Spinal connections of ventral-group bulbospinal inspiratory neurons studied with cross-correlation in the decerebrate rat

We examined the synaptic connections from ventral-group bulbospinal inspiratory neurons to upper cervical inspiratory neurons and phrenic and intercostal motoneurons in decerebrate rats using cross-correlation. Inspiratory neurons were recorded in the medulla (n=28) at the level of the obex and from the upper-cervical segments (C1 and C2) of the spinal cord (n=29) in 18 vagotomized, paralyzed, ventilated, and decerebrated rats. The neurons were identified by their inspiratory firing pattern and antidromic activation from the spinal cord at C7. Whole-nerve recordings were made using bipolar electrodes from the central cut ends of the C5 phrenic nerve and the external and internal intercostal nerves at various thoracic levels. Cross-correlation histograms were computed between these recordings to detect short time scale synchronizations indicative of synaptic connections. Cross-correlation histograms (n=20), computed between the activities of ventral-group bulbospinal inspiratory neurons and the phrenic nerve, all showed peaks (mean half-amplitude width±SD, 1.1±0.3 ms) at short latencies (mean latency±SD, 2.0±0.6 ms) suggestive of monosynaptic excitation. Cross-correlation histograms (n=33), computed between the activities of ventral-group bulbospinal inspiratory neurons and upper-cervical inspiratory neurons, displayed four (12%) peaks (mean halfamplitude width±SD, 0.9±0.1 ms) at short latencies(mean latency±SD, 1.8±0.6 ms) suggestive of monosynaptic excitation, and six (18%) peaks (mean half-amplitude width±SD, 1.4±0.4 ms) at latencies near zero suggestive of excitation fro m a common source. Cross-correlation histograms (n=34), computed between the activities of ventral-group bulbospinal inspiratory neurons and the internal and external intercostal nerves at various thoracic levels (T2-8), showed six (18%) peaks (mean half-amplitude width±SD, 2.5±0.5 ms) at short latency (mean latency±SD, 4.5±1.1 ms) suggestive of oligosynaptic connections. Cross-correlation histograms (n=42) computed between activities of intercostal nerves at various levels of the thoracic spinal cord showed central peaks suggestive of excitation from a common source. Although the size of the peaks decreased with segmental separation, the displacement of the peaks from time zero did not increase with segmental separation (mean displacement±SD, 0.6±0.6 ms) as would be expected if the common excitation resulted from a descending monosynaptic excitation by a source such as the ventral-groupbulbospinal inspiratory neurons. We conclude that all ventral-group bulbospinal inspiratory neurons make monosynaptic connections to phrenic motoneurons, a few make monosynaptic connections to upper-cervical inspiratory neurons, but connections to intercostal motoneurons are made via interneurons.     

Connections from upper cervical inspiratory neurons to phrenic and intercostal motoneurons studied with cross-correlation in the decerebrate rat

We examined the synaptic connections from upper cervical inspiratory neurons to phrenic and intercostal motoneurons in decerebrate rats using cross-correlation. Upper cervical inspiratory neurons (n=79) were recorded from the C1 and C2 segments of the spinal cord in 38 vagotomized, paralyzed, ventilated, and decerebrate rats. The neurons were identified by their inspiratory firing pattern and antidromic activation from the ipsilateral spinal cord at C7. Whole-nerve recordings were made using bipolar electrodes from the central cut ends of the C5 phrenic nerve and the external and internal intercostal nerves at various thoracic levels. Cross-correlation histograms were computed between these recordings to detect short time-scale synchronizations indicative of synaptic connections. The 55 cross-correlation histograms computed between the upper cervical inspiratory neurons and the ipsilateral phrenic nerve showed seven (13%) narrow peaks (mean half-amplitude width±SD, 1.09±0.15 ms) at short latencies (mean latency±SD, 1.29±0.26 ms) suggestive of monosynaptic excitation, and four (7%) broader peaks (mean half-amplitude width±SD, 1.50±0.17 ms) at short latencies (mean latency±SD, 1.40±0.24 ms) suggestive of oligosynaptic excitation. Another 14 (25%) cross-correlation histograms displayed a central broad peak (mean half-amplitude width±SD, 1.59±0.23 ms) suggestive of common activation. The eight cross-correlation histograms computed between the upper cervical inspiratory neurons and the contralateral phrenic nerve were featureless. The 77 cross-correlation histograms computed between the upper cervical inspiratory neurons and the internal and external intercostal nerves at various thoracic levels (T2–8) showed no peaks suggestive of synaptic connections. We conclude that some upper cervical inspiratory neurons make monosynaptic and paucisynaptic connections to phrenic motoneurons but not to intercostal motoneurons.     

Extensive monosynaptic inhibition of ventral respiratory group neurons by augmenting neurons in the Bötzinger complex in the cat

Summary Axonal projections and synaptic connectivity of expiratory B?tzinger neurons with an augmenting firing pattern (Bot-Aug neurons) to neurons in the ipsilateral ventral respiratory group (VRG) were studied in anaesthetized cats. Antidromic mapping revealed extensive axonal arborizations of Bot-Aug neurons (24 of 45) to the rostral or caudal VRG, with some having arbors in both regions. Of 234 pairs of neurons studied with intracellular recording and spike-triggered averaging, monosynaptic inhibitory postsynaptic potentials (IPSPs) were evoked in 49/221 VRG neurons by 38/98 Bot-Aug neurons. The highest incidence of monosynaptic inhibition was found in inspiratory bulbospinal neurons (10 of 23 tested). Evidence was also found for monosynaptic inhibition, by a separate group of Bot-Aug neurons, of expiratory bulbospinal neurons (12/58), while excitatory postsynaptic potentials (EPSPs) were identified in another two of these neurons. In addition, monosynaptic IPSPs were recorded from 13 of 53 identified laryngeal motoneurons, and from 14 of 100 respiratory propriobulbar neurons. Presumptive disynaptic IPSPs were recorded from 11 of the 221 VRG neurons. We conclude that Bot-Aug neurons exert widespread inhibition on all major neuron categories in the ipsilateral VRG, and should be regarded as an important element in shaping the spatiotemporal output pattern of both respiratory motoneurons and premotor neurons.     

Monosynaptic excitation of medullary inspiratory neurons by bulbospinal inspiratory neurons of the ventral respiratory group in the cat

Summary In Nembutal-anesthetized, immobilized, and artificially ventilated cats, we studied the connectivity of medullary collaterals of bulbospinal inspiratory (BS-I) neurons in the ventral respiratory group (VRG). BS-I neurons which projected to the contralateral spinal cord were isolated extracellularly and intracellular recordings were made from the respiratory neurons in the contralateral VRG. The intracellular membrane potentials were averaged using extracellular spikes of the BS-I neurons as triggers (spike-triggered averaging method). Fast-rising and short-lasting depolarizing potentials locked to the triggering spikes were obtained and shown to be unitary EPSPs induced monosynaptically by the medullary collaterals of BS-I neurons. A total of 137 pairs were analyzed and unitary EPSPs were found in 11 pairs. Four BS-I neurons and 7 inspiratory vagal motoneurons received EPSPs from the medullary collaterals of BS-I neurons. These findings suggest that 1) BS-I neurons in the VRG drive medullary motoneurons of accessory respiratory muscles and phrenic or intercostal motoneurons simultaneously, 2) BS-I neurons on both sides synchronize via the excitatory connections, and 3) the augmenting firing pattern of BS-I neurons might partly be produced by this reexcitatory connection within the population of BS-I neurons.     

Diaphragmatic and external intercostal muscle control during vomiting: behavior of inspiratory bulbospinal neurons

1. The role of dorsal and ventral respiratory group (DRG and VRG) bulbospinal inspiratory (I) neurons in the control of diaphragmatic and external intercostal (inspiratory) muscle activity during vomiting was examined by recording from these neurons during fictive vomiting in decerebrate, paralyzed cats. Fictive vomiting was defined by a characteristic series of bursts of coactivation of phrenic and abdominal muscle nerves, elicited either by electrical stimulation of abdominal vagal afferents or by emetic drugs, which would be expected to produce vomiting if the animals were not paralyzed. 2. Data were recorded from 22 DRG and 29 VRG I neurons that were antidromically activated from the fourth cervical spinal segment (C4). Only 10% (5/51) of these neurons started to fire near the beginning of phrenic discharge during fictive vomiting and thus had the appropriate discharge pattern to contribute to the initial activation of the diaphragm and coactive external intercostal muscles during vomiting. The frequency of occurrence of these Active neurons was not significantly different in the DRG (3/22) and VRG (2/29) (chi 2 test). Most remaining neurons were either totally silent (n = 7) or had only sporadic, infrequent firing (n = 16) (Silent neurons, 23/51 = 45%), or else fired near the end of phrenic discharge during fictive vomiting (End neurons, 21/51 = 41%). Two neurons were categorized as having miscellaneous (Misc) behavior. 3. No differences were found among neurons having different response patterns during fictive vomiting in regard to the following: the manner in which fictive vomiting was elicited: cell location: conduction velocity; and neuronal firing onset, rate, and pattern during respiration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inputs to intercostal motoneurons from ventrolateral medullary respiratory neurons in the cat

The investigation examined the synaptic input from medullary respiratory neurons in the nucleus retroambigualis (NRA) to external (EIM) and internal (IIM) intercostal motoneurons. Antidromic mapping revealed that 112/117 (96%) tested NRA units had axons descending into thoracic spinal cord with extensive arborizations at many thoracic segments, mainly contralaterally. The conduction velocities ranged from 10 to 105 m X s-1. The descending projections did not appear to be somatotopically arranged. Cross-correlation of the spike trains of NRA inspiratory units with the discharge of external intercostal nerves (performed usually with 4 contralateral nerves) showed significant narrow peaks only in 5 out of 40 averages. Of the 25 trigger units tested for the thoracic projection in this series of experiments, 24 were antidromically activated. Intracellular recordings were made from 52 IIMs [mean membrane potential 65.3 mV, central respiratory drive potentials (CRDPs) greater than 1 mV present in 23/52] and 53 EIM (mean membrane potential 54.3 mV, CRDPs in 31/53). During the depolarizing phase of the CRDPs, synaptic noise with frequent and apparently unitary EPSPs with amplitudes in excess of 1 mV was observed. Spike-triggered averages of synaptic noise were computed for 153 pairings between 137 NRA neurons and 105 contralateral intercostal motoneurons. Only four PSPs were revealed: two monosynaptic EPSPs between expiratory NRA units and IIMs and two probably disynaptic EPSPs between inspiratory NRA units and EIMs. When advancing the microelectrode down to the motoneuron pools, frequent recordings were made from interneurons with spontaneous respiratory discharge (inspiratory or expiratory) located dorsal and medial to the motor nuclei. The interneurons could be excited following stimulation of segmental afferents. It is concluded that monosynaptic connections between respiratory NRA neurons and intercostal motoneurons are rare (connectivity no more than approximately 4%). Segmental interneurons, interposed between the majority of descending respiratory axons and intercostal motoneurons, are likely to produce large unitary EPSPs and, thus, short-term synchronization in the discharge of intercostal motoneurons as observed by others.     

Role of upper cervical inspiratory neurons studied by cross-correlation in the cat

Summary Axonal projections and synaptic connectivity of upper cervical inspiratory neurons (UCINs) were investigated in anaesthetised cats to clarify their role as propriospinal respiratory interneurons. Antidromic mapping showed axonal collaterals near phrenic and intercostal motonuclei. Of the UCINs tested, 37% had collaterals at T3-4; 55% had ipsilateral projections and 45% had contralateral projections. Ipsilateral or contralateral cross-correlations of the activity of pairs of UCINs (one on each side of the spinal cord) with the discharge of internal intercostal, external intercostal (T3-4) or phrenic nerves revealed similar features. Those with the internal intercostal and phrenic nerves were interpreted as evidence for shared or oligosynaptic excitation, those with the external intercostal nerve as shared excitation and inhibition. No evidence for monosynaptic connections was found. Monosynaptic connections could also not be demonstrated between inspiratory intercostal neurons located near (< 0.5 mm) the UCINs collateral arborizations in T3-4, examined by cross-correlation. Afferent feedback from internal intercostal nerves (T3-4) was investigated by cross-correlating nerve stimulation with UCINs activity. Ipsilateral and contralateral cross-correlograms had similar features, providing evidence for excitation in some cases and inhibition in others. Finally, cross-correlations between ipsilateral UCINs and cervical sympathetic nerves were featureless. The results suggest that the role of UCINs as part of a respiratory propriospinal control system analagous to forelimb motor control is untenable, although they may be part of an intercostal afferent feedback loop.     

An electrophysiological investigation of propriospinal inspiratory neurons in the upper cervical cord of the cat

Summary This study was performed in order to describe the location, axonal projection and possible synaptic action of the inspiratory neurons recently described in the upper cervical cord. In 26 cats anaesthetized with Nembutal, extracellular recordings were made from 224 cervical inspiratory units which were found near the lateral border of lamina VII and formed a column extending from the caudal end of the nucleus retroambigualis at the C₁ segment to the rostral half of the C₃ segment. Most of the units (approximately 85%) could be excited antidromically from the thoracic cord. Antidromic mapping showed collateral branches to the C₅ segment in the vicinity of the phrenic nucleus, occasionally crossing the midline. No synaptic connections with phrenic motoneurones could be revealed either by cross-correlation of the activity of the cervical units with the discharge of C₅ phrenic root, or by spike-triggered averaging (STA) of the post-synaptic noise recorded intracellularly from phrenic motoneurons. Extensive branching was found in the examined T₃–T₅ segments with arborizations near the ipsilateral intercostal motor nuclei and often extending across the midline. Cross-correlation experiments did not show clear monosynaptic connections to the inspiratory intercostal motoneurons. Intracellular recording from intercostal motoneurons and STA resulted in a few (2 out of 37) small, probably disynaptic, e.p.s.p.s. It is concluded that the upper cervical neurons are involved in the control of phrenic and intercostal motoneurons, probably through a disynaptic pathway involving segmental interneurons.     

Effects of graded focal cold block in the solitary and para-ambigual regions of the medulla in the cat

Unilateral focal cold blocks in the region of the nucleus tractus solitarius and the dorsal respiratory group of neurons, DRG, of anaesthetized cats consistently caused apneustic-type breathing. There was no concomitant change in the initial rate of rise of inspiratory activity. The apneustic prolongation of inspiratory duration, TI, was most pronounced in, but was not confined to, the DRG. The apneustic effects were more marked after vagotomy. In cats with intact vagus nerves being given artificial ventilation, focal cooling at certain sites of the DRG region could produce 'unlocking' of the respiratory rhythm from that of the respiratory pump. At other sites in this region, focal cooling could selectively block the effects of the inspiration-facilitating reflex induced by deflation without blocking the inspiration-inhibiting Hering-Breuer reflex. Unilateral focal cold blocks in the region of the intermediate part of the ventral respiratory group of neurons, VRG, generally caused depression of the rate of rise of inspiratory activity, but almost never apneustic effects. All effects of unilateral focal cooling both in the DRG and VRG were bilaterally symmetrical. No systematic differences between the effects on phrenic and external intercostal inspiratory activity were found in response to focal cooling either of the DRG or VRG suggesting that differential control of phrenic and external intercostal motoneurons is not exerted mainly at the level of these medullary structures. The results suggest that the DRG and VRG areas exert somewhat different effects on the respiratory pattern: DRG appears to be more concerned with integration of vagal and other inputs contributing to the inspiratory off-switch mechanisms which, however, are not confined only to the DRG. The VRG inspiratory mechanisms, on the other hand, appear to be more involved in the gain control of the inspiratory output intensity.     

Neural organization of the pathways from the superior colliculus to trochlear motoneurons

The neural organization of the pathways from the superior colliculus (SC) to trochlear motoneurons was analyzed in anesthetized cats using intracellular recording and transneuronal labeling techniques. Stimulation of the ipsilateral or contralateral SC evoked excitation and inhibition in trochlear motoneurons with latencies of 1.1-2.3 and 1.1-3.8 ms, respectively, suggesting that the earliest components of excitation and inhibition were disynaptic. A midline section between the two SCs revealed that ipsi- and contralateral SC stimulation evoked disynaptic excitation and inhibition in trochlear motoneurons, respectively. Premotor neurons labeled transneuronally after application of wheat germ agglutinin-conjugated horseradish peroxidase into the trochlear nerve were mainly distributed ipsilaterally in the Forel's field H (FFH) and bilaterally in the interstitial nucleus of Cajal (INC). Consequently, we investigated these two likely intermediaries between the SC and trochlear nucleus electrophysiologically. Stimulation of the FFH evoked ipsilateral mono- and disynaptic excitation and contralateral disynaptic inhibition in trochlear motoneurons. Preconditioning stimulation of the ipsilateral SC facilitated FFH-evoked monosynaptic excitation. Stimulation of the INC evoked ipsilateral monosynaptic excitation and inhibition, and contralateral monosynaptic inhibition in trochlear motoneurons. Preconditioning stimulation of the contralateral SC facilitated contralateral INC-evoked monosynaptic inhibition. These results revealed a reciprocal input pattern from the SCs to vertical ocular motoneurons in the saccadic system; trochlear motoneurons received disynaptic excitation from the ipsilateral SC via ipsilateral FFH neurons and disynaptic inhibition from the contralateral SC via contralateral INC neurons. These inhibitory INC neurons were considered to be a counterpart of inhibitory burst neurons in the horizontal saccadic system.     

Control of abdominal muscles by brain stem respiratory neurons in the cat

Control of abdominal musculature by brain stem respiratory neurons was studied in decerebrate unanesthetized cats by determining 1) which brain stem respiratory neurons could be antidromically activated from the lumbar cord, from which the abdominal muscles receive part of their innervation, and 2) if lumbar-projecting respiratory neurons make monosynaptic connections with abdominal motoneurons. A total of 462 respiratory neurons, located between caudal C2 and the retrofacial nucleus (B?tzinger complex), were tested for antidromic activation from the upper lumbar cord. Fifty-eight percent of expiratory (E) neurons (70/121) in the caudal ventral respiratory group (VRG) between the obex and rostral C1 were antidromically activated from contralateral L1. Eight of these neurons were activated at low thresholds from lamina VIII and IX in the L1-2 gray matter. One-third (14/41) of the E neurons that projected to L1 could also be activated from L4-5. Almost all antidromic E neurons had an augmenting firing pattern. Ten scattered inspiratory (I) neurons projected to L1 but could not be activated from L4-5. No neurons that fired during both E and I phases (phase-spanning neurons) were antidromically activated from the lumbar cord. In order to test for possible monosynaptic connections between descending E neurons and abdominal motoneurons, cross-correlations were obtained between 27 VRG E neurons, which were antidromically activated from caudal L2 and contralateral L1 and L2 abdominal nerve activity (47 neuron-nerve combinations). Only two neurons showed a correlation with one of the two nerves tested. Although there is a large projection to the lumbar cord from expiratory neurons in the ventral respiratory group caudal to the obex, cross-correlation analyses suggest that strong monosynaptic connections between these neurons and abdominal motoneurons are scarce.     

Identification of NO/sGC signalling in the bulbospinal respiratory pathway after C2-C3 hemisection of the spinal cord in rats

Guanylyl cyclase (GC) as the effector molecule for nitric oxide (NO) plays a key role in the NO/cGMP signalling cascade. Based on these observations, our study focused on NO/sGC signalization in the bulbospinal respiratory pathway. The distribution of neuronal nitric oxide synthase (nNOS), β1 subunit of soluble guanylyl cyclase (β1sGC) and synaptophysin (SYN) was explored in the upper part of the respiratory pathway after C2-C3 hemisection of the spinal cord in male Wistar rats. Unilateral injection of Fluorogold into the phrenic nucleus (PN) at C4 level and survival of animals for 2 days revealed many Fluorogold fluorescent neurons in the ventral respiratory group (VRG) of the medulla, mostly on the contralateral side. Under physiological conditions we noted nNOS-fluorescent terminals of VRG neurons around β1sGC fluorescent motoneurons in the PN. A strong depletion of nNOS/SYN fluorescent terminals was noted 8 days after hemisection around alpha motoneurons in the PN on the contralateral side. On the side of injury, nNOS/SYN fluorescent puncta were detected around phrenic motoneurons only sporadically. Phrenic alpha motoneurons responded to C2-C3 hemisection by a loss of β1sGC positivity. The results confirm, that β1sGC immunoreactive phrenic motoneurons are innervated by nNOS positive terminals coming from the VRG.     

Excitation and inhibition of medullary inspiratory neurons by two types of burst inspiratory neurons in the cat

In Nembutal-anesthetized and artificially ventilated cats, we studied the connectivity of burst inspiratory (I) neurons in the B?tzinger complex and the ventral respiratory group (VRG) with spike-triggered averaging methods. Burst I neurons exhibited tonic (I-TON) or decrementing (I-DEC) firing patterns. Spikes of I-TON neurons induced monosynaptic EPSPs in intracellularly recorded I neurons of both the VRG and the dorsal respiratory group (DRG). Spikes of I-DEC neurons induced monosynaptic inhibitory postsynaptic potentials (IPSPs) in both VRG and DRG I neurons.     

Chemical activation of caudal medullary expiratory neurones alters the pattern of breathing in the cat.

1. The purpose of this work was to ascertain whether the activation of caudal expiratory neurones located in the caudal part of the ventral respiratory group (VRG) may affect the pattern of breathing via medullary axon collaterals. 2. We used microinjections of DL-homocysteic acid (DLH) to activate this population of neurones in pentobarbitone-anaesthetized, vagotomized, paralysed and artificially ventilated cats. Both phrenic and abdominal nerve activities were monitored; extracellular recordings from medullary and upper cervical cord respiratory neurones were performed. 3. DLH (160 mM) microinjected (10-30 nl for a total of 1.6-4.8 nmol) into the caudal VRG, into sites where expiratory activity was encountered, provoked an intense and sustained activation of the expiratory motor output associated with a corresponding period of silence in phrenic nerve activity. During the progressive decline of the activation of abdominal motoneurones, rhythmic inspiratory activity resumed, displaying a decrease in frequency and a marked reduction or the complete suppression of postinspiratory activity as its most consistent features. 4. Medullary and upper cervical cord inspiratory neurones exhibited inhibitory responses consistent with those observed in phrenic nerve activity, while expiratory neurones in the caudal VRG on the side contralateral to the injection showed excitation patterns similar to those of abdominal motoneurones. On the other hand, in correspondence to expiratory motor output activation, expiratory neurones of the Bötzinger complex displayed tonic discharges whose intensity was markedly lower than the peak level of control breaths. 5. Bilateral lignocaine blockades of neural transmission at C2-C3 affecting the expiratory and, to a varying extent, the inspiratory bulbospinal pathways as well as spinal cord transections at C2-C3 or C1-C2, did not suppress the inhibitory effect on inspiratory neurones of either the ipsi- or contralateral VRG in response to DLH microinjections into the caudal VRG. 6. The results show that neurones within the column of caudal VRG expiratory neurones promote inhibitory effects on phrenic nerve activity and resetting of the respiratory rhythm. We suggest that these effects are mediated by medullary bulbospinal expiratory neurones, which may, therefore, have a function in the control of breathing through medullary axon collaterals.     

Synchronization of ventral-group, bulbospinal inspiratory neurons in the decerebrate rat

We examined the synaptic connections between ventral-group, bulbospinal inspiratory neurons in 27 vagotomized, paralyzed, ventilated, and decerebrated rats using cross-correlation and spike-triggered averaging of intracellular potentials. The neurons were recorded in the medulla about the level of the obex and identified by their inspiratory firing pattern and antidromic activation from the spinal cord at C7. Whole C5 phrenic nerve recordings were made using bipolar electrodes from the central cut ends of the nerve. Most (108/137, 79%) inspiratory neurons discharged only during inspiration but some (29/137, 21%) also discharged during early expiration. Their intracellular membrane potentials displayed a pattern of depolarization during inspiration, repolarization during early expiration, and hyperpolarization during late expiration. Intracellular chloride iontophoresis changed the inspiratory membrane potential trajectories from augmenting to decrementing in 11 of 19 neurons tested (58%), and demonstrated the presence of both early-decrementing and late-augmenting waves of inhibitory postsynaptic potentials during expiration in 11 of 19 neurons tested (58%). Cross-correlation histograms were computed between pairs of extracellularly recorded neurons to detect short time scale synchronizations indicative of synaptic connections (26 ipsilateral; 23 contralateral). While none of the cross-correlation histograms for contralateral pairs showed peaks, most (23, 88%) of those for ipsilateral pairs showed peaks (mean half-amplitude width ± SD = 1.3 ± 0.4 ms) at time zero suggestive of common activation. Some of the latter (6, 23%) showed troughs superimposed on the central peaks (mean half-amplitude width ± SD = 0.9 ± 0.2 ms) at short latencies (mean latency ± SD = 1.8 ± 1.9 ms) suggestive of inhibition; others (8, 31%) had asymmetrical central peaks and two had bilateral peaks suggesting more complex interconnections. Averages of intracellular membrane potentials of inspiratory neurons (n = 24), triggered by action potentials of a nearby extracellularly recorded inspiratory neuron, were computed to detect synchronized postsynaptic potentials. Over half (16, 67%) showed postsynaptic potentials (mean amplitude ± SD = 201 ± 176 μV; mean half-amplitude width ± SD = 2.3 ± 0.8 ms) confirming the cross-correlation findings of common excitation. We conclude that in decerebrated rats, ventral-group inspiratory neurons projecting to the C7 spinal segment share powerful, ipsilaterally distributed excitatory inputs which enhance their synchronous activity during inspiration. They also receive inhibition during inspiration and early-decrementing and late-augmenting inhibitory inputs during expiration. Received: 25 October 1996 / Accepted: 21 March 1997     

Multimodal medullary neurons and correlational linkages of the respiratory network.

This study addresses the hypothesis that multiple sensory systems, each capable of reflexly altering breathing, jointly influence neurons of the brain stem respiratory network. Carotid chemoreceptors, baroreceptors, and foot pad nociceptors were stimulated sequentially in 33 Dial-urethan-anesthetized or decerebrate vagotomized adult cats. Neuronal impulses were monitored with microelectrode arrays in the rostral and caudal ventral respiratory group (VRG), nucleus tractus solitarius (NTS), and n. raphe obscurus. Efferent phrenic nerve activity was recorded. Spike trains of 889 neurons were analyzed with cycle-triggered histograms and tested for respiratory-modulated firing rates. Responses to stimulus protocols were assessed with peristimulus time and cumulative sum histograms. Cross-correlation analysis was used to test for nonrandom temporal relationships between spike trains. Spike-triggered averages of efferent phrenic activity and antidromic stimulation methods provided evidence for functional associations of bulbar neurons with phrenic motoneurons. Spike train cross-correlograms were calculated for 6,471 pairs of neurons. Significant correlogram features were detected for 425 pairs, including 189 primary central peaks or troughs, 156 offset peaks or troughs, and 80 pairs with multiple peaks and troughs. The results provide evidence that correlational medullary assemblies include neurons with overlapping memberships in groups responsive to different sets of sensory modalities. The data suggest and support several hypotheses concerning cooperative relationships that modulate the respiratory motor pattern. 1) Neurons responsive to a single tested modality promote or limit changes in firing rate of multimodal target neurons. 2) Multimodal neurons contribute to changes in firing rate of neurons responsive to a single tested modality. 3) Multimodal neurons may promote responses during stimulation of one modality and "limit" changes in firing rates during stimulation of another sensory modality. 4) Caudal VRG inspiratory neurons have inhibitory connections that provide negative feedback regulation of inspiratory drive and phase duration.     

The output of the hippocampus is inhibited during social behavior in the male rat

We tested the role of C5 segment inspiratory interneurons in transcribing central respiratory drive to phrenic motoneurons and mediating intersegmental reflexes by cross-correlating the spontaneous activity of 26 interneurons with that of the ipsi -and contralateral C5 phrenic nerves in decerebrate cats. There were 10 interneurons that discharged only during inspiration (phrenic burst) and 16 that discharged tonically with increased firing during inspiration. Of the cross-correlograms for 26 of the interneurons with the ipsilateral phrenic, 20 were flat and 2 had peaks centred about time zero, interpreted as a common activation of the interneurons and motoneurons. The cross-correlograms for 4 other interneurons had troughs centred about time zero, interpreted as a synchronous excitation of the interneurons and inhibition of the motoneurons. Of the cross-correlograms for 23 interneurons with the contralateral phrenic, 22 were flat and 1 had a peak centred about time zero, interpreted as a common activation of the interneuron and motoneurons. Nine of ten cross-correlograms between pairs of interneurons were flat; one had a peak centred about time zero. We conclude that, despite their inspiratory modulated discharge patterns, there is no evidence from this study that the C5 segment inspiratory interneurons convey central respiratory drive to C5 phrenic motoneurons.     

Influence of rubrospinal tract and the adjacent mesencephalic reticular formation on the activity of medullary respiratory neurons and the phrenic nerve discharge in the rabbit

Suprapontine brain sites acting on the central respiratory system have been demonstrated to give rise to inspiratory as well as expiratory facilitatory effects. In the present study the inspiratory inhibitory effect which has been reported in the cat to be elicited consistently by electrical stimulation of the rubrospinal tract and the adjacent mesencephalic reticular formation was examined in the urethane-anaesthetized rabbit. Stimulation of these sites with single electrical shocks of moderate intensity induced a short latency (onset after 3.0 ms) transient (duration: 29 ms) inhibition of the phrenic nerve activity (PHR). Short volleys of stimuli applied in mid- to late-inspiration led to a premature off-switch of inspiration. The extracellularly recorded discharge activity of the different types of medullary respiration-related units (RRU) reflected these alterations, accordingly. Axonal connections of RRU with mesencephalic structures were evaluated. Examination of orthodromic responses of medullary RRU to stimulation of this pathway revealed that most bulbospinal inspiratory neurons (10 out of 13) were paucisynaptically inhibited after short latency (at least 1.2 ms). The conduction time from bulbospinal inspiratory neurons to the recording site of PHR was 1.6 ms. Thus, a disynaptic pathway — including bulbospinal inspiratory neurons — is suggested inducing inspiratory inhibition 3.0 ms after single shock midbrain stimulation. This inhibition results in disfacilitation of phrenic motoneurons. The fact that extensive electrolytic lesions of the pneumotaxic center in rostral pons did not abolish the observed inspiratory inhibitions excludes these structures from being involved. A direct pathway from the red nucleus and the adjacent reticular formation to phrenic nuclei of the spinal cord, however, can not be excluded from being involved in the demonstrated inspiratory inhibition. The described effects may play a role in behavioral or voluntary control of respiration.     

Respiratory pre-motor control of hypoglossal motoneurons in the rat

The goal of this study was to determine the origin and transmission pathway of respiratory drive to hypoglossal motoneurons. First we recorded intracellularly from 28 antidromically activated inspiratory hypoglossal motoneurons (resting membrane potential, −50±3 mV), and found that injection of chloride ions had no discernible effect on the shape of their membrane potential trajectories. We concluded that the membrane potential trajectories of these hypoglossal motoneurons were determined primarily by inspiratory excitation. To determine the origin of this excitation we cross-correlated the extracellular discharge of medullary inspiratory neurons, including those in the hypoglossal motor nucleus, with the hypoglossal nerve discharge. We found 27 inspiratory neurons within the hypoglossal motor nucleus that were not antidromically activated from the ipsilateral hypoglossal nerve; their cross-correlograms featured either central peaks (1.7±0.2 ms) alone (n=14; 39%), or central peaks (1.3±0.2 ms) followed by troughs (1.3±0.1 ms) at short latencies (1.1±0.4 ms) (n=13; 36%), and suggest that these neurons are hypoglossal interneurons. We recorded from 238 inspiratory neurons throughout the rest of the medulla; the cross-correlograms of 19 neurons (8%), located mostly in the lateral tegmental field, displayed narrow half-amplitude peaks (1.0±0.1 ms) at short latencies (0.9±0.1 ms), which we interpreted as evidence for monosynaptic excitation of hypoglossal motoneurons.

We conclude that the respiratory control of hypoglossal motoneurons originates from inspiratory premotor neurons scattered throughout the lateral tegmental field and interneurons within the hypoglossal motor nucleus.     

Presynaptic delta opioid receptors differentially modulate rhythm and pattern generation in the ventral respiratory group of the rat

The specific role of the Delta opioid receptor (DOR), in opioid-induced respiratory depression in the ventral respiratory group (VRG) is largely unknown. Here, we sought to determine (1) the relationship between DOR-immunoreactive (ir) boutons, bulbospinal and functionally identified respiratory neurons in the VRG and (2) the effects of microinjection of the selective DOR agonist, D-Pen 2,5-enkephalin (DPDPE), into different subdivisions of the VRG, on phrenic nerve discharge and mean arterial pressure. Following injections of retrograde tracer into the spinal cord or intracellular labelling of respiratory neurons, in Sprague-Dawley rats, brainstem sections were processed for retrograde or intracellular labelling and DOR-ir. Bulbospinal neurons were apposed by DOR-ir boutons regardless of whether they projected to single (cervical or thoracic ventral horn) or multiple (cervical and thoracic ventral horn) targets in the spinal cord. In the VRG, a total of 24 +/- 5% (67 +/- 13/223 +/- 49) of neurons projecting to the cervical ventral horn, and 37 +/- 3% (96 +/- 22/255 +/- 37) of neurons projecting to the thoracic ventral horn, received close appositions from DOR-ir boutons. Furthermore, DOR-ir boutons closely apposed six of seven intracellularly labelled neurons, whilst the remaining neuron itself possessed boutons that were DOR-ir. DPDPE was microinjected (10 mM, 60 nl, unilateral) into regions of respiratory field activity in the VRG of anaesthetised, vagotomised rats, and the effects on phrenic nerve discharge and mean arterial pressure were recorded. DPDPE depressed phrenic nerve amplitude, with little effect on phrenic nerve frequency in the B?tzinger complex, pre-B?tzinger complex and rVRG, the greatest effects occurring in the B?tzinger complex. The results indicate that the DOR is located on afferent inputs to respiratory neurons in the VRG. Activation of the DOR in the VRG is likely to inhibit the release of neurotransmitters from afferent inputs that modulate the pattern of activity of VRG neurons.