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Effect of preganglionic stimulation or chronic decentralization on neurotensin-like immunoreactivity in sympathetic ganglia of the cat
Brain Research, 482 (1989) 365-370 Elsevier
365
BRE 23414
Effect of preganglionic stimulation or chronic decentralization on neurotensin-Iike immunoreactivity in sympathetic ganglia of the cat Monica M. Caverson 1, Manjit Bachoo l, John Ciriello 2 and Canio Polosa 1 1Department of Physiology, McGill University, Montreal, Que. (Canada) and 2Departmentof Physiology, Health Sciences Centre, The University of Western Ontario, London, Ont. (Canada) (Accepted 6 December 1988) Key words: Neurotensin; Radioimmunoassay;Stellate ganglion; Superior cervical ganglion; Non-cholinergictransmission; Sympathetic preganglionic neuron
In pentobarbital-anesthetized cats, supramaximal stimulation (40 Hz, 2 h) of the preganglionicinput to the acutely decentralized right stellate (RSG) or superior cervical (RSCG) ganglion resulted in a decrease in neurotensin (NT)-like immunoreactivity (IR), by 83% in the SG and by 46% in the SCG, as determined by radioimmunoassay. Chronic (7 days) decentralization of the ganglia resulted in a similar depletion of NT-like IR (SG: 86%; SCG: 76%). Supramaximal stimulation (40 Hz, 2 h) of the intact postganglionic outflow of either ganglion had no effect on NT-like IR. These data suggest that NT in the SG and SCG is present in preganglionic axons and is released by activation of these axons.
The tridecapeptide neurotensin (NT) 7"8 has been demonstrated immunohistochemically in neuronal perikarya of thoracolumbar sympathetic preganglionic neurons in the cat 15. In the same species, a dense network of NT-immunoreactive (IR) fibers has been demonstrated in sympathetic ganglia 3'x3"16, and NT has been shown to accumulate in thoracic white rami or ventral roots proximal to a ligation 16. Preganglionic stimulation of the stellate (SG) or superior cervical (SCG) ganglia with 40 Hz pulse trains, during block of cholinergic transmission, has been shown to produce slow, long-lasting effector organ responses due to excitation of ganglion cells 1" 4.10. Prolonged supramaximal stimulation (40 Hz, 2 h) of the preganglionic input to the SG of the cat results in a loss of the heart rate response mediated by non-cholinergic transmission in the SG 4. Coincident with the loss of this heart rate response is a decrease in NT-like immunoreactivity (IR) in preganglionic fibers and terminals in the SG 3. This evidence was interpreted to suggest that NT stores were depleted during preganglionic stimulation 3. In
addition, indirect evidence of NT receptors on SG cells is provided by the observation that close intra-arterial injections of NT produces excitation of ganglion cells 5. Taken together, this evidence suggests that NT functions as a transmitter in sympathetic ganglia. However, the previous immunohistochemical data 3 are only qualitative. Therefore, in the present study a radioimmunoassay for NT was used to establish that NT in the SG and SCG is contained in preganglionic axons by measuring the NT content in these ganglia after chronic decentralization and to provide an estimate of the fraction of the ganglionic NT that is lost as a consequence of prolonged preganglionic stimulation. A preliminary account of some of these data has been published 9. Experiments were done in adult cats of either sex (2.7-6.4 kg) anesthetized with sodium pentobarbital (35 mg/kg i.p. followed by 9 mg/kg i.v. every 3 h). After intubation of the trachea, the cats were paralyzed with pancuronium bromide (0.2 mg/kg i.v. followed by 0.1 mg/kg every 2 h) and artificially
Correspondence: M.M. Caverson, Department of Anatomy, Health Sciences Centre, The Universityof Western Ontario, London, Ont., Canada N6A 5C1. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
366 ventilated. End-tidal C O 2 w a s monitored (Beckman LB2 analyzer) and maintained within 4-5%. Rectal temperature was monitored and maintained at 37 + 0.5 °C by a feedback-controlled infrared heating lamp. The left femoral artery and vein were cannulated to record arterial pressure (AP) and for the administration of drugs, respectively. Heart rate (HR) was continuously recorded with a tachograph triggered by the AP pulse. The nictitating membrane on the right side was connected with silk thread to a force transducer, the output of which was continuously recorded, together with AP and HR, on a Grass model 7 polygraph. The right SG (RSG) was identified and prepared for stimulation after removal of the first 3 ribs. Orthodromic stimulation of the preganglionic input was done using bipolar silver hook electrodes placed on the T l, T 2 and T 3 white rami (WR) as well as on the sympathetic trunk just below T3WR. The postganglionic fibers from the RSG were antidromically stimulated with an electrode placed on the ventral ansa subclavia. The SG on both sides was excised immediately after a 2 h period of stimulation of the preganglionic or postganglionic fibers. In experiments in which the RSG was chronically decentralized, the ganglion was approached through the second intercostal space. The T~WR, T2WR, T3WR as well as the sympathetic trunk above T~WR and below T3WR were transected on the right side. The chest incision was closed and the animals were allowed to recover from the anesthesia and were given post-operative care. Seven days after transection the animals were anesthetized and the SG was removed on both sides. The SCG was isolated on both sides through a midline incision in the neck. Approximately 1 cm of the right cervical sympathetic trunk (RCST) was dissected free, desheathed and placed on a bipolar silver hook electrode for orthodromic stimulation of the preganglionic fibers. The right internal and external carotid and pterygopalatine nerves were isolated, desheathed and placed on an electrode for antidromic stimulation of the postganglionic fibers. Immediately following a 2 h period of stimulation of the pre- or postganglionic fibers, the SCG was excised on both sides. In additional animals, chronic decentralization of the RSCG was done by transection of the RCST. After transection, the neck
incision was closed, the animals were allowed to recover from the anesthesia and 7 days later the animals were anesthetized and the SCG on both sides was excised. Orthodromic or antidromic stimulation was done using a Grass $88 stimulator connected to a stimulus isolation unit. Stimuli consisted of square wave pulses of 0.5 ms duration, 40 Hz frequency and supramaximal intensity (10 V). After determining the intensity for maximal responses of HR and nictitating membrane (3-5 V) cholinergic transmission was blocked with hexamethonium bromide (30 mg/kg i.v. and supplemented by an infusion of 1 mg/kg/h) and atropine methylbromide (1 mg/kg i.v.). The slow responses of the HR 4 and the nictitating membrane ~'2"1° to preganglionic stimulation, due to non-cholinergic transmission in the ganglion, were monitored during a 2 h period of orthodromic stimulation. The H R and nictitating membrane response to postganglionic stimulation were monitored during a 2 h period of antidromic stimulation. NT content of the SG and SCG was measured using a NT radioimmunoassay kit (Milab, Sweden) based on the procedures outlined by Pedersen and F a h r e n k r u f 9. Immediately after orthodromic or antidromic stimulation or 7 days following decentralization, the SG and SCG were excised on both sides. All nerve bundles were cut as close to the ganglia as possible and the ganglia were immediately extracted according to the procedure outlined by Bryant and Bloom 6. The ganglia were quickly desheathed, weighed, cut into small pieces, and extracted in 1 ml of 0.5 M boiling acetic acid for 15 rain. The supernatant was removed, the pH was adjusted to 7.4 with 1 N NaOH and the extracts were stored at -70 °C until assayed. To determine whether sensory afferent fibers contributed to the NT content measured in the SG and SCG, NT in the nodose (n = 3) and in the T 2 dorsal root ganglia (n = 2) was measured in these control tissue. Samples were assayed in duplicate according to the manufacturer's instructions (Milab, Sweden). In brief, 450/~1 of NT antiserum (Milab no. R759A04-1, rabbit anti-NT; 1:128,000) was incubated with 450/A o f [125I]NT2° and 250 pl of samples, controls or standards for 72 h at 4 °C in assay buffer (0.02 M barbital buffer, pH 8.6, with 0.2% human serum
367 albumin, 0.05% thimerosal and 500 klU/ml aprotinin; Sigma Chemicals, St. Louis, MO, U.S.A.). NT (Milab, Sweden) dissolved in assay buffer was used as standards. The working range was 15.6 to 1000 pg/ml. Free NT was separated from antibody-bound NT by the addition of a 1 ml suspension containing 2.5% activated charcoal (Milab, Sweden) and 10% human serum (Sigma Chemicals, U.S.A.) in assay buffer. The bound fraction contained within the supernatant was counted in an LKB gamma-counter. The recovery was 97% at 125 pg/ml. Our values have not been corrected for loss during extraction. The sensitivity of the assay was 7.0 pg/ml with 95% confidence. Multiple serial dilutions of all unknown samples were assayed in duplicate so as to span the sensitive part of the standard curve. Only those dilutions within the sensitive range of the standard curve were used in the calculation of the results. Intra-assay variability evaluated by 6-fold estimation of standards with 15.6, 125 and 1000 pg/ml NT was 7.6%, 5.1% and 8.3%, respectively. Inter-assay variability estimated by 6-fold measurements of the same sample (125 pg/ml) in 7 assays within 12 weeks was 25.9%. The NT antibody used in this study recognizes the 1-13 amino acid sequence. For details on the cross-reactivity of the antibody see Pedersen and Fahrenkrug 19 and Pedersen et al. 2°. No cross-reactivity with secretin, gastric inhibitory peptide, vasoactive intestinal peptide, peptide histidine-isoleucine, pancreatic glucagon, and pancreatic polypeptide has been detected (Milab, Sweden) 19"2°. All values of NT-like IR are expressed as means + S.E. Comparisons between RSG and left (L)SG, RSCG and LSCG or SG(R + L) and SCG(R + L) in control ganglia were made using Student's t-test for independent variables. Comparisons between stimulated or decentralized ganglia (right side) and control ganglia (left side) were made using the Mann-Whitney test after Bartlett's test indicated an heterogeneity of variance 22. A P value of 0.05 or less was taken to indicate statistical significance. Qualitatively similar results were obtained following chronic decentralization or stimulation of the SG or SCG. Therefore, the data for both ganglia will be described together for the 4 experimental conditions: control (no stimulation, intact innervation), chronic decentralization, preganglionic (orthodro-
mic) stimulation and postganglionic (antidromic) stimulation. These data are summarized in Table I. Control conditions. The content of NT-like IR in the RSG and LSG, as well as in the RSCG and LSCG, was not significantly different. Therefore, the left ganglion was used as a control in subsequent experiments in which the right ganglion was stimulated or decentralized. Control levels of NT-like IR in the SG were approximately 7-8-fold greater than that in the SCG (Table I). Chronic decentralization. The NT-like IR in ganglia excised 7 days after decentralization was significantly (P < 0.01) lower than that in the contralateral control ganglia. The NT content was decreased by 86% in the RSG and by 76% in the RSCG (Table I). The weight of the decentralized RSG was observed to be greater than that of the control LSG. However, this effect was not observed in the decentralized RSCG compared to the intact LSCG. Preganglionic (orthodromic) stimulation. Electrical stimulation (2 h) of the preganglionic input to the RSG and RSCG resulted in a significant (P < 0.05) decrease in the NT-like IR as compared to contralateral control ganglia. The NT content was decreased by 83% in the RSG and by 46% in the RSCG (Table I). Postganglionic (antidromic) stimulation. The NTlike IR in ganglia excised after 2 h stimulation of the postganglionic fibers was not significantly different from that in control ganglia (Table I). Control tissue. The content of NT-like IR in control tissue was considerably lower than that observed in the SG or SCG under control conditions. In extracts of the nodose and the T 2 dorsal root ganglia the NT-like IR ranged from 0.7 to 5.6 pmol/g tissue. The results of this study demonstrate that stimulation of the nerves that provide preganglionic input to, but not of the nerves that are the output of, the SG and SCG produce a decrease in NT-like IR. The lack of effect of stimulation of the intact postganglionic fibers suggests that the decrease in NT-like IR is not the result of increased discharge of sympathetic ganglion cells, as postganglionic stimulation would result in antidromic excitation of these cells at a rate similar to, and possibly higher than, that produced by orthodromic stimulation 21. Moreover, since the same sensory afferents would be
368 TABLE 1 NT-like IR in SG and SCG 7 days after decentralization and after orthodromic or antidromic stimulation
Values are means ± S.E.M. RSG, right stellate ganglion; LSG, left stellate ganglion; RSCG, right superior cervical ganglion; LSCG. left superior cervical ganglion; Ortho, orthodromic; Anti, antidromic. % Difference in concentration was determined by comparing experimental (right) ganglia to control (left) ganglia. No. o f cats
Experimental condition
Ganglion weight (rag)
Content (pg/ganglion )
Concentration (pmol/g wet wt. )
5
Control RSG Control LSG Control RSCG Control LSCG
21 _+3 21 ± 3 17 ± 2 18 ± 2
3607 _+_510 3650 ± 518 399 + 80 401 ± 85
110 ± 20 118 ± 26 15 ± 4 15 + 4
Decentralized RSG Control LSG Decentralized RSCG Control LSCG
40 ± 5* 23 + 4 20 _+2 20 ± 3
792 ± 93** 3766 ± 909 112 ± 36** 462 _+26
12 ± 2** 94 _+4 4 + 2** 15 ± 2
Ortho stimulation RSG Control LSG Ortho stimulation RSCG Control LSCG
20 + 1 16 + 2 18 ± 2 18 _+3
545 _+79* 2674 + 410 152 _+24* 276 ± 45
17 ± 2* 98 + 9 5 ± 1* 10 _+_2
Anti stimulation RSG Control LSG Anti stimulation RSCG Control LSCG
17 + 2 16 ± 1 16 + 4 16 +_3
2858 + 311 2825 + 277 445 ± 112 470 _+64
8 5 5 4 6 2 2
104 + 2 106 ± 2 19 _+7 19 ± 8
% Difference in concentration -
7%
0% -86% -76% -83% -47%
-
2%
0%
* Significantlydifferent from control (P < 0.05); **significantlydifferent from control (P < 0.01).
present in both preganglionic and postganglionic nerves 18, the lack of a decrease in NT-like IR with postganglionic stimulation suggests that the decrease in NT-like IR with preganglionic stimulation is not the result of a decrease in NT in sensory afferent
can be eliminated as the source of the decreased NT-like IR. It has been shown that N T can be released from neural tissue by depolarization or high [K+], and that this release is Ca2+-dependent 14'17.
fibers. This finding is supported by previous immu-
suggest that the ganglionic N T content is associated with preganglionic axons, it may be expected that the invasion of preganglionic axon terminals by
nohistochemical data in which no changes were observed in the immunoreactivity pattern of fibers and presumed axon terminals in the SG as a result of prolonged (2 h) stimulation of the postganglionic fibers 3. In addition, the.low content of NT-like IR measured in dorsal root and nodose ganglia in the present study and that reported previously in dorsal roots ~6 suggests that sensory afferents are unlikely to contribute significantly to the NT content of the SG and SCG. The present results also demonstrate that the content of NT-like IR in the SG and SCG decreased as a result of chronic preganglionic denervation. This suggests that the ganglionic N T content is associated with preganglionic axons. Therefore, it is likely that preganglionic nerve stimulation depletes NT from preganglionic axons, since sensory axon collaterals
Since the experiments of chronic decentralization
action potentials results in the release of NT. Therefore, if the action potential frequency is high (e.g. 40 Hz), as was used in this study, the rate of release of N T may exceed the rate of replacement of this substance, or of its precursors 12, resulting in depletion. How closely the content of NT-like IR in the SG or SCG compares after preganglionic stimulation or denervation depends on how completely NT, presumably contained in the degenerating axons, has been degraded and removed, and on the relative proportion of the releasable and non-releasable fraction of the total N T pool. In the SG, the close-matching of the depletion of NT-like IR as a result of chronic decentralization and of prolonged orthodromic stimulation suggests that most of the
369 N T contained in the SG is readily releasable by preganglionic nerve stimulation. On the o t h e r hand, the decrease in NT-like I R in the S C G o b t a i n e d with o r t h o d r o m i c stimulation was 62% of that obtained with chronic decentralization, suggesting that some of the NT-like I R contained in preganglionic axons in the S C G may not be releasable. It was o b s e r v e d that the wet weight of the R S G was significantly greater than the L S G after chronic decentralization. H o w e v e r , this effect was not noted in the R S C G . The reason for this difference in wet weight is unclear. Nevertheless, the concentration of NT, which incorporates the weight of the ganglion, was significantly decreased in the R S G after chronic decentralization. A n o t h e r interesting observation in this study was the 7 - 8 - f o l d greater content of NT-like I R in control S G as c o m p a r e d to control SCG. Recent immunohistochemical data support this finding as a greater density of NT-like I R fibers and p r e s u m e d axon terminals were o b s e r v e d in the S G c o m p a r e d to the S C G 3"11'13. The reason for this difference is unclear; however, a possible explanation is that the SG receives a greater n u m b e r of preganglionic axons 1 Alkadhi, K.A. and Mclsaac, R.J., Non-nicotinic transmission during ganglionic block with chlorisondamine and nicotine, Eur. J. Pharmacol., 24 (1973) 78-85. 2 Bachoo, M., Ciriello, J., Caverson, M.M. and Polosa, C., Non-cholinergic transmission in the superior cervical ganglion of the cat, Can. Physiol., 18 (1987) 101. 3 Bachoo, M., Ciriello, J. and Poiosa, C., Effect of preganglionic stimulation on neuropeptide-like immunoreactivity in the stellate ganglion of the cat, Brain Research, 400 (1987) 377-382. 4 Bachoo, M., Isacoff, E. and Polosa, C., Slow cardioacceleration mediated by non-cholinergic transmission in the stellate ganglion of the cat, Can. J. Physiol. Pharmacol., in press. 5 Bachoo, M. and Polosa, C., Cardioacceleration produced by close intra-arterial injection of neurotensin into the stellate ganglion of the cat, Can. J. Physiol. Pharmacol., 66 (1988) 408-412. 6 Bryant, M.G. and Bloom, S.R., Measurement in tissues. In S.R. Bloom and R.G. Long (Eds.), Radioimmunoassay of Gut Regulatory Peptides, Saunders, Canada, 1982, pp. 36-41. 7 Carraway, R. and Leeman S.E., The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami, J. Biol. Chem., 248 (1973) 6854-6861. 8 Carraway, R. and Leeman, S.E., The amino acid sequence of a hypothalamic peptide, neurotensin, J. Biol. Chem., 250 (1975) 1907-1911. 9 Caverson, M.M., Bachoo, M., Ciriello, J. and Polosa, C., Effect of prolonged preganglionic stimulation on radioim-
than the SCG. Alternatively, the possibility may exist that preganglionic axons containing N T preferentially innervate SG cells. This m a y be due to factors, perhaps related to the target of ganglion cell innervation, which d e t e r m i n e the type of n e u r o p e p tide synthesized by a given sympathetic preganglionic neuron or the selective innervation of specific ganglion cells by certain preganglionic axons. In conclusion, quantitative observations r e p o r t e d in the present study, of a decrease in the content of r a d i o i m m u n o a s s a y a b l e NT-like I R in the SG and SCG after o r t h o d r o m i c stimulation or chronic decentralization of the preganglionic axons, provide evidence to suggest that N T may function as a neurotransmitter or n e u r o m o d u l a t o r at sympathetic ganglionic synapses.
This work was s u p p o r t e d by the M R C of C a n a d a , the Q u e b e c H e a r t F o u n d a t i o n , the D y s a u t o n o m i a F o u n d a t i o n and the H e a r t and Stroke F o u n d a t i o n of Ontario. M . M . C . is a C a n a d i a n H e a r t F o u n d a t i o n Research Scholar and J.C. is a C a r e e r Investigator of the H e a r t and Stroke F o u n d a t i o n of Ontario.
10 11
12
13
14
15
16
munoassayable neurotensin (NT) in the superior cervical ganglion (SCG) of the cat, Soc. Neurosci. Abstr., 14 (1988) 24. Chen, S.S., Late contraction of nictitating membrane of the dog, Am. J. Physiol., 217 (1969) 1205-1210. Ciriello, J., Bachoo, M., Caverson, M.M. and Polosa, C., Preganglionic stimulation alters the pattern of immunoreactivity to neuropeptides in the superior cervical ganglion (SCG) of the cat, Soc. Neurosci. Abstr., 14 (1988) 24. Dobner, P., Borker, D., Viller-Kamaroff, L. and McKiernan, C., Cloning and sequence analysis of cDNA for the canine neurotensin/neuromedian N precursor, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 3516-3520. Heym, C., Reinecke, M., Weike, E. and Forssman, W.G., Dopamine-beta-hydroxylase, neurotensin, substance P, vasoactive intestinal polypeptide and enkephalin immunohistochemistry of paravertebral and prevertebral ganglia in the cat, Cell Tissue Res., 235 (1984) 411-418. Iversen, L.L., Iversen, S.D., Bloom, E, Doughas, C., Brown, M. and Vale, W., Calcium-dependent release of somatostatin and neurotensin from brain 'in vitro', Nature (Lond.), 273 (1978) 161-163. Krukoff, T.L., Ciriello, J. and Calaresu, ER., Segmental distribution of peptide-like immunoreactivity in cell bodies of the thoracolumbar sympathetic nuclei of the cat, J. Comp. Neurol., 240 (1985) 90-102. Lundberg, J.M., Rokaeus, A., H6kfelt, T., Rosell, S., Brown, M. and Goldstein, M., Neurotensin-like immunoreactivity in the preganglionic sympathetic nerves and in the adrenal medulla of the cat, Acta Physiol. Scand., 114
370 (1982) 153-155. 17 Maeda, K. and Frohman, L.A., Neurotensin release by hypothalamic fragments "in vitro', Brain Research, 210 (1981) 261-269. 18 Oldfield, B.J. and McLachlan, E.M., Localization of sensory neurons traversing the stellate ganglion of the cat, J. Comp. Neurol.. 182 (1978) 915-922. 19 Pedersen, J.H. and Fahrenkrug, J., Neurotensin-like immunoreactivities in human plasma: feeding responses and metabolism, Peptides, 7 (1986) 15-20.
20 Pedersen, J.H., Stadil, F. and Fahrenkrug, J., Preparation of I25I-(Tyr3)- and 125I-(Tyrll)-neurotensin for radioimmunoassay, Scand. J. Clin. Invest., 43 (1983) 483-491. 21 Perri, V., Sacchi, O. and Casella, C., Synaptically mediated potentials elicited by the stimulation of post-ganglionic trunks in the guinea-pig superior cervical ganglion, Pfliigers Arch., 314 (1970) 55-67. 22 Zar, J.H., Biostatistical Analysis, 2nd edn., Prentice Hall, New Jersey, 1984.
365
BRE 23414
Effect of preganglionic stimulation or chronic decentralization on neurotensin-Iike immunoreactivity in sympathetic ganglia of the cat Monica M. Caverson 1, Manjit Bachoo l, John Ciriello 2 and Canio Polosa 1 1Department of Physiology, McGill University, Montreal, Que. (Canada) and 2Departmentof Physiology, Health Sciences Centre, The University of Western Ontario, London, Ont. (Canada) (Accepted 6 December 1988) Key words: Neurotensin; Radioimmunoassay;Stellate ganglion; Superior cervical ganglion; Non-cholinergictransmission; Sympathetic preganglionic neuron
In pentobarbital-anesthetized cats, supramaximal stimulation (40 Hz, 2 h) of the preganglionicinput to the acutely decentralized right stellate (RSG) or superior cervical (RSCG) ganglion resulted in a decrease in neurotensin (NT)-like immunoreactivity (IR), by 83% in the SG and by 46% in the SCG, as determined by radioimmunoassay. Chronic (7 days) decentralization of the ganglia resulted in a similar depletion of NT-like IR (SG: 86%; SCG: 76%). Supramaximal stimulation (40 Hz, 2 h) of the intact postganglionic outflow of either ganglion had no effect on NT-like IR. These data suggest that NT in the SG and SCG is present in preganglionic axons and is released by activation of these axons.
The tridecapeptide neurotensin (NT) 7"8 has been demonstrated immunohistochemically in neuronal perikarya of thoracolumbar sympathetic preganglionic neurons in the cat 15. In the same species, a dense network of NT-immunoreactive (IR) fibers has been demonstrated in sympathetic ganglia 3'x3"16, and NT has been shown to accumulate in thoracic white rami or ventral roots proximal to a ligation 16. Preganglionic stimulation of the stellate (SG) or superior cervical (SCG) ganglia with 40 Hz pulse trains, during block of cholinergic transmission, has been shown to produce slow, long-lasting effector organ responses due to excitation of ganglion cells 1" 4.10. Prolonged supramaximal stimulation (40 Hz, 2 h) of the preganglionic input to the SG of the cat results in a loss of the heart rate response mediated by non-cholinergic transmission in the SG 4. Coincident with the loss of this heart rate response is a decrease in NT-like immunoreactivity (IR) in preganglionic fibers and terminals in the SG 3. This evidence was interpreted to suggest that NT stores were depleted during preganglionic stimulation 3. In
addition, indirect evidence of NT receptors on SG cells is provided by the observation that close intra-arterial injections of NT produces excitation of ganglion cells 5. Taken together, this evidence suggests that NT functions as a transmitter in sympathetic ganglia. However, the previous immunohistochemical data 3 are only qualitative. Therefore, in the present study a radioimmunoassay for NT was used to establish that NT in the SG and SCG is contained in preganglionic axons by measuring the NT content in these ganglia after chronic decentralization and to provide an estimate of the fraction of the ganglionic NT that is lost as a consequence of prolonged preganglionic stimulation. A preliminary account of some of these data has been published 9. Experiments were done in adult cats of either sex (2.7-6.4 kg) anesthetized with sodium pentobarbital (35 mg/kg i.p. followed by 9 mg/kg i.v. every 3 h). After intubation of the trachea, the cats were paralyzed with pancuronium bromide (0.2 mg/kg i.v. followed by 0.1 mg/kg every 2 h) and artificially
Correspondence: M.M. Caverson, Department of Anatomy, Health Sciences Centre, The Universityof Western Ontario, London, Ont., Canada N6A 5C1. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
366 ventilated. End-tidal C O 2 w a s monitored (Beckman LB2 analyzer) and maintained within 4-5%. Rectal temperature was monitored and maintained at 37 + 0.5 °C by a feedback-controlled infrared heating lamp. The left femoral artery and vein were cannulated to record arterial pressure (AP) and for the administration of drugs, respectively. Heart rate (HR) was continuously recorded with a tachograph triggered by the AP pulse. The nictitating membrane on the right side was connected with silk thread to a force transducer, the output of which was continuously recorded, together with AP and HR, on a Grass model 7 polygraph. The right SG (RSG) was identified and prepared for stimulation after removal of the first 3 ribs. Orthodromic stimulation of the preganglionic input was done using bipolar silver hook electrodes placed on the T l, T 2 and T 3 white rami (WR) as well as on the sympathetic trunk just below T3WR. The postganglionic fibers from the RSG were antidromically stimulated with an electrode placed on the ventral ansa subclavia. The SG on both sides was excised immediately after a 2 h period of stimulation of the preganglionic or postganglionic fibers. In experiments in which the RSG was chronically decentralized, the ganglion was approached through the second intercostal space. The T~WR, T2WR, T3WR as well as the sympathetic trunk above T~WR and below T3WR were transected on the right side. The chest incision was closed and the animals were allowed to recover from the anesthesia and were given post-operative care. Seven days after transection the animals were anesthetized and the SG was removed on both sides. The SCG was isolated on both sides through a midline incision in the neck. Approximately 1 cm of the right cervical sympathetic trunk (RCST) was dissected free, desheathed and placed on a bipolar silver hook electrode for orthodromic stimulation of the preganglionic fibers. The right internal and external carotid and pterygopalatine nerves were isolated, desheathed and placed on an electrode for antidromic stimulation of the postganglionic fibers. Immediately following a 2 h period of stimulation of the pre- or postganglionic fibers, the SCG was excised on both sides. In additional animals, chronic decentralization of the RSCG was done by transection of the RCST. After transection, the neck
incision was closed, the animals were allowed to recover from the anesthesia and 7 days later the animals were anesthetized and the SCG on both sides was excised. Orthodromic or antidromic stimulation was done using a Grass $88 stimulator connected to a stimulus isolation unit. Stimuli consisted of square wave pulses of 0.5 ms duration, 40 Hz frequency and supramaximal intensity (10 V). After determining the intensity for maximal responses of HR and nictitating membrane (3-5 V) cholinergic transmission was blocked with hexamethonium bromide (30 mg/kg i.v. and supplemented by an infusion of 1 mg/kg/h) and atropine methylbromide (1 mg/kg i.v.). The slow responses of the HR 4 and the nictitating membrane ~'2"1° to preganglionic stimulation, due to non-cholinergic transmission in the ganglion, were monitored during a 2 h period of orthodromic stimulation. The H R and nictitating membrane response to postganglionic stimulation were monitored during a 2 h period of antidromic stimulation. NT content of the SG and SCG was measured using a NT radioimmunoassay kit (Milab, Sweden) based on the procedures outlined by Pedersen and F a h r e n k r u f 9. Immediately after orthodromic or antidromic stimulation or 7 days following decentralization, the SG and SCG were excised on both sides. All nerve bundles were cut as close to the ganglia as possible and the ganglia were immediately extracted according to the procedure outlined by Bryant and Bloom 6. The ganglia were quickly desheathed, weighed, cut into small pieces, and extracted in 1 ml of 0.5 M boiling acetic acid for 15 rain. The supernatant was removed, the pH was adjusted to 7.4 with 1 N NaOH and the extracts were stored at -70 °C until assayed. To determine whether sensory afferent fibers contributed to the NT content measured in the SG and SCG, NT in the nodose (n = 3) and in the T 2 dorsal root ganglia (n = 2) was measured in these control tissue. Samples were assayed in duplicate according to the manufacturer's instructions (Milab, Sweden). In brief, 450/~1 of NT antiserum (Milab no. R759A04-1, rabbit anti-NT; 1:128,000) was incubated with 450/A o f [125I]NT2° and 250 pl of samples, controls or standards for 72 h at 4 °C in assay buffer (0.02 M barbital buffer, pH 8.6, with 0.2% human serum
367 albumin, 0.05% thimerosal and 500 klU/ml aprotinin; Sigma Chemicals, St. Louis, MO, U.S.A.). NT (Milab, Sweden) dissolved in assay buffer was used as standards. The working range was 15.6 to 1000 pg/ml. Free NT was separated from antibody-bound NT by the addition of a 1 ml suspension containing 2.5% activated charcoal (Milab, Sweden) and 10% human serum (Sigma Chemicals, U.S.A.) in assay buffer. The bound fraction contained within the supernatant was counted in an LKB gamma-counter. The recovery was 97% at 125 pg/ml. Our values have not been corrected for loss during extraction. The sensitivity of the assay was 7.0 pg/ml with 95% confidence. Multiple serial dilutions of all unknown samples were assayed in duplicate so as to span the sensitive part of the standard curve. Only those dilutions within the sensitive range of the standard curve were used in the calculation of the results. Intra-assay variability evaluated by 6-fold estimation of standards with 15.6, 125 and 1000 pg/ml NT was 7.6%, 5.1% and 8.3%, respectively. Inter-assay variability estimated by 6-fold measurements of the same sample (125 pg/ml) in 7 assays within 12 weeks was 25.9%. The NT antibody used in this study recognizes the 1-13 amino acid sequence. For details on the cross-reactivity of the antibody see Pedersen and Fahrenkrug 19 and Pedersen et al. 2°. No cross-reactivity with secretin, gastric inhibitory peptide, vasoactive intestinal peptide, peptide histidine-isoleucine, pancreatic glucagon, and pancreatic polypeptide has been detected (Milab, Sweden) 19"2°. All values of NT-like IR are expressed as means + S.E. Comparisons between RSG and left (L)SG, RSCG and LSCG or SG(R + L) and SCG(R + L) in control ganglia were made using Student's t-test for independent variables. Comparisons between stimulated or decentralized ganglia (right side) and control ganglia (left side) were made using the Mann-Whitney test after Bartlett's test indicated an heterogeneity of variance 22. A P value of 0.05 or less was taken to indicate statistical significance. Qualitatively similar results were obtained following chronic decentralization or stimulation of the SG or SCG. Therefore, the data for both ganglia will be described together for the 4 experimental conditions: control (no stimulation, intact innervation), chronic decentralization, preganglionic (orthodro-
mic) stimulation and postganglionic (antidromic) stimulation. These data are summarized in Table I. Control conditions. The content of NT-like IR in the RSG and LSG, as well as in the RSCG and LSCG, was not significantly different. Therefore, the left ganglion was used as a control in subsequent experiments in which the right ganglion was stimulated or decentralized. Control levels of NT-like IR in the SG were approximately 7-8-fold greater than that in the SCG (Table I). Chronic decentralization. The NT-like IR in ganglia excised 7 days after decentralization was significantly (P < 0.01) lower than that in the contralateral control ganglia. The NT content was decreased by 86% in the RSG and by 76% in the RSCG (Table I). The weight of the decentralized RSG was observed to be greater than that of the control LSG. However, this effect was not observed in the decentralized RSCG compared to the intact LSCG. Preganglionic (orthodromic) stimulation. Electrical stimulation (2 h) of the preganglionic input to the RSG and RSCG resulted in a significant (P < 0.05) decrease in the NT-like IR as compared to contralateral control ganglia. The NT content was decreased by 83% in the RSG and by 46% in the RSCG (Table I). Postganglionic (antidromic) stimulation. The NTlike IR in ganglia excised after 2 h stimulation of the postganglionic fibers was not significantly different from that in control ganglia (Table I). Control tissue. The content of NT-like IR in control tissue was considerably lower than that observed in the SG or SCG under control conditions. In extracts of the nodose and the T 2 dorsal root ganglia the NT-like IR ranged from 0.7 to 5.6 pmol/g tissue. The results of this study demonstrate that stimulation of the nerves that provide preganglionic input to, but not of the nerves that are the output of, the SG and SCG produce a decrease in NT-like IR. The lack of effect of stimulation of the intact postganglionic fibers suggests that the decrease in NT-like IR is not the result of increased discharge of sympathetic ganglion cells, as postganglionic stimulation would result in antidromic excitation of these cells at a rate similar to, and possibly higher than, that produced by orthodromic stimulation 21. Moreover, since the same sensory afferents would be
368 TABLE 1 NT-like IR in SG and SCG 7 days after decentralization and after orthodromic or antidromic stimulation
Values are means ± S.E.M. RSG, right stellate ganglion; LSG, left stellate ganglion; RSCG, right superior cervical ganglion; LSCG. left superior cervical ganglion; Ortho, orthodromic; Anti, antidromic. % Difference in concentration was determined by comparing experimental (right) ganglia to control (left) ganglia. No. o f cats
Experimental condition
Ganglion weight (rag)
Content (pg/ganglion )
Concentration (pmol/g wet wt. )
5
Control RSG Control LSG Control RSCG Control LSCG
21 _+3 21 ± 3 17 ± 2 18 ± 2
3607 _+_510 3650 ± 518 399 + 80 401 ± 85
110 ± 20 118 ± 26 15 ± 4 15 + 4
Decentralized RSG Control LSG Decentralized RSCG Control LSCG
40 ± 5* 23 + 4 20 _+2 20 ± 3
792 ± 93** 3766 ± 909 112 ± 36** 462 _+26
12 ± 2** 94 _+4 4 + 2** 15 ± 2
Ortho stimulation RSG Control LSG Ortho stimulation RSCG Control LSCG
20 + 1 16 + 2 18 ± 2 18 _+3
545 _+79* 2674 + 410 152 _+24* 276 ± 45
17 ± 2* 98 + 9 5 ± 1* 10 _+_2
Anti stimulation RSG Control LSG Anti stimulation RSCG Control LSCG
17 + 2 16 ± 1 16 + 4 16 +_3
2858 + 311 2825 + 277 445 ± 112 470 _+64
8 5 5 4 6 2 2
104 + 2 106 ± 2 19 _+7 19 ± 8
% Difference in concentration -
7%
0% -86% -76% -83% -47%
-
2%
0%
* Significantlydifferent from control (P < 0.05); **significantlydifferent from control (P < 0.01).
present in both preganglionic and postganglionic nerves 18, the lack of a decrease in NT-like IR with postganglionic stimulation suggests that the decrease in NT-like IR with preganglionic stimulation is not the result of a decrease in NT in sensory afferent
can be eliminated as the source of the decreased NT-like IR. It has been shown that N T can be released from neural tissue by depolarization or high [K+], and that this release is Ca2+-dependent 14'17.
fibers. This finding is supported by previous immu-
suggest that the ganglionic N T content is associated with preganglionic axons, it may be expected that the invasion of preganglionic axon terminals by
nohistochemical data in which no changes were observed in the immunoreactivity pattern of fibers and presumed axon terminals in the SG as a result of prolonged (2 h) stimulation of the postganglionic fibers 3. In addition, the.low content of NT-like IR measured in dorsal root and nodose ganglia in the present study and that reported previously in dorsal roots ~6 suggests that sensory afferents are unlikely to contribute significantly to the NT content of the SG and SCG. The present results also demonstrate that the content of NT-like IR in the SG and SCG decreased as a result of chronic preganglionic denervation. This suggests that the ganglionic N T content is associated with preganglionic axons. Therefore, it is likely that preganglionic nerve stimulation depletes NT from preganglionic axons, since sensory axon collaterals
Since the experiments of chronic decentralization
action potentials results in the release of NT. Therefore, if the action potential frequency is high (e.g. 40 Hz), as was used in this study, the rate of release of N T may exceed the rate of replacement of this substance, or of its precursors 12, resulting in depletion. How closely the content of NT-like IR in the SG or SCG compares after preganglionic stimulation or denervation depends on how completely NT, presumably contained in the degenerating axons, has been degraded and removed, and on the relative proportion of the releasable and non-releasable fraction of the total N T pool. In the SG, the close-matching of the depletion of NT-like IR as a result of chronic decentralization and of prolonged orthodromic stimulation suggests that most of the
369 N T contained in the SG is readily releasable by preganglionic nerve stimulation. On the o t h e r hand, the decrease in NT-like I R in the S C G o b t a i n e d with o r t h o d r o m i c stimulation was 62% of that obtained with chronic decentralization, suggesting that some of the NT-like I R contained in preganglionic axons in the S C G may not be releasable. It was o b s e r v e d that the wet weight of the R S G was significantly greater than the L S G after chronic decentralization. H o w e v e r , this effect was not noted in the R S C G . The reason for this difference in wet weight is unclear. Nevertheless, the concentration of NT, which incorporates the weight of the ganglion, was significantly decreased in the R S G after chronic decentralization. A n o t h e r interesting observation in this study was the 7 - 8 - f o l d greater content of NT-like I R in control S G as c o m p a r e d to control SCG. Recent immunohistochemical data support this finding as a greater density of NT-like I R fibers and p r e s u m e d axon terminals were o b s e r v e d in the S G c o m p a r e d to the S C G 3"11'13. The reason for this difference is unclear; however, a possible explanation is that the SG receives a greater n u m b e r of preganglionic axons 1 Alkadhi, K.A. and Mclsaac, R.J., Non-nicotinic transmission during ganglionic block with chlorisondamine and nicotine, Eur. J. Pharmacol., 24 (1973) 78-85. 2 Bachoo, M., Ciriello, J., Caverson, M.M. and Polosa, C., Non-cholinergic transmission in the superior cervical ganglion of the cat, Can. Physiol., 18 (1987) 101. 3 Bachoo, M., Ciriello, J. and Poiosa, C., Effect of preganglionic stimulation on neuropeptide-like immunoreactivity in the stellate ganglion of the cat, Brain Research, 400 (1987) 377-382. 4 Bachoo, M., Isacoff, E. and Polosa, C., Slow cardioacceleration mediated by non-cholinergic transmission in the stellate ganglion of the cat, Can. J. Physiol. Pharmacol., in press. 5 Bachoo, M. and Polosa, C., Cardioacceleration produced by close intra-arterial injection of neurotensin into the stellate ganglion of the cat, Can. J. Physiol. Pharmacol., 66 (1988) 408-412. 6 Bryant, M.G. and Bloom, S.R., Measurement in tissues. In S.R. Bloom and R.G. Long (Eds.), Radioimmunoassay of Gut Regulatory Peptides, Saunders, Canada, 1982, pp. 36-41. 7 Carraway, R. and Leeman S.E., The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami, J. Biol. Chem., 248 (1973) 6854-6861. 8 Carraway, R. and Leeman, S.E., The amino acid sequence of a hypothalamic peptide, neurotensin, J. Biol. Chem., 250 (1975) 1907-1911. 9 Caverson, M.M., Bachoo, M., Ciriello, J. and Polosa, C., Effect of prolonged preganglionic stimulation on radioim-
than the SCG. Alternatively, the possibility may exist that preganglionic axons containing N T preferentially innervate SG cells. This m a y be due to factors, perhaps related to the target of ganglion cell innervation, which d e t e r m i n e the type of n e u r o p e p tide synthesized by a given sympathetic preganglionic neuron or the selective innervation of specific ganglion cells by certain preganglionic axons. In conclusion, quantitative observations r e p o r t e d in the present study, of a decrease in the content of r a d i o i m m u n o a s s a y a b l e NT-like I R in the SG and SCG after o r t h o d r o m i c stimulation or chronic decentralization of the preganglionic axons, provide evidence to suggest that N T may function as a neurotransmitter or n e u r o m o d u l a t o r at sympathetic ganglionic synapses.
This work was s u p p o r t e d by the M R C of C a n a d a , the Q u e b e c H e a r t F o u n d a t i o n , the D y s a u t o n o m i a F o u n d a t i o n and the H e a r t and Stroke F o u n d a t i o n of Ontario. M . M . C . is a C a n a d i a n H e a r t F o u n d a t i o n Research Scholar and J.C. is a C a r e e r Investigator of the H e a r t and Stroke F o u n d a t i o n of Ontario.
10 11
12
13
14
15
16
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370 (1982) 153-155. 17 Maeda, K. and Frohman, L.A., Neurotensin release by hypothalamic fragments "in vitro', Brain Research, 210 (1981) 261-269. 18 Oldfield, B.J. and McLachlan, E.M., Localization of sensory neurons traversing the stellate ganglion of the cat, J. Comp. Neurol.. 182 (1978) 915-922. 19 Pedersen, J.H. and Fahrenkrug, J., Neurotensin-like immunoreactivities in human plasma: feeding responses and metabolism, Peptides, 7 (1986) 15-20.
20 Pedersen, J.H., Stadil, F. and Fahrenkrug, J., Preparation of I25I-(Tyr3)- and 125I-(Tyrll)-neurotensin for radioimmunoassay, Scand. J. Clin. Invest., 43 (1983) 483-491. 21 Perri, V., Sacchi, O. and Casella, C., Synaptically mediated potentials elicited by the stimulation of post-ganglionic trunks in the guinea-pig superior cervical ganglion, Pfliigers Arch., 314 (1970) 55-67. 22 Zar, J.H., Biostatistical Analysis, 2nd edn., Prentice Hall, New Jersey, 1984.