Acidosis-stimulated neurons of the medullary raphe are serotonergic

Neurons of the medullary raphe project widely to respiratory and autonomic nuclei and contain co-localized serotonin, thyrotropin-releasing hormone (TRH), and substance P, three neurotransmitters known to stimulate ventilation. Some medullary raphe neurons are highly sensitive to pH and CO(2) and have been proposed to be central chemoreceptors. Here it was determined whether these chemosensitive neurons are serotonergic. Cells were microdissected from the rat medullary raphe and maintained in primary cell culture for 13-70 days. Immunoreactivity for serotonin, substance P, and TRH was present in these cultures. All acidosis-stimulated neurons (n = 22) were immunoreactive for tryptophan hydroxylase (TpOH-IR), the rate-limiting enzyme for serotonin biosynthesis, whereas all acidosis-inhibited neurons (n = 16) were TpOH-immunonegative. The majority of TpOH-IR medullary raphe neurons (73%) were stimulated by acidosis. The electrophysiological properties of TpOH-IR neurons in culture were similar to those previously reported for serotonergic neurons in vivo and in brain slices. These properties included wide action potentials (4.55 +/- 0.5 ms) with a low variability of the interspike interval, a postspike afterhyperpolarization (AHP) that reversed 25 mV more positive than the Nernst potential for K(+), prominent A current, spike frequency adaptation and a prolonged AHP after a depolarizing pulse. Thus the intrinsic cellular properties of serotonergic neurons were preserved in cell culture, indicating that the results obtained using this in vitro approach are relevant to serotonergic neurons in vivo. These results demonstrate that acidosis-stimulated neurons of the medullary raphe contain serotonin. We propose that serotonergic neurons initiate a homeostatic response to changes in blood CO(2) that includes increased ventilation and modulation of autonomic function.     

Single-unit responses of serotonergic medullary and pontine raphe neurons to environmental cooling in freely moving cats

Brain serotonin has long been implicated in the regulation of body temperature, although its precise role is not completely understood. The present study examined the effects of environmental cooling (4-8 degrees C for 2 or 4h) on the single-unit activity of serotonergic neurons recorded in the medullary raphe nuclei obscurus and pallidus and in the pontine dorsal raphe nucleus of freely moving cats. These neuronal groups have primarily descending projections to the spinal cord and ascending projections to the forebrain, respectively. Cold exposure induced shivering and piloerection, but no appreciable changes in core temperature. Of the medullary serotonergic cells studied (n=14), seven were activated and seven were unresponsive to cold exposure. For the responsive cells, the mean increase and peak effect in unit activity relative to baseline were 31% and 46%, respectively. Of the seven cold-responsive cells, the activity of four was monitored when the animals were transferred back to room temperature (23 degrees C). Within 15-30 min, the activity of these cells returned to baseline. In contrast, none of the dorsal raphe nucleus cells studied (n=14) displayed a significant change in neuronal activity during cold exposure, suggesting that these neurons do not receive afferent input from cold-sensitive cutaneous receptors or participate in thermoregulatory responses evoked by low ambient temperatures.Overall, these results suggest that a subset of medullary serotonergic neurons play a role in physiological mechanisms underlying cold defense (e.g. increases in motor output and/or autonomic outflow). On the other hand, the lack of responsiveness of serotonergic dorsal raphe nucleus neurons to cold exposure does not support a specific role for these cells in thermoregulation.     

Distinguishing characteristics of serotonin and non-serotonin-containing cells in the dorsal raphe nucleus: electrophysiological and immunohistochemical studies

The membrane properties and receptor-mediated responses of rat dorsal raphe nucleus neurons were measured using intracellular recording techniques in a slice preparation. After each experiment, the recorded neuron was filled with neurobiotin and immunohistochemically identified as 5-hydroxytryptamine (5-HT)-immunopositive or 5-HT-immunonegative. The cellular characteristics of all recorded neurons conformed to previously determined classic properties of serotonergic dorsal raphe nucleus neurons: slow, rhythmic activity in spontaneously active cells, broad action potential and large afterhyperpolarization potential. Two electrophysiological characteristics were identified that distinguished 5-HT from non-5-HT-containing cells in this study. In 5-HT-immunopositive cells, the initial phase of the afterhyperpolarization potential was gradual (tau=7.3+/-1.9) and in 5-HT-immunonegative cells it was abrupt (tau=1.8+/-0.6). In addition, 5-HT-immunopositive cells had a shorter membrane time constant (tau=21.4+/-4.4) than 5-HT-immunonegative cells (tau=33.5+/-4.2). Interestingly, almost all recorded neurons were hyperpolarized in response to stimulation of the inhibitory 5-HT(1A) receptor. These results suggested that 5-HT(1A) receptors are present on non-5-HT as well as 5-HT neurons. This was confirmed by immunohistochemistry showing that although the majority of 5-HT-immunopositive cells in the dorsal raphe nucleus were double-labeled for 5-HT(1A) receptor-IR, a small but significant population of 5-HT-immunonegative cells expressed the 5-HT(1A) receptor. These results underscore the heterogeneous nature of the dorsal raphe nucleus and highlight two membrane properties that may better distinguish 5-HT from non-5-HT cells than those typically reported in the literature. In addition, these results present electrophysiological and anatomical evidence for the presence of 5-HT(1A) receptors on non-5-HT neurons in the dorsal raphe nucleus.     

Neurochemical and electrophysiological studies on the functional significance of burst firing in serotonergic neurons

We have previously described a population of 5-hydroxytryptamine neurons which repetitively fires bursts of usually two (but occasionally three or four) action potentials, with a short (<20 ms) interspike interval within a regular low-frequency firing pattern. Here we used a paradigm of electrical stimulation comprising twin pulses (with 7- or 10-ms inter-pulse intervals) to mimic this burst firing pattern, and compared the effects of single- and twin-pulse electrical stimulations in models of pre- and postsynaptic 5-hydroxytryptamine function. Firstly, we measured the effect of direct electrical stimulation (2 Hz for 2 min) of rat brain slices on efflux of preloaded [3H]5-hydroxytryptamine. In this in vitro model, twin-pulse stimulation increased the efflux of tritium by about twice as much as did single-pulse stimulation. This effect was evident in the medial prefrontal cortex (area under the curve: 2. 59+/-0.34 vs 1.28+/-0.22% relative fractional release), as well as in the caudate-putamen (3.93+/-0.65 vs 2.17+/-0.51%) and midbrain raphe nuclei (5.42+/-1.05 vs 2.51+/-0.75%). Secondly, we used in vivo microdialysis to monitor changes in endogenous extracellular 5-hydroxytryptamine in rat medial prefrontal cortex in response to electrical stimulation (3 Hz for 10 min) of the dorsal raphe nucleus. In this model, twin-pulse stimulation of the dorsal raphe nucleus increased 5-hydroxytryptamine by approximately twice as much as did single-pulse stimulation at the same frequency (area under the curve: 50.4+/-9.0 vs 24.2+/-4.4 fmol). Finally, we used in vivo extracellular recording to follow the response of postsynaptic neurons in the rat medial prefrontal cortex to 5-hydroxytryptamine released by dorsal raphe stimulation. Electrical stimulation of the dorsal raphe nucleus (1 Hz) induced a clear-cut poststimulus inhibition in the majority of cortical neurons tested. In these experiments, the duration of poststimulus inhibition following twin-pulse stimulation was markedly longer than that induced by single-pulse stimulation (200+/-21 vs 77+/-18.5 ms).Taken together, the present in vitro and in vivo data suggest that in 5-hydroxytryptamine neurons, short bursts of action potentials will propagate along the axon to the nerve terminal and will enhance both the release of 5-hydroxytryptamine and its postsynaptic effect.     

Development of chemosensitivity of rat medullary raphe neurons

In many neonatal mammals, including humans and rats, there is a developmental increase in the ventilatory response to elevated pCO2. This maturation of central respiratory chemoreception may result from maturation of intrinsic chemosensitivity of brainstem neurons. We have examined age-related changes in chemosensitivity of neurons from the rat medullary raphe, a putative site for central chemoreception, using perforated patch-clamp recordings in vitro. In brain slices from rats younger than 12 days old, firing rate increased in 3% of neurons and decreased in 17% of neurons in response to respiratory acidosis (n = 36). In contrast, in slices from rats 12 days and older, firing rate increased in 18% of neurons and decreased in 15% of neurons in response to the same stimulus (n = 40). A tissue culture preparation of medullary raphe neurons was used to examine changes in chemosensitivity with age from three to 74 days in vitro. In cultured neurons younger than 12 days in vitro, firing rate increased in 4% of neurons and decreased in 44% of neurons in response to respiratory acidosis (n = 54). In contrast, in neurons 12 days in vitro and older, firing rate increased in 30% of neurons and decreased in 24% of neurons in response to respiratory acidosis (n = 105). In both types of chemosensitive neuron ("stimulated" and "inhibited"), the magnitudes of the changes in firing rate were greater in older neurons than in young neurons. These results indicate that the incidence and the degree of chemosensitivity of medullary raphe neurons increase with age in brain slices and in culture. This age-related increase in cellular chemosensitivity may underlie the development of respiratory chemoreception in vivo. Delays in this maturation process may contribute to developmental abnormalities of breathing, such as sudden infant death syndrome.     

Potential noradrenergic regulation of serotonergic neurons in the median raphe nucleus

Summary Pharmacological and morphological evidence suggests that the functional activity of serotonergic neurons may be regulated by catecholamines. We have attempted to reveal a potential pathway by which this interaction might occur. Rats received bilateral knife cut lesions of the ventral noradrenergic bundle which severed the A-1 and A-2 cell body contributions to this projection. Controls received a sham lesion into the cerebellum. Two weeks later all animals were sacrificed, and norepinephrine and serotonin levels were measured in discrete nuclei of the brain. Lesion placement was confirmed histofluorometrically. Serotonin levels in the median raphe nucleus were significantly reduced by 40%, but levels of serotonin were unaffected in the dorsal raphe nucleus and 8 serotonergic terminal regions. The lesions did not affect levels of norepinephrine in the locus coeruleus, cingulate cortex, or habenula. This study suggests that a noradrenergic projection to the median raphe nucleus from the A-1 and A-2 cell body groups may modulate serotonergic neuronal function.This work was presented, in part, at the Society for Neuroscience Annual Meeting, Anaheim, California, 1977     

Involvement of nitric oxide in regulation of the medullary respiratory rhythm in neonatal rats

The NADPH-diaphorase (as a neuronal NO-synthase) reactivity in the medullary structures of the respiratory rhythm (RR) generator and the role of NO in the regulation of respiratory activity in the phrenic nerve of artificially superfused semi-isolated medulla-spinal cord preparations were investigated in newborn rats. NADPH-diaphorase positive neurons were found in all nuclei of both dorsal and ventral respiratory groups of neurons. The maximal density of stained cells was present within the rostral part of the ventrolateral medulla (VLM), in the region of the lateral paragigantocellular reticular nucleus. It was found that endogenous NO mediates the mechanism of tonic inhibitory control of the RR frequency located in the rostral VLM under normal and hypoxic conditions, and appears to be involved in generation of the basic RR by the more caudal structures of VLM. It was shown that NO biosynthesis mediates the effect of NMDA receptors activation on the RR.     

5,7-Dihydroxytryptamine modifies excitability of rat dorsal raphe serotonergic neurons in vitro

Activity of serotonergic dorsal raphe neurons was recorded intracellularly in a brainstem slice preparation from rats before and after local microperfusion of the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). The initial effect of the drug consisted of a transient hyperpolarization. Over 1-2 h after drug application there was a gradual decrease in efficacy of a hyperpolarizing current pulse to evoke a transient rectification. Also, action potentials in some cells failed to evoke a spike after-hyperpolarization. These effects are related to the neurodegenerative action of 5,7-DHT.     

Evidence for regulation of water intake by median raphe serotonergic neurons

Radiofrequency lesions of the median raphe (MR) nucleus did not alter daily water intake in the rat, whereas electrolytic lesions produced a transient polydipsia only during the first day following lesioning. In contrast, microinjection of the specific serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) (8 μg/l μl) directly into the MR nucleus produced a small but non-significant drop in water consumption during the first postoperative day, followed by the development of a marked polydipsia peaking at Day 6 and declining again to approach control levels by Days 8–10. The results suggest that the transient polydipsia which occurred during the first 24-hours following electrolytic MR lesioning can be attributed to a different mechanism than the neurotoxin-induced polydipsia, perhaps to stimulation of cholinergic neurons known to lie in this area. The more slowly developing polydipsia which occurred following 5,7-DHT lesions of the MR nucleus can be attributed to destruction of serotonergic neurons. These results strongly implicate MR serotonergic neurons as inhibitory regulators of water intake mechanisms.     

Evidence for projections from the rostral medullary raphe onto medullary catecholamine neurons in the rat

Following the iontophoretic deposition of Phaseolus vulgaris leucoagglutinin (PHA-L) into the rostral medullary raphe, which included portions of the caudal nucleus raphe magnus, rostral nucleus raphe pallidus, rostral nucleus raphe obscurus and rostral nucleus reticularis paragigantocellularis, two-color immunoperoxidase staining was employed to demonstrate contiguity between PHA-L-immunoreactive (PHA-LI) varicose fibers and boutons and medullary catecholamine (CA) cells. Raphe projections were contiguous with phenylethanolamine N-methyltransferase-immunoreactive (PNMTI) neurons in the C1, C2 and C3 cell groups and with tyrosine hydroxylase-immunoreactive (THI) neurons in the A1 and A2 cell groups. Contiguity between PHA-LI processes and medullary CA cells was observed most frequently in the C1 cell group. Preliminary findings of this study have been presented previously.     

Purinergic modulation of respiration via medullary raphe nuclei in rats

The involvement of P2X receptors in raphe nuclei in respiratory control was investigated. Experiments were done on urethane anesthetized, spontaneously breathing or paralyzed and artificially ventilated adult rats. We found that microinjection of ATP (0.1-0.2 M, 10-70 nl) into raphe magnus (RM) caused dose-dependent decreases in integrated phrenic amplitude and respiratory frequency, whereas injection of ATP into raphe pallidus (RP) caused dose-dependent increases in phrenic amplitude and respiratory frequency. Microinjection of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (0.02 M, 50 nl), a broad-spectrum P2X receptor antagonist, into the RM or RP did not cause any significant change in respiration, but partially blocked the respiratory effects of ATP that was subsequently injected into the same sites within the RM or RP. These findings indicate that the ATP-P2X mediated neurotransmission could contribute to the respiratory control by affecting the activities of raphe nuclei.     

Opioidergic, GABAergic and serotonergic neurotransmission in the dorsal raphe nucleus modulates tonic immobility in guinea pigs

Tonic immobility (TI) is an innate defensive behavior that can be elicited by physical restriction and postural inversion and is characterized by a profound and temporary state of akinesis. Our previous studies demonstrated that the stimulation of serotonin receptors in the dorsal raphe nucleus (DRN) appears to be biphasic during TI responses in guinea pigs (Cavia porcellus). Serotonin released by the DRN modulates behavioral responses and its release can occur through the action of different neurotransmitter systems, including the opioidergic and GABAergic systems. This study examines the role of opioidergic, GABAergic and serotonergic signaling in the DRN in TI defensive behavioral responses in guinea pigs. Microinjection of morphine (1.1 nmol) or bicuculline (0.5 nmol) into the DRN increased the duration of TI. The effect of morphine (1.1 nmol) was antagonized by pretreatment with naloxone (0.7 nmol), suggesting that the activation of μ opioid receptors in the DRN facilitates the TI response. By contrast, microinjection of muscimol (0.5 nmol) into the DRN decreased the duration of TI. However, a dose of muscimol (0.26 nmol) that alone did not affect TI, was sufficient to inhibit the effect of morphine (1.1 nmol) on TI, indicating that GABAergic and enkephalinergic neurons interact in the DRN. Microinjection of alpha-methyl-5-HT (1.6 nmol), a 5-HT(2) agonist, into the DRN also increased TI. This effect was inhibited by the prior administration of naloxone (0.7 nmol). Microinjection of 8-OH-DPAT (1.3 nmol) also blocked the increase of TI promoted by morphine (1.1 nmol). Our results indicate that the opioidergic, GABAergic and serotonergic systems in the DRN are important for modulation of defensive behavioral responses of TI. Therefore, we suggest that opioid inhibition of GABAergic neurons results in disinhibition of serotonergic neurons and this is the mechanism by which opioids could enhance TI. Conversely, a decrease in TI could occur through the activation of GABAergic interneurons.     

Excitation of medullary respiratory neurons in REM sleep

STUDY OBJECTIVE: To study tonic inputs to medullary respiratory neurons during rapid eye movement (REM) sleep. DESIGN: Single medullary-respiratory-neuron recordings during sleep with spontaneous breathing and during apnea caused by mechanical hyperventilation. SETTING: Academic laboratory. SUBJECTS: Three tracheostomized adult cats implanted for polysomnography and extracellular microelectrode recordings. Intervention: Single medullary-respiratory-neuron recordings during spontaneous breathing and mechanical hyperventilation to apnea during non-REM (NREM) and REM sleep. RESULTS: Most but not all respiratory cells of all types (pre-inspiratory, decrementing, augmenting and late inspiratory, phase-spanning, and expiratory) were more active in REM sleep than in NREM sleep during both spontaneous breathing and apnea induced by mechanical hyperventilation. The mean discharge rate of the cells during spontaneous breathing in NREM sleep was 16.7 impulses per second and in REM sleep was 26.5 impulses per second. During ventilator-induced apnea, the mean rates were 10 impulses per second in NREM sleep and 17.5 per second during REM sleep. The increase in activity in REM sleep occurred after a delay of several seconds from the onset of REM sleep. Respiratory cells were excited at times individually and at other times simultaneously in either a reciprocal or nonreciprocal pattern. The degree of excitation of a neuron in REM sleep during ventilator-induced apnea was proportional to the degree of excitation of the neuron in REM sleep during spontaneous breathing. CONCLUSION: Medullary respiratory neurons are excited individually and collectively in REM sleep. The excitation occurs with a delay after the onset of the state and can stimulate and/or disorganize breathing.     

Dendrite bundles in nuclei raphe dorsalis and centralis superior of the rabbit: A possible substrate for local control of serotonergic neurons

A Golgi-Cox, cresyl violet, and histofluorescence study has revealed the presence of dendrite bundles in nuclei raphe dorsalis (NSR) and centralis superior (CNS) in the rabbit brain stem. In NRD, bundles were found in the midline, traversing the medial longitudinal fasciculus (MLF) and in a circumferential location around the MLF. In NCS, bundles were found oriented vertically in the midline. Serotonergic dendrites predominnted in these bundles, but non-serotonergic denddites from cells of the dorsal tegmental nucleus, adjacent reticular formation, and NCS also were present. Lond descending shafts from tanycytes on the floor of the rostral fourth ventricle were also found in the dendrite bundles of both NRD and NCS. We suggest that the dendrite bundles constitute a local neuronal system for regulating the activity of these raphe neurons.     

Differentiation of presumed serotonergic dorsal raphe neurons in relation to behavior and wake-sleep states.

Using extracellular single unit recording, either alone or in combination with microdialysis application of drugs, we examined the characteristics of presumed serotonergic dorsal raphe neurons during wake-sleep states in the freely moving cat. Recordings were made from a total of 272 neurons in the dorsal raphe nucleus. Of these, 240 (88%) were classified as serotonergic on the basis of their typical long-duration action potential, slow discharge activity, and reduced spontaneous discharge rate during paradoxical sleep compared to during slow-wave sleep. An inhibitory response to serotonergic agonists and a slow conduction velocity were seen in all neurons of this type tested or identified by stimulation of the main ascending serotonergic pathway. These presumed serotonergic dorsal raphe neurons could be subdivided into two typical previously identified groups (types I-A and I-B) and four atypical new groups (types I-C, II-A, II-B, and II-C) according to differences in firing patterns during wake-sleep states. The typical neurons were evenly distributed in the dorsal raphe nucleus and their activity was related to the level of behavioral arousal, since they discharged regularly at a high rate during waking and at progressively slower rates during slow-wave sleep, and ceased firing either during slow-wave sleep with ponto-geniculo-occipital waves and paradoxical sleep (type I-A) or only during paradoxical sleep (type I-B). In contrast, the atypical subgroups were unevenly distributed in the dorsal raphe nucleus and exhibited firing patterns distinct from those of the typical neurons, such as sustained tonic activity during paradoxical sleep (types I-C and II-C) or showing their highest rate of tonic discharge during slow-wave sleep, with suppression of discharge during both waking and paradoxical sleep (type II-B). From these data we suggest that presumed serotonergic dorsal raphe neurons play different roles in behavioral state control and that there is functional topographic organization in the dorsal raphe nucleus.     

Connections between pericruciate cortex and the medullary reticulospinal neurons in cat: an electrophysiological study

Summary The connections between the pericruciate cortex and the medullary reticulospinal (RS) neurons were studied in anesthetized cat. Intracellular recordings were made from reticulospinal neurons and the effects of stimulating different areas of the pericruciate cortex were compared. (1) EPSPs were elicited in all the 93 neurons studied which were antidromically activated by spinal stimulation and had an IS-SD notch on the ascending limb of their antidromic spikes. According to the conduction velocity (c.v.) of the axon and the minimal EPSP latency to cortical stimulation, the neurons could be divided into two groups, i.e. fast-conducting RS neurons (FRS neurons, c.v. > 45 m/s) and slow-conducting RS neurons (SRS neurons, c.v. < 45 m/s). The minimal latencies of FRS neurons were equal to or shorter than 2 ms whereas those of SRS neurons were longer than 2 ms. (2) EPSPs with short latency (< 2 ms) could be evoked in FRS neurons by stimulating a relatively wide cortical area including the major part of precruciate area 4 and area 6, with a central area of strongest excitatory effect located in area 4 slighthly medial to the tip of the cruciate sulcus. Stimulation of the postcruciate area 4 only produced long latency EPSPs. (3) By extrapolation from the cortical and peduncular latencies and the conducting distances it was revealed that the earliest part of the minimal latency EPSPs were monosynaptically evoked in FRS neurons and were mediated by fastconducting corticobulbar fibers. (4) FRS neurons could be excited by stimuli applied to both ipsilateral and contralateral pericruciate cortex. The influence from the contralateral cortex was slightly stronger.