Involvement of hypothalamic noradrenergic systems in the modulation of intestinal motility in rats

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Brain Research, 583 (1992) 332-335 © 1982 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

BRES 25266

Involvement of hypothalamic noradrenergic systems in the modulation of intestinal motility in rats B. Q

Bonaz a,b,

L.

Martin b,

E.

Beurriand a,

J.

Hostein band

C.

Feuerstein a

Laboratoire de Physiologie, Section Neurophysiologie, /nstitut Nationalde la Sante et de la Recherche Medicale, U318 and b Groupede Recherche en MotriciteDigestive, Hopita!A. Michallon, CentreHospitalier Regional Universitaire, BP 2/7, Grenoble, (France) (Accepted 7 April 1992)

Key words: Noradrenergic system; Paraventricular nucleus; Lesion ; 6-Hydroxydopamine; Intestinal motility

Selective lesions of the noradrenergic systems of the paraventricular nucleus (PVN) of the hypothalamus with 6·hydroxydopamine (6-0HDA) lengthen the periodicity of the migrating myoelectric complex (MMC), an index of intestinal motility, in rats . These lengthening effects resemble those obtained after lesions of the locus coeruleus (LC), thus suggesting that noradrenergic terminals from LC to the PVN are involved in thi s modulation.

There is compelling evidence that brain noradrenergic systems are able to modulate intestinal motility in rats , as reflected by one of its patterns: the migrating myoelectric complex (MMC). MMC is characteristic of fasting intestinal motility and is divided into three distinct phases of activity with a mean periodicity of 15 min in rats; such an activity is interrupted by a meal':", It has been previously shown that MMC period is significantly lengthened by disruption of noradrenergic systems, not only sympathetic ones, as illustrated by the effects of intraperitoneal injections of 6-hydroxydopamine (6-0HDA), a noradrenergic neurotoxic 10, but also brain noradrenergic ones, as illustrated by intracerebroventricular injections of 6-0HDAl . The aforementioned results could even conclude that the destruction of brain noradrenergic systems were effective on MMC periodicity, without alteration of peripheral sympathetic innervation. Since intracerebroventricular injections of 6-0HDA are responsible for a diffuse lesion of brain noradrenergic systems, it could only be suggested that the rostral ones should play the major modulatory role'. Effectively, selective lesions of spinal noradrenergic systems via intracisternal injections of 6-0HDA are unable to modify MMC periodicity ' , Interestingly, locus coeruleus (LC) seems to be a

major candidate for this modulation by rostral noradrenergic systems". Such an assumption relies on the consequences of selective lesions of LC, which are also responsible for a MMC lengthening effect, while lesions of the noradrenergic pathways originating in A1A2 medulla noradrenergic groups are not effect 2. Among the target sites of rostral LC projections, the paraventricular nucleus (PVN) of the hypothalamus represents a key structure, since it can control autonomic efferent systems'<". Accordingly, the aim of this study was to investigate whether noradrenergic systems projecting to the PVN might participate in the modulation of MMC patterns in rats . For this purpose, selective stereotaxic lesions of the PVN were performed with 6-0HDA. Functional effects of these lesions were evaluated by long-term electromyographic recording of intestinal motility' r', Eighteen male Wistar rats (270-290 g) were used . One group of animals (n = 12) was stereotaxically and bilaterally injected into the PVN with 6-0HDA (Sigma, St. Louis, MO ; 5 JoLg 6-0HDA hydrobromide in 1 JoLI of 0.9% NaCI containing 0.01% ascorbic acid/injection). Coordinates (in mm) were : anterior - 1, lateral ± 0.6, ventral -7.3 from dura, bregma being the zero reference point. Injections were ensured via glass mi-

Correspondence: B. Bonaz, Laboratoire de Physiologic, Section Neurophysiologie, INSERM U318, Pavilion de Neurologie, Hopital A. Michallon, CHRU, BP 217, 38043 Grenoble Cedex 09, France. Fax: (33) (76) 76765631.

333 cropipettes (50 JLm external diameter at the tip) using a previously described method''. Another group of animals (n = 6) was sham operated (controls) and received the vehicle alone at the same coordinates. One month after injections, intestinal motility was recorded during 15 days, in freely moving animals, as previously described'<. Briefly, nichrome wire electrodes (80 JLm in diameter and 90 ern in length) were implanted in the duodenojejunal wall at a distance of 5, 15 and 30 em, respectively, from the pylorus; in rats fasted for 15 h, spiking activity was amplified by an electroencephalograph (ECEM T3 +, Ozoir-laFerriere, France). After a 6-h recording, a 2 g pelleted rat diet was given to interrupt the MMC cycle. Between two recording sessions, rats had free access to food and water for 2 days. At the end of motility recordings, the efficiency of noradrenergic lesions was verified via both norepinephrine immunohistochemistry and catecholamine measurement. Those animals (n = 12, controls and lesioned) treated for norepinephrine immunohistochemistry were intracardially perfused with 5% glutaraldehyde solution; coronal sections at the level of PVN and pons-medulla were cryostat cut (20 JLm thick) at - 20°C and then further processed with the peroxidase-antiperoxidase complex method as described-!'. In the other animals (n = 6, controls and lesioned), unperfused brain was taken out and frozen; 500 JLm thick coronal sections containing the PVN were cryostat cut at - 7°C, the PVN was dissected and then homogenized in perchloric acid (0.4 N) for further catecholamine assays according to radioenzymatic procedure 1,12. A dramatic decrease of noradrenergic terminals was observed in the PVN as compared with control animals, in which the classical pattern of distribution of brain noradrenergic perikarya and terminals was observed1,2,5. Noradrenergic perikarya of pons-medulla and their terminals visualized in other locations of the brain appeared unaffected. These histochemical pat-

TABLE I Catecholamine concentrations in ng / g (mean ± S.E.M.) in the PVN from 500 urn thick frozen sections Comparisons between values from control and lesioned animals were made according to the Student's t-test. NS = non-significant.

Controls Norepinephrine Dopamine Epinephrine

right PVN left PVN right PVN left PVN right PVN left PVN

6-0HDA

1762 (±257) 593 (± 33) P < 0.01 1564 (iSI) 776 (± 68) P < 0.01 158 (±35) 156 (±3) NS 184 (±30) 204 (±8) NS 78 (± 15) 85(±19) NS 92 (±27) 89 (± 26) NS

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Fig. 1. A: mean values (± S.E.M.) of MMC period (min) determined at a distance of 5, 15 and 30 em from the pylorus (P). B: mean values (± S.E.M.) of phase I, II and III duration (min). Filled columns, are controls, and hatched columns are 6-0HDA injected animals in the PVN. Results were analyzed by a variance analysis, post hoc paired comparisons being made by a Student's r-test, as previously described'r'. Asterisks indicate values which are significantly different: * P < 0.05, ** P < 0.01, *** P < 0.001.

terns were in agreement with a significant (P < 0.01) decrease of norepinephrine content in the PVN. Conversely, dopamine and epinephrine content remained unaffected (Table I). In both groups of animals, the myoelectric activity of the duodenojejunum was typically organized as a classical MMC. However, in 6OHDA injected animals, MMC period was significantly lengthened, mean values ± S.E.M. being 20.8 ± 0.5, 17.3 ± 0.5 and 19.4 ± 1.2 vs. 17.6 ± 0.5, 14.4 ± 0.8 and 16.2 ± 0.8 min in control animals at a distance of 5, 15 and 30 cm, respectively, from the pylorus (Fig. l A), These lengthening effects were essentially due to

334 a parallel lengthening of phase II and, to a lesser extent, of phase III duration, phase I being shortened. The mean duration of MMC phases was 2.1 ± 0.2 min phase I, 12.9 ± 0.5 min phase II and 4.2 ± 0.3 min phase III, whereas it was respectively 3.0 ± 0.1 min, 9.9 ± 0.4 min and 3.2 ± 0.1 min in control animals (Fig. LB), The duration of the post-prandial interruption of the MMC was not significantly modified (127 ± 8 min, 108 ± 10 and 79 ± 10 min in 6-0HDA injected animals vs. 140 ± 6 min, 93 ± 8 min and 71 ± 10 min in control animals at a distance of 5, 15 and 30 em, respectively, from the pylorus). This study demonstrates that lesions of PVN noradrenergic systems are able to lengthen the MMC period in rats. This lengthening effect may be attributable to selective noradrenergic lesions of PVN since neither modification of dopamine and epinephrine content in the PVN nor lesion of pons-medulla noradrenergic perikarya (notably the LC) and their terminals in other locations than the PVN, was observable. One may exclude the indirect destruction of other noradrenergic perikarya via diffusion of 6-0HDA in the nearby cerebrospinal fluid of the third ventricle since very weak or even no destruction of noradrenergic systems could be detected when doses as high as 10 J-Lg of 6-0HDA were injected intracerebroventricularly". The volume and the dose of 6-0HDA injected into the PVN here are in the same order of magnitude as that previously reported by others, the norepinephrine decrease in the PVN being comparable's". A marked depletion of both norepinephrine (90%) and epinephrine (80%) has been observed in the PVN following 6-0HDA injection in the ventral noradrenergic bundle'"; however, the location and the dose (three times higher than ours) were different. The lengthening effect observed in this study is close to that obtained after selective lesions of the LC 2, mean duration (±S.E.M.) of MMC period being slightly more important in the latter case (23.6 ± 1.4 min, 19.7 ± 0.9 min and 20.5 ± 1.0 min at a distance of 5, 15 and 30 em, respectively, from the pylorus). The lengthening effect of the treatment on MMC period is only 20% of controls. It is still worth noting that control values here are superposable onto control values tn = 33) from our previous studies'<. In addition, after lesion of the LC 2 , the lengthening of MMC was never superior to 40%. Differential patterns of noradrenergic lesions are observed in the PVN depending on whether 6-0HDA is injected into the PVN itself or

into the LC: both terminals coming from AI-A2 and A6 noradrenergic perikarya" are lesioned in the former case, whereas only LC noradrenergic terminals are destroyed in the latter. In the absence of modification of MMC patterns when noradrenergic pathways com-

ing from AI-A2 are destroyed ", it is most likely that the above effects of PVN lesions are related to disruption of LC terminals projecting to the PVN. This confirms the role of the LC in the modulation of MMC, particularly its rostral projections /. Nevertheless, this does not exclude eventual interactions between AI-A2 groups and A6, both at the pons-medulla region and/or at PVN terminal site. The effects of 6-0HDA-induced PVN lesions on MMC patterns might involve hypothalamo-vagal projections'S". Indeed, MMC modifications observed in this study are essentially due to parallel modifications of phase II duration. Since phase II duration has been reported to be modulated by vagal activity8, one may suggest that these PVN noradrenergic lesions would lengthen MMC period via increased vagal activity, via hypothalamovagal interactions. This does not exclude the involvement of hypothalamic projections to the spinal cord, particularly at the level of preganglionar sympathetic neurons or the intermediate participation of CRF, from the PVN 15, which has been reported to be able to influence gut motility 3.17. Such hypothetical mechanisms remain yet to be investigated. In conclusion this study confirms that noradrenergic systems of the PVN modulate MMC patterns in rats. In light of previous data concerning the effect of selective lesions of LC 2, this effect likely involves, at least in part , the noradrenergic terminals from LC to the PVN. We thank INSERM U318 and Association Grenobloise pour Ie Developpement de la Recherche en Gastroenterologie for financial assistance. 1 Bonaz, B., Martin, L.,Beurriand, E., Manier, M., Hostein, J. and Feuerstein, C, Modulation of the migrating myoelectric complex by brain noradrenergic systems in rats, Am. 1. Physiol. iGastrointest. Liver Physiol. 25),260 (1992) G1121-G1126 . 2 Bonaz, B., Martin, L., Beurriand , E., Manier, M., Hostein, J. and Feuerstein, C. , Locus coeruleus modulates the migrating myo-

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