- Email: [email protected]
Receptor autoradiography with [3H]L-glutamate reveals the presence and axonal transport of glutamate receptors in vagal afferent neurones of the rat
European Journal of Pharmacology, 144 (1987) 413-415 Elsevier
413
EZJP 096RC
Rapid communication
Receptor autoradiography with [ 3H]L-glutamate reveals the presence and axonal transport of glutamate receptors in vagal afferent neurones of the rat Stephen J. Lewis ‘, Marion Cincotta, Anthony J.M. Verberne, Bevyn Jarrott, David Lodge 2
and Philip M. Beart * University of Melbourne, Clinical Pharmacology and Therapeutics Unii, Austin Hospital, Heidelberg, Victoria 3084, Australia and 2 Department of Physiology, Royal Veterinary College, London N WI OTU, U.K.
Received
9 November
1987, accepted
The perikarya of vagal afferent neurones are located within the inferior vagal (nodose) ganglia (Palkovits and Zaborsky, 1977). Recent evidence suggests that receptors for a variety of putative neurotransmitters/neuromodulators may be synthesized within these perikarya and then delivered by axonal transport mechanisms in the vagus nerve to their central and/or peripheral processes where they are incorporated into the terminal membranes (Laduron, 1984; Young et al., 1980). In this study we provide evidence from receptor autoradiography with [ 3H]L-glutamate that binding sites for the excitatory transmitter L-glutamate, are associated with the perikarya of vagal afferent neurones and that these receptors undergo axonal transport in the peripheral vagal trunks. Female Sprague-Dawley rats (225-250 g) were anaesthetized with an amylobarbitone-methohexitone (30 and 16.7 mg/kg i.p. respectively) mixture. In 4 rats the left nodose ganglion was exposed and the vagus nerve was tightly ligatured (siliconised silk thread, 6/O; Dynek Pty. Ltd., Australia) approximately 1 cm distal to the gan-
’ Present address: Department vascular Center, University U.S.A. * To whom all correspondence 0014-2999/87/$03.50
of Pharmacology and Cardioof Iowa, Iowa City, IA 52242, should
be addressed.
0 1987 Elsevier Science Publishers
11 November
1987
glion. In control rats (n = 4) a loosely fitting ligature was placed at the same distance from the left ganglion. Upon conclusion of the surgery, the wound was closed and the animals maintained in a warm, sterile environment. After 24 h, the animals were reanaesthetized and the vagi were sectioned a few millimetres distal to the ligature or sham-ligature, and proximal to the nodose ganglion. The pharyngeal and superior laryngeal nerves were cut near their point of connection with the ganglion (see fig. la). Each vagus nerve and associated nodose ganglion were then removed and immediately frozen in mounting medium (TissueTek, Miles Scientific, U.S.A.) at -20°C overnight. Longitudinal sections (10 pm) including the ganglion and vagus nerve, were then cut using a cryostat and thaw-mounted on to glass microscope slides for receptor autoradiography with [ 3H]L-glutamate (Cincotta et al., 1987). Tissue sections were air dried, preincubated in 50 mM Tris-citrate pH 7.0 for 20 min and dried in a stream of cool air before incubation for 10 min in 50 mM Tris-citrate pH 7.0, containing 2.5 mM Ca*’ and 20 mM Cl-, with [3H]L-glutamate (1 PM; 42 Ci/mmol, Amersham, U.K.). Rapid washing for 0.5 min in ice-cold Tris-citrate followed. Full details of this procedure will be published at a later date. Parallel sections incubated with 1 mM L-glutamate served to define non-specific binding. Thorough drying in air and over silica gel was carried out, before sections were apposed to
B.V. (Biomedical
Division)
414
NG
×
~,~L
8. Fig. 1. (A) Histology of a control nodose ganglion and vagus nerve stained with Pyronin Y. (B) Autoradiographic labelling patterns after ligation of vagus nerve and incubation with L[ ~H]L-glutamate. Arrowheads point to accumulation of silver grains. Abbreviations: N G nodose ganglion, SL superior laryngeal nerve, X vagus nerve, L ligation. Bar = 1 mm.
3H-sensitive film (Hyperfilm, Amersham) for 40 days. In the sham-ligated vagus nerve the binding of [3H]L_glutamate was almost completely associated with the nodose ganglion. The binding was near background in the vagal trunk except for a few isolated instances. Combined densitometric and histological analyses demonstrated that the majority of glutamate binding sites within the nodose ganglion were probably associated with the perikarya of vagal afferent neurones (see fig. lb). In this section of the nodose ganglia (and all of those examined) there was a widespread but uneven distribution of binding sites, with some areas showing intense labelling. In further studies where the displacement of [3H]L-glutamate by Nmethyl-D-aspartate (NMDA, 1 mM), a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (1
/xM) and kainate (1 mM) was examined by direct scintillation spectrometry, these subtype-selective glutamate agonists were found to displace respectively 26 + 4, 16 +_ 2 and 57 + 2% (all n = 4) of total [3H]L-glutamate binding. The distributions of [3H]L-glutamate binding sites within the left nodose ganglion (sections through the approximate middle of the structure) and associated peripheral vagal trunk 24 h after ligation of the vagus nerve are shown in fig. lb. In the ligated vagus nerve, the binding of [3H]Lglutamate was just as evident as in the nodose ganglion, but was now also present in the vagal trunk proximal to and to a lesser extent distal to the ligature (fig. lb). The densities of these sites~ especially proximal to the ligature, appeared to be at least as dense as that seen in the ganglion itself. The present study provides evidence that glutamate receptors are associated with the perikarya of vagal afferent neurones and that these receptors undergo bidirectional axonal flow in the peripheral vagal trunks. Moreover, our results demonstrate that all subtypes of glutamate receptors are present in the nodose ganglion/vagus system. However, kainate receptors seem to predominate and interestingly dorsal root C fibres have been reported to be selectively depolarized by kainate, but much less sensitive to quisqualate and insensitive to NMDA (Agrawal and Evans, 1986). Overall, these findings are consistent with the suggestion that receptors for neurotransmitters/neuromodulators are synthesized within the perikarya of vagal afferent neurones and then either incorporated into the perikaryal membranes and/or transported axonally toward the peripheral (and central) terminals (Laduron, 1984). Moreover, the finding that the glutamate receptors accumulate on the distal side of the vagal ligature is consistent with recent evidence that opiate receptors on peripheral vagal terminals disassociate themselves from the membranes and are then transported back toward their perikarya (kaduron, 1984). While the functional significance of the possible presence of glutamate receptors on the perikarya and peripheral terminals of vagal afferent neurones remains to be determined, this study represents the first demonstration of the axonal transport of glutamate receptors.
415
Acknowledgements This work was supported by a Programme Grant from the National Health and Medical Research Council of Australia. D. Lodge acknowledges receipt of grants from the Wellcome Trust and Ramaciotti Foundation.
References Agrawal, S.G. and R.H. Evans, 1986, The primary afferent depolarizing action of kainate in the rat, Br. J. Pharmacol. 87, 345.
Cincotta, M., F.A.O. Mendelsohn, W.J. Louis and P.M. Beart, 1987, Comparison of [3H]glutamate binding in the brain of the spontaneously hypertensive and Wistar rat, Neurosci. Lett. Suppl. 27, 63. Laduron, P.M., 1984, Axonal transport of receptors: coexistence with neurotransmitter and recycling, Biochem. Pharmacol. 33, 897. Palkovits, M. and L. Zaborszky, 1977, Neuroanatomy of central cardiovascular control. Nucleus tractus solitarii: afferent and efferent neuronal connections in relation to the baroreceptor reflex arc, Prog. Br. Res. 47, 9. Young, W.S., J.K. Wamsley, M.A. Zarbin and M.J. Kuhar, 1980, Opioid receptors undergo axonal flow, Science 210, 76.
413
EZJP 096RC
Rapid communication
Receptor autoradiography with [ 3H]L-glutamate reveals the presence and axonal transport of glutamate receptors in vagal afferent neurones of the rat Stephen J. Lewis ‘, Marion Cincotta, Anthony J.M. Verberne, Bevyn Jarrott, David Lodge 2
and Philip M. Beart * University of Melbourne, Clinical Pharmacology and Therapeutics Unii, Austin Hospital, Heidelberg, Victoria 3084, Australia and 2 Department of Physiology, Royal Veterinary College, London N WI OTU, U.K.
Received
9 November
1987, accepted
The perikarya of vagal afferent neurones are located within the inferior vagal (nodose) ganglia (Palkovits and Zaborsky, 1977). Recent evidence suggests that receptors for a variety of putative neurotransmitters/neuromodulators may be synthesized within these perikarya and then delivered by axonal transport mechanisms in the vagus nerve to their central and/or peripheral processes where they are incorporated into the terminal membranes (Laduron, 1984; Young et al., 1980). In this study we provide evidence from receptor autoradiography with [ 3H]L-glutamate that binding sites for the excitatory transmitter L-glutamate, are associated with the perikarya of vagal afferent neurones and that these receptors undergo axonal transport in the peripheral vagal trunks. Female Sprague-Dawley rats (225-250 g) were anaesthetized with an amylobarbitone-methohexitone (30 and 16.7 mg/kg i.p. respectively) mixture. In 4 rats the left nodose ganglion was exposed and the vagus nerve was tightly ligatured (siliconised silk thread, 6/O; Dynek Pty. Ltd., Australia) approximately 1 cm distal to the gan-
’ Present address: Department vascular Center, University U.S.A. * To whom all correspondence 0014-2999/87/$03.50
of Pharmacology and Cardioof Iowa, Iowa City, IA 52242, should
be addressed.
0 1987 Elsevier Science Publishers
11 November
1987
glion. In control rats (n = 4) a loosely fitting ligature was placed at the same distance from the left ganglion. Upon conclusion of the surgery, the wound was closed and the animals maintained in a warm, sterile environment. After 24 h, the animals were reanaesthetized and the vagi were sectioned a few millimetres distal to the ligature or sham-ligature, and proximal to the nodose ganglion. The pharyngeal and superior laryngeal nerves were cut near their point of connection with the ganglion (see fig. la). Each vagus nerve and associated nodose ganglion were then removed and immediately frozen in mounting medium (TissueTek, Miles Scientific, U.S.A.) at -20°C overnight. Longitudinal sections (10 pm) including the ganglion and vagus nerve, were then cut using a cryostat and thaw-mounted on to glass microscope slides for receptor autoradiography with [ 3H]L-glutamate (Cincotta et al., 1987). Tissue sections were air dried, preincubated in 50 mM Tris-citrate pH 7.0 for 20 min and dried in a stream of cool air before incubation for 10 min in 50 mM Tris-citrate pH 7.0, containing 2.5 mM Ca*’ and 20 mM Cl-, with [3H]L-glutamate (1 PM; 42 Ci/mmol, Amersham, U.K.). Rapid washing for 0.5 min in ice-cold Tris-citrate followed. Full details of this procedure will be published at a later date. Parallel sections incubated with 1 mM L-glutamate served to define non-specific binding. Thorough drying in air and over silica gel was carried out, before sections were apposed to
B.V. (Biomedical
Division)
414
NG
×
~,~L
8. Fig. 1. (A) Histology of a control nodose ganglion and vagus nerve stained with Pyronin Y. (B) Autoradiographic labelling patterns after ligation of vagus nerve and incubation with L[ ~H]L-glutamate. Arrowheads point to accumulation of silver grains. Abbreviations: N G nodose ganglion, SL superior laryngeal nerve, X vagus nerve, L ligation. Bar = 1 mm.
3H-sensitive film (Hyperfilm, Amersham) for 40 days. In the sham-ligated vagus nerve the binding of [3H]L_glutamate was almost completely associated with the nodose ganglion. The binding was near background in the vagal trunk except for a few isolated instances. Combined densitometric and histological analyses demonstrated that the majority of glutamate binding sites within the nodose ganglion were probably associated with the perikarya of vagal afferent neurones (see fig. lb). In this section of the nodose ganglia (and all of those examined) there was a widespread but uneven distribution of binding sites, with some areas showing intense labelling. In further studies where the displacement of [3H]L-glutamate by Nmethyl-D-aspartate (NMDA, 1 mM), a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (1
/xM) and kainate (1 mM) was examined by direct scintillation spectrometry, these subtype-selective glutamate agonists were found to displace respectively 26 + 4, 16 +_ 2 and 57 + 2% (all n = 4) of total [3H]L-glutamate binding. The distributions of [3H]L-glutamate binding sites within the left nodose ganglion (sections through the approximate middle of the structure) and associated peripheral vagal trunk 24 h after ligation of the vagus nerve are shown in fig. lb. In the ligated vagus nerve, the binding of [3H]Lglutamate was just as evident as in the nodose ganglion, but was now also present in the vagal trunk proximal to and to a lesser extent distal to the ligature (fig. lb). The densities of these sites~ especially proximal to the ligature, appeared to be at least as dense as that seen in the ganglion itself. The present study provides evidence that glutamate receptors are associated with the perikarya of vagal afferent neurones and that these receptors undergo bidirectional axonal flow in the peripheral vagal trunks. Moreover, our results demonstrate that all subtypes of glutamate receptors are present in the nodose ganglion/vagus system. However, kainate receptors seem to predominate and interestingly dorsal root C fibres have been reported to be selectively depolarized by kainate, but much less sensitive to quisqualate and insensitive to NMDA (Agrawal and Evans, 1986). Overall, these findings are consistent with the suggestion that receptors for neurotransmitters/neuromodulators are synthesized within the perikarya of vagal afferent neurones and then either incorporated into the perikaryal membranes and/or transported axonally toward the peripheral (and central) terminals (Laduron, 1984). Moreover, the finding that the glutamate receptors accumulate on the distal side of the vagal ligature is consistent with recent evidence that opiate receptors on peripheral vagal terminals disassociate themselves from the membranes and are then transported back toward their perikarya (kaduron, 1984). While the functional significance of the possible presence of glutamate receptors on the perikarya and peripheral terminals of vagal afferent neurones remains to be determined, this study represents the first demonstration of the axonal transport of glutamate receptors.
415
Acknowledgements This work was supported by a Programme Grant from the National Health and Medical Research Council of Australia. D. Lodge acknowledges receipt of grants from the Wellcome Trust and Ramaciotti Foundation.
References Agrawal, S.G. and R.H. Evans, 1986, The primary afferent depolarizing action of kainate in the rat, Br. J. Pharmacol. 87, 345.
Cincotta, M., F.A.O. Mendelsohn, W.J. Louis and P.M. Beart, 1987, Comparison of [3H]glutamate binding in the brain of the spontaneously hypertensive and Wistar rat, Neurosci. Lett. Suppl. 27, 63. Laduron, P.M., 1984, Axonal transport of receptors: coexistence with neurotransmitter and recycling, Biochem. Pharmacol. 33, 897. Palkovits, M. and L. Zaborszky, 1977, Neuroanatomy of central cardiovascular control. Nucleus tractus solitarii: afferent and efferent neuronal connections in relation to the baroreceptor reflex arc, Prog. Br. Res. 47, 9. Young, W.S., J.K. Wamsley, M.A. Zarbin and M.J. Kuhar, 1980, Opioid receptors undergo axonal flow, Science 210, 76.