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Excitation and inhibition of medullary inspiratory neurons by two types of burst inspiratory neurons in the cat
Neuroscience Letters, 104 (1989) 303-308
303
Elsevier Scientific Publishers Ireland Ltd. NSL 06335
Excitation and inhibition of medullary inspiratory neurons by two types of burst inspiratory neurons in the cat K a z u h i s a E z u r e l, M o t o m u M a n a b e 1 a n d K a z u y o s h i O t a k e 2 1Department ~f Neurobiology, Tokyo Metropolitan lnstitute Jor Neurosciences, Tokyo (Japan) and 2Department of Anatomy, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo (Japan)
Key words': Inspiratory neuron; Spike-triggered averaging; Excitatory postsynaptic potential; Inhibitory postsynaptic potential In Nembutal-anesthetized and artificially ventilated cats, we studied the connectivity of burst inspiratory (I) neurons in the B6tzinger 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.
There is a type of inspiratory (I) neuron which starts burst firing at the onset of the I phase of the respiratory cycle, quickly attains its peak firing frequency which then declines gradually, and stops firing prior to the end of the I phase. Such neurons are termed early-burst or decrementing (I-DEC) I neurons and are found in the ventral respiratory group (VRG) among other types of I neuron [2, 3, 11, 12], or in the caudal VRG, which contains predominantly expiratory neurons [1]. Similar I-DEC neurons are also found in the B6tzinger complex (BOT) [4, 7, 13]. Antidromic mapping tests have shown that I-DEC neurons are propriobulbar neurons and send their axons over wide areas in the VRG [4, 12] and the BOT [7]. In addition to these I-DEC neurons, we have noticed the existence of another type of burst I neuron whose activity was recorded in the same area as that of I-DEC neurons [7]. Their burst firing starts at the onset of the I phase and is fairly tonic throughout the I phase (I-TON neurons). Intracellular horseradish peroxidase (HRP) injection into I-TON neurons revealed extensive axonal branches with terminal boutons in the medulla [7]. However, we sometimes encountered burst I neurons whose firing patterns were inbetween and which were difficult to classify into either tonic or decrementing types. Correspondence." K. Ezure, Department of Neurobiology, Tokyo Metropolitan Institute for Neurosciences, Musashidai 2-6, Fuchu-shi, Tokyo 183, Japan. 0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
3O4 fherelk)re, we have had some uncertainty as to whether the I-TON and I-DEC neurons are really two difl'erent types or whether they represent a single type of burst I neurons. The present study was aimed at clarifying this point. Data were obtained from 22 adult cats, which were anesthetized with Nembutal (initially 40 mg/kg, i.p.; supplementary doses, i.v.). We only briefly describe the methods here, since almost all of the experimental procedures were the same as those described in previous papers [5, 6]. A stimulus electrode was attached to the cervical vagus nerve on one side, and two others were fixed bilaterally in the spinal cord between C4 and C5. Craniotomy was performed and the caudal part of the cerebellum was removed. During the recording session the animal was paralyzed by pancuronium bromide (Mioblock: Organon, 0.1 mg/kg/h, i.v.), and was artificially ventilated. A bilateral pneumothorax was performed and a positive end-expiration load of 1 2 c m H 2 0 was applied. Blood pressure in the femoral artery, rectal temperature, and end-tidal CO,, were monitored. Glass rnicropipettes filled with 3 M NaCI solution saturated with fast green FCF dye for extracellular recording and stimulation, and with 2 M potassium citrate for intracellular recording, were used. Activity was monitored from the C5 phrenic nerve. Brief microstimulation in the V R G and the dorsal respiratory group (DRG) areas was performed, before the spike-triggered averaging (STA) test, to see whether the trigger I neuron projected to the area of intracellular recordings. Intracellular recordings were made from I neurons in the contralateral VRG or D R G and membrane potentials of impaled neurons were averaged using extracellular spikes of the 1 neuron as triggers (for details, see refs. 5, 6). After the STA tests, we performed further microstimulation tests especially around the tracks of intracellular recordings. After fixation, serial frozen sections of the brainstem (100 ,urn thick in the frontal plane) were made. The stimulating tracks and points, as well as the site of recorded neurons, were reconstructed using the Fast green FCF marks. We studied a total of 26 burst ! neurons which showed either tonic (I-TON) (Fig. I AI) or decrementing (I-DEC) (Fig. 1B l) firing patterns; in 3 cases, however, their firing patterns were in-between (I-BET) and it was difficult to classify them into either I-DEC or I-TON neurons. Thus we sampled 5 I-TON, 18 I-DEC, and 3 I-BET neuroils. All of them were sampled in the vicinity of the retrofacial nucleus and in the vicinity of the nucleus ambiguus, i.e., the BOT and the VRG, respectively. None of the 26 I neurons except for 2 I-DEC could be antidromically activated from the spinal cord at C4 5- We made systematic antidromic stimulation tests in the medulla contralateral to the isolated burst I neurons, especially in the areas of the VRG and the D R G where I neurons were predominantly found. All of the 26 burst i neurons were antidromically activated from the V R G area. Eighteen of 22 tested were activated from the DR(] as well as the VRG area. In all of the 26 cases of VRG stimulation and m 15 of the 18 cases of D R G stimulation, antidromic latencies could be changed in a step fashion by slight movements of the stimulating electrode within single tracks or among nearby tracks, suggesting the existence of axonal branches. We found no apparent differences in antidromic activation among the I-TON, the I-BET, and the I-DEC neurons. Intracellular recordings were made from l neurons of the VRG (n = 80) and the
305
D R G (n=30). A total of 110 intracellular units whose membrane potentials were more negative than - 30 mV were analyzed; the VRG I neurons consisted of 35 bulbospinai neurons, 32 vagal motoneurons, and 13 propriobulbar (defined as non-bulbospinal and non-vagal) neurons; the D R G neurons consisted of 27 bulbospinal neurons and 3 propriobulbar neurons. Using this intracellular activity and the extracellular activity of the 26 burst I neurons, a total of 110 pairs was examined for their connectivity by the STA method. In 10 pairs, depolarizing potentials locked to the triggering spikes were revealed. Fig. 1A2 shows a depolarizing potential induced in a VRG I neuron with triggering by an I-TON neuron. The latency of this depolarization measured from the foot of the triggering spike was 1.5 ms and the time-to-peak was 1.0 ms. This depolarizing potential with a sharp and fast rising phase was presumably a monosynaptically induced EPSP, as described in previous studies [6, 9, 10, 14]. This was supported by the following observation. The triggering I-TON neuron could be antidromically activated from the area of intracellular recording, and the antidromic latency was 1.2 ms. If the latency for spike generation in the axon at the stimulated site was about 0.1 ms [8, 9], the orthodromic conduction time along the
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Fig. 1. Spike-triggered averaging was performed between a VRG 1 neuron and an I-TON neuron (AI, A2), and between a D R G I neuron and an I-DEC neuron (BI, B2). A1, BI: spike histogram (a) ofextracellularly recorded spikes (b); intracellular potential (c); rectified and integrated phrenic nerve activity (d). A2, B2: averaged triggering spike (a); averaged membrane potential (b); averaged juxtacellular potential (c). The I-TON and I-DEC neurons induced a monosynaptic EPSP (A2b) and an IPSP (B2b) respectively.
306 axon could be estimated to be 1.1 ms. Taking synaptic delay of about 0.4 ms into consideration [6, 8, 9, 14], the above results indicate that the connection was m o n o s y naptic. In 9 other cases, a similar test indicated that the observed depolarizing potentials were monosynaptic EPSPs induced by triggering spikes o f burst I neurons. The httencies o f 10 EPSPs ranged between I. I and 2.6 ms (mean _+ S.D.: 1.7_+ 0.4 ms); the times-to-peak ranged between 0.7 and 2.6 ms ( m e a n ± S . D . : 1,5_+0.4 ms). The 10 I neurons which received these excitatory postsynaptic potentials (EPSPs) consisted of 3 bulbospinal neurons (1 V R G , 2 D R G ) , 3 V R G vagal motoneurons, and 4 propriobulbar neurons (3 V R G , 1 D R G ) . Nine of the 10 I neurons exhibited increasing membrane depolarizations during the I phase: one ( V R G propriobulbar neuron) exhibited decrementing pattern of m e m b r a n e potential. The EPSPs in these 10 neurons were induced by 5 I - T O N and 2 I-BET neurons. Two I-TON neurons, including that shown in Fig. IA. induced EPSPs in both V R G and D R G I neurons. In 13 pairs, hyperpolarizing potentials locked to the triggering spikes were observed. Fig. I B2 shows a hyperpolarizing potential induced in a D R G I neuron with triggering by an I - D E C neuron. The latency o f this hyperpolarization was 1.7 ms and the time-to-peak was 1.6 ms. The latency o f antidromic activation o f the triggering I - D E C neuron from the D R G was 1.5 ms. Employing the same a r g u m e n t as above and taking synaptic delay into consideration, the hyperpolarization was suggested to be a m o n o s y n a p t i c IPSP induced by triggering I - D E C neuron. In fact, in 2 o f the 13 cases, hyperpolarizing potentials were reversed to depolarization by intracellular injection o f hyperpotarizing current, indicating that the hyperpolarizations were inhibitory postsynaptic potentials (IPSPs). The latencies o f the IPSPs ranged between 0.9 and 2.8 ms ( m e a n ± S . D . : 1.8±0.6 ms); the times-to-peak ranged
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/ Fig. 2. Distribution of burst I neurons is projected onto horizontal (A) and transverse (B) planes. There are 7 excitatory (open circles) and 7 inhibitory (closed circles) neurons. AMB: nucleus ambiguus; CX: external cuneate nucleus; DX: dorsal motor nucleus of the vagus; IO: inferior olivary nucleus: RFN: retrofacial nucleus; S: solitary tract; 5ST: spinal trigeminal tract: 7N: facial nucleus; 12N: hypoglossal nucleus.
307 between 1.0 and 2.2 ms (mean S.D.: 1.6+0.4 ms). These 13 IPSPs were induced by 9 I-DEC neurons. Two of the 9 I-DEC neurons induced IPSPs in both VRG and D R G neurons. The 13 I neurons which received IPSPs consisted o f 11 bulbospinal I neurons (8 VRG, 3 D R G ) and 2 V R G propriobulbar neurons. All of the 13 neurons exhibited augmenting firing patterns or increasing membrane depolarizations during the I phase; the augmentation in 7 neurons showed a tendency for late onset [I 5]. In Fig. 2, we show the anatomical distribution of 7 neurons which induced EPSPs and 7 of the 9 I-DEC neurons which induced IPSPs. As shown in the horizontal plane of Fig. 2A, all of them, except for one I-TON neuron, were distributed in the vicinity of the area from the caudal part of the retrofacial nucleus to the rostral part of the nucleus ambiguus. This area corresponds to the caudal part of the BOT and the rostralmost part of the VRG. In a transverse plane (Fig. 2B), they were distributed ventrolateral to the retrofacial nucleus and the nucleus ambiguus. Fig. 2B also reveals a tendency for the I-TON and I-BET neurons to be distributed more laterally than the I-DEC neurons. This study has shown clearly that the burst I neurons do not form a homogenous group, but consist of at least two types, i.e., excitatory and inhibitory neurons. Excitatory I neurons tend to exhibit tonic firing while inhibitory I neurons exhibit decrementing firing. I-TON neurons may be an important excitatory source for many of the medullary I neurons, judging from the fact that I-TON neurons excited many kinds of I neurons in both the V R G and the DRG: bulbospinal neurons, propriospinal neurons, and vagal motoneurons. This report is the first to identify a medullary origin of excitatory inputs to D R G I neurons. Merrill [12] suggested the possibility that I-DEC neurons excite each other, since electrical stimulation in the area of I-DEC neurons resulted in both antidromic and orthodromic activation of I-DEC neurons on the opposite side of the brainstem. We obtained a similar observation. However, the orthodromic activation might be induced by activating I-TON neurons which were excitatory and situated in the same area. There is the possibility of selfreexcitatory connections among I-TON neurons but not among I-DEC neurons, since 1-TON neurons have extensive axonal branches in the contralateral medulla as well as around their somata [7]. Richter's intracellular study [15] clearly demonstrated that some late onset I neurons, which form a subset of augmenting I neurons, receive inhibitory inputs during the early part of the I phase. He hypothesized that the source of the inhibition was I-DEC neurons, since the pattern of early I inhibition resembled the discharge pattern of I-DEC neurons which were already known to have extensive axonal arborizations in the ventrolateral medulla [12]. The present results agree with Richter's hypothesis. In addition, we observed inhibition not only in late onset I neurons, but also in early onset augmenting I neurons of the VRG and the D R G (see Fig. 1B1). Previously, we showed that augmenting I neurons of the V R G make self-reexcitatory connections [6] and suggested that these connections may contribute to the production of the augmenting firing, i.e., I-ramp pattern. Acting in concert with the self-reexcitatory connections between augmenting I neurons, the disinhibition of augmenting I neurons due to decrement of inhibitory action from I-DEC neurons provides a good explanation for how inspiratory augmentation is produced in the medulla.
308
We thank Drs. H. Shimazu and H. Robinson for their critical reviews of the manuscript, and Dr. Y. Kunita for her technical assistance. This work was partly supported by a Grant-in-Aid for Science Research (63570074) from the Japanese Ministry of Education, Science and Culture. 1 Arita, H., Kogo, N. and Koshiya, N., Morphological and physiological properties of caudal medullary expiratory neurons of the cat, Brain Res., 401 (1987) 258 266. 2 Bianchi. A.L.. Localisation et 6tude des neurones rcspiratoires bulbaires. Mise en jeu antidromiquc par stimulation spinal ou vagale, .1. Physiol. (Paris), 63 (1971) 5 40. 3 Bianchi, A.L. Modalitc}s de d~charge et propriOtgzs anatomofonctionelles des neurones respiratoires bulbaires, J. Phy~iol. (Paris), 68 (1974) 555 587. 4 Bianchi, A.L. and Barillot, J.('., Respiratory neurons in the region of the retrofacial nucleus: pontile, medullary, spinal and vagal projections, Neurosci. Lett., 31 (1982) 277 282. 5 Ezure, K. and Manabe, M., Decrementing expiratory neurons of the B6tzinger complex. II. Direct inhibitory synaptic linkage with ventral respiratory group neurons, Exp. Brain Res., 72 (1988) 159 166. 6 Enzure, K. and Manabe, M., Monosynaptic excitation of medullary inspiratory neurons by bulbospina~inspira~oryneuronsof~hcventralrespiratorygroupinthecat,Exp.BrainRes.~74(~989)501 511. 7 Ezure, K., Manabe, M., Otake, K., Sasaki, H. and Mannen, H., The axonal projection of two types of burst inspiratory neurons to the B6tzinger complex (BOT), Neurosci. Res., Suppl., 2 (1986) $79. 8 ftikosaka, O., lgusa, Y., Nakao, S. and Shimazu, H , Direct inhibitory synaptic linkage of pontomedullary reticular burst neurons with abducens motoneurons in the cal. Exp. Brain Res., 33 (I978) 337 352. 9 Jankowska, E. and Roberts, W.,1., Synaptic actions of single interneurones mediating reciprocal la inhibition of motoneurones, J. Physiol. (Lond.), 222 [I 972) 623 642. I0 Kirkwood, P.A. and Sears, T.A.. Monosynaptic excitation of thoracic expiratory motoneurons from lateral respiratory neurones in the medulla of the cal, J. Physiol. (Lond.), 234 (1973) 87p 89p. 11 Merrill, E.G., [nleractions between medullary respiratory neurones in cats, J. Physiol. (Lond.), 226 (1972) 72P 74P. 12 Merrill, E.G., Fmdmg a respiratory function for the medullary respiratory neurons. In R. Bellairs and E.(}. Gray (Eds.), Essays on the Nervous System, Clarendon, Oxford, 1974, pp. 451 486. 13 Merrill, E.G., Lipski, J.. Kubin. L. and Fedorko, L., Origin of the expiratory inhibition of nucleus Iractus solitarius inspiratory neurones, Brain Res., 263 (1983) 43 50. 14 Rapoport. S., Susswein, A., Uchino, Y. and Wilson. V.J., Synaptic actions of individual vestibular neurones on cat neck motoneurones, J. Physiol. {Lond.}, 272 (1977) 367 382. 15 Richter. I).W., Generation and maintenance of the respiratory rhythm, J. Exp. Biol., 100 (1982) 93 107.
303
Elsevier Scientific Publishers Ireland Ltd. NSL 06335
Excitation and inhibition of medullary inspiratory neurons by two types of burst inspiratory neurons in the cat K a z u h i s a E z u r e l, M o t o m u M a n a b e 1 a n d K a z u y o s h i O t a k e 2 1Department ~f Neurobiology, Tokyo Metropolitan lnstitute Jor Neurosciences, Tokyo (Japan) and 2Department of Anatomy, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo (Japan)
Key words': Inspiratory neuron; Spike-triggered averaging; Excitatory postsynaptic potential; Inhibitory postsynaptic potential In Nembutal-anesthetized and artificially ventilated cats, we studied the connectivity of burst inspiratory (I) neurons in the B6tzinger 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.
There is a type of inspiratory (I) neuron which starts burst firing at the onset of the I phase of the respiratory cycle, quickly attains its peak firing frequency which then declines gradually, and stops firing prior to the end of the I phase. Such neurons are termed early-burst or decrementing (I-DEC) I neurons and are found in the ventral respiratory group (VRG) among other types of I neuron [2, 3, 11, 12], or in the caudal VRG, which contains predominantly expiratory neurons [1]. Similar I-DEC neurons are also found in the B6tzinger complex (BOT) [4, 7, 13]. Antidromic mapping tests have shown that I-DEC neurons are propriobulbar neurons and send their axons over wide areas in the VRG [4, 12] and the BOT [7]. In addition to these I-DEC neurons, we have noticed the existence of another type of burst I neuron whose activity was recorded in the same area as that of I-DEC neurons [7]. Their burst firing starts at the onset of the I phase and is fairly tonic throughout the I phase (I-TON neurons). Intracellular horseradish peroxidase (HRP) injection into I-TON neurons revealed extensive axonal branches with terminal boutons in the medulla [7]. However, we sometimes encountered burst I neurons whose firing patterns were inbetween and which were difficult to classify into either tonic or decrementing types. Correspondence." K. Ezure, Department of Neurobiology, Tokyo Metropolitan Institute for Neurosciences, Musashidai 2-6, Fuchu-shi, Tokyo 183, Japan. 0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
3O4 fherelk)re, we have had some uncertainty as to whether the I-TON and I-DEC neurons are really two difl'erent types or whether they represent a single type of burst I neurons. The present study was aimed at clarifying this point. Data were obtained from 22 adult cats, which were anesthetized with Nembutal (initially 40 mg/kg, i.p.; supplementary doses, i.v.). We only briefly describe the methods here, since almost all of the experimental procedures were the same as those described in previous papers [5, 6]. A stimulus electrode was attached to the cervical vagus nerve on one side, and two others were fixed bilaterally in the spinal cord between C4 and C5. Craniotomy was performed and the caudal part of the cerebellum was removed. During the recording session the animal was paralyzed by pancuronium bromide (Mioblock: Organon, 0.1 mg/kg/h, i.v.), and was artificially ventilated. A bilateral pneumothorax was performed and a positive end-expiration load of 1 2 c m H 2 0 was applied. Blood pressure in the femoral artery, rectal temperature, and end-tidal CO,, were monitored. Glass rnicropipettes filled with 3 M NaCI solution saturated with fast green FCF dye for extracellular recording and stimulation, and with 2 M potassium citrate for intracellular recording, were used. Activity was monitored from the C5 phrenic nerve. Brief microstimulation in the V R G and the dorsal respiratory group (DRG) areas was performed, before the spike-triggered averaging (STA) test, to see whether the trigger I neuron projected to the area of intracellular recordings. Intracellular recordings were made from I neurons in the contralateral VRG or D R G and membrane potentials of impaled neurons were averaged using extracellular spikes of the 1 neuron as triggers (for details, see refs. 5, 6). After the STA tests, we performed further microstimulation tests especially around the tracks of intracellular recordings. After fixation, serial frozen sections of the brainstem (100 ,urn thick in the frontal plane) were made. The stimulating tracks and points, as well as the site of recorded neurons, were reconstructed using the Fast green FCF marks. We studied a total of 26 burst ! neurons which showed either tonic (I-TON) (Fig. I AI) or decrementing (I-DEC) (Fig. 1B l) firing patterns; in 3 cases, however, their firing patterns were in-between (I-BET) and it was difficult to classify them into either I-DEC or I-TON neurons. Thus we sampled 5 I-TON, 18 I-DEC, and 3 I-BET neuroils. All of them were sampled in the vicinity of the retrofacial nucleus and in the vicinity of the nucleus ambiguus, i.e., the BOT and the VRG, respectively. None of the 26 I neurons except for 2 I-DEC could be antidromically activated from the spinal cord at C4 5- We made systematic antidromic stimulation tests in the medulla contralateral to the isolated burst I neurons, especially in the areas of the VRG and the D R G where I neurons were predominantly found. All of the 26 burst i neurons were antidromically activated from the V R G area. Eighteen of 22 tested were activated from the DR(] as well as the VRG area. In all of the 26 cases of VRG stimulation and m 15 of the 18 cases of D R G stimulation, antidromic latencies could be changed in a step fashion by slight movements of the stimulating electrode within single tracks or among nearby tracks, suggesting the existence of axonal branches. We found no apparent differences in antidromic activation among the I-TON, the I-BET, and the I-DEC neurons. Intracellular recordings were made from l neurons of the VRG (n = 80) and the
305
D R G (n=30). A total of 110 intracellular units whose membrane potentials were more negative than - 30 mV were analyzed; the VRG I neurons consisted of 35 bulbospinai neurons, 32 vagal motoneurons, and 13 propriobulbar (defined as non-bulbospinal and non-vagal) neurons; the D R G neurons consisted of 27 bulbospinal neurons and 3 propriobulbar neurons. Using this intracellular activity and the extracellular activity of the 26 burst I neurons, a total of 110 pairs was examined for their connectivity by the STA method. In 10 pairs, depolarizing potentials locked to the triggering spikes were revealed. Fig. 1A2 shows a depolarizing potential induced in a VRG I neuron with triggering by an I-TON neuron. The latency of this depolarization measured from the foot of the triggering spike was 1.5 ms and the time-to-peak was 1.0 ms. This depolarizing potential with a sharp and fast rising phase was presumably a monosynaptically induced EPSP, as described in previous studies [6, 9, 10, 14]. This was supported by the following observation. The triggering I-TON neuron could be antidromically activated from the area of intracellular recording, and the antidromic latency was 1.2 ms. If the latency for spike generation in the axon at the stimulated site was about 0.1 ms [8, 9], the orthodromic conduction time along the
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Fig. 1. Spike-triggered averaging was performed between a VRG 1 neuron and an I-TON neuron (AI, A2), and between a D R G I neuron and an I-DEC neuron (BI, B2). A1, BI: spike histogram (a) ofextracellularly recorded spikes (b); intracellular potential (c); rectified and integrated phrenic nerve activity (d). A2, B2: averaged triggering spike (a); averaged membrane potential (b); averaged juxtacellular potential (c). The I-TON and I-DEC neurons induced a monosynaptic EPSP (A2b) and an IPSP (B2b) respectively.
306 axon could be estimated to be 1.1 ms. Taking synaptic delay of about 0.4 ms into consideration [6, 8, 9, 14], the above results indicate that the connection was m o n o s y naptic. In 9 other cases, a similar test indicated that the observed depolarizing potentials were monosynaptic EPSPs induced by triggering spikes o f burst I neurons. The httencies o f 10 EPSPs ranged between I. I and 2.6 ms (mean _+ S.D.: 1.7_+ 0.4 ms); the times-to-peak ranged between 0.7 and 2.6 ms ( m e a n ± S . D . : 1,5_+0.4 ms). The 10 I neurons which received these excitatory postsynaptic potentials (EPSPs) consisted of 3 bulbospinal neurons (1 V R G , 2 D R G ) , 3 V R G vagal motoneurons, and 4 propriobulbar neurons (3 V R G , 1 D R G ) . Nine of the 10 I neurons exhibited increasing membrane depolarizations during the I phase: one ( V R G propriobulbar neuron) exhibited decrementing pattern of m e m b r a n e potential. The EPSPs in these 10 neurons were induced by 5 I - T O N and 2 I-BET neurons. Two I-TON neurons, including that shown in Fig. IA. induced EPSPs in both V R G and D R G I neurons. In 13 pairs, hyperpolarizing potentials locked to the triggering spikes were observed. Fig. I B2 shows a hyperpolarizing potential induced in a D R G I neuron with triggering by an I - D E C neuron. The latency o f this hyperpolarization was 1.7 ms and the time-to-peak was 1.6 ms. The latency o f antidromic activation o f the triggering I - D E C neuron from the D R G was 1.5 ms. Employing the same a r g u m e n t as above and taking synaptic delay into consideration, the hyperpolarization was suggested to be a m o n o s y n a p t i c IPSP induced by triggering I - D E C neuron. In fact, in 2 o f the 13 cases, hyperpolarizing potentials were reversed to depolarization by intracellular injection o f hyperpotarizing current, indicating that the hyperpolarizations were inhibitory postsynaptic potentials (IPSPs). The latencies o f the IPSPs ranged between 0.9 and 2.8 ms ( m e a n ± S . D . : 1.8±0.6 ms); the times-to-peak ranged
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/ Fig. 2. Distribution of burst I neurons is projected onto horizontal (A) and transverse (B) planes. There are 7 excitatory (open circles) and 7 inhibitory (closed circles) neurons. AMB: nucleus ambiguus; CX: external cuneate nucleus; DX: dorsal motor nucleus of the vagus; IO: inferior olivary nucleus: RFN: retrofacial nucleus; S: solitary tract; 5ST: spinal trigeminal tract: 7N: facial nucleus; 12N: hypoglossal nucleus.
307 between 1.0 and 2.2 ms (mean S.D.: 1.6+0.4 ms). These 13 IPSPs were induced by 9 I-DEC neurons. Two of the 9 I-DEC neurons induced IPSPs in both VRG and D R G neurons. The 13 I neurons which received IPSPs consisted o f 11 bulbospinal I neurons (8 VRG, 3 D R G ) and 2 V R G propriobulbar neurons. All of the 13 neurons exhibited augmenting firing patterns or increasing membrane depolarizations during the I phase; the augmentation in 7 neurons showed a tendency for late onset [I 5]. In Fig. 2, we show the anatomical distribution of 7 neurons which induced EPSPs and 7 of the 9 I-DEC neurons which induced IPSPs. As shown in the horizontal plane of Fig. 2A, all of them, except for one I-TON neuron, were distributed in the vicinity of the area from the caudal part of the retrofacial nucleus to the rostral part of the nucleus ambiguus. This area corresponds to the caudal part of the BOT and the rostralmost part of the VRG. In a transverse plane (Fig. 2B), they were distributed ventrolateral to the retrofacial nucleus and the nucleus ambiguus. Fig. 2B also reveals a tendency for the I-TON and I-BET neurons to be distributed more laterally than the I-DEC neurons. This study has shown clearly that the burst I neurons do not form a homogenous group, but consist of at least two types, i.e., excitatory and inhibitory neurons. Excitatory I neurons tend to exhibit tonic firing while inhibitory I neurons exhibit decrementing firing. I-TON neurons may be an important excitatory source for many of the medullary I neurons, judging from the fact that I-TON neurons excited many kinds of I neurons in both the V R G and the DRG: bulbospinal neurons, propriospinal neurons, and vagal motoneurons. This report is the first to identify a medullary origin of excitatory inputs to D R G I neurons. Merrill [12] suggested the possibility that I-DEC neurons excite each other, since electrical stimulation in the area of I-DEC neurons resulted in both antidromic and orthodromic activation of I-DEC neurons on the opposite side of the brainstem. We obtained a similar observation. However, the orthodromic activation might be induced by activating I-TON neurons which were excitatory and situated in the same area. There is the possibility of selfreexcitatory connections among I-TON neurons but not among I-DEC neurons, since 1-TON neurons have extensive axonal branches in the contralateral medulla as well as around their somata [7]. Richter's intracellular study [15] clearly demonstrated that some late onset I neurons, which form a subset of augmenting I neurons, receive inhibitory inputs during the early part of the I phase. He hypothesized that the source of the inhibition was I-DEC neurons, since the pattern of early I inhibition resembled the discharge pattern of I-DEC neurons which were already known to have extensive axonal arborizations in the ventrolateral medulla [12]. The present results agree with Richter's hypothesis. In addition, we observed inhibition not only in late onset I neurons, but also in early onset augmenting I neurons of the VRG and the D R G (see Fig. 1B1). Previously, we showed that augmenting I neurons of the V R G make self-reexcitatory connections [6] and suggested that these connections may contribute to the production of the augmenting firing, i.e., I-ramp pattern. Acting in concert with the self-reexcitatory connections between augmenting I neurons, the disinhibition of augmenting I neurons due to decrement of inhibitory action from I-DEC neurons provides a good explanation for how inspiratory augmentation is produced in the medulla.
308
We thank Drs. H. Shimazu and H. Robinson for their critical reviews of the manuscript, and Dr. Y. Kunita for her technical assistance. This work was partly supported by a Grant-in-Aid for Science Research (63570074) from the Japanese Ministry of Education, Science and Culture. 1 Arita, H., Kogo, N. and Koshiya, N., Morphological and physiological properties of caudal medullary expiratory neurons of the cat, Brain Res., 401 (1987) 258 266. 2 Bianchi. A.L.. Localisation et 6tude des neurones rcspiratoires bulbaires. Mise en jeu antidromiquc par stimulation spinal ou vagale, .1. Physiol. (Paris), 63 (1971) 5 40. 3 Bianchi, A.L. Modalitc}s de d~charge et propriOtgzs anatomofonctionelles des neurones respiratoires bulbaires, J. Phy~iol. (Paris), 68 (1974) 555 587. 4 Bianchi, A.L. and Barillot, J.('., Respiratory neurons in the region of the retrofacial nucleus: pontile, medullary, spinal and vagal projections, Neurosci. Lett., 31 (1982) 277 282. 5 Ezure, K. and Manabe, M., Decrementing expiratory neurons of the B6tzinger complex. II. Direct inhibitory synaptic linkage with ventral respiratory group neurons, Exp. Brain Res., 72 (1988) 159 166. 6 Enzure, K. and Manabe, M., Monosynaptic excitation of medullary inspiratory neurons by bulbospina~inspira~oryneuronsof~hcventralrespiratorygroupinthecat,Exp.BrainRes.~74(~989)501 511. 7 Ezure, K., Manabe, M., Otake, K., Sasaki, H. and Mannen, H., The axonal projection of two types of burst inspiratory neurons to the B6tzinger complex (BOT), Neurosci. Res., Suppl., 2 (1986) $79. 8 ftikosaka, O., lgusa, Y., Nakao, S. and Shimazu, H , Direct inhibitory synaptic linkage of pontomedullary reticular burst neurons with abducens motoneurons in the cal. Exp. Brain Res., 33 (I978) 337 352. 9 Jankowska, E. and Roberts, W.,1., Synaptic actions of single interneurones mediating reciprocal la inhibition of motoneurones, J. Physiol. (Lond.), 222 [I 972) 623 642. I0 Kirkwood, P.A. and Sears, T.A.. Monosynaptic excitation of thoracic expiratory motoneurons from lateral respiratory neurones in the medulla of the cal, J. Physiol. (Lond.), 234 (1973) 87p 89p. 11 Merrill, E.G., [nleractions between medullary respiratory neurones in cats, J. Physiol. (Lond.), 226 (1972) 72P 74P. 12 Merrill, E.G., Fmdmg a respiratory function for the medullary respiratory neurons. In R. Bellairs and E.(}. Gray (Eds.), Essays on the Nervous System, Clarendon, Oxford, 1974, pp. 451 486. 13 Merrill, E.G., Lipski, J.. Kubin. L. and Fedorko, L., Origin of the expiratory inhibition of nucleus Iractus solitarius inspiratory neurones, Brain Res., 263 (1983) 43 50. 14 Rapoport. S., Susswein, A., Uchino, Y. and Wilson. V.J., Synaptic actions of individual vestibular neurones on cat neck motoneurones, J. Physiol. {Lond.}, 272 (1977) 367 382. 15 Richter. I).W., Generation and maintenance of the respiratory rhythm, J. Exp. Biol., 100 (1982) 93 107.