15.5. 1976

Speeialia

e v e n 6 weeks after p i n e a l e c t o m y also indicated the ind e p e n d e n t synthesis of N A S in this tissue 3. Using P C P A t r e a t m e n t we h a v e e x p e c t e d a decrease of N A I positive substance in t h e granule layer of cerebellum. The opposite findings seems to c o n t r a d i c t t h e established effect of P C P A on serotonin synthesis. H o w e v e r this paradoxical o b s e r v a t i o n is n o t unique. I n 7 brain areas P C P A was r e p o r t e d to decrease t h e c o n t e n t of serotonin (range 31-46%). On the other h a n d in cerebellum the serotonin was increased b y 44% 12. AC-HAJANIAN et al. 1~ discovered t h a t P C P A was n o t able to p r e v e n t the L - t r y p t o p h a n induced increase of serotonin fluorescence of the neurons of the raphe system, even t h o u g h t h e region of terminals in t h e Iorebrain was substantially depleted. I n 1974 HARVEY and GAL 14 observed no blockade of T R - 5 O H a s e in t h e septal region after PCPA. The authors of these two findings offer two different explanations of our u n e x p e c t e d results. AGHAJANIAN et al. hypothesized t h a t synthesis of serotonin b y P C P A is blocked in t h e nerve t e r m i n a l s while in the p e r i k a r y o n of t h e neurons t h e serotonin p r o d u c t i o n continues due to new synthesis of T R - 5 - O H a s e . The s a m e e x p l a n a t i o n m a y hold for granule cells. T o g e t h e r w i t h a feedback response to depletion of serotonin synthesis in the terminals of the granule ceils it could even explain an increase of serotonin synthesis in t h e granule ceils, supplying t h e substrate for increased

581

N A S synthesis. A n o t h e r possible e x p l a n a t i o n is the speculation of HARVEY and GAL 14 t h a t at least 2 T R - 5 O H a s e exist, one which is blocked b y P C P A and t h e o t h e r which is not. T h i s e x p l a n a t i o n does n o t p r o v i d e a n y reason for increase in N A S . The third possibility is an as y e t u n k n o w n metabolic p a t h w a y for N A S synthesis which does not involve T R - 5 OHase. Such a p a t h w a y m i g h t involve t h e m e t a b o l i s m of t r y p t o p h a n to t r y p t a m i n e , t h e n to N - a c e t y l t r y p t a m i n e and finally h y d r o x y l a t i o n to N A S b y an unidentified reaction. H o w e v e r such a p a t h w a y seems less probable in v i e w of recent findings of L. H s l : et al. (personal communication). T h e y observed an increase of N-acetyltransferase in the rat cerebellum after PCA t r e a t m e n t . A l t h o u g h this report correlated w i t h our findings it does n o t help to explain the m e c h a n i s m b y which N A S is synthesized in t h e granule layer of cerebellum after PCPA. W e hope t h a t further investigations will c o n t r i b u t e to the solution of this problem.

12 E. D. WEITZMAN, ~/[. RAPPORT, P. MCGREGOR a n d J. JAcoBY,

Science 760, 1361 (1968). la G. K. AGHAJANIAN, M. J. KUHAR and R. H. ROTK, Brain Res.

54,

851 (1973). 24 j. A. HARVEYand E. M. GAL, Science 783, 869 (1974).

Is G l u t a m i c A c i d the P y r a m i d a l T r a c t N e u r o t r a n s m i t t e r ? T . W . STONE ~

Department o/ Physiology, University o[ Aberdeen, Marischal College, Aberdeen A B 9 7AS (Scotland), 2 December 7975. Summary. Applied b y microiontophoresis, 1-hydroxy-3-amino-pyrrolidone-2 (HA-966) antagonized e x c i t a t i o n b y g l u t a m i e acid b u t n o t b y acetylcholine of neurones in the rat cuneate nucleus. HA-966 blocked the short latency excitation of cuneate neurones following stimulation of t h e p y r a m i d a l t r a c t on 28 of 40 cells (70%). Thus, g l u t a m a t e or a related amino-acid m a y be the n e u r o t r a n s m i t t e r released b y p y r a m i d a l t r a c t neurones. The p y r a m i d a l tracts h a v e at least two i m p o r t a n t physiological functions. T h e y are i n v o l v e d in t h e control of fine m o v e m e n t s of distal limb muscles 2, and t h e y m o d u l a t e the a m o u n t of sensory i n f o r m a t i o n reaching higher centres such as t h e cerebral c o r t e x 3. Y e t little a t t e n t i o n has been paid to t h e possible n e u r o t r a n s m i t t e r released b y the p y r a m i d a l t r a c t (PT) neurones. F r o m n e u r o c h e m i c a l analyses t h e t r a n s m i t t e r is unlikely to be acetylcholine ~, 5 or a mOlloamine such as noradrenaline 6, 7. Studies h a v e therefore been carried out using the amino-acid a n t a g o n i s t 1-hydroxy-3-amino-pyrrolidone-2 (HA-966) to d e t e r m i n e w h e t h e r an e x c i t a t o r y aminoacid could be involved. Materials and methods. A d u l t male rats were anaesthetised w i t h urethane, 1.25 g/kg. i.p. and placed on a h e a t i n g pad to m a i n t a i n t h e rectal t e m p e r a t u r e at 37-38~ T h e left cerebral c o r t e x and r i g h t cuneate nucleus were exposed and t h e n covered w i t h 5% agar in saline to reduce p n l s a t o r y m o v e m e n t s . P y r a m i d a l t r a c t fibres were excited b y single anodal pulses of 0.1 msec d u r a t i o n applied to the m o t o r or sensory areas of the c o r t e x b y a silver ball electrode. E v o k e d spike a c t i v i t y in the cuneate was considered to be m o n o s y n a p t i c a l l y induced, and therefore directly due to P T a c t i v i t y a) if t h e spike had a m i n i m u m latency not exceeding 5.0 msecS; b) if the m i n i m u m spike l a t e n c y did n o t v a r y b y more t h a n 4- 0.2 msec w i t h just threshold and 5 • threshold stimuli, and c) if the spike would Iol-

low 25-100 H z stimulation b u t n o t more t h a n 100 Hz. These criteria were i n t e n d e d to eliminate from s t u d y a n y p o l y s y n a p t i c a l l y induced spikes. Drugs were applied to single cells in t h e cuneate nucleus b y microiontophoresis using 5-barrelled micropipettes as described elsewhere", 1~ One barrel always contained 200 m M NaC1 solution for current balancing and current testing% The remaining 4 barrels were filled w i t h a selection of: sodium L-glutamate 200 m M , p H 8.0; acetylcholine chloride 200 m M , p H 5.0; atropine sulphate 100 raM, p H 5.0; 1-hydroxy-3-amino-pyrrolidone-2 (HA966) 100 m M , p H 4.5 in 0.2 N HC1. E x t r a c e l l u l a r unit a c t i v i t y was recorded b y a single electrode fixed alongside t h e m u l t i b a r r e l assembly 10. Spikes were amplified in a F e n l o w AD55 preamplifier, 1 Acknowledgment. I wish to thank Dr. J..C WATKINSfor the gift of a sample of HA-966. 2 R. PORTER, Progr. Neurobiel. 7, 1 (1970). M. WIESENDANGER,Ergebn. Physiol. 67, 72 (1969). 4 G. B. KOELLE,J. temp. Neurol. 700, 211 (1954). 9 5 W. FELDBERGand M. VoG% J. Physiol., Lend. 107, 372 (1948). 6 A. CARLSSON, B. FALCKand N.-A. HILLARP, Acta physiol, scand. 56 suppl. 196 (1962). 2 A. DAHLSTR(SMand K. FuxE, Acta physiol, scand. 62, suppl. 232 (1964). s T. W. STOtCE,ExpI. Neurol. 35, 492 (1972). 9 T. W. STONE, J. Physiol., Lend. 225, 485 (1972). 10 T. W. STONE, J. Physiol., Lend. 233, 211 (1973).

582

=_.

g

Specialia

Zll

EXPERIENTIA 32/5 Fig. 1. Records of the firing rate of a c u n e a t e neurone excited b y g l u t a m a t e , 60 nA (G) and acetylcholine, 60 nA (A). An o u t w a r d current control of 100 nA (Na +) has noeffeet on the cell. HA-966, 40 nA (H) aboIishes the g l u t a m a t e excitations w i t h o u t d e t e c t a b l y affecting the acetylcholine responses. Time: 2 min.

2m Na+

H

H

e

"

2S Ill

g

s

@

2m

H

Na+

Fig. 2. Upper trace: Record of the firing r a t e of a neurone excited b y pulses of g l u t a m a t e , 60 nA. The responses are greatly reduced b y HA-966, 40 nA (H), b u t unaffected b y an o u t w a r d current of 100 nA (Na+). Lower records: P h o t o g r a p h s of the responses of the neurone to a c t i v a t i o n of corticofugal fibres, t a k e n at the times i n d i c a t e d in the upper trace. I n a) is seen an early m o n o s y n a p t i c spike followed b y p o l y s y n a p t i c spikes. In b} and e), t a k e n w h e n g l u t a m a t e e x c i t a t i o n was blocked b y HA-966, the early spike has also been blocked, b u t the later spikes show t h a t HA-966 was not affecting spike height at this time. The early spike r e a p p e a r s as the g l u t a m a t e respoflses r e a p p e a r in d). O u t w a r d current does n o t affect the cell (e). The spikes in d) and e) are slightly smaller t h a n in a - e due to m o v e m e n t of the eleetrode. Upper t r a c e : time 2 rain. Lower records: calibrations 200 DV and 10 msec.

15.5. 1976

Specialia

a n d p a s s e d t h r o u g h a pulse s h a p i n g a n d c o u n t i n g u n i t . A c o n t i n u o u s r e c o r d of cell firing r a t e was o b t a i n e d o n a S e r v o s c r i b e p e n recorder. T h e spike a c t i v i t y was also m o n i t o r e d o n T e l e q u i p m e n t oscilloscopes. Results. T h e a b i l i t y of H A - 9 6 6 t o a n t a g o n i z e a m i n o acid e x c i t a t i o n was c o n f i r m e d b y a p p l y i n g t h e d r u g w i t h a n o u t w a r d c u r r e n t of 4 0 - 6 0 n A to cells in t h e c u n e a t e n u c l e u s w h i c h were e x c i t e d b y pulses of g l u t a m a t e l a s t i n g a b o u t 5 sec. H A - 9 6 6 r e d u c e d g l u t a m a t e e x c i t a t i o n o n 35 cells of 48 t e s t e d (73%) (Figure 1). D i f f i c u l t y was e x p e r i e n c e d in d e m o n s t r a t i n g t h e specificity of t h i s a n t a g o n i s m since n o s u b s t a n c e s c o n s i s t e n t l y e x c i t e c u n e a t e n e u r o n e s o t h e r t h a n a m i n o - a c i d s a n d some c h e l a t i n g a g e n t s 11. H o w e v e r , a few cells (6) were f o u n d w h i c h were excited b y a c e t y l c h o l i n e n , 12, a n d o n all t h e s e cells H A - 9 6 6 p r o v e d able t o r e d u c e g l u t a m a t e e x c i t a t i o n b y a t l e a s t 50% w i t h o u t a f f e c t i n g a c e t y l c h o l i n e excitat i o n to a n y d e t e c t a b l e e x t e n t (Figure 1). N [ o n o s y n a p t i c a l l y e v o k e d spikes were i n d u c e d in 40 of 112 n e u r o n e s s t u d i e d in t h e c u n e a t e nucleus. All b u t 3 of t h e s e u n i t s were e n c o u n t e r e d 600-800 > m below t h e surface of t h e c u n e a t e nucleus. T h e i o n t o p h o r e s i s of H A 966 r e s u l t e d in a b l o c k a d e of t h i s m o n o s y n a p t i c a l l y i n d u c e d a c t i v i t y in 28 of t h e 40 cells (70%). G l u t a m a t e e x c i t a t i o n was r e d u c e d b y a t l e a s t 5 0 % in all t h e s e ceils a t t h e t i m e of s y n a p t i c b l o c k a d e (Figure 2). 11 cells, i n c l u d i n g t h a t i l l u s t r a t e d in F i g u r e 2, s h o w e d p o l y s y n a p t i c a l l y i n d u c e d spikes following t h e m o n o s y n a p t i c spike. T h e l a t e r spikes were u n a f f e c t e d b y HA-966, i n d i c a t i n g t h a t t h i s a n t a g o n i s t was n o t h a v i n g a n y d i r e c t local a n a e s t h e t i c effect o n t h e cell m e m b r a n e , a n d was n o t p r o d u c i n g a generalized nonspecific b l o c k a d e of all s y n a p t i c a l l y i n d u c e d a c t i v i t y . A t r o p i n e was applied t o 6 cells a n d in n o n e of t h e s e did a n y a p p a r e n t c h a n g e of s y n a p t i c a l l y e v o k e d a c t i v i t y result. Discussion. T h e efficacy a n d specificity of t h e a n t a g o n i s m of a m i n o a c i d e x c i t a t i o n b y HA-96613 1~ h a s b e e n confirmed. The possibility must be considered that by stimulating t h e c e r e b r a l c o r t e x a c t i v i t y w a s b e i n g i n d u c e d in t h e c u n e a t e n u c l e u s over p a t h w a y s o t h e r t h a n t h e P T . T h i s is u n l i k e l y since i t h a s b e e n s h o w n t h a t corticallyi n d u c e d e x c i t a t i o n of cells in t h e dorsal c o l u m n nuclei is

Locomotory

Energetics

in a Marsupial

583

a b o l i s h e d b y s e c t i o n i n g t h e P T ~6,17. T h e P T , h o w e v e r , is k n o w n t o send collaterals to t h e s e nuclei, c a p a b l e of m o n o s y n a p t i c a l l y a c t i v a t i n g n e u r o n e s t h e r e ~8. All t h e s h o r t l a t e n c y spikes seen in t h e p r e s e n t s t u d y were e n c o u n t e r e d r e l a t i v e l y d e e p l y in t h e c u n e a t e nucleus. A P T origin for t h e s e spikes is t h e r e f o r e s u p p o r t e d b y anatomical studies showing that PT axons terminate p r e f e r e n t i a l l y in t h e d e e p e r layers of t h e d o r s a l c o l u m n nuclei ~9, 20. T h e a n t a g o n i s m b y H A - 9 6 6 of s y n a p t i c a l l y i n d u c e d spikes t h e r e f o r e suggests t h a t t h e n e u r o t r a n s m i t t e r released b y a x o n s of t h e P T m i g h t b e a n e x c i t a t o r y a m i n o acid, t h o u g h H A - 9 6 6 c a n n o t d i f f e r e n t i a t e b e t w e e n s e v e r a l a m i n o - a c i d s s u c h as g l u t a m a t e , a s p a r t a t e a n d DL-homoc y s t e a t e ~8, 15. G l u t a m a t e is a p a r t i c u l a r l y s t r o n g c a n d i d a t e since i t is p r e s e n t in h i g h c o n c e n t r a t i o n s in t h e dorsal c o l u m n nuclei 21, it is p r e s e n t in s y n a p t o s o m e s in t h e c e r e b r a l cortex22 a n d i t c a n be 'released' a t t h e c o r t i c a l surface b y s t i m u l a t i o n of some, b u t n o t all, a f f e r e n t p a t h w a y s 23. I t h a s also b e e n s h o w n t h a t some cortical n e u r o n e s excited b y t h e P T are e x t r e m e l y s e n s i t i v e t o t h e m i c r o i o n t o p h o r e s i s of g l u t a m a t e ~~ T h e s e f i n d i n g s suggest t h a t g l u t a m a t e m a y b e t h e p y r a m i d a l t r a c t n e u r o transmitter.

11 A. GALINDO,162. KRNJEVld and S. SCHWARTZ, J. Physiol., Lond. 792, 359 (1966). le F. A. STEINER and ~. MEVER, Experientia 22, 58 (1966). la j. DAvIEs and J. C. WATKINS, Nature, Loud. 238, 61 (1972). 14 j. DAVIES and J. C. WATKINS, Brain. Res. 59, 311 (1973). 15 j. DAVIES and J, C. WATKINS,Neuropharmacology 72, 637 (1973). 16 S. J. JABBUR and A. L. TOWE, J. Neurophysiol. 2d, 499 (1961). 17 ~{. LEVITT, M. CARRERAS, C. N. LIU a n d W. W. CHAMBERS,

Archo. ital. Biol. 702, 197 (1964). lS F. HARRIS, S. J. JABBUR, R. W. MORSE and A. L. TOWE, Nature,

Lond. 208, 1215 (1965). i9 F. WALBXRO, Brain 80, 273 (1957). s0 H. G. J. M. KU~ZPERS,J. Anat. 92, 198 (1958). ~1 j. L. JOHNSON and M. H. APRISON, Brain Res. 24, 285 (1970). 22 H. F. BRADFORD a n d A. S. THOMAS, J. Neuroehem. 76, 1495

(1969). 23 H. JASPER and I. KOYAMA,Electroenceph. clin. Neurophysiol. 24, 292 (1968).

(Antechinomys spenceri)

and a Rodent

(Notomys alexis)

R. V. ]SAUDINETTE,KAREN A. INTAGLEa n d R. A. D. SCOTT 1

School o/ Biological Sciences, The Flinders University o/South Australia, Bed/ord Park 5042 (South Australia), 4 December 1975. Summary. S t e a d y s t a t e o x y g e n c o n s u m p t i o n was c o m p a r e d in a r o d e n t Notomys alexis a n d a m a r s u p i a l Antechinomys spenceri. T h e m a r s u p i a l was f o u n d t o diverge f r o m p r e d i c t e d e u t h e r i a n e n e r g e t i c p a t t e r n s . N. alexis a p p e a r s t o use e n e r g y s t o r a g e as a s i g n i f i c a n t p a r t of t h e step cycle before b e c o m i n g bipedal. Aerobic scope a n d h e a t s t o r a g e d u r i n g r u n n i n g are s i m i l a r in b o t h species. NIarsupials g e n e r a l l y h a v e s t a n d a r d r a t e s of o x y g e n consumption about 30% below the predicted entherian values, b o d y t e m p e r a t u r e s 2-3 degrees lower 2 a n d r e s t i n g h e a r t r a t e s a b o u t h a l f t h a t g i v e n for e u t h e r i a n species ~. W e r e p o r t h e r e o n a c o m p a r i s o n in e n e r g y e x p e n d i t u r e d u r i n g l o c o m o t i o n b e t w e e n a m a r s u p i a l a n d a r o d e n t of s i m i l a r b o d y f o r m a n d weight. Materials and methods. R a t e s of o x y g e n c o n s u m p t i o n , b o d y t e m p e r a t u r e , s t r i d e l e n g t h a n d f r e q u e n c y were m e a s u r e d in 2 i n d i v i d u a l s of t h e c a r n i v o r o u s d a s y u r i d m a r s u p i a l Antechinomys spenceri (28.2 g a n d 31.6 g) a n d

5 s p e c i m e n s of t h e m u r i d r o d e n t Notomys alexis ( m e a n b o d y w e i g h t 27.4 g). B o t h i n h a b i t t h e A u s t r a l i a n desert, o c c u r r i n g s y m p a t r i c M l y o v e r p a r t of t h e i r range. T h e y 1 We thank P. BAVERSTOCK,C. WATTS and H. ASLIN for the use of the Antechinomys, Ms. B. WHAITE for assistance. Supported by Grants from the Nuffield Foundation, the Ingram Trust and the Flinders University Research Committee to RVB. R. E. MACMILLEN and J. E. NELSOn, Am. J. Physiol. 217, 1246 (1969). - T. J. DAWSON and A. J. HIJLBERT, Am. J. Physiol. 218, 1233 (1970). 3 j. E. KIN~EAR and G. D. BROW~, Nature, Lond. 215, 1501 (1967).

Is glutamic acid the pyramidal tract neurotransmitter?

15.5. 1976 Speeialia e v e n 6 weeks after p i n e a l e c t o m y also indicated the ind e p e n d e n t synthesis of N A S in this tissue 3. Using...
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