progressive decrease in the Na(+)/K(+)/Cl(-) co-transporter NKCC1 contributes to the maturation of neural networks. Because NKCC1 becomes activated during hypoxia, we then tested the hypothesis that NKCC1 contributes to maturation of the central O(2) chemoreflex. Double labeling experiments showed that the proportion of tyrosine hydroxylase positive neurons expressing NKCC1 in the LC decreases during development. Inactivation of NKCC1 with bumetanide bath application reversed the lung burst response to hypoxia in tadpoles. Bumetanide inhibited the response in adults. These data indicate that a structure within the pons (potentially the LC) is necessary to the central hypoxic chemoreflex and demonstrate that NKCC1 plays a role in central O(2) chemosensitivity and its maturation in this species.

Developmental profile of cholinergic and purinergic traits and receptors in peripheral chemoreflex pathway in cats.

This study describes the developmental profile of specific aspects of cholinergic and purinergic neurotransmission in key organs of the peripheral chemoreflex: the carotid body (CB), petrosal ganglion (PG) and superior cervical ganglion (SCG). Using real time RT-PCR and Western blot analyses, we assessed both mRNA and protein expression levels for choline-acetyl-transferase (ChAT), nicotinic receptor (subunits alpha3, alpha4, alpha7, and beta2), ATP and purinergic receptors (P2X2 and P2X3). These analyses were performed on tissue from 1- and 15-day-old, 2-month-old, and adult cats. During development, ChAT protein expression level increased slightly in CB; however, this increase was more important in PG and SCG. In CB, mRNA level for alpha4 nicotinic receptor subunit decreased during development (90% higher in 1-day-old cats than in adults). In the PG, mRNA level for beta2 nicotinic receptor subunit increased during development (80% higher in adults than in 1-day-old cats). In SCG, mRNA for alpha7 nicotinic receptor levels increased (400% higher in adults vs. 1-day-old cats). Conversely, P2X2 receptor protein level was not altered during development in CB and decreased slightly in PG; a similar pattern was observed for the P2X3 receptor. Our findings suggest that in cats, age-related changes in cholinergic and purinergic systems (such as physiological expression of receptor function) are significant within the afferent chemoreceptor pathway and likely contribute to the temporal changes of O2-chemosensitivity during development.

Developmental pattern of M1 and M2 muscarinic gene expression and receptor levels in cat carotid body, petrosal and superior cervical ganglion.

Using real-time reverse transcriptase polymerase chain reaction, Northern blot, and Western blot analyses, we evaluated the developmental pattern of mRNA and protein expression level of muscarinic M1 and M2 receptors in the carotid body, petrosal ganglion and superior cervical ganglion of 1-day, 15-day, 2-month-old and adult cats. mRNA expression and protein levels of tyrosine hydroxylase, the rate limiting enzyme for dopamine synthesis, were also assessed. Carotid body M1 receptor mRNA, increased significantly by approximately 100% and 300% in 2-month and adult vs. 1- and 15-day-old cats, but protein level decreased gradually being approximately 50% lower compared with 1-day-old cats. In the petrosal ganglion, muscarinic M1 receptor mRNA level was higher in 15-day-old cats vs. 1-day-old, 2-month-old and adult cats and protein levels were about 30% lower than in 1- and 15-day-old cats. In the superior cervical ganglion, muscarinic M1 receptor mRNA was approximately 50% and 80% higher in 2-month-old and adult cats than 1- and 15-day-old, but no changes in the protein level except in 15-day-old cats which was approximately 40% higher than 1-day-old. There was no change of muscarinic M2 receptor mRNA or protein level in the carotid body or petrosal ganglion. However, in the superior cervical ganglion, the significant increase of mRNA of 30% and 50% in 2-month-olds and adults, respectively was not associated with an increase in receptor protein. Tyrosine hydroxylase mRNA and protein level decreased significantly with age in the carotid body and petrosal ganglion. In the superior cervical ganglion, the age dependent increase in tyrosine hydroxylase mRNA was not associated with any changes in the protein level. These results show that the expression of muscarinic M1 and M2 receptors are age and organ-dependent in cats. Consequently, these changes may modulate chemosensory activity during development since muscarinic M1 receptor is predominantly involved in postsynaptic chemosensory activity, while muscarinic M2 receptor modulates acetylcholine and dopamine release from chemosensitive cells.

Age-dependent effect of domperidone on dopamine release by the hypoxic carotid body in the rabbit.

The aim of this study was to identify the mechanisms that regulate dopamine release (DA(r)) by the hypoxic carotid body (CB) during development. CBs sampled from adult (n = 58) and 10-day-old (n = 53) rabbits were incubated for 1 h in a medium equilibrated with 8% O(2) in N(2) without or in the presence of the specific DA D(2) receptor antagonist domperidone, 0.01, 0.1 and 1 microM. DA and its major metabolite DOPAC were measured in the CB (DA(CB)) and in the medium (DA(r)) by HPLC+ED. In adults, each concentration of domperidone significantly decreased DA(CB) and increased DA(r), compared with control (p < 0.01). In contrast, in 10-day old, only the 1 microM domperidone concentration decreased DA(CB) and increased DA(r) compared with control (p < 0.001). The data show that domperidone increases CB DA(r) in response to hypoxia in a concentration- and age-dependent manner and suggest this response depends, in part, on the functional maturation of CB DA D(2) receptors.

Plasticity in respiratory motor control: intermittent hypoxia and hypercapnia activate opposing serotonergic and noradrenergic modulatory systems.

Experimental results consistently show that the respiratory control system is plastic, such that environmental factors and experience can modify its performance. Such plasticity may represent basic neurobiological principles of learning and memory, whereby intermittent sensory stimulation produces long-term alterations (i.e. facilitation or depression) in synaptic transmission depending on the timing and intensity of the stimulation. In this review, we propose that intermittent chemosensory stimulation produces long-term changes in respiratory motor output via specific neuromodulatory systems. This concept is based on recent data suggesting that intermittent hypoxia produces a net long-term facilitation of respiratory output via the serotonergic system, whereas intermittent hypercapnia produces a net long-term depression by a mechanism associated with the noradrenergic system. There is suggestive evidence that, although both respiratory stimuli activate both modulatory systems, the balance is different. Thus, these opposing modulatory influences on respiratory motor control may provide a 'push-pull' system, preventing unchecked and inappropriate fluctuations in ventilatory drive.

Chronic intermittent hypoxia elicits serotonin-dependent plasticity in the central neural control of breathing.

We tested the hypothesis that chronic intermittent hypoxia (CIH) elicits plasticity in the central neural control of breathing via serotonin-dependent effects on the integration of carotid chemoafferent inputs. Adult rats were exposed to 1 week of nocturnal CIH (11-12% O(2)/air at 5 min intervals; 12 hr/night). CIH and untreated rats were then anesthetized, paralyzed, vagotomized, and artificially ventilated. Time-dependent hypoxic responses were assessed in the phrenic neurogram during and after three 5 min episodes of isocapnic hypoxia. Integrated phrenic amplitude (integralPhr) responses during hypoxia were greater after CIH at arterial oxygen pressures (PaO(2)) between 25 and 45 mmHg (p < 0.05), but not at higher PaO(2) levels. CIH did not affect hypoxic phrenic burst frequency responses, although the post-hypoxia frequency decline that is typical in rats was abolished. integralPhr and frequency responses to electrical stimulation of the carotid sinus nerve were enhanced by CIH (p < 0.05). Serotonin-dependent long-term facilitation (LTF) of integralPhr was enhanced after CIH at 15, 30, and 60 min after episodic hypoxia (p < 0.05). Pretreatment with the serotonin receptor antagonists methysergide (4 mg/kg, i.v.) and ketanserin (2 mg/kg, i.v.) reversed CIH-induced augmentation of the short-term hypoxic phrenic response and restored the post-hypoxia frequency decline in CIH rats. Whereas methysergide abolished CIH-enhanced phrenic LTF, the selective 5-HT(2) antagonist ketanserin only partially reversed this effect. The results suggest that CIH elicits unique forms of serotonin-dependent plasticity in the central neural control of breathing. Enhanced LTF after CIH may involve an upregulation of a non-5-HT(2) serotonin receptor subtype or subtypes.

Role of endogenous opioid system in the regulation of the stress response.

Numerous studies and reviews support an important contribution of endogenous opioid peptide systems in the mediation, modulation, and regulation of stress responses including endocrine (hypothalamopituitary-adrenal, HPA axis), autonomic nervous system (ANS axis), and behavioral responses. Although several discrepancies exist, the most consistent finding among such studies using different species and stressors is that opioids not only diminish stress-induced neuroendocrine and autonomic responses, but also stimulate these effector systems in the non-stressed state. A distinctive feature of the analgesic action of opioids is the blunting of the distressing, affective component of pain without dulling the sensation itself. Therefore, opioid peptides may diminish the impact of stress by attenuating an array of physiologic responses including emotional and affective states. The widespread distribution of enkephalin (ENK) throughout the limbic system (including the extended amygdala, cingulate cortex, entorhinal cortex, septum, hippocampus, and the hypothalamus) is consistent with a direct role in the modulation the stress responses. The predictability of stressful events reduces the impact of a wide range of stressors and ENK appears to play an important role in this process. Therefore, ENK and its receptors could represent a major modulatory system in the adaptation of an organism to stress, balancing the response that the stressor places on the central stress system with the potentially detrimental effects that a sustained stress may produce. Chronic neurogenic stressors will induce changes in specific components of the stress-induced ENKergic system, including ENK, delta- and mu-opioid receptors. This review presents evidences for adaptive cellular mechanisms underlying the response of the central stress system when assaulted by repeated psychogenic stress, and the involvement of ENK in these processes.

Carbachol effect on carotid body dopamine in vitro release in response to hypoxia in adult and pup rabbit.

Dopamine (DA) release from the adult carotid body (CB) is dependent, in part, upon CB cholinergic receptor stimulation. The aim of the present study was to determine the role of cholinergic stimulation on DA release from rabbit pup CB with reference to adult's. CBs sampled from adult (n = 52) and 10-day-old (n = 49) rabbits were incubated in vitro for 1 h in a surviving medium bubbled with either 100 or 8% O2 in N2, without (control) or in the presence of the cholinergic agonist carbachol 1 microM. In adults, DA released (DAr) in the medium was significantly larger with 1 microM carbachol compared with control in either 100 or 8% O(2) (P < 0.01). In pups, carbachol 1 microM had no effect in 100% O2 but significantly increased DAr compared with control in 8% O2 (P < 0.01). The data suggest that cholinergic mechanisms regulating DAr are not fully expressed in pup rabbit CBs, in contrast with adults and thus, exhibit maturation-related functional differences.

Stress-induced attenuation of the hypercapnic ventilatory response in awake rats.

To test the hypothesis that stress alters the performance of the respiratory control system, we compared the acute (20 min) responses to moderate hypoxia and hypercapnia of rats previously subjected to immobilization stress (90 min/day) with responses of control animals. Ventilatory measurements were performed on awake rats using whole body plethysmography. Under baseline conditions, there were no differences in minute ventilation between stressed and unstressed groups. Rats previously exposed to immobilization stress had a 45% lower ventilatory response to hypercapnia (inspiratory CO(2) fraction = 0.05) than controls. In contrast, stress exposure had no statistically significant effect on the ventilatory response to hypoxia (inspiratory O(2) fraction = 0.12). Stress-induced attenuation of the hypercapnic response was associated with reduced tidal volume and inspiratory flow increases; the frequency and timing components of the response were not different between groups. We conclude that previous exposure to a stressful condition that does not constitute a direct challenge to respiratory homeostasis can elicit persistent (> or =24 h) functional plasticity in the ventilatory control system.

Effect of chronic psychogenic stress exposure on enkephalin neuronal activity and expression in the rat hypothalamic paraventricular nucleus.

This study tested the hypothesis that the activation pattern of enkephalinergic (ENKergic) neurons within the paraventricular nucleus of the hypothalamus (PVH) in response to psychogenic stress is identical whether in response to repeated exposure to the same stress (homotypic; immobilization) or to a novel stress (heterotypic; air jet puff). Rats were assigned to either acute or chronic immobilization stress paradigms (90 min/day for 1 or 10 days, respectively). The chronic group was then subjected to an additional 90-min session of either heterotypic or homotypic stress. A single 90-min stress session (immobilization or air jet) increased PVH-ENK heteronuclear (hn) RNA expression. In chronically stressed rats, exposure to an additional stress session (whether homotypic or heterotypic) continued to stimulate ENK hnRNA expression. Acute immobilization caused a marked increase in the numbers of Fos-immunoreactive and Fos-ENK double-labeled cells in the dorsal and ventral medial parvicellular, and lateral parvicellular subdivisions of the PVH. Chronic immobilization caused an attenuated Fos response ( approximately 66%) to subsequent immobilization. In contrast, chronic immobilization did not impair ENKergic neuron activation within the PVH following homotypic or heterotypic stress. These results indicate that within the PVH, chronic psychogenic stress markedly attenuates the Fos response, whereas ENKergic neurons resist habituation, principally within the ventral neuroendocrine portion of the nucleus. This suggests an increase in ENK effect during chronic stress exposure. Homotypic (immobilization) and heterotypic (air jet) psychogenic stressors produce similar responses, including Fos, ENK-Fos, and ENK hnRNA, within each subdivision of the PVH, suggesting similar processing for painless neurogenic stimuli.

Long term facilitation of phrenic motor output.

Episodic hypoxia or electrical stimulation of carotid chemoafferent neurons elicits a sustained, serotonin-dependent augmentation of respiratory motor output known as long term facilitation (LTF). The primary objectives of this paper are to provide an updated review of the literature pertaining to LTF, to investigate the influence of selected variables on LTF via meta-analysis of a large data set from LTF experiments on anesthetized rats, and to propose an updated mechanism of LTF. LTF has been demonstrated in anesthetized and awake experimental preparations, and can be evoked in some human subjects during sleep. The mechanism underlying LTF requires episodic chemoafferent stimulation, and is not elicited by similar cumulative durations of sustained hypoxia. Meta-analysis of phrenic nerve responses following episodic hypoxia in 63 experiments on anesthetized rats (conducted by four investigators over a period of several years) indicates that phrenic LTF magnitude correlates with peak phrenic responses during hypoxia and hypercapnia, but not with the level of hypoxia during episodic exposures. Potential mechanisms underlying these relationships are discussed, and currently available data are synthesized into an updated mechanistic model of LTF. In this model, we propose that LTF arises predominantly from episodic activation of serotonergic receptors on phrenic motoneurons, activating intracellular kinases and, thus, phosphorylating and potentiating ionic currents associated with the glutamate receptors that mediate respiratory drive.

Time-dependent hypoxic ventilatory responses in rats: effects of ketanserin and 5-carboxamidotryptamine.

We hypothesized that the 5-hydroxytryptamine (5-HT) active drugs ketanserin and 5-carboxamidotryptamine (5-CT) would modulate time-dependent hypoxic phrenic and hypoglossal responses, including 1) short-term hypoxic response, 2) posthypoxia frequency decline (PHFD), and 3) long-term facilitation (LTF) of respiratory motor output. Phrenic and hypoglossal nerve activities were recorded in urethan-anesthetized, paralyzed, vagotomized, and artificially ventilated rats pretreated either with ketanserin (5-HT(2A/C) antagonist; 2 mg/kg iv), 5-CT (5-HT(1A/B) agonist; 10 microg/kg iv), or saline (sham). Rats were exposed to three 5-min episodes of hypoxia [fractional inspired O(2) (FI(O2)) = 0.11], separated by 5 min of hyperoxia (FI(O2) = 0.5). During hypoxia, ketanserin augmented phrenic but not hypoglossal burst amplitude; 5-CT had no effect. Both drugs accentuated PHFD. Ketanserin blocked phrenic LTF; hypoglossal LTF was not apparent, even in sham-treated rats. 5-CT reversed LTF, resulting in a long-lasting depression of phrenic burst frequency and amplitude without effect on hypoglossal burst amplitude. The data suggest that 1) 5-HT(2A/C) receptor activation modulates the short-term hypoxic phrenic response and PHFD and is necessary for LTF; and 2) 5-CT may affect time-dependent hypoxic ventilatory responses by reducing serotonin release via 5-HT(1A/B) autoreceptor activation.

Post-hypoxia frequency decline in rats: sensitivity to repeated hypoxia and alpha2-adrenoreceptor antagonism.

We tested the hypothesis that the post-hypoxia frequency decline of phrenic nerve activity following brief, isocapnic hypoxic episodes in rats is diminished by prior hypoxic episodes and alpha2-adrenoreceptor antagonism. Anesthetized (urethane), artificially ventilated (FIO2=0.50) and vagotomized rats were presented with two or three, 5 min episodes of isocapnic hypoxia (FIO2 approximately 0.11), separated by 30 min of control, hyperoxic conditions. Phrenic nerve discharge, end-tidal CO2, and arterial blood gases were measured before during and after hypoxia. The average maximum frequency decline, measured 5 min after the first hypoxic episode, was 26+/-7 bursts/min below pre-hypoxic baseline values (a 70+/-16% decrease). By 30 min post-hypoxia, frequency had returned to baseline. Two groups of rats were then administered either: (1) saline (sham) or (2) the alpha2-receptor antagonist, RX821002 HCl (2-[2-(2-Methoxy-1,4-benzodioxanyl)] imidazoline hydrochloride; 0.25 mg/kg, i.v.). Isocapnic hypoxia was repeated 10 min later. In sham rats, the post-hypoxia frequency decline (PHFD) was significantly attenuated relative to the initial (control) response. However, PHFD was attenuated significantly more in RX821002-treated vs. sham rats (-3+/-3 bursts/min vs. -12+/-4 bursts/min @ 5 min post hypoxia for RX821002 and sham-treated, respectively; p<0.05). We conclude that the magnitude of PHFD is dependent on the prior history of hypoxia and that alpha2 adrenoreceptor activation plays a role in its underlying mechanism.

Central respiratory pattern generation in the bullfrog, Rana catesbeiana.

There are two components to breathing pattern generation the production of the pattern of neural discharge associated with individual breaths, and the pattern in which breaths are produced to effect ventilation. Bullfrogs typically breathe with randomly distributed breaths. When respiratory drive is elevated, breathing becomes more regular and often episodic. Studies on in vitro brainstem-spinal cord preparations of the adult bullfrog and in situ preparations of decerebrate, paralyzed, unidirectionally ventilated animals suggest that output from the central rhythm generator in frogs is conditional on receiving some input and that a host of central inputs remain even in the most reduced preparations. There appear to be descending inputs from sites in the dorsal brainstem just caudal to the optic chiasma that cluster breaths into episodes, a strong excitatory input caudal to this site but rostral to the origin of the Vth cranial nerve and, possibly, segmental rhythm generators throughout the medulla that are normally entrained to produce the normal breathing pattern. The data also suggest that the shape of the discharge pattern (augmenting, decrementing) and timing of outputs (alternating vs synchronous) associated with motor outflow during each breath are also dependent on the interconnections between these various sites.

Cervical dorsal rhizotomy enhances serotonergic innervation of phrenic motoneurons and serotonin-dependent long-term facilitation of respiratory motor output in rats.

We tested the hypothesis that spinal plasticity elicited by chronic bilateral cervical dorsal rhizotomy (C3-C5; CDR) has functional implications for respiratory motor control. Surgery was performed on rats (CDR or sham-operated) 26 d before phrenic motoneurons were retrogradely labeled with cholera toxin. Rats were killed 2 d later, and their spinal cords were harvested and processed to reveal the cholera toxin-labeled phrenic motoneurons and serotonin-immunoreactive terminals. The number of serotonin-immunoreactive terminals within 5 micrometer of labeled phrenic motoneuron soma and primary dendrites increased 2.1-fold after CDR versus sham-operation. Time-dependent phrenic motor responses to hypoxia were compared among CDR, sham-operated, and control rats. Anesthetized, paralyzed, vagotomized, and artificially ventilated rats were exposed to three, 5 min episodes of isocapnic hypoxia (FiO2 = 0.11), separated by 5 min hyperoxic intervals (FiO2 = 0.5). One hour after hypoxia, a long-lasting, serotonin-dependent enhancement of phrenic motor output (long-term facilitation) was observed in both sham and control rats. After CDR, long-term facilitation was 108 and 163% greater than control and sham responses, respectively. Pretreatment of CDR rats with a 5-HT2 receptor antagonist (ketanserin tartrate, 2 mg/kg, i.v.) before episodic hypoxia prevented long-term facilitation and revealed a modest (-28 +/- 13%; p < 0.05) long-lasting depression of phrenic motor output. The results indicate that CDR: (1) increases serotonergic innervation of the phrenic motor nucleus; and (2) augments serotonin-dependent long-term facilitation of phrenic motor output. These results further suggest a form of plasticity based on changes in the capacity for neuromodulation.

The role of the nucleus isthmi in respiratory pattern formation in bullfrogs.

The nucleus isthmi (NI) is a mesencephalic structure of the amphibian brain located between the roof of the midbrain and the cerebellum. From a neuroanatomical perspective, the NI can be compared with the pons which, in mammals, contributes to the control of breathing pattern. This study tested the hypothesis that the NI plays a critical role in breathing pattern formation in the bullfrog. More specifically, we postulated that this nucleus was the site responsible for clustering breaths into distinct episodes of breathing. This hypothesis was tested by comparing the respiratory motor output of decerebrate, paralyzed and artificially ventilated bullfrogs before and after bilateral lesions of the NI by pressure microinjections of lidocaine or kainic acid (KA) into this area. Bilateral microinjections of lidocaine or KA into the NI transformed the breathing pattern from episodic (many breaths per episode) to one of evenly spaced single breaths, without affecting the amplitude of the fictive breaths. These changes in breathing pattern were associated with an overall decrease in breathing frequency and a reduction in CO2-chemosensitivity. Breathing episodes of more than one breath reappeared during hypercarbia (3.5% CO2 in air) after KA lesioning. Bilateral lesions to the NI did not affect the changes in the timing or the amplitude of the respiratory-related bursts elicited by pulmonary stretch receptor feedback, indicating that mechanoreflexes do not require NI input. We conclude that the NI is not responsible for the genesis of breathing episodes, but provides a tonic excitatory input to respiratory centers in the lower brainstem. The NI also plays an important role in either CO2 chemodetection or, more probably, integration of CO2 chemoreceptor information. This, in turn, contributes to the production of episodes of more than one breath.