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Neural responses of rat area postrema to stimuli producing nausea
Journalof the
Autonomic Nervous System ELSEVIER
Journal of the AutonomicNervous System 49 (1994) 55-60
Neural responses of rat area postrema to stimuli producing nausea Gohichi Tsukamoto
a, A k i r a A d a c h i
b,.
a Department of Oral and Maxillofacial Surgery H and b Department of Physiology, Okayama University Dental School, Shikata-cho, Okayama 700, Japan
(Received 13 October 1993;revision received and accepted 10 December 1993)
Abstract
To identify a neuron within the area postrema (AP) that participates in producing nausea, neural responses of the rat AP to noxious, excessive distension of the stomach were recorded electrophysiologically under urethane-chloralose anesthesia. There were two types of the neural responses; one is characterized by increasing the frequency of discharges responding to the stomach distension (excitatory type), while the other shows the opposite response (decreasing the frequency) to the same stimulation (inhibitory type). After this identification, the effect of LiCI or apomorphine superfused on the floor of IVth ventricle was examined to ascertain a convergence of afferents responding to chemical (LiC1 or apomorphine) as well as mechanical noxious stimulation (stomach distension) on the same AP neuron. It was revealed that the rat AP involves multimodality neurons responsive to various emetic stimuli, so indicating their participation in producing nausea. Key words: Area postrema; Nausea; Gastric distension; Lithium chloride; Apomorphine
I. Introduction
The area postrema (AP), one of the circumventricular organs, is situated on the floor of the IVth ventricle and lacks the blood-brain barrier [12]. Since ablation of the AP causes loss of the vomiting response induced by emetic substances administered intravenously [14,22] the A P has been designated the chemoreceptive trigger zone (CTZ) for vomiting [5,6,21]. It is known that the rat, used in this experiment, does not vomit. For
* Corresponding author.
this reason, the rat has not been used as the experimental animal for the purpose of analyzing the mechanism of vomiting. However, the rat A P may be regarded as participating in producing nausea, because A P lesioned rats are unable to acquire the conditioned taste aversions (CTA) [10] that have commonly been attributed to nausea or toxicity produced by the agents used as unconditioned stimuli [11]. Recently, it has been elucidated that especially in rodents, ablation of the AP affects food intake and salt preference [8], indicating other roles of the AP than CTZ. Enteroceptor neurons responsive to glucose or sodium in the internal environment were found within the rat A P [3]. This evidence indicates a possible
0165-1838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-1838(94)00007-7
56
G. Tsukamoto, A. Adachi /Journal of the Autonomic Nert'ous System 49 (1994) 55-60
role of the AP in the control of ingestion. Very little is known as yet concerning the neural responses of the AP to the emetic stimuli especially in rodents, though a few reports have been published on canines [7]. The purpose of the present study is to identify neurons within the AP in the rat that relate to initiation of nausea by analyzing neural responses of the AP to gastric distension or chemical emetic stimuli.
(HCl-apomorphine 700 /xM, NaCI 147 mM, KCI 4 mM, CaC12 2.97 mM), (3) hypertonic solution (NaC1 300 mM, KC1 4 raM, CaC12 2.97 raM). Neural responses to these stimuli were recorded using conventional electrophysiological equipment. All records were stored on magnetic tape for later analysis. The n u m b e r of impulses in each 5 s was counted by means of a pulse rate m e t e r and displayed on a pen recorder. All recording sites were marked by pontamine sky blue and examined histologically afterward.
2. Methods A total of 81 male Sprague-Dawley rats (Charles River of Japan), weighing 250-350 g, were used. To empty the stomach, the animals were fasted overnight before each experimental day. U n d e r urethane-chloralose anesthesia (urethane 0.8 g / k g , chloralose 65 m g / k g , i.p.), a cannula was inserted into the trachea to keep the airways open. A balloon connected at the other end to a tube with a syringe was placed in the stomach through the esophagus. To distend the stomach, 4 ml or 10 ml water was injected into the balloon at a rate of 2 - 3 m l / s . A distension of l0 ml, which is 2.5 times larger than the size of a normally satiated stomach (4 ml), is to be regarded as a noxious stimulus that causes an unpleasant feeling like nausea. The rat was mounted on a stereotaxic apparatus with its head tilted down. The occipital bone was removed and the cerebellum was pushed up rostrally until the dorsal surface of the AP was entirely exposed. As previously reported [1,2], a superfusion technique was employed for chemical stimulation of the AP. The dorsal surface of the IVth ventricle was superfused with standard Ringer's solution (NaCI 147 mM, KCI 4 mM, CaC12 2.97 mM). A glass pipette electrode filled with 0.5 M sodium acetate and 2% pontamine sky blue was used for recording unit discharges. U n d e r inspection through a dissecting microscope, the recording electrode was inserted into the AP until spontaneous discharges of a single unit were observed. Superfusion with Ringer's solution was switched to test solutions. Test solutions were as follows: (1) LiCI solution (LiC1 150 mM, NaC1 147 mM, KCI 4 raM, CaC1 e 2.97 raM), (2) apomorphine solution
3. Results 3.1. Responses to gastric distension
Seventy neurons were examined as summarized in Table 1. A m o n g them, 39 neurons were responsive to gastric distension. As illustrated in Fig. 1, there were two different types of neural response: one is characterized by increasing the discharge in response to this stimulation (excitatory), while the other shows a decreasing discharge in response to the same stimulus (inhibitory). Though no primary afferents have been observed where discharge rates are suppressed by gastric distension [17], the latter type of neuron, the inhibitory one (32 neurons), is more numerous than the former, the excitatory one (7 neurons) in this experiment. No topographical relationship between the types of neurons was ob-
Table 1 Response to gastric distension Upward arrows indicate excitatory responses and downward arrows indicate inhibitory responses. Horizontal arrows indicate no response. Distension Number of units 4 m! 10 ml (n = 70) 1
~ $
5 2
,1, ,L
,L $ ~
13 16 3
--->
32 31
G. Tsukamoto A. Adachi /Journal of the Autonomic Nert~ous System 49 (1994) 55-60
A
2 min
0
B
2 rnin
0
f
E 0L
_E o L
7
T~
4
30 sec
C ~
10
500 pV
] 10 ml Distension
Fig. 1. Two different types of response (inhibitory in A and excitatory in B) of A P neurons to gastric distension. Pen recording of the discharge rate vs. time in A shows complete disappearance of the discharge responses to gastric distension by 4 or 10 ml. The same recording in B represents marked increases of the discharge responses to gastric distension. Note that the magnitude of the response is positively correlate with that of gastric distension. C. Oscillograph record of discharges during 10 ml distension in A.
served. A m o n g seven neurons showing the former type of response (excitatory), five were specifically responsive to 10 ml distension regarded as a nociceptive stimulus. Two were responsive to 4 ml as well as 10 ml distension. Their discharge rates were positively correlated with the intensity. They may contribute to transfer information concerned with mechanical stretching of the gastric wall. A m o n g the inhibitory neurons, 13 were specifically responsive to 10 ml distension and 16 were responsive to 4 ml as well as 10 ml distension. However, three neurons were only responsive to 4 ml distension but not to excessive, 10 ml distension. A role of the inhibitory neurons is unknown.
3. 2. Responses to chemical stimuli Two chemicals were employed in this experiment. One was lithium chloride, which is frequently used as an unconditioned stimulus for CTA, and the other was apomorphine, which is one of the most effective chemicals to induce emesis when given via the intravenous route. (1) Lithium chloride. As presented in Table 2, a total of 55 neurons were examined. In this
57
examination, the test solution containing 150 mM LiC1 in Ringer solution, hypertonic solution, was employed except in a few cases. Since there are enteroceptor neurons that are responsive to hypertonicity in the AP [1,2], it is necessary to distinguish a neuron that is responsive to LiCI from one that is responsive to hypertonicity. Therefore, all neurons responding to LiC1 were examined whether or not they responded to hypertonicity. The results are summarized in Table 2. Two different types of response were observed: one is characterized by an increasing frequency of discharge responses to LiCI (excitatory) while the other shows a decreasing frequency (inhibitory). With regard to both types of neuron, there are neurons that are specifically responsive to LiC1. It should be noted that neurons responding to harmful, noxious chemicals are involved in the AP in addition to osmosensitive neurons. As summarized in Table 2, six neurons were identified as excitatory noxious chemosensitive neurons and nine were inhibitory ones. It is not surprising that osmosensitive neurons are responsive to hypertonicity of a test solution achieved by adding LiCI to isotonic Ringer solution. (2) Apomorphine. There has been no report dealing with an effect of apomorphine on AP neurons in the rat, although it is a very popular emetic and agent employed as the unconditioned stimulus for C T A [9]. It is confirmed that some AP neurons were responsive to apomorphine and the responses of 36 AP neurons to apomorphine applied by means of superfusion were both excitatory (eight neurons) and inhibitory (22 neurons). Table 2 Response to LiCI and hypertonicity T h e arrows indicate the same responses as defined in Table 1. LiCI
Hypertonic
$
$
N u m b e r of units (n = 55) 5 1
? -* T $ /
6
5 8 1
9 20 15
G. Tsukamoto, A. Adachi / Journal of the Autonomic Nervous System 49 (1994) 55-60
58
Table 3 Response to gastric distension, LiCI and apomorphine The arrows indicate the same responses as defined in Table 1.
A
10 ml
LiCI
~ 20 f co ffl
T
7
$
20
Number of units
10 ml
APemorphine
Number of units
T +
2 1 4
T
5
T + ~
3 1 1
$ $ --,
1 5 14
k
13
q" $ ~
1 9 3
0
4
10
LiCI
Hyper- Apetonic morphine
B It lllJ ,_l. 1'-'~""" 5 see
~ _ d l i l | J u . u . lllllll l a l l d l h _ d . i l l | t b
tTr,,.v, ,, ...... r,..,, r.,--uIP-,Pl.p, II 200 pv 10 ml Distension
Fig. 2. Excitatory responses to gastric distension and to LiCI. A. Pen recording of the discharge rate vs. time. Note that this neuron responds to 10 ml gastric distension and LiCI applied by superfusion of the IVth ventricle. No response is elicited by 4 ml distension, superfusion with hypertonic saline or apomorphine containing Ringer solution. B. Oscillograph record of discharges during 10 ml distension in A.
It is again noted that inhibitory neurons are more numerous than excitatory ones.
3.3. Convergence of various inputs elicited by mechanical and chemical irritant stimuli onto AP neurons As shown in Fig. 2, this neuron respond to gastric distension and to LiCI as well in a similar manner such as increasing the discharge rate in
A
B 2 min
f
:.'_~_~ ' ~ " ~ " ! iIIllll~lllllllP".... i 500/iV "~;~,~- -,,- ~,1111fllllllBFIsn,,....
10 sec
10 ml Distension
/~'umm~'A~'hnidalllUhi'°a'i500 pV
or
4 10 LiCI Hpertonic
~pp~m~p~Hti~jpu
10 sac
I
I_iCI
Fig. 3. Inhibitory responses to gastric distension and to LiC1, A. Pen recording of the discharge rate vs. time. Note that this neuron decreases its discharge rate in response to 10 ml gastric distension and superfusion with LiCI as well. No response is elicited by 4 ml distension or superfusion with hypertonic saline. B. Oscillograph records of discharges during 10 ml distension (upper trace) and superfusion with LiC1 containing Ringer solution (lower trace) in A.
response to the stimuli. Regarding the inhibitorytype neurons, the same convergence was observed as seen in Fig. 3. This neuron decreases the discharge rate in response to 10 mi distension, and also to superfusion with Ringer solution containing LiC1. The results obtained are summarized in Table 3. This evidence suggests that afferent inputs concerned with nociceptive information converge onto a neuron within the AP. In general, the responses to gastric distension and to emetic substances were mostly in a similar manner, that is, if the former is of the excitatory type, the latter is also excitatory and vice versa. The recording site is shown in Fig. 4.
4. Discussion
The visceral sensory afferents originating in the stomach wall project to the central nervous system via the vagal and splanchnic nerves [15]. Recent histological investigations revealed that the subdiaphragmatic vagal branches project to the ventral portion of the AP. However, gastric vagal projection to the AP in the rat has not been proved yet. [13,16]. As there are close neural connections between the AP and the nucleus tractus solitarius (NTS) [19], it is supposed that neural responses to gastric distension can be recorded trans-synaptically within the AP. Paintal [17] recorded ascending neural responses of the right cervical vagus that increased their discharge rates in response to gastric distension. No primary afferents that decreased their discharge rates in response to this stimulation were ob-
G. Tsukamoto, A. Adachi /Journal of the Autonomic Nervous System 49 (1994) 55-60
59
Fig. 4. Photomicrograph of coronal section of the medulla oblongata. Arrow indicates the marked position of the tip of the recording electrode in the AP. AP, area postrema; NTS, solitary tract nucleus; DMV, dorsal motor nucleus of the vagus. Bar = 200 /.~m.
served [17]. Therefore, the inhibitory-type response observed in this study would be postsynaptically elicited via an inhibitory interneuron that is activated by the primary afferent discovered by Paintal [17]. It should be noted that the n u m b e r of neurons which show the inhibitory-type response is much greater than that of the excitatory type in the AP as well as in the NTS [18]. The reason for this still remains obscure. C a r p e n t e r et al. [7] examined the responses of AP neurons in the dog to various neurotransmitters and bioactive peptides by means of a multiple-barreled microelectrode. They found that the responses were almost excitatory. A p o m o r p h i n e induced an excitatory response in 86% of the tested neurons but no inhibitory response. This discrepancy between our present results and Carpenter et al.'s may be explicable by the different method used to administer chemicals. The superfusion technique employed in this experiment for chemical stimulation is incapable of administering chemicals restrictedly on a target neuron but stimulates the whole AP. Therefore, it can hardly
be said that the recorded neurons were directly stimulated by the chemicals introduced. It is possible that the recorded neurons in this study are occasionally different from the receptor neurons themselves, but they might be the relay neurons postsynaptically activated by the receptor neurons. Otherwise, iontophoretic application using the multiple-barreled microelectrode is capable of stimulating a restricted neuron whose activity is recording, so there is no possibility to record the postsynaptic response via the inhibitory interneuron. It may not be chance that the inhibitory-type response was recorded in their experiment. It has been proved that AP lesioned rats are unable to acquire C T A when LiC1 is employed as an unconditioned stimulus. O u r finding that there are nausea related neurons which are responsive to LiC1 and also to excessive gastric distension may give a possible explanation for this behavioral deficit. Loss of a group of AP neurons related to the induction of nausea by ablation of the AP may result in an inability to acquire CTA.
6(1
G. Tsukamoto, A. Adachi /Journal of theAutonomic Nervous System 49 (1994) 55-60
Even in rats that fail to vomit, there is a group of neurons responsive to chemical irritant stimuli that participate in producing nausea. The receptive mechanisms of AP neurons to LiC1 as well as apomorphine are unclear. However, it is possible that the dopaminergic neural network contributes to the initiation of nausea in rats because apomorphine, a nonspecific dopamine agonist [4], elicited responses of some AP neurons in rats, as reported for dogs by Stefanini e t a l . [20].
Acknowledgement This work was supported by Grant-in-Aid for General Scientific Research 05454502 from the Ministry of Education, Science and Culture of Japan.
References [1] Adachi, A. and Kobashi, M., Chemosensitive neurons within the area postrema of the rat, Neurosci. Lett., 55 (1985) 137-140. [2] Adachi, A. and Kobashi, M., Electrophysiological analysis of chemosensitive neurons within the area postrema of the rat, Prog. Brain Res., 74 (1988) 77-83. [3] Adachi, A., Kobashi, M., Miyoshi, N. and Tsukamoto, G., Chemosensitive neurons in the area postrema of the rat and their possible functions, Brain Res. Bull., 26 (1991) 137-140. [4] Billard, W., Ruperto, V., Crosby, G., Iorio, L.C. and Barnett, A., Characterization of the binding of 3H-SCH 23390, a selective D-1 receptor antagonist ligand, in rat striatum, Life Sci., 35 (1984) 1885-1893. [5] Borison, H.L., Area postrema: chemoreceptor trigger zone for vomiting - is that all?, Life Sci., 14 (1974) 1807-1817. [6] Borison, H.L. and Wang, S.C., Physiology and pharmacology of vomiting, Pharmacol. Rev., 5 (1953) 193-230. [7] Carpenter, D.O., Briggs, D.B., Knox, A.P. and Strominger, N., Excitation of area postrema neurons by transmitters, peptides, and cyclic nucleotides, J. Neurophysiol., 59 (1988) 358-369.
[8] Contreras, R.J. and Stetson, P.W., Changes in salt intake after lesions of the area postrema and the nucleus of the solitary tract in rats, Brain Res., 211 (1981) 355-366. [9] D'Mello, G.D., Goldberg, D.M., Goldberg, S.R and Stolerman, I.P., Conditioned taste aversion and operant behavior in rats: Effects of cocaine, apomorphine and some long-acting derivatives, J. Pharmacol. Exp. Ther., 219 (1981) 60-68. [10] Edwards, G.L. and Ritter, R.C., Ablation of the area postrema causes exaggerated consumption of preferred foods in the rat, Brain Res., 216 (1981) 265-276. [11] Garcia, J. and Ervin, F.R., Gustatory-visceral and telereceptor-cutaneous conditioning: Adaptation in internal and external milieus~ Commun. Behav. Biol., (A) 1 11968) 389-415. [12] Leslie, R.A., Comparative aspects of the area postrema: fine-structural considerations help to determine its function, Cell Mol. Neurobiol., 6 (1986) 95-120. [13] Leslie, R.A., Gwyn, D.G. and Hopkins, D.A., The central distribution of the cervical vagus nerve and gastric afferent and efferent projections in the rat, Brain Res. Bull., 8 (1982) 37-43. [14] Lindstorm, P.A. and Brizzee, K.R., Relief of intractable vomiting from surgical lesions in the area postrema, J. Neurosurg., 19 (1962) 228-236. [15] Niijima, A., Afferent impulses in the vagal and splanchnic nerves of toad's stomach, and their role in sensory mechanism, Jpn J. Physiol., 12 (1962) 25-44. [16] Norgren, R. and Smith, G.P., Central distribution of subdiaphragmatic vagal branches in the rat, J. Comp. Neurol., 273 (1988) 207-223. [17] Paintal, A.S., A study of gastric stretch receptors. Their role in the peripheral mechanism of satiation of hunger and thirst, J. Physiol., 126 (1954) 255-270. [18] Raybould, H.E., Gayton, R.J. and Dockray, G.J., CNS effects of circulating CCK8: involvement of brainstem neurons responding to gastric distension, Brain Res., 342 (1985) 187-190. [19] Shapiro, R.E. and Miselis, R.R., The central neural connections of the area postrema of the rat, J. Comp. Neurol., 234 (1985) 344-364. [20] Stefanini, E. and Clement-Cormier, Y., Detection of dopamine receptors in the area postrema, Eur. J. Pharmacol., 74 (1981) 257-260. [21] Wang, S.C., Emetic and antiemetic drugs, Physiol. Pharmacol., 2 (1965) 255-328. [22] Wang, S.C. and Borison, H.L., A new concept of organization of the central emetic mechanism: recent studies on the sites of action of apomorphine, copper sulfate and cardiac glycosides, Gastroenterology, 22 (1952) 1-12.
Autonomic Nervous System ELSEVIER
Journal of the AutonomicNervous System 49 (1994) 55-60
Neural responses of rat area postrema to stimuli producing nausea Gohichi Tsukamoto
a, A k i r a A d a c h i
b,.
a Department of Oral and Maxillofacial Surgery H and b Department of Physiology, Okayama University Dental School, Shikata-cho, Okayama 700, Japan
(Received 13 October 1993;revision received and accepted 10 December 1993)
Abstract
To identify a neuron within the area postrema (AP) that participates in producing nausea, neural responses of the rat AP to noxious, excessive distension of the stomach were recorded electrophysiologically under urethane-chloralose anesthesia. There were two types of the neural responses; one is characterized by increasing the frequency of discharges responding to the stomach distension (excitatory type), while the other shows the opposite response (decreasing the frequency) to the same stimulation (inhibitory type). After this identification, the effect of LiCI or apomorphine superfused on the floor of IVth ventricle was examined to ascertain a convergence of afferents responding to chemical (LiC1 or apomorphine) as well as mechanical noxious stimulation (stomach distension) on the same AP neuron. It was revealed that the rat AP involves multimodality neurons responsive to various emetic stimuli, so indicating their participation in producing nausea. Key words: Area postrema; Nausea; Gastric distension; Lithium chloride; Apomorphine
I. Introduction
The area postrema (AP), one of the circumventricular organs, is situated on the floor of the IVth ventricle and lacks the blood-brain barrier [12]. Since ablation of the AP causes loss of the vomiting response induced by emetic substances administered intravenously [14,22] the A P has been designated the chemoreceptive trigger zone (CTZ) for vomiting [5,6,21]. It is known that the rat, used in this experiment, does not vomit. For
* Corresponding author.
this reason, the rat has not been used as the experimental animal for the purpose of analyzing the mechanism of vomiting. However, the rat A P may be regarded as participating in producing nausea, because A P lesioned rats are unable to acquire the conditioned taste aversions (CTA) [10] that have commonly been attributed to nausea or toxicity produced by the agents used as unconditioned stimuli [11]. Recently, it has been elucidated that especially in rodents, ablation of the AP affects food intake and salt preference [8], indicating other roles of the AP than CTZ. Enteroceptor neurons responsive to glucose or sodium in the internal environment were found within the rat A P [3]. This evidence indicates a possible
0165-1838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-1838(94)00007-7
56
G. Tsukamoto, A. Adachi /Journal of the Autonomic Nert'ous System 49 (1994) 55-60
role of the AP in the control of ingestion. Very little is known as yet concerning the neural responses of the AP to the emetic stimuli especially in rodents, though a few reports have been published on canines [7]. The purpose of the present study is to identify neurons within the AP in the rat that relate to initiation of nausea by analyzing neural responses of the AP to gastric distension or chemical emetic stimuli.
(HCl-apomorphine 700 /xM, NaCI 147 mM, KCI 4 mM, CaC12 2.97 mM), (3) hypertonic solution (NaC1 300 mM, KC1 4 raM, CaC12 2.97 raM). Neural responses to these stimuli were recorded using conventional electrophysiological equipment. All records were stored on magnetic tape for later analysis. The n u m b e r of impulses in each 5 s was counted by means of a pulse rate m e t e r and displayed on a pen recorder. All recording sites were marked by pontamine sky blue and examined histologically afterward.
2. Methods A total of 81 male Sprague-Dawley rats (Charles River of Japan), weighing 250-350 g, were used. To empty the stomach, the animals were fasted overnight before each experimental day. U n d e r urethane-chloralose anesthesia (urethane 0.8 g / k g , chloralose 65 m g / k g , i.p.), a cannula was inserted into the trachea to keep the airways open. A balloon connected at the other end to a tube with a syringe was placed in the stomach through the esophagus. To distend the stomach, 4 ml or 10 ml water was injected into the balloon at a rate of 2 - 3 m l / s . A distension of l0 ml, which is 2.5 times larger than the size of a normally satiated stomach (4 ml), is to be regarded as a noxious stimulus that causes an unpleasant feeling like nausea. The rat was mounted on a stereotaxic apparatus with its head tilted down. The occipital bone was removed and the cerebellum was pushed up rostrally until the dorsal surface of the AP was entirely exposed. As previously reported [1,2], a superfusion technique was employed for chemical stimulation of the AP. The dorsal surface of the IVth ventricle was superfused with standard Ringer's solution (NaCI 147 mM, KCI 4 mM, CaC12 2.97 mM). A glass pipette electrode filled with 0.5 M sodium acetate and 2% pontamine sky blue was used for recording unit discharges. U n d e r inspection through a dissecting microscope, the recording electrode was inserted into the AP until spontaneous discharges of a single unit were observed. Superfusion with Ringer's solution was switched to test solutions. Test solutions were as follows: (1) LiCI solution (LiC1 150 mM, NaC1 147 mM, KCI 4 raM, CaC1 e 2.97 raM), (2) apomorphine solution
3. Results 3.1. Responses to gastric distension
Seventy neurons were examined as summarized in Table 1. A m o n g them, 39 neurons were responsive to gastric distension. As illustrated in Fig. 1, there were two different types of neural response: one is characterized by increasing the discharge in response to this stimulation (excitatory), while the other shows a decreasing discharge in response to the same stimulus (inhibitory). Though no primary afferents have been observed where discharge rates are suppressed by gastric distension [17], the latter type of neuron, the inhibitory one (32 neurons), is more numerous than the former, the excitatory one (7 neurons) in this experiment. No topographical relationship between the types of neurons was ob-
Table 1 Response to gastric distension Upward arrows indicate excitatory responses and downward arrows indicate inhibitory responses. Horizontal arrows indicate no response. Distension Number of units 4 m! 10 ml (n = 70) 1
~ $
5 2
,1, ,L
,L $ ~
13 16 3
--->
32 31
G. Tsukamoto A. Adachi /Journal of the Autonomic Nert~ous System 49 (1994) 55-60
A
2 min
0
B
2 rnin
0
f
E 0L
_E o L
7
T~
4
30 sec
C ~
10
500 pV
] 10 ml Distension
Fig. 1. Two different types of response (inhibitory in A and excitatory in B) of A P neurons to gastric distension. Pen recording of the discharge rate vs. time in A shows complete disappearance of the discharge responses to gastric distension by 4 or 10 ml. The same recording in B represents marked increases of the discharge responses to gastric distension. Note that the magnitude of the response is positively correlate with that of gastric distension. C. Oscillograph record of discharges during 10 ml distension in A.
served. A m o n g seven neurons showing the former type of response (excitatory), five were specifically responsive to 10 ml distension regarded as a nociceptive stimulus. Two were responsive to 4 ml as well as 10 ml distension. Their discharge rates were positively correlated with the intensity. They may contribute to transfer information concerned with mechanical stretching of the gastric wall. A m o n g the inhibitory neurons, 13 were specifically responsive to 10 ml distension and 16 were responsive to 4 ml as well as 10 ml distension. However, three neurons were only responsive to 4 ml distension but not to excessive, 10 ml distension. A role of the inhibitory neurons is unknown.
3. 2. Responses to chemical stimuli Two chemicals were employed in this experiment. One was lithium chloride, which is frequently used as an unconditioned stimulus for CTA, and the other was apomorphine, which is one of the most effective chemicals to induce emesis when given via the intravenous route. (1) Lithium chloride. As presented in Table 2, a total of 55 neurons were examined. In this
57
examination, the test solution containing 150 mM LiC1 in Ringer solution, hypertonic solution, was employed except in a few cases. Since there are enteroceptor neurons that are responsive to hypertonicity in the AP [1,2], it is necessary to distinguish a neuron that is responsive to LiCI from one that is responsive to hypertonicity. Therefore, all neurons responding to LiC1 were examined whether or not they responded to hypertonicity. The results are summarized in Table 2. Two different types of response were observed: one is characterized by an increasing frequency of discharge responses to LiCI (excitatory) while the other shows a decreasing frequency (inhibitory). With regard to both types of neuron, there are neurons that are specifically responsive to LiC1. It should be noted that neurons responding to harmful, noxious chemicals are involved in the AP in addition to osmosensitive neurons. As summarized in Table 2, six neurons were identified as excitatory noxious chemosensitive neurons and nine were inhibitory ones. It is not surprising that osmosensitive neurons are responsive to hypertonicity of a test solution achieved by adding LiCI to isotonic Ringer solution. (2) Apomorphine. There has been no report dealing with an effect of apomorphine on AP neurons in the rat, although it is a very popular emetic and agent employed as the unconditioned stimulus for C T A [9]. It is confirmed that some AP neurons were responsive to apomorphine and the responses of 36 AP neurons to apomorphine applied by means of superfusion were both excitatory (eight neurons) and inhibitory (22 neurons). Table 2 Response to LiCI and hypertonicity T h e arrows indicate the same responses as defined in Table 1. LiCI
Hypertonic
$
$
N u m b e r of units (n = 55) 5 1
? -* T $ /
6
5 8 1
9 20 15
G. Tsukamoto, A. Adachi / Journal of the Autonomic Nervous System 49 (1994) 55-60
58
Table 3 Response to gastric distension, LiCI and apomorphine The arrows indicate the same responses as defined in Table 1.
A
10 ml
LiCI
~ 20 f co ffl
T
7
$
20
Number of units
10 ml
APemorphine
Number of units
T +
2 1 4
T
5
T + ~
3 1 1
$ $ --,
1 5 14
k
13
q" $ ~
1 9 3
0
4
10
LiCI
Hyper- Apetonic morphine
B It lllJ ,_l. 1'-'~""" 5 see
~ _ d l i l | J u . u . lllllll l a l l d l h _ d . i l l | t b
tTr,,.v, ,, ...... r,..,, r.,--uIP-,Pl.p, II 200 pv 10 ml Distension
Fig. 2. Excitatory responses to gastric distension and to LiCI. A. Pen recording of the discharge rate vs. time. Note that this neuron responds to 10 ml gastric distension and LiCI applied by superfusion of the IVth ventricle. No response is elicited by 4 ml distension, superfusion with hypertonic saline or apomorphine containing Ringer solution. B. Oscillograph record of discharges during 10 ml distension in A.
It is again noted that inhibitory neurons are more numerous than excitatory ones.
3.3. Convergence of various inputs elicited by mechanical and chemical irritant stimuli onto AP neurons As shown in Fig. 2, this neuron respond to gastric distension and to LiCI as well in a similar manner such as increasing the discharge rate in
A
B 2 min
f
:.'_~_~ ' ~ " ~ " ! iIIllll~lllllllP".... i 500/iV "~;~,~- -,,- ~,1111fllllllBFIsn,,....
10 sec
10 ml Distension
/~'umm~'A~'hnidalllUhi'°a'i500 pV
or
4 10 LiCI Hpertonic
~pp~m~p~Hti~jpu
10 sac
I
I_iCI
Fig. 3. Inhibitory responses to gastric distension and to LiC1, A. Pen recording of the discharge rate vs. time. Note that this neuron decreases its discharge rate in response to 10 ml gastric distension and superfusion with LiCI as well. No response is elicited by 4 ml distension or superfusion with hypertonic saline. B. Oscillograph records of discharges during 10 ml distension (upper trace) and superfusion with LiC1 containing Ringer solution (lower trace) in A.
response to the stimuli. Regarding the inhibitorytype neurons, the same convergence was observed as seen in Fig. 3. This neuron decreases the discharge rate in response to 10 mi distension, and also to superfusion with Ringer solution containing LiC1. The results obtained are summarized in Table 3. This evidence suggests that afferent inputs concerned with nociceptive information converge onto a neuron within the AP. In general, the responses to gastric distension and to emetic substances were mostly in a similar manner, that is, if the former is of the excitatory type, the latter is also excitatory and vice versa. The recording site is shown in Fig. 4.
4. Discussion
The visceral sensory afferents originating in the stomach wall project to the central nervous system via the vagal and splanchnic nerves [15]. Recent histological investigations revealed that the subdiaphragmatic vagal branches project to the ventral portion of the AP. However, gastric vagal projection to the AP in the rat has not been proved yet. [13,16]. As there are close neural connections between the AP and the nucleus tractus solitarius (NTS) [19], it is supposed that neural responses to gastric distension can be recorded trans-synaptically within the AP. Paintal [17] recorded ascending neural responses of the right cervical vagus that increased their discharge rates in response to gastric distension. No primary afferents that decreased their discharge rates in response to this stimulation were ob-
G. Tsukamoto, A. Adachi /Journal of the Autonomic Nervous System 49 (1994) 55-60
59
Fig. 4. Photomicrograph of coronal section of the medulla oblongata. Arrow indicates the marked position of the tip of the recording electrode in the AP. AP, area postrema; NTS, solitary tract nucleus; DMV, dorsal motor nucleus of the vagus. Bar = 200 /.~m.
served [17]. Therefore, the inhibitory-type response observed in this study would be postsynaptically elicited via an inhibitory interneuron that is activated by the primary afferent discovered by Paintal [17]. It should be noted that the n u m b e r of neurons which show the inhibitory-type response is much greater than that of the excitatory type in the AP as well as in the NTS [18]. The reason for this still remains obscure. C a r p e n t e r et al. [7] examined the responses of AP neurons in the dog to various neurotransmitters and bioactive peptides by means of a multiple-barreled microelectrode. They found that the responses were almost excitatory. A p o m o r p h i n e induced an excitatory response in 86% of the tested neurons but no inhibitory response. This discrepancy between our present results and Carpenter et al.'s may be explicable by the different method used to administer chemicals. The superfusion technique employed in this experiment for chemical stimulation is incapable of administering chemicals restrictedly on a target neuron but stimulates the whole AP. Therefore, it can hardly
be said that the recorded neurons were directly stimulated by the chemicals introduced. It is possible that the recorded neurons in this study are occasionally different from the receptor neurons themselves, but they might be the relay neurons postsynaptically activated by the receptor neurons. Otherwise, iontophoretic application using the multiple-barreled microelectrode is capable of stimulating a restricted neuron whose activity is recording, so there is no possibility to record the postsynaptic response via the inhibitory interneuron. It may not be chance that the inhibitory-type response was recorded in their experiment. It has been proved that AP lesioned rats are unable to acquire C T A when LiC1 is employed as an unconditioned stimulus. O u r finding that there are nausea related neurons which are responsive to LiC1 and also to excessive gastric distension may give a possible explanation for this behavioral deficit. Loss of a group of AP neurons related to the induction of nausea by ablation of the AP may result in an inability to acquire CTA.
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G. Tsukamoto, A. Adachi /Journal of theAutonomic Nervous System 49 (1994) 55-60
Even in rats that fail to vomit, there is a group of neurons responsive to chemical irritant stimuli that participate in producing nausea. The receptive mechanisms of AP neurons to LiC1 as well as apomorphine are unclear. However, it is possible that the dopaminergic neural network contributes to the initiation of nausea in rats because apomorphine, a nonspecific dopamine agonist [4], elicited responses of some AP neurons in rats, as reported for dogs by Stefanini e t a l . [20].
Acknowledgement This work was supported by Grant-in-Aid for General Scientific Research 05454502 from the Ministry of Education, Science and Culture of Japan.
References [1] Adachi, A. and Kobashi, M., Chemosensitive neurons within the area postrema of the rat, Neurosci. Lett., 55 (1985) 137-140. [2] Adachi, A. and Kobashi, M., Electrophysiological analysis of chemosensitive neurons within the area postrema of the rat, Prog. Brain Res., 74 (1988) 77-83. [3] Adachi, A., Kobashi, M., Miyoshi, N. and Tsukamoto, G., Chemosensitive neurons in the area postrema of the rat and their possible functions, Brain Res. Bull., 26 (1991) 137-140. [4] Billard, W., Ruperto, V., Crosby, G., Iorio, L.C. and Barnett, A., Characterization of the binding of 3H-SCH 23390, a selective D-1 receptor antagonist ligand, in rat striatum, Life Sci., 35 (1984) 1885-1893. [5] Borison, H.L., Area postrema: chemoreceptor trigger zone for vomiting - is that all?, Life Sci., 14 (1974) 1807-1817. [6] Borison, H.L. and Wang, S.C., Physiology and pharmacology of vomiting, Pharmacol. Rev., 5 (1953) 193-230. [7] Carpenter, D.O., Briggs, D.B., Knox, A.P. and Strominger, N., Excitation of area postrema neurons by transmitters, peptides, and cyclic nucleotides, J. Neurophysiol., 59 (1988) 358-369.
[8] Contreras, R.J. and Stetson, P.W., Changes in salt intake after lesions of the area postrema and the nucleus of the solitary tract in rats, Brain Res., 211 (1981) 355-366. [9] D'Mello, G.D., Goldberg, D.M., Goldberg, S.R and Stolerman, I.P., Conditioned taste aversion and operant behavior in rats: Effects of cocaine, apomorphine and some long-acting derivatives, J. Pharmacol. Exp. Ther., 219 (1981) 60-68. [10] Edwards, G.L. and Ritter, R.C., Ablation of the area postrema causes exaggerated consumption of preferred foods in the rat, Brain Res., 216 (1981) 265-276. [11] Garcia, J. and Ervin, F.R., Gustatory-visceral and telereceptor-cutaneous conditioning: Adaptation in internal and external milieus~ Commun. Behav. Biol., (A) 1 11968) 389-415. [12] Leslie, R.A., Comparative aspects of the area postrema: fine-structural considerations help to determine its function, Cell Mol. Neurobiol., 6 (1986) 95-120. [13] Leslie, R.A., Gwyn, D.G. and Hopkins, D.A., The central distribution of the cervical vagus nerve and gastric afferent and efferent projections in the rat, Brain Res. Bull., 8 (1982) 37-43. [14] Lindstorm, P.A. and Brizzee, K.R., Relief of intractable vomiting from surgical lesions in the area postrema, J. Neurosurg., 19 (1962) 228-236. [15] Niijima, A., Afferent impulses in the vagal and splanchnic nerves of toad's stomach, and their role in sensory mechanism, Jpn J. Physiol., 12 (1962) 25-44. [16] Norgren, R. and Smith, G.P., Central distribution of subdiaphragmatic vagal branches in the rat, J. Comp. Neurol., 273 (1988) 207-223. [17] Paintal, A.S., A study of gastric stretch receptors. Their role in the peripheral mechanism of satiation of hunger and thirst, J. Physiol., 126 (1954) 255-270. [18] Raybould, H.E., Gayton, R.J. and Dockray, G.J., CNS effects of circulating CCK8: involvement of brainstem neurons responding to gastric distension, Brain Res., 342 (1985) 187-190. [19] Shapiro, R.E. and Miselis, R.R., The central neural connections of the area postrema of the rat, J. Comp. Neurol., 234 (1985) 344-364. [20] Stefanini, E. and Clement-Cormier, Y., Detection of dopamine receptors in the area postrema, Eur. J. Pharmacol., 74 (1981) 257-260. [21] Wang, S.C., Emetic and antiemetic drugs, Physiol. Pharmacol., 2 (1965) 255-328. [22] Wang, S.C. and Borison, H.L., A new concept of organization of the central emetic mechanism: recent studies on the sites of action of apomorphine, copper sulfate and cardiac glycosides, Gastroenterology, 22 (1952) 1-12.