Acetylcholine modulates respiratory pattern: effects mediated by M3-like receptors in preBötzinger complex inspiratory neurons

Perturbations of cholinergic neurotransmission in the brain stem affect respiratory motor pattern both in vivo and in vitro; the underlying cellular mechanisms are unclear. Using a medullary slice preparation from neonatal rat that spontaneously generates respiratory rhythm, we patch-clamped inspiratory neurons in the preB?tzinger complex (preB?tC), the hypothesized site for respiratory rhythm generation, and simultaneously recorded respiratory-related motor output from the hypoglossal nerve (XIIn). Most (88%) of the inspiratory neurons tested responded to local application of acetylcholine (ACh) or carbachol (CCh) or bath application of muscarine. Bath application of 50 microM muscarine increased the frequency, amplitude, and duration of XIIn inspiratory bursts. At the cellular level, muscarine induced a tonic inward current, increased the duration, and decreased the amplitude of the phasic inspiratory inward currents in preB?tC inspiratory neurons recorded under voltage clamp at -60 mV. Muscarine also induced seizure-like activity evident during expiratory periods in XIIn activity; these effects were blocked by atropine. In the presence of tetrodotoxin (TTX), local ejection of 2 mM CCh or ACh onto preB?tC inspiratory neurons induced an inward current along with an increase in membrane conductance under voltage clamp and induced a depolarization under current clamp. This response was blocked by atropine in a concentration-dependent manner. Bath application of 1 microM pirenzepine, 10 microM gallamine, or 10 microM himbacine had little effect on the CCh-induced current, whereas 10 microM 4-diphenylacetoxy-N-methylpiperidine methiodide blocked the current. The current-voltage (I-V) relationship of the CCh-induced response was linear in the range of -110 to -20 mV and reversed at -11.4 mV. Similar responses were found in both pacemaker and nonpacemaker inspiratory neurons. The response to CCh was unaffected when patch electrodes contained a high concentration of EGTA (11 mM) or bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (10 mM). The response to CCh was reduced greatly by substitution of 128 mM Tris-Cl for NaCl in the bath solution; the I-V curve shifted to the left and the reversal potential shifted to -47 mV. Lowering extracellular Cl(-) concentration from 140 to 70 mM had no effect on the reversal potential. These results suggest that in preB?tC inspiratory neurons, ACh acts on M3-like ACh receptors on the postsynaptic neurons to open a channel permeable to Na(+) and K(+) that is not Ca(2+) dependent. This inward cation current plays a major role in depolarizing preB?tC inspiratory neurons, including pacemakers, that may account for the ACh-induced increase in the frequency of respiratory motor output observed at the systems/behavioral level.     

Mechanisms underlying regulation of respiratory pattern by nicotine in preBötzinger complex

Cholinergic neurotransmission plays a role in regulation of respiratory pattern. Nicotine from cigarette smoke affects respiration and is a risk factor for sudden infant death syndrome (SIDS) and sleep-disordered breathing. The cellular and synaptic mechanisms underlying this regulation are not understood. Using a medullary slice preparation from neonatal rat that contains the preB?tzinger Complex (preB?tC), the hypothesized site for respiratory rhythm generation, and generates respiratory-related rhythm in vitro, we examined the effects of nicotine on excitatory neurotransmission affecting inspiratory neurons in preB?tC and on the respiratory-related motor activity from hypoglossal nerve (XIIn). Microinjection of nicotine into preB?tC increased respiratory frequency and decreased the amplitude of inspiratory bursts, whereas when injected into XII nucleus induced a tonic activity and an increase in amplitude but not in frequency of inspiratory bursts from XIIn. Bath application of nicotine (0.2--0.5 microM, approximately the arterial blood nicotine concentration immediately after smoking a cigarette) increased respiratory frequency up to 280% of control in a concentration-dependent manner. Nicotine decreased the amplitude to 82% and increased the duration to 124% of XIIn inspiratory bursts. In voltage-clamped preB?tC inspiratory neurons (including neurons with pacemaker properties), nicotine induced a tonic inward current of -19.4 +/- 13.4 pA associated with an increase in baseline noise. Spontaneous excitatory postsynaptic currents (sEPSCs) present during the expiratory period increased in frequency to 176% and in amplitude to 117% of control values; the phasic inspiratory drive inward currents decreased in amplitude to 66% and in duration to 89% of control values. The effects of nicotine were blocked by mecamylamine (Meca). The inspiratory drive current and sEPSCs were completely eliminated by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in the presence or absence of nicotine. In the presence of tetrodotoxin (TTX), low concentrations of nicotine did not induce any tonic current or any increase in baseline noise, nor affect the input resistance in inspiratory neurons. In this study, we demonstrated that nicotine increased respiratory frequency and regulated respiratory pattern by modulating the excitatory neurotransmission in preB?tC. Activation of nicotinic acetylcholine receptors (nAChRs) enhanced the tonic excitatory synaptic input to inspiratory neurons including pacemaker neurons and at the same time, inhibited the phasic excitatory coupling between these neurons. These mechanisms may account for the cholinergic regulation of respiratory frequency and pattern.     

Patterns of phrenic motor output evoked by chemical stimulation of neurons located in the pre-Bötzinger complex in vivo

The pre-B?tzinger complex (pre-B?tC) has been proposed to be essential for respiratory rhythm generation from work in vitro. Much less, however, is known about its role in the generation and modulation of respiratory rhythm in vivo. Therefore we examined whether chemical stimulation of the in vivo pre-B?tC manifests respiratory modulation consistent with a respiratory rhythm generator. In chloralose- or chloralose/urethan-anesthetized, vagotomized cats, we recorded phrenic nerve discharge and arterial blood pressure in response to chemical stimulation of neurons located in the pre-B?tC with DL-homocysteic acid (DLH; 10 mM; 21 nl). In 115 of the 122 sites examined in the pre-B?tC, unilateral microinjection of DLH produced an increase in phrenic nerve discharge that was characterized by one of the following changes in cycle timing and pattern: 1) a rapid series of high-amplitude, rapid rate of rise, short-duration bursts, 2) tonic excitation (with or without respiratory oscillations), 3) an integration of the first two types of responses (i.e., tonic excitation with high-amplitude, short-duration bursts superimposed), or 4) augmented bursts in the phrenic neurogram (i.e., eupneic breath ending with a high-amplitude, short-duration burst). In 107 of these sites, the phrenic neurogram response was accompanied by an increase or decrease (>/=10 mmHg) in arterial blood pressure. Thus increases in respiratory burst frequency and production of tonic discharge of inspiratory output, both of which have been seen in vitro, as well as modulation of burst pattern can be produced by local perturbations of excitatory amino acid neurotransmission in the pre-B?tC in vivo. These findings are consistent with the proposed role of this region as the locus for respiratory rhythm generation.     

Serotonergic modulation of neurotransmission in the rat basolateral amygdala.

Whole cell patch-clamp recordings were obtained from projection neurons and interneurons of the rat basolateral amygdala (BLA) to understand local network interactions in morphologically identified neurons and their modulation by serotonin. Projection neurons and interneurons were characterized morphologically and electrophysiologically according to their intrinsic membrane properties and synaptic characteristics. Synaptic activity in projection neurons was dominated by spontaneous inhibitory postsynaptic currents (IPSCs) that were multiphasic, reached 181 +/- 38 pA in amplitude, lasted 296 +/- 27 mS, and were blocked by the GABAA receptor antagonist, bicuculline methiodide (30 microM). In interneurons, spontaneous synaptic activity was characterized by a burst-firing discharge patterns (200 +/- 40 Hz) that correlated with the occurrence of 6-cyano-7-nitroquinoxaline-2,3-dione-sensitive, high-amplitude (260 +/- 42 pA), long-duration (139 +/- 19 mS) inward excitatory postsynaptic currents (EPSCs). The interevent interval of 831 +/- 344 mS for compound inhibitory postsynaptic potentials (IPSPs), and 916 +/- 270 mS for EPSC bursts, suggested that spontaneous IPSP/Cs in projection neurons are driven by burst of action potentials in interneurons. Hence, BLA interneurons may regulate the excitability of projection neurons and thus determine the degree of synchrony within ensembles of BLA neurons. In interneurons 5-hydroxytryptamine oxalate (5-HT) evoked a direct, dose-dependent, membrane depolarization mediated by a 45 +/- 6.9 pA inward current, which had a reversal potential of -90 mV. The effect of 5-HT was mimicked by the 5-HT2 receptor agonist, alpha-methyl-5-hydroxytryptamine (alpha-methyl-5-HT), but not by the 5-HT1A receptor agonist, (+/-) 8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT), or the 5-HT1B agonist, CGS 12066A. In projection neurons, 5-HT evoked an indirect membrane hyperpolarization ( approximately 2 mV) that was associated with a 75 +/- 42 pA outward current and had a reversal potential of -70 mV. The response was independent of 5-HT concentration, blocked by TTX, mimicked by alpha-methyl-5-HT but not by 8-OH-DPAT. In interneurons, 5-HT reduced the amplitude of the evoked EPSC and in the presence of TTX (0.6 microM) reduced the frequency of miniature EPSCs but not their quantal content. In projection neurons, 5-HT also caused a dose-dependent reduction in the amplitude of stimulus evoked EPSCs and IPSCs. These results suggest that acute serotonin release would directly activate GABAergic interneurons of the BLA, via an activation of 5-HT2 receptors, and increase the frequency of inhibitory synaptic events in projection neurons. Chronic serotonin release, or high levels of serotonin, would reduce the excitatory drive onto interneurons and may act as a feedback mechanism to prevent excess inhibition within the nucleus.     

Pre-B?tzinger neurons with preinspiratory discharges "in vivo" express NK1 receptors in the rat.

Substance P stimulates respiration, in part by a direct action on the pre-B?tzinger complex (preB?tC). This region of the medulla oblongata contains neurons that are strongly immunoreactive for the neurokinin-1 receptor (NK1R-ir), and a recent theory has postulated that these cells might be the adult form of excitatory interneurons that are essential for respiratory rhythmogenesis in neonates. Here we sought to determine whether preB?tC respiratory neurons are indeed NK1R-ir in the adult rat. Preinspiratory (pre-I) neurons were recorded in the preB?tC region of halothane-anesthetized rats. Most pre-I cells could be antidromically activated from the contralateral side of the medulla (7 of 10; latency 1.3 +/- 0.2 ms), suggesting that most of them were propriomedullary neurons rather than respiratory motoneurons or bulbospinal cells. Thirty-two pre-I neurons including seven cells with contralateral projection were labeled with biotinamide using the juxtacellular method. Eleven cells (34.4%) were NK1R-ir, including three of the seven pre-I cells that were antidromically activated from the contralateral side. In 3 control rats we labeled 20 preB?tC neurons with patterns of discharge other than pre-I and found that none were detectably NK1R-ir. In conclusion, some of the intensely NK1R-ir neurons of the adult preB?tC region are indeed respiratory interneurons as suggested by Gray et al. The subtype of NK1R identified by the antibody is detectable only in a small minority of preB?tC respiratory cells, most notably in pre-I interneurons. Given prior anatomical evidence, these NK1R-ir pre-I interneurons are most likely glutamatergic. The data are consistent with the possibility that the NK1R-ir pre-I interneurons of the adult preB?tC could be the adult form of a class of inspiratory neurons that are rhythmogenic in the neonate (either the pacemakers and/or an excitatory subtype of follower neurons).     

Cholinergic neurotransmission in the preBötzinger Complex modulates excitability of inspiratory neurons and regulates respiratory rhythm

We investigated whether there is endogenous acetylcholine (ACh) release in the preBötzinger Complex (preBötC), a medullary region hypothesized to contain neurons generating respiratory rhythm, and how endogenous ACh modulates preBötC neuronal function and regulates respiratory pattern. Using a medullary slice preparation from neonatal rat, we recorded spontaneous respiratory-related rhythm from the hypoglossal nerve roots (XIIn) and patch-clamped preBötC inspiratory neurons. Unilateral microinjection of physostigmine, an acetylcholinesterase inhibitor, into the preBötC increased the frequency of respiratory-related rhythmic activity from XIIn to 116±13% (mean±S.D.) of control. Ipsilateral physostigmine injection into the hypoglossal nucleus (XII nucleus) induced tonic activity, increased the amplitude and duration of the integrated inspiratory bursts of XIIn to 122±17% and 117±22% of control respectively; but did not alter frequency. In preBötC inspiratory neurons, bath application of physostigmine (10 μM) induced an inward current of 6.3±10.6 pA, increased the membrane noise, decreased the amplitude of phasic inspiratory drive current to 79±16% of control, increased the frequency of spontaneous excitatory postsynaptic currents to 163±103% and decreased the whole cell input resistance to 73±22% of control without affecting the threshold for generation of action potentials. Bath application of physostigmine concurrently induced tonic activity, increased the frequency, amplitude and duration of inspiratory bursts of XIIn motor output. Bath application of 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 2 μM), a M3 muscarinic acetylcholine receptor (mAChR) selective antagonist, increased the input resistance of preBötC inspiratory neurons to 116±9% of control and blocked all of the effects of physostigmine except for the increase in respiratory frequency. Dihydro-β-erythroidine (DH-β-E; 0.2 μM), an 4β2 nicotinic receptor (nAChR) selective antagonist, blocked all the effects of physostigmine except for the increase in inspiratory burst amplitude. In the presence of both 4-DAMP and DH-β-E, physostigmine induced opposite effects, i.e. a decrease in frequency and amplitude of XIIn rhythmic activity. These results suggest that there is cholinergic neurotransmission in the preBötC which regulates respiratory frequency, and in XII nucleus which regulates tonic activity, and the amplitude and duration of inspiratory bursts of XIIn in neonatal rats. Physiologically relevant levels of ACh release, via mAChRs antagonized by 4-DAMP and nAChRs antagonized by DH-β-E, modulate the excitability of inspiratory neurons and excitatory neurotransmission in the preBötC, consequently regulating respiratory rhythm.     

Modulation of expiratory motor output evoked by chemical activation of pre-Bötzinger complex in vivo

We have previously demonstrated that chemical stimulation of the pre-B?tzinger complex (pre-B?tC) in the anesthetized cat produces either phasic or tonic excitation of phrenic nerve discharge. This region is characterized by a mixture of inspiratory-modulated, expiratory-modulated, and phase-spanning (including pre-inspiratory (pre-I)) neurons; however, its influence on expiratory motor output is unknown. We, therefore, examined the effects of chemical stimulation of the pre-B?tC on expiratory motor output recorded from the caudal iliohypogastric (lumbar, L(2)) nerve. We found that unilateral microinjection of DL-homocysteic acid (DLH; 10 mM; 10-20 nl) into 16 sites in the pre-B?tC enhanced lumbar nerve discharge, including changes in timing and patterning similar to those previously reported for phrenic motor output. Both increased peak amplitude and frequency of phasic lumbar bursts as well as tonic excitation of lumbar motor activity were observed. In some cases, evoked phasic lumbar nerve activity was synchronized in phase with phrenic nerve discharge. These findings demonstrate that chemical stimulation of the pre-B?tC not only excites inspiratory motor activity but also excites expiratory motor output, suggesting a role for the pre-B?tC in generation and modulation of inspiratory and expiratory rhythm and pattern.     

Neuromodulation and the orchestration of the respiratory rhythm

The respiratory system is continuously modulated by numerous aminergic and peptidergic substances that act at all levels of integration: from the sensory level to the level of central networks and motor nuclei. The same neuronal networks receive inputs from multiple modulators released locally as well as from distal nuclei. All parameters of respiratory control are controlled by multiple neuromodulators. By partly converging onto similar G-proteins and second messenger systems, acetylcholine, norepinephrine, histamine, serotonin (5-HT), dopamine, ATP, substance P, cholecystokinin (CCK) can increase frequency, regularity and amplitude of respiratory activity. Yet, the same modulator can also exert differential effects on respiratory activity by acting on different receptors partly in the same neurons. In the pre-B?tzinger complex (pre-B?tC) modulators can differentially modulate frequency and amplitude in different types of pacemaker neurons. Similarly motoneurons located in different motor nuclei receive differential amplitude modulation from different modulators. Thus, modulators are capable of orchestrating and modulating different parameters of respiratory activity by differentially targeting different cellular targets. A disturbance in modulatory control may lead to Sudden Infant Death Syndrome (SIDS) and erratic breathing.     

Modulation of gasp frequency by activation of pre-Bötzinger complex in vivo

Under hyperoxic conditions, both chemical stimulation of neurons and focal hypoxia in the pre-B?tzinger complex (pre-B?tC) in vivo modify the eupneic pattern of inspiratory motor output by eliciting changes in the patterning and timing of phrenic bursts, which includes both phasic and tonic excitation. The influence of this region on the gasping pattern of phrenic motor output produced during severe brain hypoxia is unknown. We therefore examined the effects of chemical stimulation of neurons (DL-homocysteic acid; DLH; 10 mM; < or =20 nl) and focal hypoxia (sodium cyanide; NaCN; 1 mM; < or =20 nl) in the pre-B?tC on hypoxia-induced gasping in chloralose-anesthetized, vagotomized, mechanically ventilated cats. Unilateral microinjection of DLH into the pre-B?tC during hypoxia-induced gasping increased phrenic burst frequency by approximately 630% (P < 0.01) over baseline frequency due predominantly to a reduction in T(E) (from 28.9 +/- 6.2 to 5.2 +/- 1.8 s; mean +/- SE; P < 0.01). No significant changes in T(I) or rate of rise between hypoxia-induced gasps and the DLH-induced bursts were observed; the effects on peak amplitude of integrated phrenic nerve discharge were variable. Similar responses were evoked by unilateral microinjection of NaCN into the pre-B?tC. These findings demonstrate that both activation of pre-B?tC neurons and focal hypoxia in the pre-B?tC not only influence the eupneic pattern of phrenic motor output but also modify the expression of hypoxia-induced gasping in vivo. These findings also provide additional support to the concept of intrinsic hypoxic chemosensitivity of the pre-B?tC.     

The Effects of Serotonin on Functionally Diverse Isolated Lamprey Spinal Cord Neurons

The experiments reported here showed that application of serotonin (5-hydroxytryptamine, 5-HT) (100 M) did not induce any significant current through the membranes of any of the spinal neurons studied (n= 62). At the same time, the membranes of most motoneurons and interneurons (15 of 18) underwent slight depolarization (2–6 mV) in the presence of 5-HT, which was not accompanied by any change in the input resistance of the cells. Depolarization to 10–20 mV occurred in some cells (3 of 18) of these functional groups, this being accompanied by 20–60% decreases in input resistance. The same concentration of 5-HT induced transient low-amplitude depolarization of most sensory spinal neurons (dorsal sensory cells), this changing smoothly to long-term hyperpolarization by 2–7 mV. The input resistance of the cell membranes in these cases showed no significant change (n= 8). Data were obtained which provided a better understanding of the mechanism by which 5-HT modulates the activity of spinal neurons. Thus, 5-HT facilitates chemoreceptive currents induced by application of NMDA to motoneurons and interneurons, while the NMDA responses of dorsal sensory cells were decreased by 5-HT. 5-HT affected the post-spike afterresponses of neurons. 5-HT significantly decreased the amplitude of afterhyperpolarization arising at the end of the descending phase of action potentials in motoneurons and interneurons and increased the amplitude of afterdepolarization in these types of cells. In sensory spinal neurons, 5-HT had no great effect on post-spike afterresponses. The results obtained here support the suggestion that 5-HT significantly modulates the activity of spinal neurons of different functional types. 5-HT facilitates excitation induced by subthreshold depolarization in motoneurons and some interneurons, facilitating the generation of rhythmic discharges by decreasing afterhyperpolarization. In sensory cells, 5-HT enhances inhibition due to hyperpolarization, suppressing NMDA currents. The differences in the effects of 5-HT on functionally diverse neurons are presumed to be associated with the combination of different types of 5-HT receptors on the membranes of these types of spinal neurons.     

Glutamate neurotransmission is not required for, but may modulate, hypoxic sensitivity of pre-Bötzinger complex in vivo

Focal hypoxia in the pre-B?tzinger complex (pre-B?tC) in vivo elicits excitation of inspiratory motor output by modifying the patterning and timing of phrenic bursts. Hypoxia, however, has been reported to enhance glutamate release in some regions of the brain, including the medullary ventral respiratory column; thus the pre-B?tC-mediated hypoxic respiratory excitation may result from, or be influenced by, hypoxia-induced activation of ionotropic glutamate [i.e., excitatory amino acid (EAA)] receptors. To test this possibility, the effects of focal pre-B?tC hypoxia [induced by sodium cyanide (NaCN)] were examined before and after blockade of ionotropic EAA receptors [using kynurenic acid (KYN)] in this region in chloralose-anesthetized, vagotomized, mechanically ventilated cats. Before blockade of ionotropic EAA receptors, unilateral microinjection of NaCN (1 mM; 10-20 nl) into the pre-B?tC produced either phasic or tonic excitation of phrenic nerve discharge. Unilateral microinjection of KYN (50-100 mM; 40 nl) decreased the amplitude and frequency of basal phrenic nerve discharge; however, subsequent microinjection of NaCN, but not DL-homocysteic acid (DLH, a glutamate analog), still produced excitation of phrenic motor output. Under these conditions, the NaCN-induced excitation included frequency modulation (FM) of phasic phrenic bursts, and in many cases, augmented and/or fractionated phrenic bursts. These findings show that the hypoxia-sensing function of the in vivo pre-B?tC, which produces excitation of phrenic nerve discharge, is not dependent on activation of ionotropic glutamate receptors, but ionotropic glutamate receptor activation may modify the expression of the focal hypoxia-induced response. Thus these findings provide additional support to the concept of intrinsic hypoxic sensitivity of the pre-B?tC.     

Pre-Bötzinger complex functions as a central hypoxia chemosensor for respiration in vivo

Recently, we identified a region located in the pre-B?tzinger complex (pre-B?tC; the proposed locus of respiratory rhythm generation) in which activation of ionotropic excitatory amino acid receptors using DL-homocysteic acid (DLH) elicits a variety of excitatory responses in the phrenic neurogram, ranging from tonic firing to a rapid series of high-amplitude, rapid rate of rise, short-duration inspiratory bursts that are indistinguishable from gasps produced by severe systemic hypoxia. Therefore we hypothesized that this unique region is chemosensitive to hypoxia. To test this hypothesis, we examined the response to unilateral microinjection of sodium cyanide (NaCN) into the pre-B?tC in chloralose- or chloralose/urethan-anesthetized vagotomized, paralyzed, mechanically ventilated cats. In all experiments, sites in the pre-B?tC were functionally identified using DLH (10 mM, 21 nl) as we have previously described. All sites were histologically confirmed to be in the pre-B?tC after completion of the experiment. Unilateral microinjection of NaCN (1 mM, 21 nl) into the pre-B?tC produced excitation of phrenic nerve discharge in 49 of the 81 sites examined. This augmentation of inspiratory output exhibited one of the following changes in cycle timing and/or pattern: 1) a series of high-amplitude, short-duration bursts in the phrenic neurogram (a discharge similar to a gasp), 2) a tonic excitation of phrenic neurogram output, 3) augmented bursts in the phrenic neurogram (i.e., eupneic breath ending with a gasplike burst), or 4) an increase in frequency of phrenic bursts accompanied by small increases or decreases in the amplitude of integrated phrenic nerve discharge. Our findings identify a locus in the brain stem in which focal hypoxia augments respiratory output. We propose that the respiratory rhythm generator in the pre-B?tC has intrinsic hypoxic chemosensitivity that may play a role in hypoxia-induced gasping.     

Heterogeneous actions of serotonin on interneurons in rat visual cortex

The effects of serotonin (5-HT) on excitability of two cortical interneuronal subtypes, fast-spiking (FS) and low threshold spike (LTS) cells, and on spontaneous inhibitory postsynaptic currents (sIPSCs) in layer V pyramidal cells were studied in rat visual cortical slices using whole-cell recording techniques. Twenty-two of 28 FS and 26 of 35 LTS interneurons responded to local application of 5-HT. In the group of responsive neurons, 5-HT elicited an inward current in 50% of FS cells and 15% of LTS cells, an outward current was evoked in 41% of FS cells and 81% of LTS cells, and an inward current followed by an outward current in 9% of FS cells and 4% LTS cells. The inward and outward currents were blocked by a 5-HT(3) receptor antagonist, tropisetron, and a 5-HT(1A) receptor antagonist, NAN-190, respectively. The 5-HT-induced inward and outward currents were both associated with an increase in membrane conductance. The estimated reversal potential was more positive than -40 mV for the inward current and close to the calculated K(+) equilibrium potential for the outward current. The 5-HT application caused an increase, a decrease, or an increase followed by a decrease in the frequency of sIPSCs in pyramidal cells. The 5-HT(3) receptor agonist 1-(m-chlorophenyl) biguanide increased the frequency of larger and fast-rising sIPSCs, whereas the 5-HT(1A) receptor agonist (+/-)8-hydroxydipropylaminotetralin hydrobromide elicited opposite effects and decreased the frequency of large events. These data indicate that serotonergic activation imposes complex actions on cortical inhibitory networks, which may lead to changes in cortical information processing.     

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

Rhythmically active medullary slice preparations isolated from neonatal rats (postnatal days 0-3, P0-P3) were used to study the modulation of respiraory rhythmogenesis and hypoglossal (XII) nerve discharge by serotonin (5-hydroxytryptamine, 5-HT) and noradrenaline (NA). 5-HT, NA and their respective receptor agonists and antagonists were applied either to the bathing medium or focally via pressure injection into regions encompassing the pre-Bötzinger complex or XII motoneurons. The effects of endogenously released 5-HT were also studied by chemical stimulation of neurons within the raphe obscurus. The frequency of respiratory burst discharge was increased when 5-HT was applied: (1) to the bathing medium (37±16%; 30 µM; P < 0.05); (2) via pressure injection into the region of the pre-Bötzinger complex (22 ± 14%; < 25 pmol; P < 0.05); or (3) endogenously released in response to activation of neurons within the raphe obscurus via pressure injection of (R,S)-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydro-bromide (AMPA, 34 ± 15%; P < 0.05) or 5-HT (33 ± 5%; P < 0.05). All of these effects were antagonized by bath application of methysergide (30-40 µM). NA caused a reduction of respiratory burst frequency when applied to the bathing medium (40 ± 15%; 100 µM; P < 0.05) or when pressure injected into the region of the pre-Bötzinger complex (22 ± 11 %; < 25 pmol; P < 0.05). These effects were blocked by the bath application of the a₂-receptor antagonist idazoxan (2 µM). 5-HT and NA both caused an augmentation of tonic discharge of XII nerves when applied either to the bathing medium or via pressure injection into the XII motoneuron pool. The 5-HT-induced XII nerve tonic discharge was mimicked by the 5-HT₂ receptor agonist R(-)2-(2,5-dimethoxy-4-iodophenyl) (DOI.HC1, 5 µM) and blocked by the 5-HT₂ receptor antagonist ketanser-ine tartrate (30-40 µM). The NA-induced XII nerve tonic discharge was mimicked by the α₁-receptor agonist phenylephrine HC1 (500 µM) and blocked by the α₁-receptor antagonist prozasin HC1 (@#@ 1 µM).     

Pharmacology of nicotinic receptors in preBötzinger complex that mediate modulation of respiratory pattern

Nicotine regulates respiratory pattern by modulating excitatory neurotransmission affecting inspiratory neurons within the preB?tzinger Complex (preB?tC). The nicotinic acetylcholine receptor (nAChR) subtypes mediating these effects are unknown. Using a medullary slice preparation from neonatal rat, we recorded spontaneous respiratory-related rhythm from the hypoglossal nerve (XIIn) and patch-clamped inspiratory neurons in the preB?tC simultaneously. The alpha7 nAChR antagonists alpha-bungarotoxin or methyllycaconitine (MLA) had little effect on the actions of low concentrations of nicotine (0.5 microM), which included an increase in respiratory frequency; a decrease in amplitude of XIIn inspiratory bursts; a tonic inward current associated with an increase in membrane noise; an increase in the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs), and; a decrease in the amplitude of inspiratory drive current in voltage-clamped preB?tC inspiratory neurons. These nicotinic actions were completely reversed by dihydro-beta-erythroidine (DH-beta-E) or hexamethonium and reduced by D-tubocurarine. Comparable concentrations of RJR-2403 (0.5-1 microM), an agonist selective for alpha4beta2 nAChRs, increased respiratory frequency to 186% and decreased the amplitude of XIIn inspiratory bursts to 83% of baseline. In voltage-clamped preB?tC inspiratory (including pacemaker) neurons, RJR-2403 induced a tonic inward current of -15.2 pA associated with an increase in membrane noise, increased the frequency to 157% and amplitude to 106% of spontaneous EPSCs, and decreased the amplitude of inspiratory drive current to 80% of baseline. MLA had little effect on RJR-2403 actions, while DH-beta-E completely reversed them. These results suggest that the predominant subtype of nAChRs in preB?tC in neonatal rats that mediates the modulation of respiratory pattern by low concentrations of nicotine is an alpha4beta2 combination and not an alpha7 subunit homomer. We do not exclude the possibility that co-assembly of alpha4beta2 with other subunits or other nAChR subtypes are also expressed in preB?tC neurons. The parallel changes in the cellular and systems level responses induced by different nicotinic agonists and antagonists support the idea that modulation of excitatory neurotransmission affecting preB?tC inspiratory neurons is a mechanism underlying the cholinergic regulation of respiratory pattern (). This study provides a useful model system for evaluating potential therapeutic cholinergic agents for their respiratory effects and side effects.     

Models of respiratory rhythm generation in the pre-B?tzinger complex. III. Experimental tests of model predictions.

We used the testable predictions of mathematical models proposed by Butera et al. to evaluate cellular, synaptic, and population-level components of the hypothesis that respiratory rhythm in mammals is generated in vitro in the pre-B?tzinger complex (pre-B?tC) by a heterogeneous population of pacemaker neurons coupled by fast excitatory synapses. We prepared thin brain stem slices from neonatal rats that capture the pre-B?tC and maintain inspiratory-related motor activity in vitro. We recorded pacemaker neurons extracellularly and found: intrinsic bursting behavior that did not depend on Ca(2+) currents and persisted after blocking synaptic transmission; multistate behavior with transitions from quiescence to bursting and tonic spiking states as cellular excitability was increased via extracellular K(+) concentration ([K(+)](o)); a monotonic increase in burst frequency and decrease in burst duration with increasing [K(+)](o); heterogeneity among different cells sampled; and an increase in inspiratory burst duration and decrease in burst frequency by excitatory synaptic coupling in the respiratory network. These data affirm the basis for the network model, which is composed of heterogeneous pacemaker cells having a voltage-dependent burst-generating mechanism dominated by persistent Na(+) current (I(NaP)) and excitatory synaptic coupling that synchronizes cell activity. We investigated population-level activity in the pre-B?tC using local "macropatch" recordings and confirmed these model predictions: pre-B?tC activity preceded respiratory-related motor output by 100-400 ms, consistent with a heterogeneous pacemaker-cell population generating inspiratory rhythm in the pre-B?tC; pre-B?tC population burst amplitude decreased monotonically with increasing [K(+)](o) (while frequency increased), which can be attributed to pacemaker cell properties; and burst amplitude fluctuated from cycle to cycle after decreasing bilateral synaptic coupling surgically as predicted from stability analyses of the model. We conclude that the pacemaker cell and network models explain features of inspiratory rhythm generation in vitro.     

Voltage-dependent calcium currents in trigeminal motoneurons of early postnatal rats: modulation by 5-HT receptors

Trigeminal motoneurons relay the final output signals generated within the oral-motor pattern generating circuit(s) to muscles for execution of various motor patterns. In recent years, these motoneurons were shown to possess voltage dependent nonlinear membrane properties that allow them to actively participate in sculpting their final output. A complete understanding of the factors controlling trigeminal motoneuronal (TMN) discharge during oral-motor activity requires, at a minimum, a detailed understanding of the palette of ion channels responsible for membrane excitability and a determination of whether these ion channels are targets for modulation. Toward that end, we studied in detail the properties of calcium channels in TMNs and their susceptibility to modulation by 5-HT in rat brain slices. We found that based on pharmacological and voltage-dependent properties, high-voltage-activated (HVA) N-type [omega-conotoxin GVIA (omega-CgTX)]-sensitive, and to a lesser extent P/Q-type [omega-agatoxin IVA (omega-Aga IVA)]-sensitive, calcium channels make up the majority of the whole cell calcium current. 5-HT (5.0 microM) decreased HVA current by 31.3 +/- 2.2%, and the majority of this suppression resulted from reduction of current flow through N- and P/Q-type calcium channels. In contrast, 5-HT had no effect on low-voltage-activated (LVA) current amplitude in TMNs. HVA calcium current inhibition was mimicked by 5-CT, a 5-HT1 receptor agonist, and by R(+)-8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT), a specific 5-HT1A agonist. The effects of 5-HT were blocked by the 5-HT1A antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine hydrobromide (NAN-190) but not by ketanserin, a 5-HT(2/1C) antagonist. Under current clamp, omega-CgTX and 5-HT were most effective in suppressing the mAHP and both increased the spike frequency and input/output gain in response to current injection. Calcium current modulation by 5-HT1A receptors likely is an important mechanism to fine tune the input/output gain of TMNs in response to small incoming synaptic inputs and accounts for some of the previously reported effects of 5-HT on TMN excitability during tonic and burst activity during oral-motor behavior.     

Respiratory interneurons of the lower cervical (C4-C5) cord: membrane potential changes during fictive coughing,vomiting, and swallowing in the decerebrate cat

The possible roles of interneurons in the C4-C5 cervical spinal cord in conveying central drives to phrenic motoneurons during different behaviour patterns were investigated using intracellular recordings in decerebrate, paralysed, artificially ventilated cats. Eleven cells were tentatively classified as respiratory interneurons since they: (i) could not be antidromically activated from the ipsilateral whole intrathoracic phrenic nerve, and (ii) exhibited large membrane potential changes during eupnea (7.3 mV±3.6, range 2–13.5 mV) or non-respiratory behaviour patterns. Six neurons depolarized in phase with phrenic discharge; four others depolarized during the expiratory phase; one neuron exhibited depolarization during the end of both expiration and inspiration. A variety of responses was observed during fictive coughing, vomiting, and swallowing. The results are consistent with C4-C5 expiratory interneurons conveying inhibition to phrenic motoneurons during different behaviour patterns. The responses of inspiratory and multiphasic neurons suggest that the roles of these interneurons are mode complex than simply relaying central excitatory or inhibitory drive to phrenic motoneurons.     

Swimming in Aplysia brasiliana: behavioral and cellular effects of serotonin

1. Aplysia brasiliana is a marine mollusk that swims by repeated metachronal flapping movements of its bilateral fleshy parapodia. Animals with bilateral cerebropedal connective (CPC) lesions do not swim when suspended above the substrate, although tonic CPC stimulation can elicit normal parapodial flapping. Although the parapodial opener-phase (POP) cells, a previously identified group of neurons, fire synchronous bursts of efferent spikes in-phase with parapodial opening movements in both intact animals and dissected preparations, they are not likely to be primary parapodial motoneurons. These cells receive one or more large, apparently monosynaptic excitatory postsynaptic potentials (EPSPs) during CPC stimulation that are effective in producing the swimming motor program (SMP). 2. In suspended CPC-lesioned animals, injections of serotonin (5-HT) that produce an average hemolymph concentration of 10(-5) M induced full-amplitude parapodial flapping. Selected episodes of flapping were similar in frequency to normal suspended swimming. 3. In suspended CPC-lesioned animals, 5-HT injections elicited an apparently normal swimming motor program that was associated with synchronous bursts of large-amplitude efferent spikes in the parapodial nerves. In many semi-intact preparations, exposing the circumoesophageal ganglia to 5-HT elicited a similar rhythmic motor program, but usually at a lower frequency than during normal swimming or during tonic CPC stimulation. 4. In isolated-ganglion preparations, bath application of 5-HT produced immediate depolarization and tonic firing of individual POP neurons, followed by smooth and regular bursting in the apparent absence of synaptic input. In such preparations, the motor program elicited by bath-applied 5-HT differed from the one elicited by tonic CPC stimulation in that the 5-HT-elicited rhythmic bursting usually was not synchronous in different POP neurons. Tonic CPC stimulation during bath applications of 5-HT produced immediate synchronization of bursts among the POP neurons. 5. Hyperpolarization (or depolarization) of a POP neuron during bath application of 5-HT increased (or decreased) the burst period, but membrane polarization did not change the burst period elicited during tonic CPC stimulation.     

Persistent sodium current,membrane properties and bursting behavior of pre-bötzinger complex inspiratory neurons in vitro

We measured persistent Na(+) current and membrane properties of bursting-pacemaker and nonbursting inspiratory neurons of the neonatal rat pre-B?tzinger complex (pre-B?tC) in brain stem slice preparations with a rhythmically active respiratory network in vitro. In whole-cell recordings, slow voltage ramps ( g(NaP)/g(Leak) in nonpacemaker cells (P < 0.0002). We conclude that I(NaP) is ubiquitously expressed by pre-B?tC inspiratory neurons and that bursting pacemaker behavior within the heterogeneous population of inspiratory neurons is achieved with specific ratios of these two conductances, g(NaP) and g(Leak).