Involvement of adenosinergic A1 systems in the occurrence of respiratory perturbations encountered in newborns following an in utero caffeine exposure. a study on brainstem-spinal cord preparations isolated from newborn rats

Involvement of adenosinergic A1 systems in the occurrence of respiratory perturbations encountered in newborns following an in utero caffeine exposure has been investigated on pontomedullary-spinal cord, caudal pons-medullary-spinal cord and medullary-spinal cord preparations isolated from newborn rats. According to the drinking fluid of dams (tap water or 0.02% caffeine), two groups of preparations were distinguished, no-caffeine and caffeine. In the no-caffeine group, adenosine A1 receptor activation induces a decrease in respiratory frequency (Rf) in caudal pons-medullary-spinal cord and medullary-spinal cord preparations whereas, in presence of the rostral pons, an increase is observed. A parallel Fos detection indicates that this discrepancy may be due to the excitatory action of the medial parabrachial nucleus at the rostral pontine level that surpasses inhibitory influence of the adenosine A1 receptor activation at the medullary level particularly in the ventrolateral reticular nucleus of the medulla. In caffeine group, an increase in the baseline Rf in presence of the pons and no change in medullary-spinal cord preparations have been observed. Depending on Fos detection, we assume that the medial parabrachial nucleus is the main region involved in the exaggeration of Rf. Moreover, adenosine A1 receptor activation was modified by in utero caffeine exposure with an overcharge of the Rf increase in pontomedullary-spinal cord preparations and an exaggeration of the Rf decrease in medullary-spinal cord preparations. Based on Fos detection, we link the overcharge in Rf of pontomedullary spinal cord preparations to an increase in the medial parabrachial nucleus neuronal activity. Similarly, exaggeration of Rf decrease observed without the pons is linked with a decrease in activity of the ventrolateral reticular neurons. This study brings evidence for the involvement of adenosinergic A1 systems in the occurrence of respiratory perturbations in newborns following in utero caffeine exposure and the importance of rostral pons in the adenosinergic A1 modulation of the respiratory control.     

Carbachol microinjections in the mediodorsal pontine tegmentum are unable to induce paradoxical sleep after caudal pontine and prebulbar transections in the cat.

In 7 cats, total transections of the brainstem at the caudal pontine or the prebulbar level led to preparations which presented neither behavioral nor electrophysiological signs of paradoxical sleep (PS) throughout their survival periods (17-30 days). Carbachol microinjections in the mediodorsal pontine tegmentum (MDPT), which induced PS in the intact cat, were no longer able to induce it in the transected animals. Rapid eye movement (REM) and pontogeniculo-occipital (PGO)-like bursts were evoked by carbachol microinjections in the pontine magnocellular tegmental field (FTM) of cats transected at the prebulbar level, as in the intact cat. Only REM bursts were obtained by the same injections in caudal pontine transected cats. It is concluded that (1) the pons is insufficient to generate PS; (2) complex reciprocal interactions with the medulla are necessary for the generation of this state of sleep; and (3) the production of long REM and PGO bursts is controlled by the caudal pontine tegmentum.     

Evidence that ventilatory rhythmogenesis in the frog involves two distinct neuronal oscillators

In Rana catesbeiana the upper airways are used for two distinct yet highly coordinated ventilatory behaviours: buccal ventilation and lung inflation cycles. How these behaviours are generated and coordinated is unknown. The purpose of this study was to identify putative rhythmogenic brainstem loci involved in these ventilatory behaviours. We surveyed the isolated postmetamorphic brainstem to determine sites where local depolarization, produced by microinjecting the non-NMDA glutamate receptor agonist, AMPA, augmented the ventilatory motor patterns. Two sites were identified: a caudal site, at the level of cranial nerve (CN) X, where AMPA injections caused increased buccal burst frequency but abolished lung bursts, and a rostral site, between the levels of CN VIII and IX, where injections increased the frequency of both types of ventilatory bursts. These two sites were further examined using GABA microinjections to locally inhibit cells. GABA injected into the caudal site suppressed the buccal rhythm but the lung rhythm continued, albeit at a different frequency. When GABA was injected into the rostral site the lung bursts were abolished but the buccal rhythm continued. When the two sites were physically separated by transection, both rostral and caudal brainstem sections were capable of rhythmogenesis. The results suggest the respiratory network within the amphibian brainstem is composed of at least two distinct but interacting oscillators, the buccal and lung oscillators. These putative oscillators may provide a promising experimental model for studying coupled oscillators in vertebrates.     

Mapping of alpha-neo-endorphin- and neurokinin B-immunoreactivity in the human brainstem

We have studied the distribution of alpha-neo-endorphin- or neurokinin B-immunoreactive fibres and cell bodies in the adult human brainstem with no prior history of neurological or psychiatric disease. A low density of alpha-neo-endorphin-immunoreactive cell bodies was only observed in the medullary central gray matter and in the spinal trigeminal nucleus (gelatinosa part). Alpha-neo-endorphin-immunoreactive fibres were moderately distributed throughout the human brainstem. A high density of alpha-neo-endorphin-immunoreactive fibres was found only in the solitary nucleus (caudal part), in the spinal trigeminal nucleus (caudal part), and in the gelatinosa part of the latter nucleus. Neurokinin B-immunoreactive cell bodies (low density) were found in the periventricular central gray matter, the reticular formation of the pons and in the superior colliculus. The distribution of the neurokinin-immunoreactive fibres was restricted. In general, for both neuropeptides the density of the immunoreactive fibres was low. In the human brainstem, the proenkephalin system was more widely distributed than the prodynorphin system, and the preprotachykinin A system (neurokinin A) was more widely distributed than the preprotachykinin B system (neurokinin B).     

Erythropoietin and its antagonist regulate hypoxic fictive breathing in newborn mice

Clinical use of erythropoietin in adult and newborn patients has revealed its involvement in neuroprotection, neurogenesis, and angiogenesis. More recently, we showed in adult mouse, that brain erythropoietin interacts with the major brainstem centers associated with respiration to enhance the ventilatory response to acute and chronic conditions of physiological hypoxia (e.g., as occurring at high altitude). However, whether brain erythropoietin is involved in breathing regulation in newborns remains unknown. In this study, en bloc brainstem-spinal cord preparations were obtained from mice at postnatal day 4. After various periods (30, 60, or 90 min) of incubation with 0, 25, or 250 U of erythropoietin, preparations were superfused with artificial cerebrospinal fluid bubbled with normoxic or hypoxic gas mixtures. The electrophysiological fictive breathing produced by axons at the C4 ventral root was next recorded. Our results show that erythropoietin attenuates the hypoxia-mediated decrease of the central respiratory activity and improves post-hypoxic recovery. Additional analysis revealed that the soluble erythropoietin receptor (the endogenous erythropoietin antagonist) dramatically decreases neural hypoxic respiratory activity, confirming the specific erythropoietin effect on respiratory drive. These results imply that erythropoietin exerts main modulation and maintenance of respiratory motor output during hypoxic and post-hypoxic challenges in 4-days old mice.     

Facilitation of respiratory rhythm by a mu-opioid agonist in newborn rat pons-medulla-spinal cord preparations

We investigated the effect of a mu-opioid agonist, DAGO, on the respiratory frequency of pons-medulla-spinal cord preparations from newborn rats. Bath application of a low concentration of DAGO (0.2 microM) facilitated respiratory rhythm in pons-medulla-spinal cord preparations, whereas it induced respiratory depression in medulla-spinal cord preparations (without pons). At a higher concentration (1.0 microM), at which the inspiratory burst generation in the medulla was strongly depressed, the respiratory rhythm in half of the pons-medulla-spinal cord preparations increased and then decreased, thus showing a biphasic response. In the other half of these preparations, only the facilitatory effect was observed. The burst rate of pre-inspiratory neurons in the rostral ventrolateral medulla was also facilitated by DAGO application. Such facilitation of the respiratory rhythm is probably due to disinhibition of a pontine inhibitory system. Our findings also suggest the existence of a pontine excitatory system, which is depressed by the pontine inhibitory system under control conditions.     

Baroreceptor-mediated inhibition of respiration after peripheral and central administration of a 5-HT1A receptor agonist in neonatal piglets

Inhibition of neurones in the ventral medulla accentuates the respiratory inhibition associated with acute blood pressure elevation in piglets. Activation of presynaptic 5-HT(1A) receptors inhibits serotonergic neurones in the ventral medulla and caudal raphé, and we tested the hypothesis that administration of 8-hydroxydipropylaminotetralin (8-OH-DPAT), a 5-HT(1A) agonist, within the rostroventral medulla and caudal raphé would enhance baroreceptor-mediated inhibition of respiratory activity in decerebrate, neonatal piglets. Baroreceptor stimulation was achieved by inflating a balloon in the distal aorta to elevate carotid blood pressure. After two to four control trials of baroreceptor stimulation, each piglet was given either a single intravenous (i.v.) dose of 10 microg kg(-1) 8-OH-DPAT or treated by adding 10 or 30 mm 8-OH-DPAT to the dialysate for approximately 10 min to inhibit serotonergic neurones, after which the baroreceptor stimulation trials were repeated. Baroreceptor stimulation reduced respiratory activity, particularly the respiratory frequency, which diminished from 35.7 +/- 3.3 to 33.8 +/- 3.1 breaths min(-1) (P < 0.02) and, following i.v. 8-OH-DPAT, baroreceptor-mediated inhibition of respiratory output was significantly accentuated (P < 0.05); the respiratory frequency declined from 34.5 +/- 3.6 to 26.5 +/- 2.9 breaths min(-1). Increasing aortic blood pressure reduced the respiratory frequency (P < 0.01), but focal dialysis of 10 or 30 mm 8-OH-DPAT had, on average, no effect on the ventilatory inhibition associated with an acute elevation of blood pressure. We conclude that activation of 5-HT(1A) receptors after systemic administration of 8-OH-DPAT enhanced baroreflex-mediated inhibition of ventilation, but this effect cannot be attributed to 5-HT(1A) receptor activation within the rostroventral medulla and caudal raphé.     

Serotonergic modulation of the respiratory rhythm generator at birth: an in vitro study in the rat.

In order to investigate the mechanisms through which serotonin (5-HT) modulates the activity of the respiratory rhythm generator, respiratory activity was recorded from cervical ventral roots of the superfused isolated brainstem-spinal cord preparation of the newborn rat. Replacing the normal bathing medium by a medium containing 5-HT (30 microM) increased the respiratory frequency by 70% of the control value. Intact pontomedullary structures are necessary for this effect to take place, however, since the 5-HT-induced increases in respiratory frequency were no longer observed after elimination (section and electrolytic lesion) of the caudal ventro-lateral pons containing the A5 areas. Local applications of 5-HT (dual bath, microdialysis and microinjection experiments) revealed, however, that 5-HT acts at the medullary level and that its effects are not due to a diffuse action on all the neurons of the medullary respiratory centers but to a specific action focusing on structures located in the rostral ventro-lateral medulla.     

The diving response depresses ventilation in man

When the face of diving animals comes into contact with water, the diving response is elicited, resulting in bradycardia, peripheral vasoconstriction and ventilatory depression. The cardiovascular aspects of the diving response have been described in man; little is known about the ventilatory depression in man. Compresses with water of various temperatures were applied to the cheeks of 16 normal volunteers and of two hyperventilating subjects. Ventilation was measured with a spirometer or with a pneumatic thoracic transducer: an accordion shaped balloon, strapped around the thorax. When the ventilation was measured with the spirometer via mouth-piece and nose-clip, the diving response caused ventilatory depressions to 60-70% of control. Water temperatures of 10, 20 and 37 degrees C had no significantly different effects from one another on ventilation. When the face of the subjects was left free of nose-clip and mouth-piece and the ventilation was measured by a thoracic transducer, the diving response caused a ventilatory depression to approximately 60% of control. The ventilatory depression did not adapt within 5 min. The two subjects who hyperventilated reacted qualitatively and quantitatively the same as the other subjects.     

Distribution of androgen receptor immunoreactivity in the brainstem of male rats

Gonadal steroids such as testosterone and estrogen are necessary for the normal activation of male rat sexual behavior. The medial preoptic area (MPOA), an important neural substrate regulating mating, accumulates steroids and also expresses functional androgen receptors (AR). The MPOA is intimately connected with other regions implicated in copulation, such as the bed nucleus of the stria terminalis and medial amygdala. Inputs to the MPOA arise from several areas within the brainstem, synapsing preferentially onto steroid sensitive MPOA cells which are activated during sexual activity. Given that little is known about the distribution of AR protein in the brainstem of male rats, we mapped the distribution of AR expressing cells in the pons and medulla using immunocytochemistry. In agreement with previous reports, AR immunoreactivity (AR-ir) was detected in ventral spinal motoneurons and interneurons. In addition, AR-ir was detected in areas corresponding to the solitary tract, lateral paragigantocellular and alpha and ventral divisions of the gigantocellular reticular nuclei, area postrema, raphe pallidus, ambiguus nucleus, and intermediate reticular nucleus. Several regions within the pons contained AR-ir, such as the tegmental and central gray, parabrachial nucleus, locus coeruleus, Barrington's nucleus, periaqueductal gray, and dorsal raphe. In contrast with in situ hybridization studies, auditory and somatosensory areas were AR-ir negative, and, except for very light staining in the prepositus nucleus, areas carrying vestibular information did not display AR-ir. Additionally, cranial nerve motoneurons of the hypoglossal, facial, dorsal vagus, and spinal trigeminal did not display AR-ir in contrast to previous reports. The data presented here indicate that androgens may influence numerous cell groups within the brainstem. Some of these probably constitute a steroid sensitive circuit linking the MPOA to motoneurons in the spinal cord via androgen responsive cells in the caudal ventral medulla.     

Respiratory responses to intermittent hypoxia in unsedated piglets: relation to substance P binding in brainstem

Respiratory responses to single intermittent hypoxia (5 min 21% O(2), 5 min 8% O(2) X6) in 5-6, 10-11, 21-22 and 26-27 day-old piglets, and to recurrent six daily intermittent hypoxia in 10-11 and 26-27 day-old piglets were assessed. Substance P binding in the piglets' brainstem immediately after the last hypoxic episode was measured. All piglets hyperventilated during hypoxia. Weight adjusted inspired ventilation, tidal volume and instantaneous flow decreased with age. The oldest piglets uniquely displayed attenuated ventilation and tidal volume during the sixth versus first hypoxic episode with single intermittent hypoxia, and reduced inspired ventilation and tidal volume during the first hypoxic episode on the sixth daily hypoxia compared to single hypoxia. By contrast, substance P binding was greatly reduced in the solitary, hypoglossal, paraambigual and lateral reticular brainstem nuclei of both younger and older piglets following either single or recurrent intermittent hypoxia. Thus, the reduction in membrane-bound neurokinin receptors by intermittent hypoxia, presumably consequent to endogenously released substance P, does not exclusively determine whether the ventilatory response to that hypoxia will be attenuated or not.     

Respiratory rhythm of brainstem-spinal cord preparations: Effects of maturation, age, mass and oxygenation

We examined the effect of age, mass and the presence of the pons on the longevity (length of time spontaneous respiratory-related activity is produced) of brainstem–spinal cord preparations of neonatal rodents (rats and hamsters) and the level of oxygenation in the medulla respiratory network in these preparations. We found the longevity of the preparations from both species decreased with increasing postnatal age. Physical removal of the pons increased respiratory frequency and the longevity of the preparation. However, tissue oxygenation at the level of the medullary respiratory network was not affected by removal of the pons or increasing postnatal age (up to postnatal day 4). Taken together, these data suggest that the effect of removing the pons on respiratory frequency and the longevity of brainstem–spinal cord preparations with increasing postnatal age are primarily due to postnatal development and appear to be unrelated to mass or changes in levels of tissue oxygenation.     

The human raphe nuclei and the serotonergic system

The raphe nuclei are distributed near the midline of the brainstem along its entire rostro-caudal extension. The serotonergic neurons are their main neuronal components, although a proportion of them lie in subdivisions of the lateral reticular formation. They develop from mesopontine and medullary primordia, and the resulting grouping into rostral and caudal clusters is maintained into adulthood, and is reflected in the connectivity. Thus, the mesencephalon and rostral pons, neurons within the rostral raphe complex (caudal linear, dorsal raphe, and median raphe nuclei) project primarily to the forebrain. By contrast, in the caudal pons and medulla oblongata, neurons within the caudal raphe complex (raphe magnus, raphe obscurus, raphe pallidus nuclei and parts of the adjacent lateral reticular formation) project to the brainstem nuclei and to the spinal cord. The median raphe and dorsal raphe nuclei provide parallel and overlapping projections to many forebrain structures with axon fibers exhibiting distinct structural and functional characteristics. The caudal group of the serotonergic system projects to the brainstem, and, by three parallel projections, to the dorsal, intermediate and ventral columns in the spinal cord. The serotonergic axons arborize over large areas comprising functionally diverse targets. Some projections form classical chemical synapses while many do not, thus contributing to the so-called paracrine or volume transmission. The serotonergic projections participate in the regulation of different functional (motor, somatosensory, limbic) systems; and have been associated with a wide range of neuropsychiatric and neurological disorders. Finally, recent experimental data support the role of serotonin in modulating brain development, such that a dysfunction in serotonergic transmission during early life could lead to long lasting structural and functional alterations.     

Propriospinal neurons are sufficient for bulbospinal transmission of the locomotor command signal in the neonatal rat spinal cord

We recently showed that propriospinal neurons contribute to bulbospinal activation of locomotor networks in the in vitro neonatal rat brainstem–spinal cord preparation. In the present study, we examined whether propriospinal neurons alone, in the absence of long direct bulbospinal transmission to the lumbar cord, can successfully mediate brainstem activation of the locomotor network. In the presence of staggered bilateral spinal cord hemisections, the brainstem was stimulated electrically while recording from lumbar ventral roots. The rostral hemisection was located between C1 and T3 and the contralateral caudal hemisection was located between T5 and mid-L1. Locomotor-like activity was evoked in 27% of the preparations, which included experiments with staggered hemisections placed only two segments apart. There was no relation between the likelihood of developing locomotor-like activity and the distance separating the two hemisections or specific level of the hemisections. In some experiments, where brainstem stimulation alone was ineffective, neurochemical excitation of propriospinal neurons (using 5-HT and NMDA) at concentrations subthreshold for producing locomotor-like activity, promoted locomotor-like activity in conjunction with brainstem stimulation. In other experiments, involving neither brainstem stimulation nor cord hemisections, the excitability of propriospinal neurons in the cervical and/or thoracic region was selectively enhanced by bath application of 5-HT and NMDA or elevation of bath K⁺ concentration. These manipulations produced locomotor-like activity in the lumbar region. In total, the results suggest that propriospinal neurons are sufficient for transmission of descending locomotor command signals. This observation has implications for regeneration strategies aimed at restoration of locomotor function after spinal cord injury.     

Possible involvement of neurons in locus coeruleus in inhibitory effect on glossopharyngeal expiratory activity in a neonatal rat brainstem-spinal cord preparation in vitro

In this study, we found that a certain motor branch of glossopharyngeal (IX) motor nerves stably exhibits not only inspiratory activity but also expiratory activity with pons removal in neonatal rat brainstem-spinal cord preparations in vitro. Because this finding indicates that IX expiratory activity is masked by an inhibitory mechanism operating in the pons, we sought to determine the candidate neurons that exert an inhibitory effect on IX expiratory activity. IX expiratory activity was observed when only the pons was perfused with noradrenaline (NA) or clonidine (alpha2 adrenergic receptor agonist), but not when NA and yohimbine (alpha2 adrenergic receptor antagonist) were perfused together. IX expiratory activity was also observed following the removal of the dorsal pons but not the ventral pons. The local administration of clonidine into the bilateral locus coeruleus (LC) evoked burst discharges during the expiratory phase in the IX motor rootlet. These results suggest that neurons in the LC that possess an alpha2 adrenergic receptor on the membrane surface exert a tonic inhibitory effect on IX expiratory activity in neonatal rat brainstem-spinal cord preparations.     

The effect of hypocapnia (PaCO2 27 mmHg) on CaM kinase IV activity, Bax/Bcl-2 protein expression and DNA fragmentation in the cerebral cortex of newborn piglets

The present study tests the hypothesis that a PaCO(2) of 27 mmHg for 1 hr results in increased neuronal nuclear Ca(++)/calmodulin-dependent protein kinase IV (CaM kinase IV) activity, pro-apoptotic protein expression and DNA fragmentation in the cerebral cortex of newborn piglets. Hypocapnic (HC) and normocapnic newborn piglets were studied. Tissue levels of ATP and phosphocreatine (PCr) were lower in the HC group. CaM kinase IV activity and Bax protein density were higher in the HC group. Bcl-2 protein density was the same in both groups, resulting in an increased ratio of Bax/Bcl-2 in the HC group. Density of nuclear DNA fragments was greater in the HC group and varied inversely with ATP and PCr levels. We conclude that hypocapnia (PaCO(2) 27 mmHg) results in increased expression of pro-apoptotic proteins and fragmentation of nuclear DNA in newborn piglets.     

Respiratory neural activity responses to chemical stimuli in newborn rats: reversible transition from normal to 'secondary' rhythm during asphyxia and its implication for 'respiratory like' activity of isolated medullary preparation

To clarify a possible origin of 'respiratory like' rhythmic activities observed in in vitro brainstem preparation, the phrenic (Phr) and cranial nerve (XII or IX) inspiratory activities were analyzed in halothane-anesthetized, vagotomized and artificially ventilated newborn (2--6 days after birth) and young adult rats (30--50 days) during altered chemical stimuli and prolonged asphyxia at 25 degrees C. The newborn rat showed regular rhythmic inspiratory discharges of short duration, and their responses to CO(2) and hypoxia did not differ from those seen in adult rats. In the newborn rat the Phr and cranial nerve inspiratory discharges increased first, then respiratory frequency decreased and finally ceased completely for approximately 1--2 min during asphyxia. Thereafter, 'secondary' rhythmic inspiratory activity emerged at a slower rate with decremental inspiratory discharge profile, which persisted for a period more than 40 min of asphyxia. A normal respiratory activity recovered after resumption of artificial ventilation. Though young adult rats exhibited similar sequential changes in respiratory activity during asphyxia, the 'secondary' rhythmic activity persisted for a period of several min only. The pattern of 'secondary' respiratory activity corresponded well with that of rhythmic activities seen in the isolated medullary block preparation of newborn rat. 'Respiratory like' activity found in isolated medullary preparations of newborn animals may arise from a mechanism that generates 'secondary' (or so called 'gasping' type) rhythmic inspiratory activity during prolonged asphyxia in in vivo preparations.     

Brainstem sites for the carbachol elicitation of the hippocampal theta rhythm in the rat

The effects of brainstem microinjections of carbachol on the hippocampal theta rhythm were examined in urethane anesthetized rats. The two most effective theta-eliciting sites with carbachol were the nucleus pontis oralis (RPO) and the acetylcholine-containing pedunculopontine tegmental nucleus (PPT) of the dorsolateral pontine tegmentum. RPO injections generated theta at mean latencies of 38.5±70.8 s and for mean durations of 12.9±5.1 min. Five of seven RPO injections gave rise to theta virtually instantaneously, i.e., before the completion of the injection. PPT injections generated theta at mean latencies of 1.7±1.1 min and for mean durations of 11.9±6.0 min. Injections rostral or caudal to RPO in the caudal midbrain reticular formation (RF) or the caudal pontine RF (nucleus pontis caudalis) generated theta at considerably longer latencies (generally greater than 5 min) or were without effect. Medullary RF injections essentially failed to alter the hippocampal EEG. The finding that theta was produced at very short latencies at RPO suggests that RPO, the putative brainstem source for the generation of theta, is modulated by a cholinergic input. The further demonstration that theta was also very effectively elicited with PPT injections suggests this acetylcholine-containing nucleus of the dorsolateral pons may be a primary source of cholinergic input to RPO in the generation of theta. The hippocampal theta rhythm is a major event of REM sleep. The present results are consistent with earlier work showing that each of the other major events of REM sleep, as well as the REM state, are cholinergically activated at the level of the pontine tegmentum.     

Distribution of dopamine-immunoreactive fibers in the rat brainstem

We describe the distribution of axons immunoreactive for dopamine in pons and medulla oblongata of rat under normal conditions or after inhibition of monoamine oxidase or dopamine beta-hydroxylase. In the pons of non-treated animal, fairly dense plexuses of dopamine-immunoreactive varicose fibers were found in the locus coeruleus, dorsal parabrachial and dorsal raphe nuclei, central gray and reticular formation dorsal to the superior olive. In the medulla oblongata, the immunoreactive fibers were abundant in the dorsal vagal complex, lateral paragigantocellular nucleus, midline raphe nuclei and spinal trigeminal nucleus. Monoamine oxidase inhibition made it possible to increase the intensity of immunoreactivity and consequently the number of labeled fibers in these areas, indicating that dopamine is perpetually oxidized by monoamine oxidase, and consequently in low concentration under normal conditions. Sparse dopamine-immunoreactive fibers were observed in the pontine gray, motor trigeminal nucleus, inferior olive and major axon bundles such as the dorsal and ventral tegmental bundles, where numerous noradrenergic fibers have been reported. In axons of these areas, intense dopamine-immunoreactivity was seen only after inhibition of dopamine-beta-hydroxylase. It appears that dopamine is released and oxidized in response to autonomic changes such as hypoxia, hemorrhage, and cardiovascular variation in the caudal brainstem, as we have described elsewhere.