Evidence for a serotonergically mediated sympathoexcitatory response to stimulation of medullary raphe nuclei

The cardiovascular role of serotonin (5-HT) containing neurons in the midline medullary raphe nuclei was studied in anesthetized cats. High frequency electrical stimulation of nucleus (n.) raphe (r.) pallidus, n.r. obscurus and n.r. magnus produced both pressor and depressor responses. Single shock stimulation of pressor sites produced an excitatory evoked potential of sympathetic nervous discharge (SND) recorded from the inferior cardiac nerve. Conversely, single shock stimulation of vasodepressor sites resulted in a computer-summed inhibition of SND. The mean conduction velocity in the sympathoexcitatory medullo-spinal pathway to sympathetic preganglionic neurons was calculated to be 1.24 m/s. The 5-HT antagonists methysergide and metergoline blocked the excitation of sympathetic activity evoked from medullary raphe nuclei. In contrast, these agents failed to alter the sympathoexcitatory response to electrical stimulation of lateral medulla pressor sites or the sympathoinhibitory response elicited by raphe stimulation. The 5-HT uptake inhibitor chlorimipramine increased the duration of the sympathoexcitatory response evoked from the raphe but not from the lateral medulla. Finally, mid-collicular transection did not effect the excitation of sympathetic activity elicited by stimulation of medullary raphe nuclei. These data suggest that serotonergic neurons in the midline medullary raphe nuclei provide an excitatory input to sympathetic neurons in the spinal cord.     

Nucleus raphe obscurus evokes 5-HT-1A receptor-mediated modulation of respiratory neurons

We analysed in vivo the synaptic mechanisms underlying serotonin-mediated depression of expiratory neuronal discharges and phrenic nerve activity. We report that nucleus raphe obscurus stimulation not only abolishes phrenic nerve activity, but also hyperpolarizes the membrane potential, depresses periodic synaptic drive potentials and thus action potential discharges in caudal medullary expiratory neurons. These effects originate from pre- and post-synaptic inhibitory processes that involve 5-HT-1A receptor activation.     

Spinal 5-HT2A receptors regulate cutaneous sympathetic vasomotor outflow in rabbits and rats; relevance for cutaneous vasoconstriction elicited by MDMA (3,4-methylenedioxymethamphetamine, "Ecstasy") and its reversal by clozapine

We determined whether spinal 5-hydroxytryptamine 2A (5-HT2A) receptors contribute to resting cutaneous sympathetic vasomotor activity, and to increases in activity elicited by electrical stimulation of the medullary raphe/parapyramidal region, and whether these receptors are involved in the cutaneous vasoconstricting action of systemically administered MDMA (3,4-methylenedioxymethamphetamine, "Ecstasy") and its reversal by clozapine. Experiments were conducted in urethane-anesthetized rabbits and rats. Administration of the 5-HT2A antagonist, trans-4-((3Z)3-[(2-Dimethylaminoethyl)oxyimino]-3-(2-fluorophenyl)propen-1-yl)-phenol, hemifumarate (SR 46349B, 0.1 mg/kg, i.v.) inhibited resting ear pinna sympathetic vasomotor nerve discharge and reduced the extent to which raphe/parapyramidal electrical stimulation caused ear pinna (rabbit) and tail (rat) artery blood flow to fall. Clozapine (0.125-0.5 mg/kg, i.v.) also reduced the fall in ear pinna blood flow elicited by raphe/parapyramidal stimulation. In rabbits, after inactivation of raphe/parapyramidal function by local microinjection of muscimol (1 nmol in 100 nl), the 5-HT2A agonist R(-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI, 50 microg/kg, i.v.) increased ear pinna sympathetic nerve activity from 3+/-2% to 129+/-5% of pre-muscimol levels (P<0.01, n=6), and this increase was abolished by section of the ipsilateral cervical sympathetic nerve trunk. MDMA (2 mg/kg, i.v.) after muscimol decreased ear pinna blood flow from 33+/-10 to 2+/-1 cm/s (P<0.01, n=5) and increased ear pinna sympathetic nerve activity from 8+/-4% to 120+/-41% of pre-muscimol levels (P<0.01, n=6). The MDMA-elicited increase in nerve activity was abolished by SR 46349B. Data suggest that spinal 5-HT2A receptors contribute to sympathetically induced cutaneous vasoconstriction regulated by raphe/parapyramidal neurons in the brainstem, and that these receptors contribute to the cutaneous vasoconstricting action of MDMA and its reversal by clozapine.     

Effect of electrical and chemical stimulation of the raphe obscurus on phrenic nerve activity in the cat

The effect of electrical and chemical (L-glutamate) stimulation of the raphe obscurus on phrenic nerve activity was examined in the cat. Phrenic nerve activity was recorded from a C5 nerve root in anesthetized, paralyzed and artificially ventilated cats. Neural discharge was quantitated by integrating the phrenic nerve activity. The respiratory frequency was determined from the integrated nerve signal. Focal electrical stimulation (18-144 microA; 5-40 Hz; 100 microseconds pulse duration) resulted in significant (P less than 0.05) increases in both integrated phrenic nerve (IPN) amplitude and respiratory frequency. These changes were dependent upon current intensity and frequency of stimulation. The largest increases in IPN amplitude and respiratory frequency were 47 +/- 17% and 146 +/- 8%, respectively. To insure that the changes in integrated phrenic nerve activity (IPNA) were the result of stimulation of cell bodies and not axons of passage, L-glutamate (100, 200 nmol) was microinjected (100 nl) into the raphe obscurus. Significant (P less than 0.05) dose-related changes occurred in integrated phrenic nerve amplitude with an increase of 44 +/- 13% at 100 nmol and 80 +/- 13% at 200 nmol L-glutamate. No significant increase in respiratory frequency was observed with L-glutamate microinjection. The results suggest that the raphe obscurus may be involved in respiratory control.     

Glutamate stimulation of raphe pallidus attenuates the cardiopulmonary reflex in anaesthetised rats

Serotonin has been implicated as having a modulatory action on NTS neurones mediating vagal inputs controlling the cardiorespiratory system. Since raphe pallidus and raphe obscurus both send serotonergic projections to the NTS, we have investigated a neuromodulatory role for these structures on the cardiopulmonary reflex. A multibarrelled microelectrode positioned around the level of the area postrema was placed at varying depths into mid-line brainstem structures and the effect of glutamate stimulation on the cardiopulmonary reflex tested. Excitatory chemical stimulation in the region of raphe pallidus, but not raphe obscurus, attenuated significantly the respiratory and bradycardic components of the cardiopulmonary reflex. This attenuation was reversed by an NTS microinjection of RS-39604, a selective 5-HT, receptor antagonist. We propose that neurones in raphe pallidus that project to the NTS can release serotonin which acts via 5-HT4 receptors to attenuate the reflex phrenic nerve activity and heart rate components of the cardiopulmonary reflex.     

Effect of electric and chemical stimulation of the raphe obscurus on phrenic nerve activity in the cat

The effect of electrical and chemical (l-glutamate) stimulation of the raphe obscurus on phrenic nerve activity was examined in the cat. Phrenic nerve activity was recorded from a C5 nerve root in anesthetized, paralyzed and artificially ventilated cats. Neural discharge was quantitated by integrating the phrenic nerve activity. The respiratory frequency was determined from the integrated nerve signal. Focal electrical stimulation (18–144 μA; 5–40 Hz; 100 μs pulse duration) resulted in significant (P < 0.05) increases in both integrated phrenic nerve (IPN) amplitude and respiratory frequency. These changes were dependent upon current intensity and frequency of stimulation. The largest increases in IPN amplitude and respiratory frequency were47 ± 17%and146 ± 8%, respectively. To insure that the changes in integrated phrenic nerve activity (IPNA) were the result of stimulation of cell bodies and not axons of passage,l-glutamate (100, 200 nmol) was microinjected (100 nl) into the raphe obscurus. Significant (P < 0.05) dose-related changes occurred in integrated phrenic nerve amplitude with an increase of44 ± 13% at 100 nmol and80 ± 13% at 200 nmoll-glutamate. No significant increase in respiratory frequency was observed withl-glutamate microinjection. The results suggest that the raphe obscurus may be involved in respiratory control.     

Serotonin-mediated excitation of recurrent laryngeal and phrenic motoneurons evoked by stimulation of the raphe obscurus

Short-latency averaged responses in the recurrent laryngeal nerve (RLN) and C₅ phrenic nerve to electrical stimulation (2.5–80 μA; 2.5–160 Hz; 150 μs pulse duration) of the medullary nucleus raphe obscurus (RO) were investigated in anaesthetized, paralyzed and artificially ventilated cats. The response evoked in RLN by stimulation within RO was excitatory and consisted of a single peak. Characteristics of this response in RLN were compared with those of the delayed excitatory response in C₅ phrenic nerve, which we previously showed to be elicited by stimulation within RO. Mean latency to onset for the excitatory response in RLN was5.7 ± 0.3ms, while the delayed excitatory response in C₅ phrenic nerve occurred at7.0 ± 0.3ms. The excitatory response in both could be evoked when stimulation was applied during inspiration as well as during expiration. The stimulus threshold varied between 2.5 and 5 μA for evoked the inspiratory-phase response in each nerve. The magnitude of this response in RLN and in C₅ phrenic nerve was directly related to current intensity and was dependent upon stimulus frequency. Intravenous administration of the serotonin receptor antagonist, methysergide (0.1–2.4 mg/kg) caused significant dose-related reductions in the response in each nerve. In summary, characteristics of the evoked response in RLN and phrenic nerve are similar in several ways. Both responses are: (1) excitatory in nature, (2) elicited at small stimulus currents, (3) affected similarly by increasing stimulus current and frequency, (4) elicited by stimulation during inspiration and expiration, and (5) mediated at least in part by activation of pathways using serotonin as a neurotransmitter.     

Prolonged augmentation of respiratory discharge in hypoglossal motoneurons following superior laryngeal nerve stimulation

Experiments were conducted to investigate long-lasting effects of brief superior laryngeal nerve (SLN) stimulation on respiratory discharge in the hypoglossal nerve. In paralyzed, decerebrate and artificially ventilated cats, SLN stimulation (Hz, 3-5, s, 3-5 times threshold for inhibition of phrenic nerve discharge) immediately increase hypoglossal activity. Following stimulation, the amplitude of respiratory activity in the hypoglossal nerve was augmented (478 +/- 205%), and slowly decayed to prestimulus levels with a time constant of 106 +/- 16 s. In contrast, phrenic nerve activity was completely inhibited during the SLN stimulation and for several seconds thereafter. After activity resumed, phrenic burst frequency remained depressed (33 +/- 6%). Stimulation of the carotid sinus nerve elicited similar effects on hypoglossal nerve activity. Intracellular recordings from hypoglossal motoneurons indicated that SLN stimulation increased central respiratory drive potentials (CRDPs) following a stimulus train, but had inconsistent effects on resting membrane potential. Intracellular depolarizing current pulses (5-15 nA; 2 s) had no prolonged effects on membrane potential or CRDPs. The possible role of serotonin in prolonged augmentation of hypoglossal activity following SLN stimulation was investigated. Intracellular injection of horseradish peroxidase (HRP) into hypoglossal motoneurons and immunohistochemistry for serotonin revealed some close appositions between serotonin immunoreactive boutons and HRP-labeled neurons, but such appositions were sparse. Pretreatment with methysergide had little effect on prolonged augmentation of hypoglossal discharge following SLN stimulation. These results indicate that: (1) SLN stimulation causes prolonged augmentation of hypoglossal activity probably via increased synaptic inputs to hypoglossal motoneurons; and (2) serotonin is not necessary in the underlying mechanism.     

Effects of baclofen and gamma-aminobutyric acid on different types of medullary respiratory neurons

Effects of baclofen and gamma-aminobutyric acid on medullary respiratory neurons were investigated. Medullary inspiratory neurons of the dorsal and ventral respiratory groups were stimulated by baclofen, 0.5-2 mg/kg, and depressed by doses greater than 4-6 mg/kg. Expiratory neurons were depressed by doses of baclofen which increased phrenic nerve activity. Microelectrophoresis of baclofen (5 mM, pH 3) depressed medullary inspiratory neurons. It is suggested that low i.v. doses of baclofen increase inspiratory activity by disinhibition of medullary neurons whereas higher doses directly depress medullary inspiratory neurons.     

Theophylline-induced respiratory recovery following cervical spinal cord hemisection is augmented by serotonin 2 receptor stimulation

Cervical spinal cord hemisection leads to a disruption of bulbospinal innervation of phrenic motoneurons resulting in paralysis of the ipsilateral hemidiaphragm. We have previously demonstrated separate therapeutic roles for theophylline, and more recently serotonin (5-HT) as modulators to phrenic nerve motor recovery; mechanisms that likely occur via adenosine A1 and 5-HT2 receptors, respectively. The present study was designed to specifically determine if concurrent stimulation of 5-HT2 receptors may enhance motor recovery induced by theophylline alone. Adult female rats (250-350 g; n=7 per group) received a left cervical (C2) hemisection that resulted in paralysis of the ipsilateral hemidiaphragm. Twenty-four hours later rats were given systemic theophylline (15 mg/kg, i.v.), resulting in burst recovery in the ipsilateral phrenic nerve. Theophylline-induced recovery was enhanced with the 5-HT2A/2C receptor agonist, (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI; 1.0 mg/kg). DOI-evoked augmentation of theophylline-induced recovery was attenuated following subsequent injection of the 5-HT2 receptor antagonist, ketanserin (2.0 mg/kg). In a separate group, rats were pretreated with ketanserin, which did not prevent subsequent theophylline-induced respiratory recovery. However, pretreatment with ketanserin did prevent DOI-induced augmentation of the theophylline-evoked phrenic nerve burst recovery. Lastly, using immunocytochemistry and in situ hybridization, we showed for the first time a positive co-localization of adenosine A1 receptor mRNA and immunoreactivity with phrenic motoneurons of the cervical ventral horns. Taken together, the results of the present study suggest that theophylline may induce motor recovery likely at adenosine A1 receptors located at the level of the spinal cord, and the concurrent stimulation of converging 5-HT2 receptors may augment the response.     

Raphe pallidus modulates Bötzinger complex-induced inhibition of the phrenic nerve activity in rats

The raphe pallidus (RPa) and Bötzinger complex (BötC) represent two important nuclei which project to spinal phrenic motor neurons. Stimulation of the RPa produces facilitative effects on respiratory activity, whereas stimulation of the BötC induces inhibitory effects on respiratory activity. In the present study, we examined the modulatory effects of serotonergic (5‐hydroxytryptamine, 5‐HT) RPa neurons on the inhibitory response of the phrenic nerve activity elicited from the BötC in rats. Experiments were performed on spontaneously breathing, urethane‐anesthetized adult rats. Either high‐frequency stimulation or glutamatergic chemical activation of the RPa region significantly attenuated the BötC‐induced inhibition of the phrenic nerve. This attenuation showed a post‐stimulation time and intensity dependency. Pharmacological experiments showed that intravenous injection of methysergide, a broad‐spectrum antagonist of 5‐HT receptors, markedly reduced the respiratory facilitation induced by electrical stimulation of the RPa. Furthermore, microinjections of methysergide into the cerebrospinal fluid around the phrenic motor nucleus (PMN) region at spinal cord segments C4 and C5 significantly decreased the RPa‐related attenuation effects on BötC‐evoked inhibition of phrenic nerve discharge. These results suggest that RPa serotonergic neurons could modulate the inhibition of phrenic nerve activity induced by BötC. Moreover, as the relevant 5‐HT receptors for RPa’s modulatory effects are located in the cervical spinal cord, 5‐HT may, in part, function as a modulator to suppress the BötC neuronal activity via direct RPa‐PMN and BötC‐PMN convergent projection pathways to phrenic motoneurons.     

Activation of a latent respiratory motor pathway by stimulation of neurons in the medullary chemoreceptor area of the rat

Previous studies have demonstrated that during respiratory stress (hypercapnia and hypoxia), a latent crossed respiratory pathway can be activated to produce hemidiaphragm recovery following an ipsilateral C2 spinal cord hemisection. The present study investigates the effects of ventral medullary chemoreceptor area stimulation by microinjection of (1S,3R)-aminocyclopentanedicarboxylic acid (ACPD), a glutamate metabotropic receptor agonist, on activating the latent pathway following left C2 spinal cord hemisection in rats in which end-tidal CO2 was maintained at a constant level. Experiments were conducted on anesthetized, vagotomized, paralyzed, and artificially ventilated rats in which phrenic nerve activity was recorded bilaterally. Before drug injection, the phrenic nerve contralateral to hemisection showed vigorous respiratory-related activity, but the phrenic nerve ipsilateral to hemisection showed no discernible respiratory-related activity. ACPD (1-100 nl, 1 mM) was injected directly into the region of the retrotrapezoid nucleus (RTN), a known medullary chemoreceptor area. Microinjection of ACPD into the right RTN increased respiratory-related activity in the right phrenic nerve (contralateral to hemisection). ACPD (>5 nl, 1 mM) microinjection also significantly induced respiratory recovery in the phrenic nerve ipsilateral to hemisection in a dose-dependent manner. The present study indicates that respiratory recovery can be achieved by stimulation of respiratory circuitry without increasing CO2 levels.     

Effects of baclofen and γ-aminobutyric acid on different types of medullary respiratory neurons

Effects of baclofen and γ-aminobutyric acid on medullary respiratory neurons were investigated. Medullary inspiratory neurons of the dorsal and ventral respiratory groups were stimulated by baclofen, 0.5–2 mg/kg, and depressed by doses greater than 4–6 mg/kg. Expiratory neurons were depressed by doses of baclofen which increased phrenic nerve activity. Microelectrophoresis of baclofen (5 mM, pH 3) depressed medullary inspiratory neurons. It is suggested that low i.v. doses of baclofen increase inspiratory activity by disinhibition of medullary neurons whereas higher doses directly depress medullary inspiratory neurons.     

Distribution of serotonin 2A and 2C receptor mRNA expression in the cervical ventral horn and phrenic motoneurons following spinal cord hemisection

Cervical spinal cord injury leads to a disruption of bulbospinal innervation from medullary respiratory centers to phrenic motoneurons. Animal models utilizing cervical hemisection result in inhibition of ipsilateral phrenic nerve activity, leading to paralysis of the hemidiaphragm. We have previously demonstrated a role for serotonin (5-HT) as one potential modulator of respiratory recovery following cervical hemisection, a mechanism that likely occurs via 5-HT2A and/or 5-HT2C receptors. The present study was designed to specifically examine if 5-HT2A and/or 5-HT2C receptors are colocalized with phrenic motoneurons in both intact and spinal-hemisected rats. Adult female rats (250-350 g; n = 6 per group) received a left cervical (C2) hemisection and were injected with the fluorescent retrograde neuronal tracer Fluorogold into the left hemidiaphragm. Twenty-four hours later, animals were killed and spinal cords processed for in situ hybridization and immunohistochemistry. Using (35)S-labeled cRNA probes, cervical spinal cords were probed for 5-HT2A and 5-HT2C receptor mRNA expression and double-labeled using an antibody to Fluorogold to detect phrenic motoneurons. Expression of both 5-HT2A and 5-HT2C receptor mRNA was detected in motoneurons of the cervical ventral horn. Despite positive expression of both 5-HT2A and 5-HT2C receptor mRNA-hybridization signal over phrenic motoneurons, only 5-HT2A silver grains achieved a signal-to-noise ratio representative of colocalization. 5-HT2A mRNA levels in identified phrenic motoneurons were not significantly altered following cervical hemisection compared to sham-operated controls. Selective colocalization of 5-HT2A receptor mRNA with phrenic motoneurons may have implications for recently observed 5-HT2A receptor-mediated regulation of respiratory activity and/or recovery in both intact and injury-compromised states.     

Synaptic basis of orbital cortically induced rhythmical masticatory activity of trigeminal motoneurons in immobilized cats

Synaptic basis of the centrally induced masticatory rhythm was studied by intracellular recording from jaw closer and opener motoneurons during repetitive stimulation of the orbital gyrus (OG) of the encéphale isoléof immobilized cats, with the following results.

(1) Repetitive OG stimulation induced a rhythmical alternation of hyperpolarizing and depolarizing potentials in jaw closer motoneurons. Rhythmical efferent burst discharges induced in the digastric nerve by OG stimulation coincided with the hyperpolarizing phase. The hyperpolarizing potential was reversed to a depolarizing potential by intracellular Cl⁻ injection, while the depolarizing potential was not reversed to hyperpolarization, indicating that the hyperpolarizing and depolarizing potentials consisted at least mainly of IPSPs and EPSPs, respectively.

(2) Repetitive OG stimulation induced rhythmical depolarizing potentials superimposed by bursts in jaw opener motoneurons, corresponding in time with the rhythmical efferent bursts in the digastric nerve. Synaptic activation noise was increased coincidentally with the depolarizing potential, indicating that EPSPs were involved in the degeration of the depolarizing potential. No or little hyperpolarization if any was observed between successive depolarizing potentials.

(3) Repetitive OG stimulation induced efferent burst discharges in the digastric nerve with the masticatory rhythm unaccompanied by any spike potential in the supratrigeminal neurons responsible for peripherally evoked inhibition of jaw closer motoneurons, indicating that the peripheral inhibitory mechanisms do not play an essential role in the central generation of the masticatory rhythm by OG stimulation.

Identification of serotonergic and sympathetic neurons in medullary raphe nuclei

The purpose of the present study was to identify midline medullary serotonin (5-HT) neurons and to determine if these neurons were distinct from previously identified sympathoinhibitory and sympathoexcitatory neurons. Identification of medullary 5-HT neurons was based on electrophysiological and pharmacological similarities to dorsal raphe 5-HT neurons. Sympathoinhibitory and sympathoexcitatory neurons were characterized by an irregular discharge pattern which was temporally related to inferior cardiac sympathetic nerve discharge (SND) and to the cardiac cycle. Sympathoinhibitory neurons increased their discharge rate and the discharge of sympathoexcitatory neurons decreased during baroreceptor reflex activation. A third type of neuron fired in an extremely regular fashion (as judged by interspike interval analysis), fired at a rate of 1.1 spikes/s and had spike durations of approximately 2 ms. The discharges of regularly firing neurons were not temporally related to SND and were not affected during baroreceptor reflex activation. Regularly firing neurons and sympathoinhibitory neurons could be antidromically activated by electrical stimulation of the intermediolateral cell column of the spinal cord. Axonal conduction velocity of sympathoinhibitory neurons (2.4 m/s) was significantly greater than that for regularly firing neurons (1.3 m/s). Regularly firing neurons were completely inhibited by low doses of the 5-HT1A agonist 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT) (i.e. 2 micrograms/kg, i.v.) while much higher doses of the drug failed to affect the discharges of sympathoinhibitory and sympathoexcitatory neurons. Microiontophoretic application of 5-HT and 8-OH-DPAT profoundly depressed the firing of regularly discharging neurons. Based on the striking similarities between regularly firing medullary neurons and dorsal raphe 5-HT neurons it is concluded that the regularly firing neurons were 5-HT-containing neurons. Furthermore, these medullary 5-HT neurons are distinct from sympathoinhibitory and sympathoexcitatory neurons.     

Central respiratory effects versus neuromuscular actions of nerve agents

The relative contributions of peripheral neuromuscular and central nervous system components in the respiratory failure following acute exposure to organophosphorous anticholinesterase (AChE) agents remain unclear. We examined the effects of the nerve agents, soman, sarin, tabun, and VX on diaphragm contraction, diaphragm electromyogram (EMG), phrenic nerve activity, medullary respiratory-related unit activity, and airflow in the cat. The agents were infused at the rate of 1 LD50 per 15 minutes until respiratory arrest, at which time the phrenic nerve was stimulated supramaximally to test diaphragmic contraction. We found that one of the first signs of respiratory distress is disruption of the normal firing pattern of the medullary respiratory-related neurons. This is followed by changes in phrenic nerve activity, diaphragm EMG, diaphragm contraction and airflow. At the time of respiratory arrest, the medullary respiratory-related units and the phrenic nerve have stopped firing. Immediately following cessation of spontaneous respiration, the diaphragm was tested by stimulating the phrenic nerve with 2 msec pulses of 500 msec duration at 10 Hz and at 100 Hz. Stimulation at 10 Hz consistently produced a frequency-following contraction, while stimulation at 100 Hz produced a tetanic contraction. We conclude that loss of central respiratory drive is the predominant cause of nerve agent-induced respiratory failure, as the diaphragm muscle still contracts tetanically when challenged with a 100 Hz train for 500 msec at the time of respiratory arrest.     

Effects of the serotonin synthesis inhibitor p-CPA on the expression of the crossed phrenic phenomenon 4 h following C2 spinal cord hemisection

The present study assesses the effects of para-chlorophenylalanine (p-CPA), a serotonin-depleting drug, on the recovery of respiratory-related activity in the phrenic nerve induced by asphyxia 4 h following ipsilateral C2 hemisection in young adult rats. HPLC analysis was used to quantify levels of serotonin (5-HT), dopamine (DA), norepinephrine, and the 5-HT metabolite, 5-hydroxyindoleacetic acid, in the C4 segment of the spinal cord, all of which were significantly lower in p-CPA-treated hemisected rats compared to hemisected controls receiving saline. Hemisection alone was found to significantly increase 5-HT levels and significantly decrease DA levels compared to normal controls. Eight of eight saline-injected rats expressed recovery of respiratory-related activity in the ipsilateral phrenic nerve during asphyxia 4 h following hemisection, while only 4/8 rats in the p-CPA-treated group expressed recovery in the ipsilateral nerve. Quantification of integrated phrenic nerve wave-forms indicated that the mean amplitude of respiratory-related activity in the ipsilateral phrenic nerve was significantly lower in p-CPA-treated rats than in saline controls. In addition, saline controls demonstrated significant increases in mean respiratory frequency and mean amplitude of contralateral phrenic nerve activity during asphyxia, compared to normocapnia. However, p-CPA-treated rats did not express significant differences in either mean respiratory frequency or mean amplitude of integrated respiratory wave-forms during asphyxia, compared to normocapnia. The results suggest that p-CPA treatment attenuates the recovery of respiratory-related activity in the phrenic nerve 4 h following ipsilateral C2 hemisection and attenuates asphyxia-induced increases in respiratory frequency and respiratory burst amplitude recorded from the contralateral phrenic nerve.     

Functional reconnections established by central respiratory neurons regenerating axons into a nerve graft bridging the respiratory centers to the cervical spinal cord

The present work investigated, in adult rats, the long-term functional properties and terminal reconnections of central respiratory neurons regenerating axons within a peripheral nerve autograft bridging two separated central structures. A nerve graft was first inserted into the left medulla oblongata, in which the respiratory centers are located. Three months later, a C3 left hemisection was performed, and the distal tip of the graft was implanted into the C4 left spinal cord at the level of the phrenic nucleus, a natural central inspiratory target. Six to eight months after medullary implantation, the animals (n = 12) were electrophysiologically investigated to test 1) the phrenic target reinnervation by analyzing the phrenic responses elicited by bridge electrical stimulation and 2) the bridge innervation by unitary recordings of the spontaneous activity of regenerated axons within the nerve bridge. In the control group (n = 6), the medullary site of implantation corresponded to the dorsolateral medulla, a region known to be an unsuitable site for inducing respiratory axonal regrowth after nerve grafting. Stimulation of the nerve bridge never elicited phrenic nerve response, and no respiratory units were found within the nerve bridge. In the experimental group (n = 6), the proximal tip of the nerve bridge was implanted within the ventrolateral medulla at the level of the respiratory centers. Electrical stimulation of the nerve bridge induced phrenic nerve responses that reflected a postsynaptic activation of the phrenic target. Subsequent unitary recordings from teased fibers within the bridge revealed the presence of regenerated inspiratory fibers exhibiting discharge patterns typical of medullary inspiratory neurons, which normally make synaptic contacts with the inspiratory phrenic target. These results indicate that, when provided with an appropriate denervated target, central respiratory neurons with regenerated axons along a nerve bridge can remain functional for a long period and can make precise and specific functional reconnections with central homotypic target neurons.     

Inspiratory neurons with decrementing firing pattern in raphe nuclei of feline medulla

Extracellular spikes of single inspiratory (I) neurons with decrementing firing pattern were recorded in the medullary raphe nuclei in decerebrated or Nembutal anesthetized cats. A total of 23 neurons with decrementing firing patterns during the I phase were recorded in the raphe obscurus and pallidus at the levels of 2.0-4.0 mm rostral to the obex. The respiratory neurons fired in the I phase during a brief stop of the ventilator, indicating that their respiratory-related activities were central in origin. The effect of electrical stimulation of the recording site of the respiratory neuron on diaphragm EMG was examined: both the diaphragm EMG activity and the respiratory frequency were increased. None of six neurons tested for projections to the cervical spinal cord was antidromically activated by electrical stimulation. The present results suggest that cat I-decrementing neurons in the medullary raphe nuclei receive inputs from the central respiratory rhythm generator and may modify the respiratory activity of supraspinal neural structures.