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The role of GABA in learning in the cockroach?
Comp. gen. Pharmac., 1972, 3~ 469-472. [Scientechnica (Publishers) Ltd.]
469
THE ROLE OF GABA IN LEARNING IN THE COCKROACH? L. D. L E A K E AND I. B. T A Y L O R Department of Biological Sciences, Portsmouth Polytechnic
(Received x3 flu/y, i972) ABSTRACT i. Injection o f N H , O H (hydroxylamine) and AOAA (amino-oxyacetate) into headless cockroaches decreased learning time. ~. INH (isonicotinic acid hydrazide) increased learning time, while semicarbide had no effect. 3. It is inferred that learning is accompanied by an increase in GABA levels. 4- A model, based on this assumption, is proposed to account for a possible role of GABA in the learning process. It postulates simultaneous facilitation of the specified (learning) pathway by ACh and inhibition of alternative pathways by GABA.
A OROUP of cockroach preparations subjected to electric shocks only when a selected leg falls below a predetermined position (P animals) receives significantly fewer shocks when retested than preparations subjected to the same series of shocks at r a n d o m (R animals). T h e y are thus said to have 'learnt' to avoid the noxious stimulus. Such learning has been demonstrated in intact and headless preparations a n d in isolated ganglia (Horridge, i962a, b, 1964; Hoyle, I965; Eisenstein and Cohen, 1965). Cockroaches placed u n d e r experimental conditions, but in the absence of electric shocks (U animals), receive significantly fewer shocks than ' P ' but are indistinguishable in their responses from ' R ' animals (Taylor, I972 ). T h e behaviour of individuals from ' U ' , 'P', and ' R ' groups shows great variability and therefore it is necessary to perform experiments with large n u m b e r s of animals. Kerkut, Oliver, Rick, and Walker (x97o) showed that the behavioural evidence of learning could be correlated with cholinesterase (ChE) activity. I n addition, anticholinesterases decreased learning time while cholinergic substances caused an increase. Thus changes in acetylcholine (ACh) levels a p p e a r to be involved in learning. While A C h is assumed to be the excitatory transmitter in the cockroach, the inhibitory transmitter is t h o u g h t to be 7 - a m i n o h u t y r i c
acid (GABA). Oliver, Taberner, Rick, and K e r k u t (197 I) suggested that during learning there was a fall in GABA, and in the activity of the enzyme glutamic acid decarboxylase (GAD) responsible for its production from glutamic acid. I n view of the availability of certain c o m p o u n d s which alter G A B A levels in vivo (Baxter, i97o), a pharmacological a p p r o a c h to G A B A was a d o p t e d to verify this. MATERIALS AND METHODS The technique used was based on that devised by ttorridge (1962b). Each pair of adult cockroaches (Periplanetaamericana) was chosen to be of the same sex and of comparable size. The left metathoracic leg was left intact, and all other legs, the head and anal cerci were removed. A record of the animal's performance was obtained using an SE 3oo6 U.V. recorder. The conditioning stimulus, applied at the rate of i per second, was of 2 mseconds duration and 4 ° V. intensity. (This voltage gave a significantly shorter learning time, 15"4o~-5"32 minutes, as opposed to 38.26-t-8.39 minutes with a 58-V. stimulus, using learning criterion 5--see below.) Learning time was established using a newly devised learning criterion (L.C.5). This depends on behaviour over a period of 5 minutes, split up into i o intervals each of 3o seconds duration. These intervals are assigned a value of + i when the animal receives ~<~ shocks, a value of -- i when they contain >~IO shocks, and a value of o when they contain 3-9 shocks. These values are summed for the 5-minute period, and if the total is ~> + 4 then the animal is said to have learnt. This method contrasts with the learning criterion
47 °
LEAKE
AND
(L.C.3) used by other workers, where learning is specified by the animal receiving less than 4 shocks in any period of 3 minutes. L.C. 5 reflects the same relationship as L.C.3 in experiments showing the effect of intensity of stimulus on learning time, but it also registers learning more rapidly, and is more consistent with subjective impressions of learning time (Taylor, i972 ) . Drugs used were: hydroxylamine. HC1 (NH~OH), Amino-oxyacetic acid hemi-HC1 (AOAA), isonicotinic acid hydrazide (INH), and semicarbazide (SCA). The doses used were adapted from data on mammals (Baxter, 197o ) . Drugs were made up in cockroach Ringer (Kerkut and others, I97O ) and injected in o.I ml. aliquots into the cockroach haemocoel. Controls were injected with o.I ml. Ringer. Pairs of animals, control (C) and test (T), were trained simultaneously. At least 8 pairs were used in each series.
TAYLOR
Comp. gen. Pharmac.
decrease l e a r n i n g time significantly (at 5 p e r cent level). I N H , w h i c h was expected to r e d u c e G A B A , was found to increase l e a r n i n g t i m e significantly. S C A was expected to decrease G A B A b u t h a d no effect on learning. T h i s last result can be e x p l a i n e d b y assuming the d r u g to be inactive in the cockroach. Such a n o m a l i e s a r e b y no m e a n s unusual, with N H 2 O H e l e v a t i n g G A B A in the b r a i n s of cats, rats, a n d m o n k e y s b u t not in mice (Baxter a n d R o b e r t s , 196o ) . As the d r u g effects a r e c o r r e l a t e d in two different directions (i.e., raising a n d lowering G A B A ) it seems unlikely t h a t side-effects o f such s t r u c t u r a l l y u n r e l a t e d drugs could be
Table L--EFFECT ONTHE COCKROACH LEARNING PREPARATION OF SOMEDRUGS WHICHARE EXPECTED TO AFFECT G A B A
DOSE (mg. per kg.)
DRUG
NH,OH AOAA INH SCA
TIME OF A C T I O N (hours)
75 ioo 20o 3o0
I'5 2.o i .o I-O
LEVELS
LEARNING TIMES (mean ~zS.E.) (minutes) Control (C)
Test (T)
C--T
4I'91 5_7"o6 27"96 5_5"7o 24'4o ± 7"05 I6.21 5_4.I6
2'835_1"49 7"oi ±3"8o 39'73 5- IO'84 i6-I I 5_7"10
39"I i7"62 2o'95 5-6'43 - 14"33 5_7"37 O'8 5-7'I I
Table//.--TEST TO SHOW IF DRUG EFFECTS ARE INDEPENDENT OF ASSOCIATION
RETEST SCORES (meannumberof shocks in 7 minutes±S.E.) DRUG
NH,OH AOAA
Random (R)
Positional (P)
R--P
124-I3 5-26-72 176'25 5-55"7
54.38 5_2o.86 32"~5 5-8'29
68.5o ±2o.28 I45'e5 5-49"8~
RESULTS Differences b e t w e e n the l e a r n i n g times o f C a n d T a n i m a l s were c a l c u l a t e d a n d a n a l y s e d using W i l c o x o n ' s p a i r e d s a m p l e test
( Table I). I f a d r u g d e c r e a s e d l e a r n i n g time it was then injected into b o t h m e m b e r s o f a p a i r . O n e was t r e a t e d as a P a n d the o t h e r as an R a n i m a l , a n d the n u m b e r o f shocks received b y each on retest was found (Table H). DISCUSSION DRUG EFFECTS
NH2OH and AOAA were expected to elevate GABA levels, and were found to
responsible for the results. T h u s it can be c o n c l u d e d t h a t G A B A levels, like A C h , a r e inversely r e l a t e d to l e a r n i n g time. T h i s r e l a t i o n s h i p could be due to a n y o f a v a r i e t y of physiological effects o f G A B A , e.g., s t i m u l a t i o n o f c a r b o h y d r a t e m e t a b o l i s m or p r o t e i n synthesis (see Baxter, 197 o, for general review). W h i c h o f these is responsible awaits e x p e r i m e n t a l d e t e r m i n a t i o n , b u t the a x i o m that both GABA and ACh are exerting their effects in their c a p a c i t y o f s y n a p t i c t r a n s m i t ters will be assumed here to allow a m e a n i n g ful c o m p a r i s o n b e t w e e n the two. A s s u m i n g r e g u l a t i o n of G A B A levels to be involved in learning, such r e g u l a t i o n could
1972, 3
ROLE OF GABA IN LEARNING
take place via control of substrate, co-factor, ionic environment, compartmentalization or enzyme configuration (i.e., allosteric or covalent alteration. Such control would be disrupted by in vitro techniques. Further, GABA levels in vitro are dependent to a large extent on the extraction techniques used, and are notorious for leading to inconsistent results (Chmelar, Hais, and Hodanova, 1964; Baxter, 197o ) . Thus, the in vitro changes in GABA found during learning (Oliver and others, I97I ) do not necessarily contradict the results of this paper. LEARNING MODELS
A model, based on the facilitated-pathway hypothesis, was formulated to explain some of the changes involved in the learning process of cockroaches (Kerkut and others, 197o ). This involved facilitation of the avoidance pathway as a result of decreases in cholinesterase activity, brought about by specific afferent reinforcement. This model is supported by the correlation of ChE activity with the behavioural evidence of learning during the extinction period, but it is inadequate for present purposes on three g r o u n d s : - i. It does not explicitly implicate changes in GABA levels. 2. It does not consider the role of the postulated learning process in the context of general behaviour. 3. It is vague as to the mechanisms leading to specific facilitation. The implications that increases in both ACh and GABA m a y take place during learning can be explained by considering that a specified pathway can be facilitated in two ways: (a) facilitation of the specified pathway itself; and (b) inhibition of alternative pathways. ACh increases could be involved in the former, whilst GABA increases could be involved in the latter. By this extension of the model, objection I is overcome. Such a dual process also allows objection 2 to be answered. Assuming that only ACh facilitation was involved, ACh in the avoidance pathway would have to build up to a level sufficient to exceed the degree of facilitation of the alternative pathways. Some of these pathways m a y show a high degree of
47 I
facilitation if they are of considerable survival value (e.g., pathways for leg searching movements in the absence of a solid surface). In view of variability, both in untrained and learnt preparations, leg behaviour can best be explained on the basis o f ' canalization ' of a variable input by the pathways involved. Thus, after removal from the training situation, the highly facilitated learnt pathway would interfere with the resumption of normal behaviour by tending to canalize the input away from ' normal ' pathways. This would be of poor adaptive value if the innate and previously learnt alternative pathways were more appropriate. Interference for a period of 3 days could hardly be tolerated, but, if it were overcome by further ACh facilitation of the alternative pathways, learning generally would tend to result eventually in synaptic ratios of I : I and a loss of specificity in response. I f GABA inhibition of alternative pathways does take place during learning, the appropriate pathway need only be facilitated by ACh to a small extent. T h e extent of ACh facilitation could depend on the importance of the response. I f GABA levels return rapidly to normal on removal from the training situation, then, if the importance and ACh facilitation of the alternative pathways is high, they can be resumed. The learning engram will, nevertheless, remain for 3 days in the form of the elevated ACh levels. If, during this time, the preparation is retested, the GABA levels in the alternative pathways m a y be rapidly built up until avoidance behaviour can reappear via the previously ' m a p p e d out ' pathway. Overcoming objection 3 requires a fuller model to account for the specificity of the process of ACh facilitation and GABA inhibition. Appropriate and inappropriate pathways can, by definition, be distinguished as follows : - I. The ' firing ' of an inappropriate pathway is accompanied by the reception of the ' n o x i o u s ' stimulus (in this case an electric shock). 2. T h e ' firing ' of an appropriate (in this case avoidance) pathway is not accompanied by t h e ' noxious ' stimulus.
472
LEAKE AND TAYLOR
Clearly, if G A B A levels can be increased only in the presence of both the fired state and the noxious stimulus, inappropriate pathways will be specifically inhibited. T h e fired state is characterized by a different ionic environment in the neurons involved, and the noxious stimulus could be specified by leading to the transitory release or synthesis o f some factor ' X '. Thus, if factor X could increase G A B A levels only in the presence of the fired ionic environment, inappropriate pathways would be specifically inhibited. Similar processes could account for the specificity of A C h facilitation. T h e appropriate pathways can be specified in two ways: (a) in the resting state when the noxious stimulus is received; (b) in the fired state when the noxious stimulus ceases. These lead to two schemes for A C h facilitation. I. Factor ' Y ' (which m a y or m a y not be the same as factor X) is released/synthesized on reception of the noxious stimulus, and elevates A C h in the presence of the resting ionic environment. 2. Factor Y specifies the cessation of the noxious stimulus. This could occur if its release is dependent on a two-stage process, the first stage of which is initiated by the noxious stimulus, the second being prevented by another such stimulus. Factor Y in the presence of the fired ionic environment could elevate A C h levels. Scheme I is less specific, and would tend to reinforce the effect of G A B A inhibition in eliciting ' original ' behaviour. These ' original ' pathways would become suppressed by G A B A if inappropriate, and only the avoidance pathways would be consistently facilitated. Scheme 2 does not lead to A C h facilitation of new pathways en masse, and the elicitation of'original' behaviour depends solely on G A B A inhibition of the n o r m a l pathways. This scheme is more specific with only the appropriate p a t h w a y being facilitated by ACh. A dual-process model of this type can explain the specificity of learning, is
consistent with observed experimental results, and illustrates the operation of learning as a constituent of general behaviour. Which, if any, o f the alternatives presented in the speculative account above applies to the true learning process in the cockroach preparation remains to be determined experimentally. No mention has been m a d e as to a possible role of R N A and protein synthesis in the learning process. However, these pathways m a y be involved in a mechanism underlying that outlined above, and their possible involvement is not regarded as contradictory to it. REFERENCES BAXTER, C. F. (x97o), ' The nature of GABA ', in Handbook of Neurochemistry (ed. Lajtha), Vol. 3, pp. 289-334 . New York: Plenum Press. BAXTER, C. F., and ROBERTS, E. (I96O), ' Demonstration of thiosemicarbazide induced convulsions in rats with elevated brain levels of GABA ', Proc. Soc. exp. Biol. Med., xo4~ 426-427. CHMELAR, V., HAIS, I. M., and HODANOVA, M. (1964) , ' G A B A content in rat brain processed under different temperature conditions ', Acta biochim, polon. H~ 327-335. EISENSTEIN, E. M., and COHEN, l~[. J. (I965), ' Learning in an isolated prothoracic ganglion ', Anita. Behav. , x3, Io4-1o8. HORRIDOE, G. A. (I962a), 'Learning of leg position in headless insects ', Nature, Lond., x939 697-698. HORRIDGE, G. A. (I962b), 'Learning of leg position of the ventral nerve cord in headless insects ', Proc. R. Soc. B., x57~ 33-52. HORRIDGE, G. A. (1964) , ' T h e electrophysiological approach to learning in isolatable ganglia ', Anita. Behav., suppl., x~ x63-i82. HOYLE, G. (1965), ' Neurophysiological studies on learning in headless insects ', in The Physiology of the Insect Central dVervous System. (ed. Treherne and Beament), pp. 203-232. New York: Academic Press. KERKUT, G. A., OLIVER, G. W. O., RICK, J. T., and WALKER, g. J. (I97O), ' T h e effects of drugs on learning in a simple preparation ', Comp. gen. Pharmac., z~ 437-484 . OLIVER, G. W. O., TABERNER,P. V., RICK, J. T., and KERKUT, G. A. (t971), ' Changes in GABA level, GAD and ChE activity in CNS of an insect during learning ', Comp. Biochem. Physiol., 38~ 529-535. TAYLOR, I. B. (I97~), unpublished data. Key Word Index: GABA, cockroach, learning, amino-oxyacetate, hydroxylamine.
469
THE ROLE OF GABA IN LEARNING IN THE COCKROACH? L. D. L E A K E AND I. B. T A Y L O R Department of Biological Sciences, Portsmouth Polytechnic
(Received x3 flu/y, i972) ABSTRACT i. Injection o f N H , O H (hydroxylamine) and AOAA (amino-oxyacetate) into headless cockroaches decreased learning time. ~. INH (isonicotinic acid hydrazide) increased learning time, while semicarbide had no effect. 3. It is inferred that learning is accompanied by an increase in GABA levels. 4- A model, based on this assumption, is proposed to account for a possible role of GABA in the learning process. It postulates simultaneous facilitation of the specified (learning) pathway by ACh and inhibition of alternative pathways by GABA.
A OROUP of cockroach preparations subjected to electric shocks only when a selected leg falls below a predetermined position (P animals) receives significantly fewer shocks when retested than preparations subjected to the same series of shocks at r a n d o m (R animals). T h e y are thus said to have 'learnt' to avoid the noxious stimulus. Such learning has been demonstrated in intact and headless preparations a n d in isolated ganglia (Horridge, i962a, b, 1964; Hoyle, I965; Eisenstein and Cohen, 1965). Cockroaches placed u n d e r experimental conditions, but in the absence of electric shocks (U animals), receive significantly fewer shocks than ' P ' but are indistinguishable in their responses from ' R ' animals (Taylor, I972 ). T h e behaviour of individuals from ' U ' , 'P', and ' R ' groups shows great variability and therefore it is necessary to perform experiments with large n u m b e r s of animals. Kerkut, Oliver, Rick, and Walker (x97o) showed that the behavioural evidence of learning could be correlated with cholinesterase (ChE) activity. I n addition, anticholinesterases decreased learning time while cholinergic substances caused an increase. Thus changes in acetylcholine (ACh) levels a p p e a r to be involved in learning. While A C h is assumed to be the excitatory transmitter in the cockroach, the inhibitory transmitter is t h o u g h t to be 7 - a m i n o h u t y r i c
acid (GABA). Oliver, Taberner, Rick, and K e r k u t (197 I) suggested that during learning there was a fall in GABA, and in the activity of the enzyme glutamic acid decarboxylase (GAD) responsible for its production from glutamic acid. I n view of the availability of certain c o m p o u n d s which alter G A B A levels in vivo (Baxter, i97o), a pharmacological a p p r o a c h to G A B A was a d o p t e d to verify this. MATERIALS AND METHODS The technique used was based on that devised by ttorridge (1962b). Each pair of adult cockroaches (Periplanetaamericana) was chosen to be of the same sex and of comparable size. The left metathoracic leg was left intact, and all other legs, the head and anal cerci were removed. A record of the animal's performance was obtained using an SE 3oo6 U.V. recorder. The conditioning stimulus, applied at the rate of i per second, was of 2 mseconds duration and 4 ° V. intensity. (This voltage gave a significantly shorter learning time, 15"4o~-5"32 minutes, as opposed to 38.26-t-8.39 minutes with a 58-V. stimulus, using learning criterion 5--see below.) Learning time was established using a newly devised learning criterion (L.C.5). This depends on behaviour over a period of 5 minutes, split up into i o intervals each of 3o seconds duration. These intervals are assigned a value of + i when the animal receives ~<~ shocks, a value of -- i when they contain >~IO shocks, and a value of o when they contain 3-9 shocks. These values are summed for the 5-minute period, and if the total is ~> + 4 then the animal is said to have learnt. This method contrasts with the learning criterion
47 °
LEAKE
AND
(L.C.3) used by other workers, where learning is specified by the animal receiving less than 4 shocks in any period of 3 minutes. L.C. 5 reflects the same relationship as L.C.3 in experiments showing the effect of intensity of stimulus on learning time, but it also registers learning more rapidly, and is more consistent with subjective impressions of learning time (Taylor, i972 ) . Drugs used were: hydroxylamine. HC1 (NH~OH), Amino-oxyacetic acid hemi-HC1 (AOAA), isonicotinic acid hydrazide (INH), and semicarbazide (SCA). The doses used were adapted from data on mammals (Baxter, 197o ) . Drugs were made up in cockroach Ringer (Kerkut and others, I97O ) and injected in o.I ml. aliquots into the cockroach haemocoel. Controls were injected with o.I ml. Ringer. Pairs of animals, control (C) and test (T), were trained simultaneously. At least 8 pairs were used in each series.
TAYLOR
Comp. gen. Pharmac.
decrease l e a r n i n g time significantly (at 5 p e r cent level). I N H , w h i c h was expected to r e d u c e G A B A , was found to increase l e a r n i n g t i m e significantly. S C A was expected to decrease G A B A b u t h a d no effect on learning. T h i s last result can be e x p l a i n e d b y assuming the d r u g to be inactive in the cockroach. Such a n o m a l i e s a r e b y no m e a n s unusual, with N H 2 O H e l e v a t i n g G A B A in the b r a i n s of cats, rats, a n d m o n k e y s b u t not in mice (Baxter a n d R o b e r t s , 196o ) . As the d r u g effects a r e c o r r e l a t e d in two different directions (i.e., raising a n d lowering G A B A ) it seems unlikely t h a t side-effects o f such s t r u c t u r a l l y u n r e l a t e d drugs could be
Table L--EFFECT ONTHE COCKROACH LEARNING PREPARATION OF SOMEDRUGS WHICHARE EXPECTED TO AFFECT G A B A
DOSE (mg. per kg.)
DRUG
NH,OH AOAA INH SCA
TIME OF A C T I O N (hours)
75 ioo 20o 3o0
I'5 2.o i .o I-O
LEVELS
LEARNING TIMES (mean ~zS.E.) (minutes) Control (C)
Test (T)
C--T
4I'91 5_7"o6 27"96 5_5"7o 24'4o ± 7"05 I6.21 5_4.I6
2'835_1"49 7"oi ±3"8o 39'73 5- IO'84 i6-I I 5_7"10
39"I i7"62 2o'95 5-6'43 - 14"33 5_7"37 O'8 5-7'I I
Table//.--TEST TO SHOW IF DRUG EFFECTS ARE INDEPENDENT OF ASSOCIATION
RETEST SCORES (meannumberof shocks in 7 minutes±S.E.) DRUG
NH,OH AOAA
Random (R)
Positional (P)
R--P
124-I3 5-26-72 176'25 5-55"7
54.38 5_2o.86 32"~5 5-8'29
68.5o ±2o.28 I45'e5 5-49"8~
RESULTS Differences b e t w e e n the l e a r n i n g times o f C a n d T a n i m a l s were c a l c u l a t e d a n d a n a l y s e d using W i l c o x o n ' s p a i r e d s a m p l e test
( Table I). I f a d r u g d e c r e a s e d l e a r n i n g time it was then injected into b o t h m e m b e r s o f a p a i r . O n e was t r e a t e d as a P a n d the o t h e r as an R a n i m a l , a n d the n u m b e r o f shocks received b y each on retest was found (Table H). DISCUSSION DRUG EFFECTS
NH2OH and AOAA were expected to elevate GABA levels, and were found to
responsible for the results. T h u s it can be c o n c l u d e d t h a t G A B A levels, like A C h , a r e inversely r e l a t e d to l e a r n i n g time. T h i s r e l a t i o n s h i p could be due to a n y o f a v a r i e t y of physiological effects o f G A B A , e.g., s t i m u l a t i o n o f c a r b o h y d r a t e m e t a b o l i s m or p r o t e i n synthesis (see Baxter, 197 o, for general review). W h i c h o f these is responsible awaits e x p e r i m e n t a l d e t e r m i n a t i o n , b u t the a x i o m that both GABA and ACh are exerting their effects in their c a p a c i t y o f s y n a p t i c t r a n s m i t ters will be assumed here to allow a m e a n i n g ful c o m p a r i s o n b e t w e e n the two. A s s u m i n g r e g u l a t i o n of G A B A levels to be involved in learning, such r e g u l a t i o n could
1972, 3
ROLE OF GABA IN LEARNING
take place via control of substrate, co-factor, ionic environment, compartmentalization or enzyme configuration (i.e., allosteric or covalent alteration. Such control would be disrupted by in vitro techniques. Further, GABA levels in vitro are dependent to a large extent on the extraction techniques used, and are notorious for leading to inconsistent results (Chmelar, Hais, and Hodanova, 1964; Baxter, 197o ) . Thus, the in vitro changes in GABA found during learning (Oliver and others, I97I ) do not necessarily contradict the results of this paper. LEARNING MODELS
A model, based on the facilitated-pathway hypothesis, was formulated to explain some of the changes involved in the learning process of cockroaches (Kerkut and others, 197o ). This involved facilitation of the avoidance pathway as a result of decreases in cholinesterase activity, brought about by specific afferent reinforcement. This model is supported by the correlation of ChE activity with the behavioural evidence of learning during the extinction period, but it is inadequate for present purposes on three g r o u n d s : - i. It does not explicitly implicate changes in GABA levels. 2. It does not consider the role of the postulated learning process in the context of general behaviour. 3. It is vague as to the mechanisms leading to specific facilitation. The implications that increases in both ACh and GABA m a y take place during learning can be explained by considering that a specified pathway can be facilitated in two ways: (a) facilitation of the specified pathway itself; and (b) inhibition of alternative pathways. ACh increases could be involved in the former, whilst GABA increases could be involved in the latter. By this extension of the model, objection I is overcome. Such a dual process also allows objection 2 to be answered. Assuming that only ACh facilitation was involved, ACh in the avoidance pathway would have to build up to a level sufficient to exceed the degree of facilitation of the alternative pathways. Some of these pathways m a y show a high degree of
47 I
facilitation if they are of considerable survival value (e.g., pathways for leg searching movements in the absence of a solid surface). In view of variability, both in untrained and learnt preparations, leg behaviour can best be explained on the basis o f ' canalization ' of a variable input by the pathways involved. Thus, after removal from the training situation, the highly facilitated learnt pathway would interfere with the resumption of normal behaviour by tending to canalize the input away from ' normal ' pathways. This would be of poor adaptive value if the innate and previously learnt alternative pathways were more appropriate. Interference for a period of 3 days could hardly be tolerated, but, if it were overcome by further ACh facilitation of the alternative pathways, learning generally would tend to result eventually in synaptic ratios of I : I and a loss of specificity in response. I f GABA inhibition of alternative pathways does take place during learning, the appropriate pathway need only be facilitated by ACh to a small extent. T h e extent of ACh facilitation could depend on the importance of the response. I f GABA levels return rapidly to normal on removal from the training situation, then, if the importance and ACh facilitation of the alternative pathways is high, they can be resumed. The learning engram will, nevertheless, remain for 3 days in the form of the elevated ACh levels. If, during this time, the preparation is retested, the GABA levels in the alternative pathways m a y be rapidly built up until avoidance behaviour can reappear via the previously ' m a p p e d out ' pathway. Overcoming objection 3 requires a fuller model to account for the specificity of the process of ACh facilitation and GABA inhibition. Appropriate and inappropriate pathways can, by definition, be distinguished as follows : - I. The ' firing ' of an inappropriate pathway is accompanied by the reception of the ' n o x i o u s ' stimulus (in this case an electric shock). 2. T h e ' firing ' of an appropriate (in this case avoidance) pathway is not accompanied by t h e ' noxious ' stimulus.
472
LEAKE AND TAYLOR
Clearly, if G A B A levels can be increased only in the presence of both the fired state and the noxious stimulus, inappropriate pathways will be specifically inhibited. T h e fired state is characterized by a different ionic environment in the neurons involved, and the noxious stimulus could be specified by leading to the transitory release or synthesis o f some factor ' X '. Thus, if factor X could increase G A B A levels only in the presence of the fired ionic environment, inappropriate pathways would be specifically inhibited. Similar processes could account for the specificity of A C h facilitation. T h e appropriate pathways can be specified in two ways: (a) in the resting state when the noxious stimulus is received; (b) in the fired state when the noxious stimulus ceases. These lead to two schemes for A C h facilitation. I. Factor ' Y ' (which m a y or m a y not be the same as factor X) is released/synthesized on reception of the noxious stimulus, and elevates A C h in the presence of the resting ionic environment. 2. Factor Y specifies the cessation of the noxious stimulus. This could occur if its release is dependent on a two-stage process, the first stage of which is initiated by the noxious stimulus, the second being prevented by another such stimulus. Factor Y in the presence of the fired ionic environment could elevate A C h levels. Scheme I is less specific, and would tend to reinforce the effect of G A B A inhibition in eliciting ' original ' behaviour. These ' original ' pathways would become suppressed by G A B A if inappropriate, and only the avoidance pathways would be consistently facilitated. Scheme 2 does not lead to A C h facilitation of new pathways en masse, and the elicitation of'original' behaviour depends solely on G A B A inhibition of the n o r m a l pathways. This scheme is more specific with only the appropriate p a t h w a y being facilitated by ACh. A dual-process model of this type can explain the specificity of learning, is
consistent with observed experimental results, and illustrates the operation of learning as a constituent of general behaviour. Which, if any, o f the alternatives presented in the speculative account above applies to the true learning process in the cockroach preparation remains to be determined experimentally. No mention has been m a d e as to a possible role of R N A and protein synthesis in the learning process. However, these pathways m a y be involved in a mechanism underlying that outlined above, and their possible involvement is not regarded as contradictory to it. REFERENCES BAXTER, C. F. (x97o), ' The nature of GABA ', in Handbook of Neurochemistry (ed. Lajtha), Vol. 3, pp. 289-334 . New York: Plenum Press. BAXTER, C. F., and ROBERTS, E. (I96O), ' Demonstration of thiosemicarbazide induced convulsions in rats with elevated brain levels of GABA ', Proc. Soc. exp. Biol. Med., xo4~ 426-427. CHMELAR, V., HAIS, I. M., and HODANOVA, M. (1964) , ' G A B A content in rat brain processed under different temperature conditions ', Acta biochim, polon. H~ 327-335. EISENSTEIN, E. M., and COHEN, l~[. J. (I965), ' Learning in an isolated prothoracic ganglion ', Anita. Behav. , x3, Io4-1o8. HORRIDOE, G. A. (I962a), 'Learning of leg position in headless insects ', Nature, Lond., x939 697-698. HORRIDGE, G. A. (I962b), 'Learning of leg position of the ventral nerve cord in headless insects ', Proc. R. Soc. B., x57~ 33-52. HORRIDGE, G. A. (1964) , ' T h e electrophysiological approach to learning in isolatable ganglia ', Anita. Behav., suppl., x~ x63-i82. HOYLE, G. (1965), ' Neurophysiological studies on learning in headless insects ', in The Physiology of the Insect Central dVervous System. (ed. Treherne and Beament), pp. 203-232. New York: Academic Press. KERKUT, G. A., OLIVER, G. W. O., RICK, J. T., and WALKER, g. J. (I97O), ' T h e effects of drugs on learning in a simple preparation ', Comp. gen. Pharmac., z~ 437-484 . OLIVER, G. W. O., TABERNER,P. V., RICK, J. T., and KERKUT, G. A. (t971), ' Changes in GABA level, GAD and ChE activity in CNS of an insect during learning ', Comp. Biochem. Physiol., 38~ 529-535. TAYLOR, I. B. (I97~), unpublished data. Key Word Index: GABA, cockroach, learning, amino-oxyacetate, hydroxylamine.