Excitation of cerebral cortical neurons by various polypeptides

EXPERIMENTAL

Excitation

NEUROLOGY

43, 414-423 (1974)

of Cerebral J. W.

Department

of

Cortical PHILLIS

Physiology,

Neurons AND

J. J.

by Various

LIMACHER

Polypeptides

1

College of Medic&se, University of Saskatchewan, Saskatoolz, Canada Received January 16,1974

Several polypeptides, including Substance P, bradykinin, physalaemin and angiotensin, have been applied iontophoretically onto single neurons in the cerebral cortex of rats. The polypeptides excited many of the spontaneously active neurons tested, including identified Betz cells. Substance P was the most potent excitant, bradykinin and physalaemin were somewhat less potent, and angiotensin had only a weak excitant action. Depressant effects were observed on a few neurons. Atropine antagonized the excitant effects of acetylcholine but not those of Substance P or bradykinin. These results, taken in conjunction with other findings on the presence of some of these compounds in brain, suggest that the polypeptides deserve further consideration as possible excitatory synaptic transmitters.

INTRODUCTION Interest in the actions of polypeptides on cerebral neurons has been rekindled by the recent commercial availability of synthetic peptides, identical to the naturally occurring compounds, and the description of potent excitant effects of several of these compounds on neurons in the isolated amphibian spinal cord (11, 17). Substance P, discovered by von Euler and Gaddum (4) is present in the brain and spinal cord where it is localized predominantly in the phylogenetically older regions (15, 18, 23). It is present in high concentrations in the dorsal roots and dorsal columns of the spinal cord and in the nuclei gracilis and cuneatus, and Lembeck (14) has suggested that it may be the transmitter at the first sensory neuron. Substance P is also present in the cerebral cortex, where it has been isolated in the subcellular fraction containing synaptic vesicles ( 10). An inactivating protease is found 1 Supported by the Canadian Medical Research Council. J. Limacher is a recipient of a C.M.R.C. Fellowship, We wish to thank Dr. R. de Castiglione of Farmitalia for gifts of bombesin and eledoisin. 414 Copyright All rights

Q 1974 by Academic Press, Inc. of reproduction in any form reserved.

POLYPEPTIDE

EXCITATION

OF NEURONS

415

in the microsomal fraction of brain tissue (10). A Substance P-like peptide is released from the cat cerebral cortex, the rate of release being enhanced by picrotoxin (21). Angiotensin and renin have been isolated from rat and dog brains where they are concentrated in the brainstem and hypothalamus (6). Brain tissue contains angiotensin-forming enzymes (7) and inactivating enzymes (1) . Most of the angiotensin in brain is angiotensin I, although angiotensin II is also present. Subcellular fractionation has shown that renin-like activity is concentrated in synaptosomal preparations (16). Bradykinin, eledoisin, physalaemin and bombesin have been detected in the skin of amphibia and in some invertebrate tissues (3). There is little information about their presence in mammalian brain although the isolation of a bradykinin-like peptide has been reported (8). Physalaemin and bradykinin have potent excitatory actions on the isolated amphibian spinal cord (11). We have examined the actions of several of these polypeptides on neurons in the cerebral cortex, and a preliminary report of the results has already appeared (20). METHODS Eleven male Sprague-Dawley rats (250-350 gm) were used in these experiments. After initiation of anesthesia with an intravenous injection of sodium thiopental into the dorsal tail vein, a tracheotomy was performed and a tracheal cannula inserted. The animal was then placed in a stereotaxic frame and anesthesia maintained with a mixture of methoxyflurane, nitrous oxide and oxygen. Body temperature was controlled at 36-38 C with an electric heating pad and rectal probe. A small hole was drilled through the parietal bone overlying the somatosensory cortex and a narrow incision through the dura mater exposed the cerebral cortex. The surface of the cortex, dura, exposed muscle and skin were then covered with a thin layer of 4% agar in Ringer solution to prevent drying. In five animals, a second hole was drilled ipsilaterally 0.5 mm lateral to the midline just anterior to the lambdoidal ridge (at stereotaxic coordinate AP 12, according to the atlas of Fifkova and Marsala (5). A bipolar concentric stimulating electrode was inserted through this hole to a depth of 11.5-12.0 mm. This electrode was used to stimulate Betz cell axons in the pyramidal tract. A neuron was considered to be a Betz cell if the antidromic invasion of an action potential subsequent to pyramidal tract stimulation occurred with a constant latency and if it followed frequencies in excess of lOO/sec. The recording of neuronal activity and iontophoresis of drug solutions were accomplished with seven-barrelled micropipettes with overall tip diameters of 6-S pm. The central recording barrel and one side barrel were

416

PHILLIS

AND

LIMACHER

filled with 2 M NaCI. The remaining barrels were filled with various combinations of the following solutions : angiotensin II (0.1 M, Beckman), bradykinin triacetate (0.01 M and 0.01 M in 165 mM NaCI, Sigma), bombesin dichlorhydrate (0.01 M and 0.01 M in 165 mM NaCl, Farmitalia), eledoisin (0.01 M, Farmitalia) , an eldoisin-related peptide (0.01 M, Sigma), physalaemin (0.001 M in 165 mM NaCl, Calbiochem), Substance P (0.008 M, Beckman), acetylcholine chloride (0.2 M, Sigma), atropine sulphate (0.1 M, Parke Davis), The polypeptide solutions were adjusted to pH 5-6 and the polypeptides passed as cations. The physalaemin samples had been lyophilized with mannitol and to ascertain that this substance was not influencing cell excitability, several barrels were filled with a 1.1 M mannitol solution in 165 mM NaCl. All electrodes were filled by centrifugation immediately prior to use. Spontaneously active, antidromically activated (Betz cells) and glutamate-fired neurons were examined in all layers of the cerebral cortex. An effect of a polypeptide was considered to be genuine if it was reversible, repeatable and not mimicked by a current control. On some occasions current neutralization was also employed. Recording procedures were similar to those described by Jordan and Phillis (9). RESULTS The results obtained in these experiments are summarized in Table 1, in which the neuronal types tested have been divided into unidentified neurons and identified Betz cells. The group of unidentified neurons has been further subdivided into glutamate-excited cells and spontaneously active cells. Ninety-nine Betz cells and 267 unidentified neurons were tested with various combinations of substances. Although slight depressant actions of all the polypeptides were noted on both glutamate-excited and on some spontaneously firing neurons, this effect, when it occurred, was weak, transient and usually difficult to distinguish from the effects of anodal control current. Therefore, because of the uncertainty as to whether such effects represented a genuine alteration in neuronal excitability, they have been excluded from Table 1 (with the exception of several clear-cut instances of depression observed with bombesin and eledoisin) . Excitant actions of the polypeptides were manifested on many spontaneously active unidentified neurons and identified Betz cells. Most of these cells were located at depths in excess of 500 pm. Substance P. This substance was the most potent excitant among the polypeptides tested in these experiments. Its excitant action was evident on 62 (91%) of the 68 spontaneously firing neurons tested and on 29 (94%) of the 31 Betz cells. Excitation frequently occurred after a latency

acid

sequences

Angiotensin II : Asp-Arg-Val-Tyr-Ile-His-Pro-Phe Bombesin: Pyr-Glu-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Gly-His-Leu-Met-NH~ Bradykinin: Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Eledoisin: Pyr-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2 Eledoisin-related peptide: Lys-Phe-Ile-Gly-Leu-Met-NH2 Physalaemin: Pyr-Asn-Pro-Asn-Arg-Phe-Ile-Gly-Leu-Met-NH2 Substance P: Arg-Pro-Lys-Pro-Gin-Phe-Phe-Gly-Leu-Met-NH*

* Amino

8

37

0

0

P

Substance

1 22 3.5 214

0 0 0

00

II

Nil

TABLE TO VARIOUS

1

62 (91%)

0

1

60

103 (26%) (30%) 59 (91%)

0 3 0

1 (70/c) 19 (56%) 47 (76%)

6

5

227

13 12 15

Nil

POLYPEPTIDES*

Spontaneously Active Excitation Depression

Cells

NEURONS

Unidentified

OF CORTICAL

Eledoisin Eledoisin-related peptide Physalaemin

Angiotensin Bombesin Bradykinin

Non-Spontaneously Active Excitation

RESPONSES

29 (94%)

12 (71%:)

29 (88%) 14 (67%)

4 (33%) 11 (SO%‘,) 38 (91%)

Excitation

Betz

Cells

2

5

4 7

8 11 4

Nil

8 z

E c jt +I 3 z

ri

: 2 ‘d g cj

418

PHILLIS

AND

LIMACHER

FIG. 1. Ratemeter records from a spontaneously firing cerebral cortical neuron (depth 6.52 pm). Increasing applications of Substance P (SP) evoked progressively more pronounced increases in the firing rate of the neuron. A control current of 40 nA passed through the NaCl-containing barrel was without effect. In this, and subsequent figures, the ordinate shows firing rate in impulses per set, while the horizontal bars above the records indicate periods of drug application.

of 7-15 set and often continued for one or more minutes after the application had terminated. No excitant effects were seen on nonspontaneous neurons. The effects of increasing amounts of Substance P on the firing frequency of an unidentified spontaneously active cortical neuron (depth 652 w) are illustrated in Fig. 1. The threshold current for excitation was about 10 nA and with increasing application currents Substance P caused more pronounced excitation. The effects of repeated application with the same SP -

50

40j-

r-

30

SP 40

SP40 -

Na+30

SP

30

Na+40

SP40

--

--

--

Na+40

SP

40

FIG. 2. Three examples of tachyphylaxis to repeated doses of Substance ent cortical neurons. Control current applications were without effect.

P

on differ-

POLYPEPTIDE

100

SP40 -

EXCITATION Nat40 --

ACH40

419

OF NEURONS SP40 -

ACM40

FIG. 3. Records from a cerebral cortical Betz cell (depth 655 pm, antidromic invasion latency 2.4 msec). Substance P (40 nA) and acetylcholine (ACH, 40 nA) initially excited the neuron. After an application of atropine (60 nA for 90 set) the action of ACH but not that of Substance P was abolished.

current appeared to diminish on several of the ceIIs tested with Substance P, possibly because tachyphylaxis was occurring. Three examples of this phenomenon are presented in Fig. 2. Tachyphylaxis to the effects of other polypeptides following application of Substance P was not observed. Many of the cells, including the Betz cells, that were excited by Substance P (and the other polypeptides) were also excited by acetylcholine (ACh). The Betz cell illustrated in Fig. 3 responded to Substance P (40 nA) and ACh (40 nA) but not to a control anodal current (40 nA). Atropine (60 nA for 90 set) was then applied and when the spontaneousfiring frequency had recovered 2 min later, the action of ACh but not of Substance P was abolished. A total of 12 identified pyramidal tract cells and 11 unidentified spontaneously active cells excited by both SP and ACh (and in some instances by bradykinin as well) were tested with atropine. In each instance the ACh excitation was abolished and the polypeptide excitation unaffected. Bradykinin and Physalaenhz. Bradykinin and physalaemin were somewhat less potent excitants than Substance P but were clearly more active than bombesin, eledoisin and angiotensin II. Bradykinin excited 47 (76%) of the 62 spontaneously firing neurons tested and 38 (91%) of the 42 pyramidal tract cells. Bradykinin excitation was consistently of long duration, lasting for several minutes. An example of bradykinin action on a Betz cell is illustrated in Fig. 4. On this occasion firing returned to the pre-bradykinin frequency some 5 min after the cessation of bradykinin application. Bradykinin was tested before and after atropine on nine Betz cells which were initially excited by ACh. Atropine blocked ACh excitation but not the effect of bradykinin. Physalaemin excited 59 (91%) of the 6.5 spontaneously firing neurons and 12 (71%) of the 17 Betz cells tested. No effects were observed on

420

PHILLIS

AND

LIMACHER

50

0 I

L

I lmin

FIG. 4. Bradykinin (BR, 60 nA) and physalaemin (PHY, 60 nA) had pronounced excitant effects on this Betz cell (depth 1184 pm, antidromic invasion latency 3.0 msec). Angiotensin (ANG, 80 nA) had a very weak excitant action.

neurons that did not display spontaneousactivity. Examples of physalaemin excitation are presented in Figs. 4 and 5. Its potency was comparable to that of bradykinin but the excitation did not have the long duration of that evoked by bradykinin. The effects of increasing amounts of physalaemin on the firing frequency of an unidentified spontaneously active cortical neuron (depth 1076 pm) are illustrated in Fig. 5. A current of 15 nA passed through the physalaemin barrel was just threshold for producing excitation. Larger currents evoked correspondingly more marked excitations. Eledoisin, Eledoisin-Related Peptide and Bombesin. These three peptides had excitant actions that were comparable to those of Substance P and physalaemin in duration, but less potent. Bombesin excited 19 (56%) of the 34 unidentified spontaneously firing neurons tested and depressedthree of them. It excited 11 (50%) of the 22 Betz cells onto which it was applied. Eledoisin excited ten (26%) of the 38 spontaneously firing neurons tested and depressedsix of them. It excited 29 (88%) of the 33 pyramidal PHY

30

PHY 60

PHY 90 -

Na+so

lmin

FIG. 5. Excitation of a spontaneously active neuron (depth 1076 pm) by increasing applications of physalaemin. Control current (60 nA) application had no effect.

POLYPEPTIDE

EXCITATION

OF NEURONS

421

tract cells tested. Comparable results were obtained with the eledoisinrelated peptide. Angiotensin II. This was the least potent polypeptide tested. An example of a very weak excitant action of angiotensin II (80 nA) on a Betz cell which was quite strongly excited by both physalaemin and bradykinin (60 nA) is presented in Fig. 4. Angiotensin excited only 1 (7%) of the 14 unidentified spontaneously firing neurons on which it was tested and 4 (33%) of the 12 Betz cells. DISCUSSION The findings presented in this paper demonstrate that several polypeptides have quite powerful excitatory actions on cerebral cortical neurons. A few instances of clear-cut depression of glutamate-induced or spontaneous firing were also observed and on many other glutamate-excited neurons there were less convincing indications of a weak depressant action. The neurons excited by the polypeptides, including the pyramidal tract cells, were characteristically spontaneously active and the majority of them were also excited by ACh. However, some neurons which responded to the polypeptides were not excited by ACh. No instances of excitation of quiescent (not spontaneously firing) neurons were observed. The relationship between spontaneity of cortical neurons and AChsensitivity was first described by Krnjevid and Phillis (13) and the finding that these neurons (which include the Betz cells) were also excited by the polypeptides suggested a possible relationship between ACh-sensitivity and polypeptide-sensitivity. Angiotensin has been reported to enhance ACh release from the cerebral cortex (2), raising the possibility of a presynaptic ACh-releasing site of action for the peptides. Alternatively it was possible that the polypeptides acted on the same postsynaptic receptor as ACh. The experiments with atropine reported in this paper, however, clearly establish that the action of the polypeptides is independent of either an action on cholinergic presynaptic terminals or on the ACh receptors, as atropine blocked the action of ACh but not that of the polypeptides. Another possibility that demands consideration is that a pressor action of the polypeptides on blood vessels in the vicinity of the microelectrode tip could result in the development of local ischaemia and consequent neuronal depolarization. Such an explanation seems to be excluded by reports that Substance P, bradykinin, physalaemin and angiotensin I and II have pronounced excitant actions on the isolated superfused amphibian spinal cord (11, 17). Of the polypeptides evaluated in this report, Substance P merits the most serious consideration as a synaptic transmitter acting on cerebral cortical

422

PHILLIS

AND

LIMACHER

neurons. It was the most potent excitant tested and it is known to be present in the synaptic vesicle fraction of cerebral cortical homogenates (10). Furthermore, it is detectable in perfusates of the cat somatosensory cortex, where its rate of release is enhanced by the convulsant, picrotoxin (21). Much of the interest in Substance P as a synaptic transmitter in the central nervous system has been focussed on the possibility that it is released by primary afferent fibers synapsing on relay neurons in the nuclei gracilis and cuneatus (14). During the course of the present investigation, a preliminary report was published on its actions when applied iontophoretically on cuneate neurons (12). Substance P had a delayed, long-lasting excitant action on some of these neurons and depressed others. However, the authors concluded that the low excitant potency and long duration of action made it unlikely that Substance P was the transmitter released by the terminals of primary afferent fibers, Substance P had a rather pronounced excitatory action on cerebral cortical neurons, usually associated with a relatively short latency of onset. It was usually more potent than ACh which is generally considered to be an excitatory transmitter acting on pyramidal cells in the cerebral cortex (19, 22) and hence Substance P must also be given serious consideration as a cerebral cortical transmitter. Evidence that the other polypeptides tested in this survey are involved in synaptic transmission in the brain is currently lacking and must await further studies on their presence and distribution. However, angiotensin and bradykinin have recently been identified in cerebral tissues and will undoubtedly receive increasing attention in the future. Physalaemin and eledoisin have been isolated from the skin of certain frogs and both are known to have similar chemical and biological characteristics to Substance P. REFERENCES 1.

and N. MARKS. 1971.Inactivation,studiesof angiotensin 27 : 1352-1353. ELIE, R., and J. C. PANISSET. 1970. Effect of angiotensin and atropine on the spontaneous release of acetylcholine from cat cerebral cortex. Brain Res. 17: 297-305. ERSPAMER, V. 1971. Biogenic amines and active polypeptides of the amphibian skin. Amu. Rev. Pharmac. 11: 327-350. EULER, U. S. VON and J. H. GADDUM. 1931. An unidentified depressor substance in certain tissue extracts. J. Physiol. 72: 74-87. FIFKOV~, E., and J. MARSALA. 1967. Stereotaxic atlases for cat, rabbit and rat, pp. 653-73’1. In. “Electrophysiological Methods in Biological Research.” J. BureS, M. PetrLn, J. Zachar, [Eds.]. Academic Press, New York. FISCHER-FERRARO, C., V. E. NAHMOD, D. J. GOLDSTEIN, and S. FINKIELMAN. 1971. Angiotensin and renin in rat and dog brain. J. Exp. Med. 133 : 353361. ABRASR,

L., R. WALTER,

II by purified enzymes. Experientia

2.

3. 4. 5. 6.

POLYPEPTIDE

EXCITATION

OF NEURONS

423

D., J. MINNICH, P. GRANGER, K. HAYDUK, H. M. BRECHT. 1971. Angiotenin-forming enzyme in brain tissue. Science 173: 64-65. 8. TORI, S. 1968. The presence of bradykinin-like polypeptide, kinin-releasing and destroying activity in brain. Jab. J. Physiol. 18: 772-787. 9. JORDAN, L. M., and J. W. PHILLIS. 1972. Acetylcholine inhibition in the intact and chronically isolated cerebral cortex. &it. J. Pharmucol. 45 : 584595. 10. KATAOKA, K. 1962. The subcellular distribution of Substance P in the nervous tissues. Jap. J. Physiol. 12: 81-96. 11. KONISHI, S., and M. OTSUKA. 1971. Actions of certain polypeptides on frog spinal neurons. Jap. J. Pharmac. 21: 685-687. 12. KRNJEVIC, K., and M. E. MORRIS. 1973. Depolarizing action of Substance P in the cuneate nucleus of the cat. Abst. Sot. Neuroscience 60.5. 13. KRNJEVI~, K., and J. W. PHILLIS. 1963. Acetylcholine-sensitive cells in the cerebral cortex. J. Physiol. (London) 166 : 296-327. 14. LEMBECK, F. 1953. Zur Frage der zentralen Ubertragung afferenter Impulse. III. Das Vorkommen und die Bedeutung der Substanz P in der dorsalen Wurzeln des Ruckenmarks. Arch. Exp. Pathol. Pharmakol. 219: 197-213. 15. LEMBECK, F., and G. ZETZER. 1971. Substance P, pp. 29-71. In “International Encyclopedia of Pharmacology and Therapeutics, Section 72,” “Pharmacology of Naturally Occurring Polypeptides and Lipid-soluble Acids,” J. M. Walker [Ed.]. Pergamon Press, Oxford. 16. MINNICH, J. L., D. GRANTEN, A. BARBEAU, and J. GENEST. 1972. Subcellular localization of cerebral renin-like activity, pp. 432-435. In “Hypertension,” J. Genest, and E. Koiew [Eds.]. Springer-Verlag, Berlin. 17. OTSUKA, M., S. KONISHI, and T. TAKAHASHI. 1972. The presence of a motoneuron-depolarizing peptide in bovine dorsal roots of spinal nerves. Proc. Jab. 7. GRANTEN,

Acad.

48 : 342-346.

18. PERNOW, B. 1953. Studies on Substance P, purification, occurrence, and biological actions. Acta Physiol. Scand. 29: Suppl. 105 : l-90. 19. PHILLIS, J. W. 1970. “The Pharmacology of Synapses.” Pergamon Press, Oxford. 20. PHILLIS, J. W., and J. J. LIMACHER. 1974. Substance P excitation of cerebral cortical Betz cells. Brai% Res. 69: 158-163. 21. SHAW, J. E., and P. W. RAMWELL. 1968. Release of a Substance P polypeptide from the cerebral cortex. Amer. J. Physiol. 215: 262-267. 22. SPEHLMANN, R. 1971. Acetylcholine and the synaptic transmission of non-specific impulses to the visual cortex. Brair, 94: 139-150. 23. ZETLER, G. 1970. Biologically active peptides (Substance P), pp. 135-148. In Vol. IV, Control mechanisms in the nervous “Handbook of Neurochemistry,” system, A. Lajtha [Ed.]. Plenum Press, New York.