- Email: [email protected]
Electrophoretic study of pipecolic acid, a biogenic imino acid, in the mammalian brain
Brain Research, 193 (1980) 6Oh; 613 ~i Elsevier/North-Holland Biomedical Pres~
608
Electrophoretic study of pipecolic acid, a biog,enic imino acid, in the mammalian brain
YOSHITOSHI KASI~, KAZUO TAKAHAMA, TADATOSHi HASHIMOTO, JUN KAISAKU, YOSHIRO OKANO and TAKESHI MIYATA
Department of Chemico-Pharmacology, Faculty of Pharmaceutical Sciences, Kumamoto University, I-5 Ohe-honmachi, Kumamoto 862 (Japan) (Accepted March 6th, 1980)
Key words: pipecolic acid - - imino acid - - GABA uptake inhibitor - - neuromodulator -- microelectrophoresis - - unit recording - - hippocampal pyramidal cell
Pipecolic acid (PA), one of the imino acids, is a normal constituent in the mammalian brain. It is said that PA is a major intermediate of lysine metabolism in the rat brain. Biochemical studies have suggested that PA may be involved in the regulation of synaptic mechanism in the CNS. Moreover, the pathopbysiological significance of PA has been also suggested by some investigators. However, there has so far been no good evidence based on the comprehensive electrophysiological experiments. Using unit recording and microelectrophoretic technique, the action of PA on single neuron activities in the rat brain was examined. PA depressed the firing of 88 out of l 15 cortical neurons tested. Only 2 were excited and 25 remained unaffected. All the identified hippocampal pyramidal neurons examined were uniformly inhibited. It has been reported that PA inhibits the uptake of GABA into the brain slices and enhances the release of GABA from the slices. Thus, it is likely that the inhibitory response due to PA may have some connections with GABAergic transmission. On the other hand, it remains to be clarified whether the specific PA sensitive receptors exist in the brain. Our findings provide a clue to the elucidation of the presumed synaptic involvement of PA in the CNS. D u r i n g the course of studies on the physiological role of piperidine 11, a n o r m a l b r a i n c o n s t i t u e n t possessing synaptoropic actions, our a t t e n t i o n has been focused on pipecolic acid (PA), because this i m i n o acid is not only a possible immediate precursor of piperidine 11 but also shows p o t e n t pharmacological actions 18. Intraventricutar a d m i n i s t r a t i o n of P A produces ptosis, h y p o t o n i a a n d sedation accompanied by suppression of fighting behavior in mice. I n addition, a d m i n i s t r a t i o n into the cerebellum a n d h i p p o c a m p u s of cats with chronically implanted c a n n u l a e and electrodes caused p r o m i n e n t depressions both in E E G and behavior. PA is also a n o r m a l constituent in the m a m m a l i a n b r a i n 1°. Some investigators d e m o n s t r a t e d the in vitro '~4 a n d in vivo z f o r m a t i o n of PA from L-lysine in the brain. Based o n tracer studies, Chang3, 4 reported that P A is a p r o b a b l e m a j o r intermediate of lysine m e t a b o l i s m in the rat brain. A n o t h e r line of biochemical studies, carried out by some groups including ourselves, d e m o n s t r a t e d that [aH]PA is taken up into the brain slices lz or s y n a p t o s o m a l fractions17, 21 in a m o d e of high affinity a n d in a temperature a n d s o d i u m - d e p e n d e n t m a n n e r . The uptake process is o u a b a i n - t2,21 a n d d i n i t r o p h e n o l sensitivelL F u r t h e r m o r e , N o m u r a et al. 20 and our own study 1~ i n d e p e n d e n t l y found
609 that radioactive PA taken up into the brain slices is released in high K ÷ medium and that the release is significantly inhibited by perfusion with Ca~+-free medium. These findings suggest that PA should be involved in the regulation of synaptic mechanism in the central nervous system. Moreover, PA seems to have a connection to a neurological disease in infants such as hyperpipecolatemia s,27 which seems to be due to inborn error of pipecolic acid metabolism. However, there has so far been no good evidence based on a comprehensive electrophysiological experiment. As the initial step for elucidation of the role of PA in synaptic mechanism, the effects of PA on the activity of the cortical and hippocampal pyramidal neurons were investigated by the use of single unit recording and microelectrophoretic technique. Forty-two male Wistar rats, weighing 180-230 g, fed ad libitum, were anesthetized with a-chloralose (60 mg/kg, i.p.) plus urethane (800 mg/kg, i.p.). After each animal was fixed with the aid of a stereotaxic instrument, the left fronto-parietal cerebral cortex was carefully exposed; the cortical surface was covered with 2 ~o Agar dissolved in Tyrode solution to prevent drying and to suppress pulsations of the brain. The body temperature was automatically maintained at 37.0 q- 1.0 °C. The unit recording and microelectrophoresis were carried out according to the method of Oomura et a123. Glass microelectrodes filled with 2 M sodium acetate, DC resistance 8-10 Mf~, used for recording unit activities, were cemented under a microscope to 7-barrelled micropipettes with polystyrene (Q-DOPE). The micropipettes whose overall outside tip diameter did not exceed 1 #m were used. Since the electrical resistance of the pipettes might change on flowing of the current, a constant current device was employed. The pipettes contained the following solutions: L-PA (0.1-0.5 M in 0.165 M NaCI solution, pH 6.0-6.7), monosodium L-glutamate (2.0 M, pH 7.4), sodium chloride (0.165 M) and sucrose (0.4 M in 0.1 M NaCI solution). To minimize the possibility that any particular effects may occur with the biased pipettes, at least 5 different micropipettes were used. The electrophoretic currents ejected were within the range of 5-70 nA. The coordinates of sites for injection-recording and for stimulation were taken from the brain atlas of K~Snig and Klippel t3. Hippocampal pyramidal cells were identified by depth beneath the cortical surface, a typical firing pattern 9, and the characteristic inhibitory 'pause't, 5 and antidromic potential t due to fimbrial stimulation. Extracellular unit activity was monitored on an oscilloscope (Tectronix: Model 5103N), and processed by the conventional method. After the experiment, the stimulating site in the fimbria was confirmed histologically. Almost all cells used were spontaneously firing with the rate of 10--20 spikes/sec; however, in a few cells, the firing was maintained by the continuous ejection of Lglutamate with a low steady current. PA was successfully applied to 115 cortical cells. A typical example of a single cortical neuron inhibited by PA is shown in Fig. 1A. Out of the 115 neurons, 88 decreased and only 2 increased the rate of firing. The remaining 25 were unaffected. The neurons affected by PA did not respond to Na ÷ or sucrose application, indicating that the effects of PA were neither due to current flow nor due to local changes in osmotic pressure. The effects caused by PA were accompanied by little change in the
610
3°I , 2O
~ ~o
Sw (/)
0
or"
-
-
~ PA 10
5 30
15
r
Na*
PA 5
15
5
5
5
5
5
-
v
ft. 20
10
PA 5
5
10
20
40
N~ 40
Suc 40
• 30sec
Fig. 1. Inhibitory effect of pipecolic acid on the firing rate of a cortical (A) and a hippocampal pyramidal neuron (B). Pipecolic acid was applied with the increasing currents for the period of time indicated by the line under each ratemeter record. To confirm that PA response is due to neither current flow nor to local changes in osmotic pressure, Na ~ and sucrose were also applied. amplitude of unit firings. Low currents such as 5-15 nA were usually sufficient to produce the inhibitory effect, i.e. the rate of firings gradually decreased with the latency of 2-3 sec and recovered to the pre-application level 5-10 sec after the current was turned off. As shown in Fig. IA, the effect was reproducible for at least 5 repetitive applications without alzparent changes in magnitude and duration. In addition, the effect was dose-dependent; the complete suppression of the firing, often followed by a slight rebound excitation, occurred with higher currents. The time course of the excitatory response to PA was similar to that of the inhibitory one, though the former was less reproducible. The response to PA was also studied on pyramidal neurons of the dorsal hippocampus. Fig. 1B illustrates the inhibitory effect of PA applied with increasing doses for the same periods of time. All the 16 neurons tested were uniformly inhibited by the application of PA with currents as low as 5-15 nA. The latency and duration of the response were almost the same length as was the case in the cortical neurons. The response was reproducible and dose-dependent in the pyramidal neurons as well as the cortical neurons. In this type of experiment, care must be taken to minimize the effects of pH of test solution, since H + affects the unit activity when a test solution of low pH is used 14. In the present study, pH of the solution containing PA was higher than 6.0. Thus, the effects observed by PA appeared to be the chemical ones, as endorsed also by the control experiments using Na + and sucrose. The results presented above provide the first findings of the effect of PA on single
i
611 neuron activity, and demonstrate that electrophoreticaUy-applied PA exerted significant depressant effects on both cerebral cortical and hippocampal pyramidal neurons. In addition to this predominant effect, a few of the cortical neurons tested were stimulated or unaffected by PA. Electrophoretic current required for production of the substantial effect was relatively small, usually 10-15 nA, though as small a current as 5 nA was sufficient to produce the excitatory response to glutamate. Judging from the current intensity applied, the inhibitory effect of PA appeared to be potent. This finding seems to be on almost the same line as our previous results obtained from the intraventricular and intracerebral administrations of PA. There are 3 conceivable interpretations for the mechanisms of the inhibitory effect of PA as follows: (1) direct actions on receptors for GABA, noradrenaline and so forth and/or on unknown receptors or receptive sites including ones specific for PA; (2) indirect effects via actions on the synaptic events such as biosynthesis, release and inactivation of transmitter substances; and (3) an action through its active metabolites. Out of these possibilities, the third seems to be excluded, because: (1) the latency of the effect is relatively short; (2) a-aminoadipic acid, a major metabolite of PA, is one of excitatory amino acids 7, and (3) piperidine, another metabolite of PA, produces predominantly the excitation of the cortical and hippocampal neurons z6. The first and second described above may be applicable for explanation of the mechanisms of the inhibitory action produced by PA. However, PA is unlikely to act directly on the GABA receptor. Segal et al. 25 has proposed that the physiologically active conformation of GABA is the fully extended one with the spatial separation of amino and carboxy groups at a distance of 0.42-0.48 nm. Given their assertion, the PA molecule is probably difficult to fit the GABA receptor for initiation of the inhibitory action, since the geometrical separation of the two groups in PA ranges from 0.2 to 0.3 nm, that is, shorter than that of GABA. The inhibitory action of microelectrophoretic PA is not difficult to correlate with recent biochemical findings. PA has been found to inhibit the uptake of [3H]GABA into the brain slices12,19 and also to facilitate the release of the compound from the slices12,z2. It is well known that GABA may function as a neuronal inhibitory transmitter in the supraspinal regions such as the cortex, hippocampus and cerebellum. Taken together, it seems likely that the inhibitory respose to PA may be produced by affecting the GABAergic transmission, i.e. by inhibiting the uptake of endogenous GABA and/or by facilitating the release of the inhibitory transmitter. Curtis et al. 6 and Lodge et al. 15 have demonstrated that a series of GABA uptake inhibitors such as nipecotic acid, a structural analog of pipecolic acid, depress the firing of single neurons and enhance the response to electrophoretic GABA. Furthermore, it has been reported that iontophoretic nipecotic acid potentiates the inhibitory action of synaptically-released GABA in rats 16. These observations are relevant to our speculation mentioned above. In this context, it should be noted that all the identified hippocampal pyramidal neurons, which receive the GABAergic inputs from the adjacent basket cells, were uniformly inhibited by the application of PA. There seems no available information regarding the mechanism of the excita-
612 tory effect of PA, observed in a small percentage of the cortical cells tested. A t present, it is too early to suggest a physiological role for PA as a n e u r o m o d u lator or a n e u r o t r a n s m i t t e r . It also remains to be clarified whether the specific PAsensitive receptors exist in the cortex a n d hippocampus. Nevertheless, the present observation provides a clue to the elucidation of a role of PA in the synaptic mechanism. This work was supported by grants from the ministry of education, Japan. We sincerely t h a n k Prof. Y. O o m u r a for valuable instruction in u n i t recordings a n d electrophoretic techniques, and Dr. N. Akaike for instruction in basic techniques for electrophysiological experiments. We also t h a n k Mr. S. Ishizaka for his kind guidance c o n c e r n i n g p r e p a r a t i o n of a c o n s t a n t current and other devices, a n d are t h a n k f u l to Mr. K. Satoh for histological examination.
1 Andersen, P., Eccles, J. C. and L~yning, Y., Pathway of postsynaptic inhibition in the hippocampus, J. Neurophysiol., 27 (1964) 608-609. 2 Chang, Y. F., Lysine metabolism in the rat brain: Blood-brain barrier transport, formation of pipecolic acid and human hyperpipecolatemia, J. Neurochem., 30 (1978) 355-360. 3 Chang, Y. F., Pipecolic acid pathway: the major lysine metabolic route in the rat brain, Biochem. biophys. Res. Commun., 69 (1976) 174-180. 4 Chang, Y. F., Lysine metabolism in the rat brain: the pipe.colic acid forming pathway, J. Neurochem., 30 (1978) 347-354. 5 Curtis, D. R., Felix, D. and McLeUan, H., GABA and hippocampal inhibition, Brit. J. Pharma. col., 40 (1970) 881-883. 6 Curtis, D. R., Game, C. J. A. and Lodge, D., The in vivo inactivation of GABA and other inhibitory amino acids in the cat nervous system, Exp. Brain Res., 25 (1976) 413~28. 7 Curtis, D. R. and Watkins, J. C., Acidic amino acids with strong excitatory actions on mammalian neurones, J. Physiol. (Lond.), 166 (1963) 1-14. 8 Gatfield, P. D., Taller, R. T., Hinton, G. G., Wallace, A. C., Abdelnour, G. M. and Haust, M. D., Hyperpipecolatemia. A new metabolic disorder associated with neuropathy and hepatomegaly; a case study, Canad. med. Ass. J., 99 (1968) 1215-1233. 9 Kandel, E. R. and Spencer, W. A., Electrophysiology of hippocampal neurons II. After-potentials and repetitive firing, J. Neurophysiol., 24 (1961) 243-259. 10 Kasd, Y., Kataoka, M., Miyata, T. and Okano, Y., Pipecolic acid in the dog brain, LiJe Sci., 13 (1973) 867-873. 11 Kas6, Y. and Miyata, T., Neurobiology of piperidine: its relevance to CNS function. In E. Costa, E. Giacobini and R. Paoletti (Eds.), First and Second Messengers - - New Vistas, Advances in Biochemical Psychopharmacology, Vol. 15, Raven Press, New York, 1976, pp. 5-16. 12 Kasd, Y., Miyata, T., Hirata, A., Morimoto, H., Okano, Y. and Takahama, K., Pharmacological studies on alicyclic amines XXVI : uptake and release of piperidine and pipecolic acid in the brain slice, Folia pharmacol, jap., in press. 13 K6nig, J. F. R. and Klippel, R. A., The Rat Brain, Williams and Wilkins, Baltimore, 1963. 14 Krnjevic, K. and Phillis, J. M., Iontophoretic studies of neurones in the mammalian cerebral cortex, J. Physiol. (Lond.), 165 (1963) 274-304. 15 Lodge, D., Johnston, G. A. R., Curtis, D. R. and Brand, S. J., Effects of the Areca nut constituents arecaidine and guvacine on the action of GABA in the cat central nervous system, Brain Research, 136 (1977) 513-522. 16 Matthews, W. D. and McCafferty, G. P., Enhancement of hippocampal recurrent inhibition by inhibitors of GABA uptake, Pharmacologist, 21 (1979) 149p 17 Meek, J. L., Uptake and metabolism of piperidine and pipecolic acid in brain, Fed. Proc., 33 (1974) 468p. 18 Miyata, T., Kamata, K., Noguchi, M., Okano, Y. and Kasd, Y., Pharmacological studies on
613 alicyclic amines XV: Intracerebral administration of pipecolic acid, Jap. J. PharmacoL, 23 Suppl. (1973) 81p. 19 Nomura, Y., Okuma, Y. and Segawa, T., Influence of piperidine and pipecolic acid on the uptake of monoamines, GABA and glycine into Pz fractions of the rat brain and the spinal cord, J. Pharm. Dyn., 1 (1978) 251-255. 20 Nomura, Y., Okuma, Y., Segawa, T., Schmidt-Glenewinkel, T. and Giacobini, E., A calciumdependent, high potassium-induced release of pipecolic acid from rat brain slices, J. Neurochem., 33 (1979) 803-805. 21 Nomura, Y., Schmidt-Glenewinkel, T. and Giacobini, E., Uptake of piperidine and pipecolic acid by synaptosomes from mouse brain, Neurochem. Res., in press. 22 Okuma, Y., Nomura, Y. and Segawa, T., The effect of piperidine and pipecolic acid on high potassium-induced release of noradrenaline, serotonin and GABA from rat brain slices, J. Pharm. Dyn., 2 (1979) 261-265. 23 Oomura, Y., Sugimori, M., Nakamura, T. and Yamada, Y., Contribution of electrophysiological techniques to the understanding of central control systems. In G. J. Mogenson and F. R. Calaresu (Eds.), Neuronal Integration of Physiological Mechanisms and Behaviour, University of Toronto Press, Toronto, 1975, pp. 375-395. 24 Schmidt-Glenewinkel, T., Nomura, Y. and Giacobini, E., The conversion of lysine into piperidine, cadaverine, and pipecolic acid in the brain and other organs of the mouse, Neurochem. Res., 2 (1977) 619-637. 25 Segal, M., Sims, K. and Smissman, E., Characterization of an inhibitory receptor in rat hippocampus: a microiontophoretic study using conformationally restricted amino acid analogues, Brit. J. Pharmacol., 54 (1975) 181-188. 26 Takahama, K., Kaisaku, J., Miyata, T., Okano, Y. and Kas6, Y., Pharmacological studies on alicyclic amines XXV: effect of microiontophoretically applied piperidine on brain neuron, Jap. J. Pharmacol., Suppl., 29 (1979) 48. 27 Thomas, G. E., Haslam, R. H. A., Batshaw, M. L., Capute, A. J., Neidengard, L. and Lansom, J. L., Hyperpipecolic acidemia associated with hepatomegaly, mental retardation, optic nerve dysplasia and progressive neurological disease, Clin. Genetics, 8 (1975) 376-382.
608
Electrophoretic study of pipecolic acid, a biog,enic imino acid, in the mammalian brain
YOSHITOSHI KASI~, KAZUO TAKAHAMA, TADATOSHi HASHIMOTO, JUN KAISAKU, YOSHIRO OKANO and TAKESHI MIYATA
Department of Chemico-Pharmacology, Faculty of Pharmaceutical Sciences, Kumamoto University, I-5 Ohe-honmachi, Kumamoto 862 (Japan) (Accepted March 6th, 1980)
Key words: pipecolic acid - - imino acid - - GABA uptake inhibitor - - neuromodulator -- microelectrophoresis - - unit recording - - hippocampal pyramidal cell
Pipecolic acid (PA), one of the imino acids, is a normal constituent in the mammalian brain. It is said that PA is a major intermediate of lysine metabolism in the rat brain. Biochemical studies have suggested that PA may be involved in the regulation of synaptic mechanism in the CNS. Moreover, the pathopbysiological significance of PA has been also suggested by some investigators. However, there has so far been no good evidence based on the comprehensive electrophysiological experiments. Using unit recording and microelectrophoretic technique, the action of PA on single neuron activities in the rat brain was examined. PA depressed the firing of 88 out of l 15 cortical neurons tested. Only 2 were excited and 25 remained unaffected. All the identified hippocampal pyramidal neurons examined were uniformly inhibited. It has been reported that PA inhibits the uptake of GABA into the brain slices and enhances the release of GABA from the slices. Thus, it is likely that the inhibitory response due to PA may have some connections with GABAergic transmission. On the other hand, it remains to be clarified whether the specific PA sensitive receptors exist in the brain. Our findings provide a clue to the elucidation of the presumed synaptic involvement of PA in the CNS. D u r i n g the course of studies on the physiological role of piperidine 11, a n o r m a l b r a i n c o n s t i t u e n t possessing synaptoropic actions, our a t t e n t i o n has been focused on pipecolic acid (PA), because this i m i n o acid is not only a possible immediate precursor of piperidine 11 but also shows p o t e n t pharmacological actions 18. Intraventricutar a d m i n i s t r a t i o n of P A produces ptosis, h y p o t o n i a a n d sedation accompanied by suppression of fighting behavior in mice. I n addition, a d m i n i s t r a t i o n into the cerebellum a n d h i p p o c a m p u s of cats with chronically implanted c a n n u l a e and electrodes caused p r o m i n e n t depressions both in E E G and behavior. PA is also a n o r m a l constituent in the m a m m a l i a n b r a i n 1°. Some investigators d e m o n s t r a t e d the in vitro '~4 a n d in vivo z f o r m a t i o n of PA from L-lysine in the brain. Based o n tracer studies, Chang3, 4 reported that P A is a p r o b a b l e m a j o r intermediate of lysine m e t a b o l i s m in the rat brain. A n o t h e r line of biochemical studies, carried out by some groups including ourselves, d e m o n s t r a t e d that [aH]PA is taken up into the brain slices lz or s y n a p t o s o m a l fractions17, 21 in a m o d e of high affinity a n d in a temperature a n d s o d i u m - d e p e n d e n t m a n n e r . The uptake process is o u a b a i n - t2,21 a n d d i n i t r o p h e n o l sensitivelL F u r t h e r m o r e , N o m u r a et al. 20 and our own study 1~ i n d e p e n d e n t l y found
609 that radioactive PA taken up into the brain slices is released in high K ÷ medium and that the release is significantly inhibited by perfusion with Ca~+-free medium. These findings suggest that PA should be involved in the regulation of synaptic mechanism in the central nervous system. Moreover, PA seems to have a connection to a neurological disease in infants such as hyperpipecolatemia s,27 which seems to be due to inborn error of pipecolic acid metabolism. However, there has so far been no good evidence based on a comprehensive electrophysiological experiment. As the initial step for elucidation of the role of PA in synaptic mechanism, the effects of PA on the activity of the cortical and hippocampal pyramidal neurons were investigated by the use of single unit recording and microelectrophoretic technique. Forty-two male Wistar rats, weighing 180-230 g, fed ad libitum, were anesthetized with a-chloralose (60 mg/kg, i.p.) plus urethane (800 mg/kg, i.p.). After each animal was fixed with the aid of a stereotaxic instrument, the left fronto-parietal cerebral cortex was carefully exposed; the cortical surface was covered with 2 ~o Agar dissolved in Tyrode solution to prevent drying and to suppress pulsations of the brain. The body temperature was automatically maintained at 37.0 q- 1.0 °C. The unit recording and microelectrophoresis were carried out according to the method of Oomura et a123. Glass microelectrodes filled with 2 M sodium acetate, DC resistance 8-10 Mf~, used for recording unit activities, were cemented under a microscope to 7-barrelled micropipettes with polystyrene (Q-DOPE). The micropipettes whose overall outside tip diameter did not exceed 1 #m were used. Since the electrical resistance of the pipettes might change on flowing of the current, a constant current device was employed. The pipettes contained the following solutions: L-PA (0.1-0.5 M in 0.165 M NaCI solution, pH 6.0-6.7), monosodium L-glutamate (2.0 M, pH 7.4), sodium chloride (0.165 M) and sucrose (0.4 M in 0.1 M NaCI solution). To minimize the possibility that any particular effects may occur with the biased pipettes, at least 5 different micropipettes were used. The electrophoretic currents ejected were within the range of 5-70 nA. The coordinates of sites for injection-recording and for stimulation were taken from the brain atlas of K~Snig and Klippel t3. Hippocampal pyramidal cells were identified by depth beneath the cortical surface, a typical firing pattern 9, and the characteristic inhibitory 'pause't, 5 and antidromic potential t due to fimbrial stimulation. Extracellular unit activity was monitored on an oscilloscope (Tectronix: Model 5103N), and processed by the conventional method. After the experiment, the stimulating site in the fimbria was confirmed histologically. Almost all cells used were spontaneously firing with the rate of 10--20 spikes/sec; however, in a few cells, the firing was maintained by the continuous ejection of Lglutamate with a low steady current. PA was successfully applied to 115 cortical cells. A typical example of a single cortical neuron inhibited by PA is shown in Fig. 1A. Out of the 115 neurons, 88 decreased and only 2 increased the rate of firing. The remaining 25 were unaffected. The neurons affected by PA did not respond to Na ÷ or sucrose application, indicating that the effects of PA were neither due to current flow nor due to local changes in osmotic pressure. The effects caused by PA were accompanied by little change in the
610
3°I , 2O
~ ~o
Sw (/)
0
or"
-
-
~ PA 10
5 30
15
r
Na*
PA 5
15
5
5
5
5
5
-
v
ft. 20
10
PA 5
5
10
20
40
N~ 40
Suc 40
• 30sec
Fig. 1. Inhibitory effect of pipecolic acid on the firing rate of a cortical (A) and a hippocampal pyramidal neuron (B). Pipecolic acid was applied with the increasing currents for the period of time indicated by the line under each ratemeter record. To confirm that PA response is due to neither current flow nor to local changes in osmotic pressure, Na ~ and sucrose were also applied. amplitude of unit firings. Low currents such as 5-15 nA were usually sufficient to produce the inhibitory effect, i.e. the rate of firings gradually decreased with the latency of 2-3 sec and recovered to the pre-application level 5-10 sec after the current was turned off. As shown in Fig. IA, the effect was reproducible for at least 5 repetitive applications without alzparent changes in magnitude and duration. In addition, the effect was dose-dependent; the complete suppression of the firing, often followed by a slight rebound excitation, occurred with higher currents. The time course of the excitatory response to PA was similar to that of the inhibitory one, though the former was less reproducible. The response to PA was also studied on pyramidal neurons of the dorsal hippocampus. Fig. 1B illustrates the inhibitory effect of PA applied with increasing doses for the same periods of time. All the 16 neurons tested were uniformly inhibited by the application of PA with currents as low as 5-15 nA. The latency and duration of the response were almost the same length as was the case in the cortical neurons. The response was reproducible and dose-dependent in the pyramidal neurons as well as the cortical neurons. In this type of experiment, care must be taken to minimize the effects of pH of test solution, since H + affects the unit activity when a test solution of low pH is used 14. In the present study, pH of the solution containing PA was higher than 6.0. Thus, the effects observed by PA appeared to be the chemical ones, as endorsed also by the control experiments using Na + and sucrose. The results presented above provide the first findings of the effect of PA on single
i
611 neuron activity, and demonstrate that electrophoreticaUy-applied PA exerted significant depressant effects on both cerebral cortical and hippocampal pyramidal neurons. In addition to this predominant effect, a few of the cortical neurons tested were stimulated or unaffected by PA. Electrophoretic current required for production of the substantial effect was relatively small, usually 10-15 nA, though as small a current as 5 nA was sufficient to produce the excitatory response to glutamate. Judging from the current intensity applied, the inhibitory effect of PA appeared to be potent. This finding seems to be on almost the same line as our previous results obtained from the intraventricular and intracerebral administrations of PA. There are 3 conceivable interpretations for the mechanisms of the inhibitory effect of PA as follows: (1) direct actions on receptors for GABA, noradrenaline and so forth and/or on unknown receptors or receptive sites including ones specific for PA; (2) indirect effects via actions on the synaptic events such as biosynthesis, release and inactivation of transmitter substances; and (3) an action through its active metabolites. Out of these possibilities, the third seems to be excluded, because: (1) the latency of the effect is relatively short; (2) a-aminoadipic acid, a major metabolite of PA, is one of excitatory amino acids 7, and (3) piperidine, another metabolite of PA, produces predominantly the excitation of the cortical and hippocampal neurons z6. The first and second described above may be applicable for explanation of the mechanisms of the inhibitory action produced by PA. However, PA is unlikely to act directly on the GABA receptor. Segal et al. 25 has proposed that the physiologically active conformation of GABA is the fully extended one with the spatial separation of amino and carboxy groups at a distance of 0.42-0.48 nm. Given their assertion, the PA molecule is probably difficult to fit the GABA receptor for initiation of the inhibitory action, since the geometrical separation of the two groups in PA ranges from 0.2 to 0.3 nm, that is, shorter than that of GABA. The inhibitory action of microelectrophoretic PA is not difficult to correlate with recent biochemical findings. PA has been found to inhibit the uptake of [3H]GABA into the brain slices12,19 and also to facilitate the release of the compound from the slices12,z2. It is well known that GABA may function as a neuronal inhibitory transmitter in the supraspinal regions such as the cortex, hippocampus and cerebellum. Taken together, it seems likely that the inhibitory respose to PA may be produced by affecting the GABAergic transmission, i.e. by inhibiting the uptake of endogenous GABA and/or by facilitating the release of the inhibitory transmitter. Curtis et al. 6 and Lodge et al. 15 have demonstrated that a series of GABA uptake inhibitors such as nipecotic acid, a structural analog of pipecolic acid, depress the firing of single neurons and enhance the response to electrophoretic GABA. Furthermore, it has been reported that iontophoretic nipecotic acid potentiates the inhibitory action of synaptically-released GABA in rats 16. These observations are relevant to our speculation mentioned above. In this context, it should be noted that all the identified hippocampal pyramidal neurons, which receive the GABAergic inputs from the adjacent basket cells, were uniformly inhibited by the application of PA. There seems no available information regarding the mechanism of the excita-
612 tory effect of PA, observed in a small percentage of the cortical cells tested. A t present, it is too early to suggest a physiological role for PA as a n e u r o m o d u lator or a n e u r o t r a n s m i t t e r . It also remains to be clarified whether the specific PAsensitive receptors exist in the cortex a n d hippocampus. Nevertheless, the present observation provides a clue to the elucidation of a role of PA in the synaptic mechanism. This work was supported by grants from the ministry of education, Japan. We sincerely t h a n k Prof. Y. O o m u r a for valuable instruction in u n i t recordings a n d electrophoretic techniques, and Dr. N. Akaike for instruction in basic techniques for electrophysiological experiments. We also t h a n k Mr. S. Ishizaka for his kind guidance c o n c e r n i n g p r e p a r a t i o n of a c o n s t a n t current and other devices, a n d are t h a n k f u l to Mr. K. Satoh for histological examination.
1 Andersen, P., Eccles, J. C. and L~yning, Y., Pathway of postsynaptic inhibition in the hippocampus, J. Neurophysiol., 27 (1964) 608-609. 2 Chang, Y. F., Lysine metabolism in the rat brain: Blood-brain barrier transport, formation of pipecolic acid and human hyperpipecolatemia, J. Neurochem., 30 (1978) 355-360. 3 Chang, Y. F., Pipecolic acid pathway: the major lysine metabolic route in the rat brain, Biochem. biophys. Res. Commun., 69 (1976) 174-180. 4 Chang, Y. F., Lysine metabolism in the rat brain: the pipe.colic acid forming pathway, J. Neurochem., 30 (1978) 347-354. 5 Curtis, D. R., Felix, D. and McLeUan, H., GABA and hippocampal inhibition, Brit. J. Pharma. col., 40 (1970) 881-883. 6 Curtis, D. R., Game, C. J. A. and Lodge, D., The in vivo inactivation of GABA and other inhibitory amino acids in the cat nervous system, Exp. Brain Res., 25 (1976) 413~28. 7 Curtis, D. R. and Watkins, J. C., Acidic amino acids with strong excitatory actions on mammalian neurones, J. Physiol. (Lond.), 166 (1963) 1-14. 8 Gatfield, P. D., Taller, R. T., Hinton, G. G., Wallace, A. C., Abdelnour, G. M. and Haust, M. D., Hyperpipecolatemia. A new metabolic disorder associated with neuropathy and hepatomegaly; a case study, Canad. med. Ass. J., 99 (1968) 1215-1233. 9 Kandel, E. R. and Spencer, W. A., Electrophysiology of hippocampal neurons II. After-potentials and repetitive firing, J. Neurophysiol., 24 (1961) 243-259. 10 Kasd, Y., Kataoka, M., Miyata, T. and Okano, Y., Pipecolic acid in the dog brain, LiJe Sci., 13 (1973) 867-873. 11 Kas6, Y. and Miyata, T., Neurobiology of piperidine: its relevance to CNS function. In E. Costa, E. Giacobini and R. Paoletti (Eds.), First and Second Messengers - - New Vistas, Advances in Biochemical Psychopharmacology, Vol. 15, Raven Press, New York, 1976, pp. 5-16. 12 Kasd, Y., Miyata, T., Hirata, A., Morimoto, H., Okano, Y. and Takahama, K., Pharmacological studies on alicyclic amines XXVI : uptake and release of piperidine and pipecolic acid in the brain slice, Folia pharmacol, jap., in press. 13 K6nig, J. F. R. and Klippel, R. A., The Rat Brain, Williams and Wilkins, Baltimore, 1963. 14 Krnjevic, K. and Phillis, J. M., Iontophoretic studies of neurones in the mammalian cerebral cortex, J. Physiol. (Lond.), 165 (1963) 274-304. 15 Lodge, D., Johnston, G. A. R., Curtis, D. R. and Brand, S. J., Effects of the Areca nut constituents arecaidine and guvacine on the action of GABA in the cat central nervous system, Brain Research, 136 (1977) 513-522. 16 Matthews, W. D. and McCafferty, G. P., Enhancement of hippocampal recurrent inhibition by inhibitors of GABA uptake, Pharmacologist, 21 (1979) 149p 17 Meek, J. L., Uptake and metabolism of piperidine and pipecolic acid in brain, Fed. Proc., 33 (1974) 468p. 18 Miyata, T., Kamata, K., Noguchi, M., Okano, Y. and Kasd, Y., Pharmacological studies on
613 alicyclic amines XV: Intracerebral administration of pipecolic acid, Jap. J. PharmacoL, 23 Suppl. (1973) 81p. 19 Nomura, Y., Okuma, Y. and Segawa, T., Influence of piperidine and pipecolic acid on the uptake of monoamines, GABA and glycine into Pz fractions of the rat brain and the spinal cord, J. Pharm. Dyn., 1 (1978) 251-255. 20 Nomura, Y., Okuma, Y., Segawa, T., Schmidt-Glenewinkel, T. and Giacobini, E., A calciumdependent, high potassium-induced release of pipecolic acid from rat brain slices, J. Neurochem., 33 (1979) 803-805. 21 Nomura, Y., Schmidt-Glenewinkel, T. and Giacobini, E., Uptake of piperidine and pipecolic acid by synaptosomes from mouse brain, Neurochem. Res., in press. 22 Okuma, Y., Nomura, Y. and Segawa, T., The effect of piperidine and pipecolic acid on high potassium-induced release of noradrenaline, serotonin and GABA from rat brain slices, J. Pharm. Dyn., 2 (1979) 261-265. 23 Oomura, Y., Sugimori, M., Nakamura, T. and Yamada, Y., Contribution of electrophysiological techniques to the understanding of central control systems. In G. J. Mogenson and F. R. Calaresu (Eds.), Neuronal Integration of Physiological Mechanisms and Behaviour, University of Toronto Press, Toronto, 1975, pp. 375-395. 24 Schmidt-Glenewinkel, T., Nomura, Y. and Giacobini, E., The conversion of lysine into piperidine, cadaverine, and pipecolic acid in the brain and other organs of the mouse, Neurochem. Res., 2 (1977) 619-637. 25 Segal, M., Sims, K. and Smissman, E., Characterization of an inhibitory receptor in rat hippocampus: a microiontophoretic study using conformationally restricted amino acid analogues, Brit. J. Pharmacol., 54 (1975) 181-188. 26 Takahama, K., Kaisaku, J., Miyata, T., Okano, Y. and Kas6, Y., Pharmacological studies on alicyclic amines XXV: effect of microiontophoretically applied piperidine on brain neuron, Jap. J. Pharmacol., Suppl., 29 (1979) 48. 27 Thomas, G. E., Haslam, R. H. A., Batshaw, M. L., Capute, A. J., Neidengard, L. and Lansom, J. L., Hyperpipecolic acidemia associated with hepatomegaly, mental retardation, optic nerve dysplasia and progressive neurological disease, Clin. Genetics, 8 (1975) 376-382.