- Email: [email protected]
Chemical inactivation of the nucleus tractus solitarius abolished hepatojejunal reflex in the rat
! ....
"•
Journal of the Autonomic Nervous
~
ELSEVIER
System
Journal of the Autonomic Nervous System 48 (1994) 207-212
Chemical inactivation of the nucleus tractus solitarius abolished hepatojejunal reflex in the rat Hironobu
M o r i t a *, K a z u t a k a T a n a k a , H i r o s h i H o s o m i
Department of Physiology, Kagawa Medical School, Kagawa 761-07, Japan Received 3 September 1993: revision received and accepted 16 November 1093
Abstract
Jejunal electrolyte absorption was measured in the jejunal loop of anesthetized rats during an infusion of 9¢;NaCI solution via the portal vein. Net absorption of Na and CI, and osmolality were significantly depressed by the portal 9% NaCl infusion. The decrease in net absorption was completely abolished by hepatic denervation. In addition, bilateral chemical inactivation of the nucleus tractus solitarius (NTS) by microinjection of kainic acid abolished the depressing effect of the portal venous infusion of 9% NaCI. These findings indicate that NTS is inw)lved in the central pathway of the hepatojejunal reflex. Key words: Hepatojejunal reflex; Jejunal absorption; Hepatic nerve: Nuclcus tractus solitarius; Portal vein: Hypertonic NaCI
It has been well documented that the liver contains many receptors, including the baroreceptor, osmoreceptor, and NaCI receptor [10]. Recently, we have demonstrated that a portal venous infusion of hypertonic NaCI solution elicits a decrease in jejunal absorption of NaCI [7]. The afferent pathway of this reflex is the hepatic nerves and the efferent pathway is the cholinergic fibers to the jejunum [7]. However, the central mechanisms of the hepatojejunal reflex are still unclear. Horseradish peroxidase and electrophysiological studies demonstrated that the hepatic vagal afferents project upon the nucleus tractus
* Corresponding author. Tel.: 11878-98-5111, ext. 2432: Fax: 0878-98-8346.
solitarius (NTS) [1-5,9]. Kobashi and Adachi [4,5] demonstrated that some neurons in the NTS responded to an intraportal infusion of hypertonic saline. From these observations, there is a possibility that NTS may be involved in the central pathway of the hepatojejunal reflex. To test this hypothesis, the hepatojejunal reflex was examined in rats with a chemical inactivation of the NTS by microinjection of kainic acid (KA). All experiments were conducted in 24 male Wister Kyoto rats weighing 220-250 g (8-10 weeks old). Rats were deprived of food 24 h before experiments. Water remained available throughout the food-deprivation period. Rats were anesthetized with inhalation of ether, then catheters for administration of urethanc (0.5 g / k g ) and a-chloralose (30 m g / k g ) and for mea-
0165-1838;/94/$07.011 ~ 1994 Elsevier Science B.V. All rights teselved SSD1 0 1(~5- 1838103)E111 43-S
2(18
H. Morita et al. /Journal of the Autonomic" Nerl'ous System 48 (1994) 207-212
suring arterial pressure were inserted into the femoral vein and artery. The trachea was intubated. Through the central laparotomy, the jejunal loop was made, which was 20 cm long and started from 3 cm distal to the duodeno-jejunal junction. The Mop was intubated at both ends. The proximal tube was for the infusion of the solution and the distal tube was for the collection of the solution. A non-occlusive catheter was inserted into the portal vein. In eight of the 24 rats, hepatic denervation was performed by section of anterior and posterior hepatic plexuses and stripping off the hepatic artery, portal vein, and bile duct. The connective tissue around these vessels and duct was severed. Then a coating of 5c~ phenol in 70% ethanol solution was applied to these areas. The hepatic nerve branches on the gastrohepatic ligament were also severed. The intestine was returned to the abdominal cavity. The tubes and catheter were exteriorized, and the incision was closed. Then the rat was placed in a stereotaxic frame with the head inclined downward by 45 ° (Summit Medical, Tokyo). To examine the jejunal absorption, Ringer's solution (Na 147.0, CI 155.5, K 4.0, Ca 4.5 in m E q / l ) at 37°C was perfused from the proximal tube, at a constant rate of 0.3 m l / m i n for 30 rain, using a syringe p u m p (Terufusion model STC 523, Terumo). Phenol red (25 r a g / l ) was added to the Ringer's solution as a non-absorbable volume marker. Perfused fluid was collected into a test tube from the distal tube. Na and C1 concentrations of infused and collected fluid were measured with a flame photometer and CI counter (Hitachi No. 750). Osmolality was measured with an o s m o m e t e r (Fiske OS Osmometer). Phenol red concentration was determined with a spect r o p h o t o m e t e r (Hitachi 320) at 558 nm, p H 8.4. The volume of the collected fluid was also measured. The calculated collected volume was determined by 9 ml (infused v o l u m e ) × phenol red concentration of the infused f l u i d / p h e n o l red concentration of the collected fluid. The yield of phenol red was calculated by measured collected v o l u m e / c a l c u l a t e d collected volume. If the yield of phenol red was less than 0.95, the data were not included in the text. The calculated collected volume was used as the collected volume. Net
absorption of fluid, Na, and CI, and osmolality were calculated as the differences between absolute values of the infused fluid and the collected fluid. A 30-60-min equilibration period was observed after the operation. Net absorption of the jejunal loop was measured during a portal infusion of 0.9% or 9% NaCI solution at a constant rate of 0.01 m l / 1 0 0 g b . w . / m i n for 30 rain. The order of the infusions was randomized. A 30-rain equilibration period was observed between the infusions. After the end of the second absorption experiment an occipital craniotomy was performed, The caudal portion of the 4th ventricle was exposed by incising the dura and arachnoid. A glass micropipette with tip diameter of 50-100 /.~m was filled with artificial cerebrospinal fluid (aCSF: Na 152, K 3.7, Mg 1.2, Ca 1.8, C1 140, H 2 P O 4 1.2, SO 4 1.2, HCO3 15, glucose 10 (in mM), 0.5% methylene blue) or KA (100 n g / # l dissolved in aCSF) was then placed in a micromanipulator. The micropipette was lowered to the calamus scriptorius, which served as the rostalcaudal (AP) and lateral (L) zero reference point. In all studies, the pipette was positioned over the intermediate portion of NTS (AP + 0.5 mm: L +0.5 mm) and lowered 0.8 mm beneath the dorsal surface of the brainstem. Because the hepatic afferents predominantly terminate to the caudal-intermediate parts of the NTS [1,2,9], this part was selected for inactivation. Bilateral injection of aCSF (n = 8) or KA (n -- 16) in a volume of 0.3 /~1 was performed at a rate of 0.3 # l / r a i n using a Harvard infusion p u m p (Harvard Apparatus, model 1140-001). A l-h equilibration period was observed, then net absorption of the jejunal loop was measured during portal infusion of 0.9% or 9% NaC1 solution. For histological examination of the brainstem the rat was anesthetized and killed by perfusion of 10% formalin through the heart. The brainstem was removed and 50-/.tm frozen sections were taken through the NTS and stained with neutral red, then histological verification of the site of injection was made using a standard atlas [8]. If the dorsal motor nucleus of the vagus was stained with methylene blue, the data were not included in the text.
H. Morita et al. /Journal of the Autonomic Nervous System 48 (1994) 207-212
All the values presented are mean _+ SEM. To evaluate the effect of intraportal infusion of 0.9% or 9% NaCI solution and microinjection of aCSF or KA into the NTS, net absorption was compared using a two-way analysis of variance. When the F ratio exceeded the critical value, Tukey's test was applied to test the significance of the differences among the mean values. Statistical significance was considered at P < 0.05. All data presented have passed the criteria about phenol red yield and histological examination. The portal infusion of 9% NaC1 solution for 30 rain increased mean arterial pressure from 105 + 2 to 118 +_ 2 mm Hg (Table 1). The portal infusion of 9% NaC1 solution decreased net absorption of electrolytes and osmolality. This response was not affected by the microinjection of aCSF into the NTS (Fig. 1). Furthermore, bilateral microinjection of aCSF into the NTS did not affect net absorption of electrolytes and osmolality during both portal infusions of 0.9% and 9% NaCI solution. Unilateral inactivation of NTS by a microinjection of KA transiently decreased arterial pressure. Arterial pressure was increased by the subsequent contralateral inactivation of NTS, then gradually returned to the pre-injection level. One
2A,.7~ <51E~
Table 1 Effects of intraportal infusion of 0.9% or 9% NaC[ solution on mean arterial pressure (mm Hg) before and after microinjection of KA or aCSF into the NTS Intact (aCSF) Before 0.9% NaCI During 0.9% NaCI
104_+3 108+2
Before 9% NaCI During 9% NaCI Before KA or aCSF After KA or aCSF Before 0.9% NaCI During 0.9% NaCI Before 9% NaCI During 9% NaCI
1115+2 118_+2" 118+2 115+2 111 + 4 115-+3 118+2 126-+3 *
Intact (KA) 96_+5 99_+5 96_+6 116-+6" 104-+8 128_+9" 100±8 104+8 1t13-+7 120+6 *
Hepatic denervation 92+5 t~3+4 88+5 1111+5" 1113_+4 136_+7" 102+5 105+4 101)+5 117+ 6 *
Values presented are mean + SEM. Intact (aCSF): intact rats with a microinjection of artificial cerebrospinal fluid into the NTS. Intact (KA): intact rats with a microinjection of kainic acid into the NTS. Hepatic denervation: hepatic denervated rats with a microinjection of kainic acid. * P < 0.05, significantly different from the Before value.
hour after the bilateral injection of KA into NTS, the absorption experiment was started. Before the bilateral inactivation of the NTS, the portal infusion of 9% NaC1 solution decreased net absorption of electrolytes and osmolality. Bilateral
~°'250125 ~~.~aCSF intoNTS
'~°~
o
209
.-
o
0.9%9%aC0'9°/°9%SFinoN~" "
"
° oo o
0.9%9% 0.9%9%
=" 0.9%9*/0 0.9%9%
o
0
Fig. 1. Effects of bilateral microinjection of artificial cerebrospinal fluid (aCSF) into the NTS on net fluid, Na, and CI absorption and osmolality absorption in the rat jejunum. Jejunal absorption was measured during intraportal infusion of 0.9c~ or 9% NaCI solution. Bars represent mean _+ SEM (n = 8). * P < 0.05.
H. Morita et al. /Journal o[ the Autonomic Nert'ous System 48 (1994) 207-212
210 A
o~
2
KAinto NTS_
_*"~KA into NTS
oE250[
g,
~ 12s
~= o
~
o
z
== t 25
~ 200
0
z
0 0.9% 9%
0.9% 9%
0
z
0.9% 9%
0.9% 9%
Fig. 2. Effects of bilateral microinjection of kainic acid (KA) into the NTS on net fluid, Na, CI and osmolality absorption in the rat jejunum. Jejunal absorption was measured during intraportal infusion of 0.9% or 9% NaCI solution. Bars represent mean + SEM (n = 8). * P < 0.05.
inactivation of the NTS completely abolished the decreases in net absorption of electrolytes and osmolality induced by the portal infusion of 9% NaCI solution (Fig. 2). To examine the afferent pathway of the decrease in net absorption induced by the portal
._~
2
KA into NTS
infusion of 9% NaCl solution, net absorption was examined in eight rats with hepatic denervation. In rats with hepatic denervation, the portal infusion of 9% NaCl had no effect on net jejunal absorption either before or after bilateral injection of KA into the NTS (Fig. 3).
.-~ 250
"
0 ~ .'~ ,'7
~
125
~, o
.=
o
._~ 250
;"
o
== t25
~ 200
o
o 0.9% 9%
0.9% 9%
z
0 0.9% 9%
0.9% 9%
Fig. 3. Effects of bilateral microinjection of kainic acid (KA) into the NTS on net fluid, Na, CI and osmolality absorption in the jejunal loop in rats with hepatic denervation. Jejunal absorption was measured during intraportal infusion of 0.9¢~4 or 9% NaCI solution. Bars represent mean _+ SEM (n = 8). * P < 0.05.
ft. Morita et al. /Journal ~" the Autonomic Nert'ous System 48 (1994) 207-212
In the present study, hypertonic NaCI infusion in the portal vein increased mean arterial pressure and decreased net jejunal absorption of fluid, Na, C1, and osmolality. An increase in arterial pressure itself depresses intestinal absorption through the baroreceptor reflex [11]. However, the decrease in net absorption observed in the present study was not due to the increase in arterial pressure for the following reason. The decrease in net absorption induced by the portal venous infusion of hypertonic NaC1 was not observed in rats with hepatic denervation, although arterial pressure increased. Thus, the depression of net absorption was mediated by the hepatic nerves, not by the baroreceptors. These findings are consistent with our previous study in dogs [7]. The depression of net absorption was also abolished by the microinjection of KA into the bilateral NTS. It appears that the effects of microinjection of KA into the NTS were due to an inactivation of nerve cell bodies in the NTS region. The previous study from our laboratory demonstrated that the efferent pathway of the hepatojejunal reflex was the cholinergic fibers to the jejunum, since the reflex was abolished by pretreatment of atropine [7]. Thus, it is also possible that an inactivation of the dorsal motor nucleus of the vagus, the origin of the parasympathetic preganglionic fibers, abolishes the hepatojejunal reflex by interrupting the efferent pathway. This interpretation may not be true, since methylene blue was restricted in the NTS region but the dorsal motor nucleus of the vagus was not stained by methylene blue, i.e., the dorsal motor nucleus of the vagus was structurally intact. However, there is a possibility that the dorsal motor nucleus of the vagus was functionally damaged and inactivated. Martin et al. [6] reported that electrical and chemical stimulation of the dorsal motor nucleus of the vagus reduced intestinal absorption. If the dorsal motor nucleus of the vagus were inactivated, net jejunal absorption might increase through a withdrawal of the vagus tone, and the increase in jejunal absorption should be abolished by a pretreatment of atropine. However, the previous study from our laboratory demonstrated that the increase in jejunal absorption secondary to the microinjection of KA into
211
the NTS was not abolished by atropine but was completely abolished by yohimbine [12]. These observations suggest that microinjection of KA into the NTS produced a localized NTS inactivation and the dorsal motor nucleus of the vagus might be functionally intact. In conclusion, the present study demonstrated that the inactivation of the NTS abolished the hepatojejunal reflex. Thus, the NTS may be involved in the central pathway of the hepatojejunal reflex. It will now be important to ascertain the neural pathway betwccn the NTS and the cholinergic fibers to the jejunum, the efferent pathway of the hepatojejunal reflex.
Acknowledgements This study was supported in part by Research Grant for Cardiovascular Disease 3A-1 from the Ministry of Health and Welfare of Japan and Research Grant from The Salt Science Research Foundation 920B.
References [1] Adachi, A., Electrophysiological study of hepatic vagal projection to the medulla, Neurosci. Len., 24 (1981) 19-23. [2] Adachi, A., Projection of the hepatic vagal nerve in the medulla oblongata, J. Auton. Nerv. Syst., 111 (1984) 287293. [3] Kobashi, M. and Adachi, A., Convergence of hepatic osmoreceptive inputs on sodium-responsive units within the nucleus of the solitary tract of thc rat, J. Neurophysiol., 54 (1985) 212-219. [4] Kobashi, M. and Adachi, A., Projection of nucleus tractus solitarius units influenced by hepatoportal afferent signal to parabrachial nucleus, J. Auton. Nerv. Syst., 16 (1986) 153-158. [5] Kobashi, M. and Adachi, A., A hepatoportal osmoreceptire afferent projection from nucleus tractus solitarius to caudal ventrolatera[ medulla, Brain Res., 24 (199(I) 775 778. [6] Martin, K., Kong, T.H., Renehan, W., Schurr, A., Dong. W., Zhang, X. and Fogel, R., Identification and function of brain stem neurons regulating rat ileal water absorptkm, Am. J. Physiol., 257 (1989) G266 (_}273. [7] Morita, l-t.. Ohyama, H., Hagiike, M., Horiba, T., Miyakc. K., Yamanouchi, H., Matsushita, K. and Hosomi, II.. Effccts of portal infusion of hypertonic solution on jeju
212
H. Morita et al. /Journal of the Autonomic Nert'ous System 48 (1994) 207-212
nal electrolyte transport in anesthetized dogs, Am. J. Physiol., 259 (1990) R 1289- R 1294. [8] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic coordinates, Academic Press, New York, 1986. [9] Rogers, R.C. and Hermann, G.E., Central connections of the hepatic branch of the vagus nerve: a horseradish peroxidase histochemical study, J. Auton. Nerv. Syst., 7 (1983) 165-174. [1(1] Sawchenko, P.E. and Friedman, M.I., Sensory functions of the liver - a review, Am. J. Physiol.~ 236 (1979) R5- R2(1.
[11] Sj6vall, H., Butcher, P., Biber, B. and Martner, J., Carotid sinus baroreceptor modulation of fluid transport and blood flow in the feline jejunum. Am. J. Physiol., 25(1 (1986) G736-G741. [12] Tanaka, K., Morita, H., Suwaki, H., Hosokawa, K and Hosomi, H., Effects of microinjection of kainic acid into the nucleus tractus solitarius on fluid and NaCI absorption across the jejunum, J. Auton. Nerv. Syst. (1994) in press.
"•
Journal of the Autonomic Nervous
~
ELSEVIER
System
Journal of the Autonomic Nervous System 48 (1994) 207-212
Chemical inactivation of the nucleus tractus solitarius abolished hepatojejunal reflex in the rat Hironobu
M o r i t a *, K a z u t a k a T a n a k a , H i r o s h i H o s o m i
Department of Physiology, Kagawa Medical School, Kagawa 761-07, Japan Received 3 September 1993: revision received and accepted 16 November 1093
Abstract
Jejunal electrolyte absorption was measured in the jejunal loop of anesthetized rats during an infusion of 9¢;NaCI solution via the portal vein. Net absorption of Na and CI, and osmolality were significantly depressed by the portal 9% NaCl infusion. The decrease in net absorption was completely abolished by hepatic denervation. In addition, bilateral chemical inactivation of the nucleus tractus solitarius (NTS) by microinjection of kainic acid abolished the depressing effect of the portal venous infusion of 9% NaCI. These findings indicate that NTS is inw)lved in the central pathway of the hepatojejunal reflex. Key words: Hepatojejunal reflex; Jejunal absorption; Hepatic nerve: Nuclcus tractus solitarius; Portal vein: Hypertonic NaCI
It has been well documented that the liver contains many receptors, including the baroreceptor, osmoreceptor, and NaCI receptor [10]. Recently, we have demonstrated that a portal venous infusion of hypertonic NaCI solution elicits a decrease in jejunal absorption of NaCI [7]. The afferent pathway of this reflex is the hepatic nerves and the efferent pathway is the cholinergic fibers to the jejunum [7]. However, the central mechanisms of the hepatojejunal reflex are still unclear. Horseradish peroxidase and electrophysiological studies demonstrated that the hepatic vagal afferents project upon the nucleus tractus
* Corresponding author. Tel.: 11878-98-5111, ext. 2432: Fax: 0878-98-8346.
solitarius (NTS) [1-5,9]. Kobashi and Adachi [4,5] demonstrated that some neurons in the NTS responded to an intraportal infusion of hypertonic saline. From these observations, there is a possibility that NTS may be involved in the central pathway of the hepatojejunal reflex. To test this hypothesis, the hepatojejunal reflex was examined in rats with a chemical inactivation of the NTS by microinjection of kainic acid (KA). All experiments were conducted in 24 male Wister Kyoto rats weighing 220-250 g (8-10 weeks old). Rats were deprived of food 24 h before experiments. Water remained available throughout the food-deprivation period. Rats were anesthetized with inhalation of ether, then catheters for administration of urethanc (0.5 g / k g ) and a-chloralose (30 m g / k g ) and for mea-
0165-1838;/94/$07.011 ~ 1994 Elsevier Science B.V. All rights teselved SSD1 0 1(~5- 1838103)E111 43-S
2(18
H. Morita et al. /Journal of the Autonomic" Nerl'ous System 48 (1994) 207-212
suring arterial pressure were inserted into the femoral vein and artery. The trachea was intubated. Through the central laparotomy, the jejunal loop was made, which was 20 cm long and started from 3 cm distal to the duodeno-jejunal junction. The Mop was intubated at both ends. The proximal tube was for the infusion of the solution and the distal tube was for the collection of the solution. A non-occlusive catheter was inserted into the portal vein. In eight of the 24 rats, hepatic denervation was performed by section of anterior and posterior hepatic plexuses and stripping off the hepatic artery, portal vein, and bile duct. The connective tissue around these vessels and duct was severed. Then a coating of 5c~ phenol in 70% ethanol solution was applied to these areas. The hepatic nerve branches on the gastrohepatic ligament were also severed. The intestine was returned to the abdominal cavity. The tubes and catheter were exteriorized, and the incision was closed. Then the rat was placed in a stereotaxic frame with the head inclined downward by 45 ° (Summit Medical, Tokyo). To examine the jejunal absorption, Ringer's solution (Na 147.0, CI 155.5, K 4.0, Ca 4.5 in m E q / l ) at 37°C was perfused from the proximal tube, at a constant rate of 0.3 m l / m i n for 30 rain, using a syringe p u m p (Terufusion model STC 523, Terumo). Phenol red (25 r a g / l ) was added to the Ringer's solution as a non-absorbable volume marker. Perfused fluid was collected into a test tube from the distal tube. Na and C1 concentrations of infused and collected fluid were measured with a flame photometer and CI counter (Hitachi No. 750). Osmolality was measured with an o s m o m e t e r (Fiske OS Osmometer). Phenol red concentration was determined with a spect r o p h o t o m e t e r (Hitachi 320) at 558 nm, p H 8.4. The volume of the collected fluid was also measured. The calculated collected volume was determined by 9 ml (infused v o l u m e ) × phenol red concentration of the infused f l u i d / p h e n o l red concentration of the collected fluid. The yield of phenol red was calculated by measured collected v o l u m e / c a l c u l a t e d collected volume. If the yield of phenol red was less than 0.95, the data were not included in the text. The calculated collected volume was used as the collected volume. Net
absorption of fluid, Na, and CI, and osmolality were calculated as the differences between absolute values of the infused fluid and the collected fluid. A 30-60-min equilibration period was observed after the operation. Net absorption of the jejunal loop was measured during a portal infusion of 0.9% or 9% NaCI solution at a constant rate of 0.01 m l / 1 0 0 g b . w . / m i n for 30 rain. The order of the infusions was randomized. A 30-rain equilibration period was observed between the infusions. After the end of the second absorption experiment an occipital craniotomy was performed, The caudal portion of the 4th ventricle was exposed by incising the dura and arachnoid. A glass micropipette with tip diameter of 50-100 /.~m was filled with artificial cerebrospinal fluid (aCSF: Na 152, K 3.7, Mg 1.2, Ca 1.8, C1 140, H 2 P O 4 1.2, SO 4 1.2, HCO3 15, glucose 10 (in mM), 0.5% methylene blue) or KA (100 n g / # l dissolved in aCSF) was then placed in a micromanipulator. The micropipette was lowered to the calamus scriptorius, which served as the rostalcaudal (AP) and lateral (L) zero reference point. In all studies, the pipette was positioned over the intermediate portion of NTS (AP + 0.5 mm: L +0.5 mm) and lowered 0.8 mm beneath the dorsal surface of the brainstem. Because the hepatic afferents predominantly terminate to the caudal-intermediate parts of the NTS [1,2,9], this part was selected for inactivation. Bilateral injection of aCSF (n = 8) or KA (n -- 16) in a volume of 0.3 /~1 was performed at a rate of 0.3 # l / r a i n using a Harvard infusion p u m p (Harvard Apparatus, model 1140-001). A l-h equilibration period was observed, then net absorption of the jejunal loop was measured during portal infusion of 0.9% or 9% NaC1 solution. For histological examination of the brainstem the rat was anesthetized and killed by perfusion of 10% formalin through the heart. The brainstem was removed and 50-/.tm frozen sections were taken through the NTS and stained with neutral red, then histological verification of the site of injection was made using a standard atlas [8]. If the dorsal motor nucleus of the vagus was stained with methylene blue, the data were not included in the text.
H. Morita et al. /Journal of the Autonomic Nervous System 48 (1994) 207-212
All the values presented are mean _+ SEM. To evaluate the effect of intraportal infusion of 0.9% or 9% NaCI solution and microinjection of aCSF or KA into the NTS, net absorption was compared using a two-way analysis of variance. When the F ratio exceeded the critical value, Tukey's test was applied to test the significance of the differences among the mean values. Statistical significance was considered at P < 0.05. All data presented have passed the criteria about phenol red yield and histological examination. The portal infusion of 9% NaC1 solution for 30 rain increased mean arterial pressure from 105 + 2 to 118 +_ 2 mm Hg (Table 1). The portal infusion of 9% NaC1 solution decreased net absorption of electrolytes and osmolality. This response was not affected by the microinjection of aCSF into the NTS (Fig. 1). Furthermore, bilateral microinjection of aCSF into the NTS did not affect net absorption of electrolytes and osmolality during both portal infusions of 0.9% and 9% NaCI solution. Unilateral inactivation of NTS by a microinjection of KA transiently decreased arterial pressure. Arterial pressure was increased by the subsequent contralateral inactivation of NTS, then gradually returned to the pre-injection level. One
2A,.7~ <51E~
Table 1 Effects of intraportal infusion of 0.9% or 9% NaC[ solution on mean arterial pressure (mm Hg) before and after microinjection of KA or aCSF into the NTS Intact (aCSF) Before 0.9% NaCI During 0.9% NaCI
104_+3 108+2
Before 9% NaCI During 9% NaCI Before KA or aCSF After KA or aCSF Before 0.9% NaCI During 0.9% NaCI Before 9% NaCI During 9% NaCI
1115+2 118_+2" 118+2 115+2 111 + 4 115-+3 118+2 126-+3 *
Intact (KA) 96_+5 99_+5 96_+6 116-+6" 104-+8 128_+9" 100±8 104+8 1t13-+7 120+6 *
Hepatic denervation 92+5 t~3+4 88+5 1111+5" 1113_+4 136_+7" 102+5 105+4 101)+5 117+ 6 *
Values presented are mean + SEM. Intact (aCSF): intact rats with a microinjection of artificial cerebrospinal fluid into the NTS. Intact (KA): intact rats with a microinjection of kainic acid into the NTS. Hepatic denervation: hepatic denervated rats with a microinjection of kainic acid. * P < 0.05, significantly different from the Before value.
hour after the bilateral injection of KA into NTS, the absorption experiment was started. Before the bilateral inactivation of the NTS, the portal infusion of 9% NaC1 solution decreased net absorption of electrolytes and osmolality. Bilateral
~°'250125 ~~.~aCSF intoNTS
'~°~
o
209
.-
o
0.9%9%aC0'9°/°9%SFinoN~" "
"
° oo o
0.9%9% 0.9%9%
=" 0.9%9*/0 0.9%9%
o
0
Fig. 1. Effects of bilateral microinjection of artificial cerebrospinal fluid (aCSF) into the NTS on net fluid, Na, and CI absorption and osmolality absorption in the rat jejunum. Jejunal absorption was measured during intraportal infusion of 0.9c~ or 9% NaCI solution. Bars represent mean _+ SEM (n = 8). * P < 0.05.
H. Morita et al. /Journal o[ the Autonomic Nert'ous System 48 (1994) 207-212
210 A
o~
2
KAinto NTS_
_*"~KA into NTS
oE250[
g,
~ 12s
~= o
~
o
z
== t 25
~ 200
0
z
0 0.9% 9%
0.9% 9%
0
z
0.9% 9%
0.9% 9%
Fig. 2. Effects of bilateral microinjection of kainic acid (KA) into the NTS on net fluid, Na, CI and osmolality absorption in the rat jejunum. Jejunal absorption was measured during intraportal infusion of 0.9% or 9% NaCI solution. Bars represent mean + SEM (n = 8). * P < 0.05.
inactivation of the NTS completely abolished the decreases in net absorption of electrolytes and osmolality induced by the portal infusion of 9% NaCI solution (Fig. 2). To examine the afferent pathway of the decrease in net absorption induced by the portal
._~
2
KA into NTS
infusion of 9% NaCl solution, net absorption was examined in eight rats with hepatic denervation. In rats with hepatic denervation, the portal infusion of 9% NaCl had no effect on net jejunal absorption either before or after bilateral injection of KA into the NTS (Fig. 3).
.-~ 250
"
0 ~ .'~ ,'7
~
125
~, o
.=
o
._~ 250
;"
o
== t25
~ 200
o
o 0.9% 9%
0.9% 9%
z
0 0.9% 9%
0.9% 9%
Fig. 3. Effects of bilateral microinjection of kainic acid (KA) into the NTS on net fluid, Na, CI and osmolality absorption in the jejunal loop in rats with hepatic denervation. Jejunal absorption was measured during intraportal infusion of 0.9¢~4 or 9% NaCI solution. Bars represent mean _+ SEM (n = 8). * P < 0.05.
ft. Morita et al. /Journal ~" the Autonomic Nert'ous System 48 (1994) 207-212
In the present study, hypertonic NaCI infusion in the portal vein increased mean arterial pressure and decreased net jejunal absorption of fluid, Na, C1, and osmolality. An increase in arterial pressure itself depresses intestinal absorption through the baroreceptor reflex [11]. However, the decrease in net absorption observed in the present study was not due to the increase in arterial pressure for the following reason. The decrease in net absorption induced by the portal venous infusion of hypertonic NaC1 was not observed in rats with hepatic denervation, although arterial pressure increased. Thus, the depression of net absorption was mediated by the hepatic nerves, not by the baroreceptors. These findings are consistent with our previous study in dogs [7]. The depression of net absorption was also abolished by the microinjection of KA into the bilateral NTS. It appears that the effects of microinjection of KA into the NTS were due to an inactivation of nerve cell bodies in the NTS region. The previous study from our laboratory demonstrated that the efferent pathway of the hepatojejunal reflex was the cholinergic fibers to the jejunum, since the reflex was abolished by pretreatment of atropine [7]. Thus, it is also possible that an inactivation of the dorsal motor nucleus of the vagus, the origin of the parasympathetic preganglionic fibers, abolishes the hepatojejunal reflex by interrupting the efferent pathway. This interpretation may not be true, since methylene blue was restricted in the NTS region but the dorsal motor nucleus of the vagus was not stained by methylene blue, i.e., the dorsal motor nucleus of the vagus was structurally intact. However, there is a possibility that the dorsal motor nucleus of the vagus was functionally damaged and inactivated. Martin et al. [6] reported that electrical and chemical stimulation of the dorsal motor nucleus of the vagus reduced intestinal absorption. If the dorsal motor nucleus of the vagus were inactivated, net jejunal absorption might increase through a withdrawal of the vagus tone, and the increase in jejunal absorption should be abolished by a pretreatment of atropine. However, the previous study from our laboratory demonstrated that the increase in jejunal absorption secondary to the microinjection of KA into
211
the NTS was not abolished by atropine but was completely abolished by yohimbine [12]. These observations suggest that microinjection of KA into the NTS produced a localized NTS inactivation and the dorsal motor nucleus of the vagus might be functionally intact. In conclusion, the present study demonstrated that the inactivation of the NTS abolished the hepatojejunal reflex. Thus, the NTS may be involved in the central pathway of the hepatojejunal reflex. It will now be important to ascertain the neural pathway betwccn the NTS and the cholinergic fibers to the jejunum, the efferent pathway of the hepatojejunal reflex.
Acknowledgements This study was supported in part by Research Grant for Cardiovascular Disease 3A-1 from the Ministry of Health and Welfare of Japan and Research Grant from The Salt Science Research Foundation 920B.
References [1] Adachi, A., Electrophysiological study of hepatic vagal projection to the medulla, Neurosci. Len., 24 (1981) 19-23. [2] Adachi, A., Projection of the hepatic vagal nerve in the medulla oblongata, J. Auton. Nerv. Syst., 111 (1984) 287293. [3] Kobashi, M. and Adachi, A., Convergence of hepatic osmoreceptive inputs on sodium-responsive units within the nucleus of the solitary tract of thc rat, J. Neurophysiol., 54 (1985) 212-219. [4] Kobashi, M. and Adachi, A., Projection of nucleus tractus solitarius units influenced by hepatoportal afferent signal to parabrachial nucleus, J. Auton. Nerv. Syst., 16 (1986) 153-158. [5] Kobashi, M. and Adachi, A., A hepatoportal osmoreceptire afferent projection from nucleus tractus solitarius to caudal ventrolatera[ medulla, Brain Res., 24 (199(I) 775 778. [6] Martin, K., Kong, T.H., Renehan, W., Schurr, A., Dong. W., Zhang, X. and Fogel, R., Identification and function of brain stem neurons regulating rat ileal water absorptkm, Am. J. Physiol., 257 (1989) G266 (_}273. [7] Morita, l-t.. Ohyama, H., Hagiike, M., Horiba, T., Miyakc. K., Yamanouchi, H., Matsushita, K. and Hosomi, II.. Effccts of portal infusion of hypertonic solution on jeju
212
H. Morita et al. /Journal of the Autonomic Nert'ous System 48 (1994) 207-212
nal electrolyte transport in anesthetized dogs, Am. J. Physiol., 259 (1990) R 1289- R 1294. [8] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic coordinates, Academic Press, New York, 1986. [9] Rogers, R.C. and Hermann, G.E., Central connections of the hepatic branch of the vagus nerve: a horseradish peroxidase histochemical study, J. Auton. Nerv. Syst., 7 (1983) 165-174. [1(1] Sawchenko, P.E. and Friedman, M.I., Sensory functions of the liver - a review, Am. J. Physiol.~ 236 (1979) R5- R2(1.
[11] Sj6vall, H., Butcher, P., Biber, B. and Martner, J., Carotid sinus baroreceptor modulation of fluid transport and blood flow in the feline jejunum. Am. J. Physiol., 25(1 (1986) G736-G741. [12] Tanaka, K., Morita, H., Suwaki, H., Hosokawa, K and Hosomi, H., Effects of microinjection of kainic acid into the nucleus tractus solitarius on fluid and NaCI absorption across the jejunum, J. Auton. Nerv. Syst. (1994) in press.