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Iontophoretic application of acetylcholine and gaba onto insect central neurones
Comp. B,ochem. Physiol., 1969, Vol. 31, pp. 611 to 633. Pergamon Press. Pnnted m Great Bntmn
IONTOPHORETIC APPLICATION OF ACETYLCHOLINE AND GABA* ONTO INSECT CENTRAL NEURONES G. A. K E R K U T , R. M. P I T M A N and R. J. W A L K E R Department of Physiology and Biochemistry, Southampton University (Received 16 April 1969)
AI~tract--1. Electrical activity showing action potentials, EPSP and IPSP can be recorded with micro-electrodes from the nerve cell bodies in the ganglia of the
cockroach. 2. The sensitivity of the nerve cell to iontophoretically applied acetylcholine is 1"31 × 10 -13 mole. It is of the same order of sensitivity to acetylcholine as shown by mollusc nerve cells. 3. The sensitivity of the nerve cell to iontophoretically applied GABA is 1'05 × 10-la mole. 4. A full summary is given on page 630.
INTRODUCTION THE PHARMACOLOGYof the insect central nervous system has been studied by many previous authors (Mikalonis & Brown, 1941 ; Lowenstein, 1942; Tobias et al., 1946; Roeder, 1948; Chang & Kearns, 1955; Twarog &'Roeder, 1956, 1957; O'Brien, 1957; Smallman & Pal, 1957; Iyatomi & Kanehisa, 1958; O'Brien & Fisher, 1958; Yamasaki & Narahashi, 1958, 1959, 1960; Colhoun, 1958a, b, c, 1963; Kanehisa, 1961; Treherne, 1962, 1966; Smith & Treherne, 1963, 1965; Rowe, 1964, 1969; Treherne & Smith, 1965a, b; Callec & Boistel, 1967; Boistel, 1968). One problem has been the relative insensitivity of insect neurones to acetylcholine, the neurones requiring 10-2-10-5 M acetylcholine to bring about excitation. The present paper describes the use of intracellular micro-electrodes on cockroach neurones and the effects of drugs on these neurones. The drugs were chosen to indicate possible transmitters in the cockroach central nervous system. The results from micro-electrodes are compared with those using suction electrodes. The application of drugs to the bathing medium is compared with iontophoretic application of drugs. A preliminary account of this work has already been published (Kerkut et al., 1968). *Abbreviations: ACh, acetylcholine; GABA, gamma aminobutyric acid; EPSP, excitatory post synaptic potential; IPSP, inhibitory post synaptic potential. 611
612
G . A. KERKUT,R. M . PITMAN AND R. J. WALKER MATERIALS
AND
METHODS
Dissection A d u l t cockroaches, Periplaneta americana, were o b t a i n e d f r o m dealers a n d k e p t u n d e r w a r m c o n d i t i o n s w i t h food a n d w a t e r supply. I n t h e p r e p a r a t i o n of t h e isolated m e t a t h o r a c i c ganglion, t h e h e a d a n d all t h e legs except t h e left m e t a t h o r a c i c leg were r e m o v e d f r o m t h e cockroach. T h e a n i m a l was p i n n e d down, v e n t r a l surface u p p e r m o s t , o n t o a wax block. T h e exoskeleton was r e m o v e d f r o m a b o v e the first t h o r a c i c ganglion, t h e m e s o t h o r a c i c g a n g h o n , t h e m e t a t h o r a c i c g a n g h o n a n d f r o m t h e u p p e r surface of t h e m e t a t h o r a c : c leg. A small a m o u n t of 5 ~o m e t h y l e n e b l u e was p u t o n t o t h e exposed area to stare t h e n e r v e s a n d m a k e t h e m m o r e visible. V i e w i n g f r o m above w : t h a b i n o c u l a r microscope, t h e m o s t p o s t e r i o r p o r t i o n of t h e a b d o m i n a l n e r v e cord visible was g r i p p e d w : t h fine forceps, cut a n d g e n t l y lifted away f r o m t h e a m m a l w h i l s t t h e u n d e r l y i n g tissue was carefully cut away f r o m t h e n e r v e c o r d a n d f r o m t h e left fifth n e r v e w h e r e it p a s s e d into t h e leg. T h e o t h e r s e g m e n t a l n e r v e s were s e v e r e d n e a r t h e g a n g h o n . T h e m e t a t h o r a c i c g a n g l i o n was m this way s e p a r a t e d f r o m t h e a n i m a l w i t h a l e n g t h of fifth n e r v e dissected o u t as far as t h e m i d d l e of the coxa. T h e t h o r a c i c n e r v e c o r d was s e v e r e d at t h e c o n n e c t i v e b e t w e e n t h e m e s o t h o r a c i c a n d rectat h o r a c i c g a n g h o n a n d t h e fifth n e r v e severed at t h e coxal region. Care was t a k e n to leave as m u c h as possible of t h e t r a c h e a l system over t h e g a n g l i o n i n t a c t so t h a t o x y g e n a t i o n of t h e g a n g l i o n was m a i n t a i n e d .
External recording: suction electrode T h e isolated g a n g l i o n was t r a n s f e r r e d to t h e e x p e r i m e n t a l b a t h w h e r e it was p l a c e d o n filter p a p e r m o i s t e n e d w i t h insect R i n g e r solution. T h e n e r v e s h e a t h over t h e v e n t r a l surface of t h e g a n g l i o n was r e m o v e d b y a m e t h o d similar to t h a t d e s c r i b e d b y T w a r o g & R o e d e r (1956). W h e n d e s h e a t h i n g was c o m p l e t e d , a n y l o n p e r f u s i o n c a n n u l a was p l a c e d so t h a t it was a b o v e t h e g a n g h a b u t a v o i d e d t h e o p e n e n d s of t h e tracheal system, a n d R i n g e r solution was p e r f u s e d over t h e p r e p a r a t i o n . T h e fifth n e r v e was d r a w n into t h e b a r r e l of a suct:on e l e c t r o d e a n d t h e p r e p a r a t i o n allowed 10 rain to settle down. T h e suction electrode led d i r e c t to a 502A T e k t r o n i x oscilloscope a n d t h e o u t p u t of this was c o n n e c t e d to a n E K C O N 522c rate m e t e r a n d a S e r v o s c r l b e recorder. T h e rate m e t e r was set at a c o u n t i n g rate of 100/sec a n d t h e t i m e c o n s t a n t of 80 sec to s m o o t h o u t a n y fluctuations in t h e firing rate m the fifth nerve.
Intracellular recor&ng : micro-electrodes I n general it p r o v e d easier to p e n e t r a t e t h e ceils of t h e s i x t h a b d o m i n a l g a n g h o n t h a n t h e cells m t h e m e t a t h o r a c l c g a n g h o n . T h e isolated g a n g l i o n a n d n e r v e c o r d was p l a c e d o n a glass slide a n d t h e c o n n e c t i v e s fixed b y m e a n s of elastic b a n d s . T h e n e r v e s h e a t h over t h e dorsal surface was r e m o v e d w i t h fine forceps (this has to b e done very carefully o t h e r w i s e t h e n e r v e cells w d l b e damaged). T h e p r e p a r a t i o n was p l a c e d in a b a t h a n d o b s e r v e d w l t h a b i n o c u l a r m i c r o s c o p e at m a g n i f i c a t i o n s × 20 to × 80. I t was usually possible to see t h e large n e r v e cell bodies in t h e d e s h e a t h e d area. T h e m i c r o - e l e c t r o d e s were p u l l e d o n a vertical N a r a s h i g e electrode puller. T h e y were n o r m a l l y filled w i t h 1 M p o t a s s i u m acetate s o l u t i o n b y b o d i n g u n d e r r e d u c e d pressure. T h e electrodes h a d a resistance of b e t w e e n 20 a n d 80 M ~ . T h e electrode led to a M e d i s t o r c a t h o d e follower a n d t h e n to a T e k t r o n i x 502A oscdloscope. T h e results were r e c o r d e d o n a G r a s s P7 P o l y g r a p h or were filmed f r o m t h e oscilloscope. A b r i d g e circuit was u s e d at t h e i n p u t of t h e c a t h o d e follower to allow t h e cell to b e h y p e r p o l a r l z e d or s t i m u l a t e d t h r o u g h t h e electrode. T h e i o n t o p h o r e t i c electrodes were filled w i t h m o l a r solutions of a c e t y l c h o h n e or G A B A a n d h a d a resistance b e t w e e n 5 a n d 14 M ~ . T h e l o n t o p h o r e t i c electrodes were b r o u g h t close to t h e r e c o r d i n g electrode. A b a c k i n g c u r r e n t was u s e d to stop t h e d r u g diffusing o u t of t h e electrode. D u r i n g s t l m u l a t i o n , a pulse of k n o w n c u r r e n t s t r e n g t h a n d
FIG. 1. Cell body m cockroach ganglion penetrated with a micro-electrode and marked with Procion yellow. The cell fluoresces (white m photograph) m u.v. hght and shows the position of the micro-electrode
ACH AND G A B A ON INSECT C N S NEURONES
613
duration was passed through the iontophoretic electrode to carry the drug out onto the cell membrane. T h e experimental bath was made of perspex with a front wall of glass. T h e volume of the hath was reduced to 10 ml by filling the space not required with wax. T h e solutions were changed by means of glass tubes entering the bath. T h e solutions were washed through the bath at a rate of 6 ml/min. T h i r t y mlllilitres of Ringer was used to wash away applied drugs. Tests with coloured solutions indicated that 15 ml would wash out the bath. T h e following agonists of acetylcholine were used: acetylcholine, carbachol, pilocarpme, nicotine. Antmhohne esterases: eserine, neostigmine, edrophonium. Antagonists: tubocurarine, atropine. T h e chemicals were made up in wt/vol, except for nicotine and neostigrmne which were made up vol/vol. T h e solutions were made in Ringer solution. T h e Ringer solution was similar to that used by Yamasakl & Narahashi (1958, 1959, 1960) but m place of the 2'0 m M phosphate buffer which tends to precipitate the calcium, we used 1"0 Tris-HC1 buffer. T h e composition of the Ringer solutions is given in Table 1. T h e CaC12 concentration was also increased to 9'0 m M from 1"8 raM. TABLE 1--THE COMPOSITION OF THE DIFFERENT RINGER SOLUTIONSUSED
A NaC1 KC1 CaC18 Tris-HCl NaAc KAc CaAc2 Trls-Ac
214 3"1 9"0 1"0
B -3"1 9"0 284"4
C
D
44"3 40 9"0 1"0 214 3"1 9"0 1"0
Values are expressed in mM/l. A. Normal Ringer solution. B. Sodium-free solution. C. Ringer solution in which the potassium concentration has been increased to 40 m M by replacement of sodium. D. Chloride-free Ringer solution. RESULTS
1. Micro-electrode recording T h e n e r v e cells on t h e m i d l i n e of t h e d o r s a l surface o f t h e g a n g l i a p r o v e d to b e easy to p e n e t r a t e w i t h m i c r o - e l e c t r o d e s . I t was p o s s i b l e t o see t h e e l e c t r o d e a p p r o a c h i n g t h e surface o f t h e cell b o d y , a n d s o m e t i m e s to see t h e n e u r o n e d i m p l e as t h e e l e c t r o d e p e n e t r a t e d . I n s o m e e x p e r i m e n t s t h e e l e c t r o d e was filled w i t h P r o c i o n Y e l l o w a n d l a t e r p u l s e d so t h a t t h e n e r v e cell filled w i t h t h e dye. T h e y e l l o w cell b o d y o f t h e n e u r o n e c o u l d b e seen in t h e f r e s h p r e p a r a t i o n . H i s t o l o g i c a l s e c t i o n s o f t h e s e g a n g l i a u n d e r u.v. l i g h t s h o w e d t h e d y e in t h e cell b o d y (Fig. 1) a n d i n d i c a t e d t h a t t h e e l e c t r o d e t i p h a d b e e n in t h e cell b o d y . U s i n g t h i s t e c h n i q u e a n d l o n g e r d y e infusion, o n e can m a p o u t t h e p a t h w a y s o f t h e axons f r o m t h e s e cells ( S t r e t t o n & K r a v i t z , 1968).
614
G. A. I~RKUT, R. M. PXTMANAND R. J. WALiC_eR
T h e cell bodies had a resting potential of 54.5 + 1.9 mV (n = 10). M a n y of the cells showed action potentials of 81.0+ 1 . 9 m V ( n = 10) and a positive after potential of 14.9 + 1.5 mV. Figure 2 shows the shape of a single action potential at a fast time sweep whilst Fig. 3 shows a series of action potentials. T h e duration of the action potential was between 2 and 3 msec. Preparations could show 30
--
0
-40
j
-80 5 m sec
FIo. 2. Action potentials from a nerve cell body in the cockroach CNS.
50mY
3
sec
FIG. 3. Series of action potentials from a cell body in the cockroach CNS. spontaneous activity for 3 hr or more. T h e use of the word "spontaneous" only implies that the activity was not directly attributable to any specific or known input excitation. Approximately 15 per cent of the penetrated cells showed spontaneous action potentials. Over 500 cells have been penetrated. T h e silent
ACH AND GABA ON INSECTCNS NEURONES
615
cells could be stimulated to activity either through the bridge circuit or by applying drugs. The nerve cell bodies thus appear to be both chemically and electrically excitable.
2. Action of acetylcholine (ACh) An iontophoretic electrode filled with 1 M ACh was brought up close to the penetrated cell. A backing current prevented the ACh from diffusing out and stimulating the cell. Figure 4 shows the results of an experiment where different
D
80nA
I10 nA
IH_ 150 nA
220 nA
50 mV I 500
nA
3 sec
FIG. 4. I o n t o p h o r e t i c application of acetylcholine onto the nerve cell body. As the iontophoretic current increased, so the a m o u n t of acetylcholine applied to the nerve cell b o d y increased and the depolarization increased. T h e n u m b e r refers to the current applied. T h e retaining current = 100 nA. T h e A C h electrode resistance was 14 M f ] .
amounts of ACh (as shown by the increasing iontophoretic current) were applied to the surface of the impaled nerve cell. There was a depolarization when the ACh was applied and the amount of depolarization and the number of action potentials increased as the amount of acetylcholine was increased. Figure 5 shows
G. A. KERKUT,R. M. PITMAN AND R. J. WALKER
616
the relationship between the amount of A C h applied (in t e r m s of the iontophoretic current) and the depolarization of the nerve cell. It is possible to calculate the amount of A C h that is liberated f r o m the iontophoretic electrode. Such calculations show that assuming the transport n u m b e r of A C h to be 0.5, the threshold amount 32 - -
I0--
8 --
6
-
4
-
2
--
/
d o
0
jrJl
2"r5
3o
Ionfophoref~c
/
/
,
40
50 current,
o
f
I
I
1
60
70
80
nA
FIG. 5. Dose-response curve showing the relationship between the amount of acetylcholine applied to the nerve cell (iontophoretic current) and the depolarization of the cell. Retaining current = 60 nA. ACh electrode resistance = 12 M ~ . of A C h was 1.31 x 10 -13 mole. T h i s value is of the same order as that for neurones in other animals and tests using the same iontophoretic electrode on cockroach nerve cells and nerve cells in the brain of the snail Helix aspersa, showed that there was little difference in the sensitivity of the neurones of these two animals to ACh. T h e s e experiments show that the sensitivity of the cockroach neurones to iontophoretically applied A C h is m u c h the same as neurones in other animals and that insect neurones are not less sensitive to ACh.
3. Iontophoretic application of GABA G A B A hyperpolarizes the nerve cells. W h e n an iontophoretic pipette filled with 1 M G A B A was brought up close to an impaled nerve cell and current passed t h r o u g h the pipette, G A B A hyperpolarized the nerve cell and could stop action potentials. T h e degree of hyperpolarization is proportional to the amount of G A B A applied (Fig. 6) and a dose-response curve for the application of G A B A is shown in Fig. 7.
ACH ANDGABA ON INSECTCNS NEURONES
617
The threshold to GABA, assuming a transport number of 0.5, was 1.05 × 10-13 mole.
!~,._.[/~. 90 nA
150 nA
lOOmY 5 sec
250 nA
450 nA
700 nA FIG. 6. Iontophoretic application of G A B A onto the nerve cell body. As the a m o u n t of G A B A applied increased so the hyperpolarization increased. It also stopped the action potentials. Retaining current = 100 nA. G A B A electrode resistance = 12 M ~ .
4. Ionic basis of GABA hyperpolarization Alteration in potassium concentration. The addition of GABA hyperpolarizes the neurone. This could be brought about either by increasing the membrane permeability to potassium ions or to chloride ions or to both. Figure 8 shows the effect of increasing the external potassium ion concentration from 3 mM to 40 mM. When GABA was applied to the bath of standard Ringer solution there was a hyperpolarization of the nerve cell by 6 mV. In the high potassium solution there was still a hyperpolarization following addition of GABA but it was only 4 mV. It should be noted that increasing the external potassium concentration to 40 mM had depolarized the membrane potential from - 5 3 mV to - 4 5 mV. Replacing
G. A. KERKUT,R. M. PITMANAND R. J. WALKER
618
normal Ringer around the nerve restored the membrane potential to - 50 mV and the GABA hyperpolarization was once more 6 mV. Increasing the potassium ion concentration by about ten times has a small effect on the GABA potential.
12
/
I0
/
/
_q
/ -
/
je 0
2-5
o 50
I00
Ionfophoret,c currenf,
2O0
300
I
600
nA
FIG. 7. Dose response curve showing the relationship between the amount of GABA applied to the nerve cell and the hyperpolarization. Retaining current = 60 hA. GABA electrode resistance = 9 Mr2.
Alteration in chloride concentration. Figure 9 shows the effect of changing the external Ringer to one that is chloride free, the chloride ions being replaced by acetate. In normal Ringer which contains 236 m M chloride ions, addition of GABA to the preparation hyperpolarizes the cell by 6 mV. When the Ringer is replaced by chloride-free Ringer, addition of GABA now brings about a 10 mV depolarization, i.e. the effect of GABA has been reversed. I f the chloride-free Ringer is replaced by normal Ringer then GABA once more brings about a hyperpolarization of 6 mV. T h e effect of removing the chloride ions in the external solution was most marked and reversible. I f the micro-electrodes are filled with potassium chloride solution, the chloride ions diffuse into the nerve cell and increase the internal chloride ion concentration. In such cases the response to GABA changes. Figure 10 shows such a response; a 1 M KC1 filled 30-Mf~ micro-electrode was inserted into the cell. Application of
ACH AND GABA ON INSECTCNS NEtrRONF~
619
50/~g of GABA within 30 sec of insertion of the electrode caused the cell to hyperpolarize by 2.5 mV (the normal hyperpolarization in cells penetrated with a potassium acetate filled electrode was 6 mV). Three minutes after impalement, application of GABA caused the neurone to depolarize by 2.5 mV and after 50 sec A "L
....
"
H
.,
,
R.R - 5 3 50 ~g
GABA
,I,
I
I
R P--45 20 mm 40mM K+
50/zg GABA
I0 rnV]
4sec RR - 5 0 20 rain Wash
k._ 50Fg GABA
FIG. 8. Effect of changing external potassium ion concentration on the GABA hyperpolarization. Changing the external K from 3 mM to 40 mM slightly reduced the effect of GABA. to fire one action potential. After 6 min GABA depolarized tile membrane potentials by 4 mV and an action potential fired after 33 sac. When GABA was added 9 min after impalement, the membrane depolarized by 6.5 mV and after 10 sec fired an action potential. Injection of chloride ions thus rapidly reversed the effect of GABA from a hyperpolarization to a depolarization. During the experiment the membrane potential remained at - 55 mV. These experiments show that the effect of GABA is greatly affected by alteration in the chloride concentration and slightly affected by alteration in the potassium concentration.
G. A. KERKUT, R. M. PITMAN AND R. J. WALKER
620 A
r ,filter !tf r
ll
@
'i
i
50 ,u.g GABA
IOmV [ _ _ 5sec
II t
1,,~
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50/zg GABA 20 mm CL- Free
~-5o ~ ' I 50~g GABA IO rnin Wash Fro. 9. Effect of reducing the external chloride ion concentration of the response to GABA. Reducing the external chloride ion concentration to zero changed the response to G A B A from a hyperpolarlzation to a depolarization. On restoring the normal chloride level (C) the effect of G A B A was partially restored.
5. Ionic basis of ACh depolarization The depolarization following addition of acetylcholine could be due to acetylcholine increasing the membrane permeability to sodium ions. The effect of removing sodium ions on this response is shown in Fig. 11. The normal Ringer solution contains 214 mM sodium ions and this was replaced by a Ringer solution in which all the sodium ions had been replaced by Tris (Table 1).
ACH AND GABA ON INSECT CNS NEURONES
621
I/2 rain after inser'hon
R. P -- 55
/~'50Fg
GABA
3 mln affer inserhon
RP--52
l 50/zg GABA
6 mln affer inset,ion
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/
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R.P-55 ~ 50~xg GABA
!
D
9 rnli3 after in msertmn R P--55
I0 mV I
5sec
i
l
50 Fg GAB
FIG. 10. Effect of increasing internal chloride ion concentration on the response to GABA. Inserting the KCl-filled micro-electrode into the cell allowed chloride ions to diffuse into the cell and changed the response to GABA from a hyperpolarization (A) to a depolarization (B-D) as the internal chloride concentration increased.
Figure l l A shows the response of the neurone in normal Ringer when 500 Fg of ACh is applied to the bath. The membrane potential was - 62 inV. On adding ACh there was a depolarization of 12 mV and the cell fired a series of action potentials at the rate of 2/sec. When the Ringer solution was changed to a sodiumfree solution, there was a hyperpolarization of the membrane potential by 16 mV over 10 min. [This would indicate that the membrane potential is not at the potassium equilibrium potential but is at a value less negative than this due to a relatively high permeability to sodium ions: Hodgkin & Horowicz (1959).]
622
G. A. KERKUT, R. M. PITMAN AND R. J. WALKER
Addition of ACh brought about a depolarization of 4 mV and it took much longer to reach this level (25 sec instead of the 15 sec as in Fig. llA). When the normal Ringer was replaced, the response to ACh was once more a quick depolarization leading to many action potentials (Fig. 11C). A ,Jr
'
i
,. ,
F j
f
,
, , ,
,
,
,
R P -62
I 500jag ACh I0 mV 5 sec
R P--78
IO
mM
No +
Free
|
500/zcj
ACh.
I
C
rI
I0
mtn
500
H-g
r
ACh
Wash
FIG. 11. Effect of reducing the external s o d i u m ion concentration on the response to acetylcholine. Removal of external s o d i u m reduced the effect of acetylcholine.
The observed effect of the sodium-free solution was not due to the hyperpolarization of the cell since if the cell in normal Ringer was artificially hyperpolarized by 16 mV through a bridge circuit, addition of 500/zg ACh brought about a fast depolarization. The slow depolarization seen in sodium-free solution could be due to the presence of some residual sodium ions still present around the preparation (Chamberlain & Kerkut, 1969). 6. Post synaptic activity in insect central neurones In many of the penetrated cells it was possible to see indications of postsynaptic activity. The positions of the interneurones that were acting presynaptically have not as yet been determined.
ACH ANDGABA ON INSEC'TCNS NEURONm
623
Figure 12A shows a cell with action potentials and also base-line activity indicative of EPSP. The EPSP are more clearly shown in Fig. 12B which shows the depolarizing potentials as a series of upward spikes. Figure 12C and D show a similar series of IPSP recorded in the cell body.
A
I0 mV
[ 2 see
5mV 3 sec
I0 mV
I sec
----¢--,-----'-v---D
I0 mV
200 msec
Fzc. 12. EPSP and IPSP recorded from insect nerve cell body in the ganglion. A, recording showing action potentials and background EPSP; B, EPSP; C, IPSP; D, IPSP on faster time-scale. The postsynaptic potentials indicate that the site of the synapse is relatively close to the penetrated nerve cell body. Such preparations allow study of the action of drugs and ions on the synaptic systems of the insect CNS and work is in progress on the determination of known pathways that will allow stimulation of either the EPSP or the IPSP.
624
G . A . KERKUT, R. M. PITMAN AND R. J. WALKER
7. Action of acetylcholine agonists T h e relative sensitivity of the cockroach preparation to acetylcholine and related compounds can be tested by seeing how the activity of the preparation changes following application of the drug. In the following series of experiments the drug was applied to the ganglion and the activity was measured in the fifth thoracic nerve by means of a suction electrode. Figure 13 shows the activity in the fifth nerve and the effect of adding 1 mg/ml ACh on this activity. The preparation shows a single unit firing (Fig. 13A); D
AP's/sec ~ _ 5 mm B A
I0
mg/ml ACh
L
100/~g/mL
I00/~g/mL
CCh
IOp.g/mL
PI LO.
NIC.
FIG. 14. Rate meter curves showing the activity m the fifth nerve following apphcation of A, acetylchohne; B, carbachol; C, pilocarpme or D, nicotine. 5 AP's/sec
] 5mm
A
B
IO/.zg/ml ES
C
IOO tzg/ml
IO /~g/mL
NEOSTIG
EDROPH
FIG. 15. Rate meter curves showing the activity in the fifth nerve following application of anticholine esterases; A, eserine; B, neostlgmine and C, edrophonmm.
Normal
/ mln Img/ml
ACh
6
3 mln
IO mm
15 mln
20
mln 0 5mV
L I set
FIG. 13. Actlvlty of nerves m the fifth nerve from the metathoraclc ganghon. When acetylcholme was added to the ganglion there was an mcrease of actnlty The time mdlcates mmutes after the addltmn of the recorded m the fifth nerve acetylcholme.
625
ACI{ AND GABA ON INSECT C N S N~URONES
addition of ACh brought about an increase in activity, the peak being reached after 20 min. In this case ACh was continuously perfused over the ganglion. In the next example the drug was applied once and the response noted. Figure 14 shows the responses of the preparations to the application of ACh, carbachol, pilocarpine A
"
.
.
.
.
.
. .
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FIO. 16. The effect of tubocurarine on the electrical activity in the fifth nerve. B - F are the activity 1, 3, 6, 10 and 15 rain after the application of tubocurarine.
626
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Fro. 17. The effect of A, 500 ~g acetylchol~ne; B, 5 Fg carbachol; C, 0"5 ~g n i c o t i n e ; D , 50 ~ g m e c h o l i n e ; E, 50 ~ g pilocarpLue; o n t h e m t r a c e l l u l a r activity of t h e n e r v e cell.
ACR AND GABA ON INSECTCNS NEURONF.S
627
and nicotine. The responses are shown in terms of rate of activity over time. The records show the response to 10 rag ACh, 100 Fg carbachol, 100 Fg pilocarpine and 10 Fg nicotine. The preparation was most sensitive to nicotine > carbachol > pilocarpine > acetylcholine. Figure 15 shows the response of the preparation to addition of anticholine esterases. The preparation responded to 10 Fg eserine, 100 Fg neostigmine and 100/zg edrophonium. The preparation was most sensitive to eserine>edrophonium > neostigmine. Tubocurarine inhibited the activity of the preparation. Addition of 100 Fg tubocurarine stopped all activity in the preparation (Fig. 16) the effect being seen 10 min after the addition of the drug. The response was not reversible. Similar results were obtained from intracellular studies. Figure 17 shows the effect of adding 500 Fg ACh, 5 tzg carbachol and 0.5 Fg nicotine to the preparation. The cell was most senskive to nicotine. When nicotine was applied at low concentrations (0.05 tzg/ml) the cell showed a brief exckation and the response could
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FIG. 18. The effect of A, adrenaline; B, noradrenaline; and C, dopamine on the intraceUular activity of the nerve cell
628
G. A.
KERKUT,
R. M. PITMANANDR. J. WALKER
be repeated with further application of nicotine. The preparation was excited by adrenaline, noradrenaline and dopamine (Fig. 18). Glycine inhibited activity. Table 2 shows the relative concentrations of the drugs required to be added to the bath in order to get an effect. The values are not those acting on the membrane surface and are merely given to indicate the relative effectiveness of the drugs. TABLE 2--THE
RELATIVE EFFECTIVE CONCENTRATIONS OF DRUGS TESTED ON THE NEURONES OF THE SIXTH ABDOMINAL GANGLION
Compounds Acetylcholine Carbachol Nicotine Mecholine Atropine Tubocurarine Eserine Adrenaline Noradrenaline Dopamme Glycine GABA
Action Depolarizes and excites Depolarizes and excites Depolarizes and e x c i t e s Depolarizes and e x c i t e s Antagonises acetylchohne and pilocarpine Antagonises nicotine Potentmtes acetylchohne Depolarizes and excites Depolarizes and excites Depolarizes and excites Hyperpolarizes and inhibits Hyperpolarizes and inhibits
Threshold 50/zg-5 mg 5/zg 0"05-0'5/~g 50-500/zg 10/zg/ml 100/zg/ml 1"0/xg/ml 500/zg 500/zg 500/zg 50-500/zg 5 ttg
The drugs are not apphed lontophoretlcally and the amounts are added to the bath and only indicate relative effecuveness. DISCUSSION Intracellular recordings from nerve cell bodies in insect C N S The electrophysiological investigations with micro-electrodes of the insect CNS has been rather limited and unrewarding compared with those on the Molluscan CNS neurones. In theory the insect central neurones should have been better experimental material since it is easier to set up and study reflex activity in insects, and insects have more interesting behaviour than that shown by gastropod molluscs. An explanation of the backwardness of the studies on insects is partly given by the initial experimental difficulties in setting up the preparation and partly due to a belief or rumour that the insect cell bodies were silent and a long way away from the site of synaptic activity. Thus the insect CNS would not apparently repay the time and effort that might be put into a study of the electrical activity of the neurones. Examination of the literature shows that some workers have been able to obtain recordings from insect central neurones. Thus Hagiwara & Watanabe (1956) have recorded from cicada central neurones and obtained good action potentials, whilst Rowe (1964, 1969), Callec & Boistel (1967) and Kerkut et al. (1968) obtained activity from cockroach central neurones.
ACH
AND
GABA
ON
INSECT
CNS
NEURONES
629
The main drawback is the initial difficulty in seeing the insect cell bodies and obtaining good penetration with micro-electrodes. We have found that we get much better activity and longer-lasting preparations when the micro-electrodes are filled with potassium acetate instead of potassium chloride and this may be part of the previous difficulty, in that many of the previous workers did use KCl-filled micro-electrodes. The present study on the use of micro-electrodes on neurones in the insect CNS indicates that many such neurones are electrically active and that it should be possible to develop preparations that show synaptic activity. Action of acetylcholine and G A B A on insect neurones
Previous studies on insect central nerve cells have indicated that the neurones were relatively insensitive to ACh. In our experiments we have shown that iontophoretic application of ACh directly onto the neurone gives a good quick response and that the sensitivity of the neurone is of the order of 10 -13 mole. This compares very favourably with estimations of the sensitivity of other preparations; the mollusc neurone was sensitive to 10 -14 mole (Gerschenfeld & Stefani, 1966). The crayfish motor end-plate was senskive to 10 -14 mole GABA (Takeuchi & Takeuchi, 1964). The frog nerve muscle junction was sensitive to addition of 10 -15 mole (del Castillo & Katz, 1955). We have used the same iontophoretic pipette for adding ACh onto cockroach neurones and snail neurones and have found little difference in the sensitivity of the two preparations. On the other hand, when we apply the ACh onto the whole ganglia and record from single neurone or from peripheral axon, we find that a much higher concentration of acetylcholine is required to bring about an effect, the threshold level being about 10 -5 g/ml. Callec & Boistel (1967) applied acetylcholine by means of a pipette placed close to the recording electrode and found that the nerve cell responded to application of 10 -8 g/ml. The most feasible explanation of the high concentration of ACh that has to be applied to the whole ganglion to get an effect is that provided by Treherne & Smith (1965a, b) and Smith & Treherne (1965). It is likely that the high concentration of choline esterase in the insect nerve cord breaks down most of the applied ACh so that the final concentration at the nerve cell membrane is low. Treherne & Smith (1965a) using labelled acetylcholine found that penetration into the intact cockroach ventral nerve cord was quite rapid. They calculated that when the cord was soaked in 10 -2 M ACh solution, the extracellular concentration was approximately 8.1 × 10 -5 M. The low potency of ACh is thus due to a biochemical barrier: the high concentration of choline esterase. This is supported by comparative pharmacological studies. Carbachol which is not destroyed by the choline esterase, is more effective than acetylcholine when applied to the whole ganglion. Thus one of the drawbacks to the acceptance of acetylcholine as a natural excitatory transmitter in the cockroach, namely the high experimental concentration
630
G . A . KERKUT, R. M. PITMAN AND R. J. WALKER
that had to be applied in order to get an effect, now no longer holds. The insect neurone is as sensitive as other neurones (mollusc or vertebrate) to applied acetylcholine provided that the ACh is applied directly and iontophoretically onto the cell membrane. The other evidence in favour of ACh as an insect CNS transmitter is as follows. The cockroach nerve cord has a high ACh content. Colhoun (1958a, b, c) found that the brain and suboesophageal ganglia contained 135.2/~g/g ACh. The ventral nerve cord contained 63.2/zg/g. The thoracic ganglia contained 95.4/zg/g and the terminal abdominal ganglia contained 63.0/zg/g. The ACh content in the thoracic ganglia was more than three times that in the connective. It is in the ganglia that most of the synaptic connections are made and this supports the idea that ACh is the excitatory transmitter. The ACh in the insect CNS is more than twice the level obtained by Brown & Feldberg (1936) for the vertebrate sympathetic ganglion. Tobias et al. (1946), Smallman & Pal (1957) and Colhoun (1958b) have demonstrated the presence of the enzyme choline acetyl transferase in the insect nerve cord. The level in the insect ganglia was eight times higher than that present in the guinea-pig cervical ganglia. There is similar evidence that GABA could be the main inhibitory transmitter in the insect CNS. Ray (1964) showed that the nerve cord of Periplaneta americana contained 2.5 mM/kg of GABA. This is the same order of activity as that in the mammalian brain. We have evidence that the nerve cord can convert labelled glutamate to labelled GABA, thus there is a glutamate decarboxylase present in the nerve cord (Huggins et al., 1967). Usherwood & Grundfest (1965) found that GABA mimics the effect of stimulating the inhibitory axon at the neuro-muscular junction of Romalea microptera and Schistocerca gregaria. Suga & Katsuki (1961) and Gahery & Boistel (1965) showed that GABA would inhibit the electrical activity in the insect CNS. We have found that cockroach central neurones are sensitive to iontophoretic application of GABA. The effect of GABA is to increase the postsynaptic membrane permeability to chloride ions. Glycine can also bring about an inhibition of the cockroach neurones but the level required is some hundred times more than that for GABA. The evidence at present is reasonably good that acetylcholine is an excitatory transmitter at insect central neurones and that GABA is an inhibitory transmitter. There is some evidence that the catecholamines could play a part in normal transmitter systems but this requires more experimental evidence. SUMMARY 1. Intracelhilar recordings from nerve cells in the isolated cockroach nerve cord showed that the cells had a resting potential of 54 _+1.9 mV; action potential of 81 + 1.9 mV and a positive after potential of 14.9 _+ 1.5 mV. The duration of the action potential was 2-3 msec. The position of the penetrated cell has been marked with Procion yellow.
A C H AND G A B A ON INSECT C N S NEURONES
631
2. U p to 15 per cent o f the nerve cells studied s h o w e d s p o n t a n e o u s action potentials and the activity can last for 3 h r in an impaled neurone. 3. T h e preparations can s h o w b o t h E P S P and I P S P . 4. I o n t o p h o r e t i c application of acetylcholine depolarized the nerve cell. T h e threshold to acetylcholine was 1.31 × 10 -13 mole. 5. I o n t o p h o r e t i c application of G A B A hyperpolarized the nerve cells. T h e threshold was 1.05 × 10 -13 mole. 6. G A B A increased the permeability of the m e m b r a n e to chloride ions and slightly to potassium ions. Acetylcholine increased the m e m b r a n e permeability to s o d i u m ions. 7. T h e effectiveness of acetylcholine agonists is n i c o t i n e > c a r b a c h o l > p i l o carpine > acetylcholine. 8. T h e effectiveness of anticholine esterases was e s e r i n e > e d r o p h o n i u m > neostigmine. 9. D o p a m i n e , noradrenaline and adrenaline excite the cells whilst glycine inhibits activity. Acknowledgements--Dr. Robert Pitman is a Research Fellow of the Agricultural Research Council. We are indebted to I.C.I. (Dyestuff Division) for a gift of Procion yellow and to Dr. Niall Horn for his advice and assistance throughout this investigation.
REFERENCES
BOISTELJ. (1968) The synaptic transmission and related phenomena in insects. Adz,. Insect Physiol. 5, 1-64. BROWN G. L. & FELOBERC W. (1936) The action of potassium on the superior cervical ganglion of the cat. ~. Physml., Lond. 86, 290-305. CALLEC J. • BOISTEL J. (1967) Les effets de l'acetylcholine aux mveaux synaptique et somatique dans le cas dernier ganglion abdominal de la Blatte, Periplaneta americana. C. r. Soc. Biol. 161,442-446. DEL CASTILLOJ. & KATZ B. (1955) On the locahsation of acetylchohne receptors, o~. Physiol., Lond. 128, 157-181. CHAMBERLAIN S. G. & KERKUT G. A. (1969) Voltage clamp analysis of the sodium and calcium inward currents in snail neurones. Comp. Bwchem. Physwl. 28, 787-803. CHANG S. C. ~%KEARNSC. W. (1955) The occurrence of acetylchohne in the nerve tissue of American cockroaches. Prog. 3rd Ann. Meet. Ent. Soc. Amer. 30, 30-31. COLHOUN E. H. (1958a) Acetylcholine in Periplaneta americana.--I. Acetylcholine levels m nervous tissue. ~. Insect Physiol. 2, 108-116. COLHOUN E. H. (1958b) Acetylcholine in Periplaneta americana--II. Acetylcholine and nervous activity. ~. Insect Physiol. 2, 117-127. COLHOUNE. H. (1958c) Distribution of choline acetylase in insect conductive tissue. Nature, Lond. 182, 1378. COLHOUN E. H. (1963) The physiological significance of acetylcholine in insects and observations upon other pharmacologmally active substances. Adv. Insect Physiol. 1, 1-46. GAHERYY. & BOISTELJ. (1965) Study of some pharmacological substances whmh modify the electrical activity of the sixth abdominal ganglion of the cockroach Periplaneta americana. In The Physiology of the Insect Central Nervous System (Edited by TREHERNE J. E. & BEAMENTJ. W. L.), pp. 73-78. Academic Press, London and New York. 2I
632
G.A. KERKUT,R. M. PITMANANDR. J. WALKER
GERSCHENFELD H. ~ . & STEFANIE. (1966) An electrophysxologlcal studyof 5HT receptors of neurones in the molluscan nervous system. J. Physiol., Lond. 185, 684-700. HAGIWARAS. 6c WATANABEA. (1956) Discharges in motoneurones of Cicadas. J. cell. comp. Physzol. 47,415-428. HODGKIN A. L. & HOROWlCZ P. (1959) T h e influence of potassium and chloride ions on the membrane potentials of single muscle fibres. J. Physiol., Lond. 148, 127-160. HUGCINS A. K., RICK J. T. & KERKUTG. A. (1967) A comparative study of the intermediary metabolism of L glutamate m muscle and nervous tissue. Comp. Biochem. Physml. 21, 23-30. IYATOMI K. & KANEHISAK. (1958) Locahsatlon of choline esterase in the American cockroach. Jap.J. appl. Ent. Zool. 2, 1-10. KANEHISA K. (1961) Studies on the cholinesterase in insects, especially in relation to the mode of action of organophosphorus insecticides. Bull. 2 Lab. Appl. Ent. Fac. of Agric.,
Nagoya Umv.Japan. KERKUT G. A., PITMAN R. M. & WALKER R. J. (1968) Electrical activity in insect nerve cell bodies. Life Scz. 7, 1,605-608. LOWENSTEIN O. (1942) A method of physiological assay of pyrethrum extracts. Effect of pyrethrum on cockroach nerve cord. Nature, Lond. 150, 760-762. MIKALONIS S. J. & BRowN R. H. (1941) Acetylchohne and choline esterase in the insect central nervous system. J. cell. comp. Physml. 18, 401-403. O'BRIEN R. D. (1957) Esterase in the serru intact cockroach. Ann. ent. Soc. ~tm. 50, 223-229. O'BRIEN R. D. & FISHER R. W. (1958) T h e relation between xonisation and toxicity to insects for some neuropharmacological compounds. J. econ. Ent. 51, 169-175. RAg J. W. (1964) T h e free amino acid pool of the cockroach (Perlplaneta americana) central nervous system and the effect of insecticides. J. Insect Physiol. 10, 587-598. ROEDER K. D. (1948) T h e effect of antlchollnesterases and related substances on nervous actlvlty in the cockroach. Bull.Johns Hopkins Hosp. 83, 587-599. R o w e E. C. (1964) Microelectrode records from an insect thoracic ganglion. Diss. Abstr. 25, 2766. ROWE E. C. (1969) Micro-electrode records from a cockroach thoracic ganglion. Comp. Bzochem. Phystol 30, 529-540. SMALLMAN B. N. • PAL R. (1957) T h e activxty and intracellular distribution of choline acetylase in insect nervous tissue. Bull. ent. Soc. Amer. 3, 25. SMITH D. S. & TREHERNE J. E. (1963) Functional aspects of the organisatlon of the insect nervous system. Adz'. Insect Physiol. 1,401-484. SMITH D. S. & TREHERNE J. E. (1965) T h e electron microscopic localisation of choline esterase activity In the central nervous system of an insect Periplaneta americana. J. ceil. Btol. 26, 445-465. STRETTON A. O. W. & KRAVITZ E. A. (1968) Neuronal geometry. Determination with a technique ofmtracellular dye rejection. Science162, 132-134. SUOA N. & KATSUKIY. (1961) Pharmacological studies on the auditory synapse m a grasshopper. J. exp. Biol. 38, 759-770. TAKEUCHI A. & TAKEUCHI N. (1964) T h e effect on crayfish muscle of lontophoretically applied glutamate. J. Physzol., Lond. 170, 296-317. TOBIAS J. M., KOLLROSJ. J. & SAVIT J. (1946) Acetylcholine and related substances in the cockroach, fly and crayfish and the effect of D D T . J. cell. comp. Physiol. 28, 159-182. TREHERNE J. E. (1962) Some effects of the ionic composition of the extracellular fluid on the electrical activity of the cockroach abdominal nerve cord. J. exp. Biol. 39, 631-641. TREHERNE J. E. (1966) The Neurochemistry of Arthropods. Cambridge University Press. TREHERNE J. E. & SMITH D S. (1965a) T h e penetration of acetylchohne into the central nervous tissue of an insect (Per~planeta americana). J exp. Bml. 43, 13-21. TREHERNE J. E. & SMITH D. S. (1965b) T h e metabolism of acetylchohne in the intact central nervous system of an insect (Perzplaneta amerzcana). J. exp. Biol. 43, 441-454.
ACH AND GABA ON INSECT CNS NEURONE...q
633
TWAROG B. M. & ROEDER K. D. (1956) Properties of the connective tissue sheath of the cockroach abdominal nerve cord. Biol. Bull. mar. biol. Lab., Woods Hole 111, 2, 278-286. TWAROG B. M. & ROEDER K. D. (1957) Pharmacological observations on the desheathed last abdominal ganglion of the cockroach. Ann. ent. Soc. Am. 50, 231-237. USHERWOOD P. N. R. & GRUNDFESTH. (1965) Peripheral inhibition in skeletal muscles of insects..7. Neurophysiol. 28, 497-518. YAMASAKIT. & NARAHASHIT. (1958) Synaptic transmission in the cockroach. Nature, Lond. 182, 1805-1806. YAMASAKI T. & NARAHASHI T (1959) The effects of potassmm and sodium ions on the resting and action potentials of the cockroach giant axon..7. Insect Physiol. 3, 146-158. YAMASAKIT. & NARAHASHIT. (1960) Synaptic transmission in the last abdominal ganglion of the cockroach..7. Insect Physiol. 4, 1-13.
Key Word Index--Insect CN S ; nerve pharmacology; cockroach; Periplaneta americana; iontophoretlc application of drugs; acetylcholine GABA; mcotme; mecholine; carbachol; eserme; edrophonium; tubocurarine; adrenaline; noradrenaline; dopamlne; ionic basis of drug action in insects.
IONTOPHORETIC APPLICATION OF ACETYLCHOLINE AND GABA* ONTO INSECT CENTRAL NEURONES G. A. K E R K U T , R. M. P I T M A N and R. J. W A L K E R Department of Physiology and Biochemistry, Southampton University (Received 16 April 1969)
AI~tract--1. Electrical activity showing action potentials, EPSP and IPSP can be recorded with micro-electrodes from the nerve cell bodies in the ganglia of the
cockroach. 2. The sensitivity of the nerve cell to iontophoretically applied acetylcholine is 1"31 × 10 -13 mole. It is of the same order of sensitivity to acetylcholine as shown by mollusc nerve cells. 3. The sensitivity of the nerve cell to iontophoretically applied GABA is 1'05 × 10-la mole. 4. A full summary is given on page 630.
INTRODUCTION THE PHARMACOLOGYof the insect central nervous system has been studied by many previous authors (Mikalonis & Brown, 1941 ; Lowenstein, 1942; Tobias et al., 1946; Roeder, 1948; Chang & Kearns, 1955; Twarog &'Roeder, 1956, 1957; O'Brien, 1957; Smallman & Pal, 1957; Iyatomi & Kanehisa, 1958; O'Brien & Fisher, 1958; Yamasaki & Narahashi, 1958, 1959, 1960; Colhoun, 1958a, b, c, 1963; Kanehisa, 1961; Treherne, 1962, 1966; Smith & Treherne, 1963, 1965; Rowe, 1964, 1969; Treherne & Smith, 1965a, b; Callec & Boistel, 1967; Boistel, 1968). One problem has been the relative insensitivity of insect neurones to acetylcholine, the neurones requiring 10-2-10-5 M acetylcholine to bring about excitation. The present paper describes the use of intracellular micro-electrodes on cockroach neurones and the effects of drugs on these neurones. The drugs were chosen to indicate possible transmitters in the cockroach central nervous system. The results from micro-electrodes are compared with those using suction electrodes. The application of drugs to the bathing medium is compared with iontophoretic application of drugs. A preliminary account of this work has already been published (Kerkut et al., 1968). *Abbreviations: ACh, acetylcholine; GABA, gamma aminobutyric acid; EPSP, excitatory post synaptic potential; IPSP, inhibitory post synaptic potential. 611
612
G . A. KERKUT,R. M . PITMAN AND R. J. WALKER MATERIALS
AND
METHODS
Dissection A d u l t cockroaches, Periplaneta americana, were o b t a i n e d f r o m dealers a n d k e p t u n d e r w a r m c o n d i t i o n s w i t h food a n d w a t e r supply. I n t h e p r e p a r a t i o n of t h e isolated m e t a t h o r a c i c ganglion, t h e h e a d a n d all t h e legs except t h e left m e t a t h o r a c i c leg were r e m o v e d f r o m t h e cockroach. T h e a n i m a l was p i n n e d down, v e n t r a l surface u p p e r m o s t , o n t o a wax block. T h e exoskeleton was r e m o v e d f r o m a b o v e the first t h o r a c i c ganglion, t h e m e s o t h o r a c i c g a n g h o n , t h e m e t a t h o r a c i c g a n g h o n a n d f r o m t h e u p p e r surface of t h e m e t a t h o r a c : c leg. A small a m o u n t of 5 ~o m e t h y l e n e b l u e was p u t o n t o t h e exposed area to stare t h e n e r v e s a n d m a k e t h e m m o r e visible. V i e w i n g f r o m above w : t h a b i n o c u l a r microscope, t h e m o s t p o s t e r i o r p o r t i o n of t h e a b d o m i n a l n e r v e cord visible was g r i p p e d w : t h fine forceps, cut a n d g e n t l y lifted away f r o m t h e a m m a l w h i l s t t h e u n d e r l y i n g tissue was carefully cut away f r o m t h e n e r v e c o r d a n d f r o m t h e left fifth n e r v e w h e r e it p a s s e d into t h e leg. T h e o t h e r s e g m e n t a l n e r v e s were s e v e r e d n e a r t h e g a n g h o n . T h e m e t a t h o r a c i c g a n g l i o n was m this way s e p a r a t e d f r o m t h e a n i m a l w i t h a l e n g t h of fifth n e r v e dissected o u t as far as t h e m i d d l e of the coxa. T h e t h o r a c i c n e r v e c o r d was s e v e r e d at t h e c o n n e c t i v e b e t w e e n t h e m e s o t h o r a c i c a n d rectat h o r a c i c g a n g h o n a n d t h e fifth n e r v e severed at t h e coxal region. Care was t a k e n to leave as m u c h as possible of t h e t r a c h e a l system over t h e g a n g l i o n i n t a c t so t h a t o x y g e n a t i o n of t h e g a n g l i o n was m a i n t a i n e d .
External recording: suction electrode T h e isolated g a n g l i o n was t r a n s f e r r e d to t h e e x p e r i m e n t a l b a t h w h e r e it was p l a c e d o n filter p a p e r m o i s t e n e d w i t h insect R i n g e r solution. T h e n e r v e s h e a t h over t h e v e n t r a l surface of t h e g a n g l i o n was r e m o v e d b y a m e t h o d similar to t h a t d e s c r i b e d b y T w a r o g & R o e d e r (1956). W h e n d e s h e a t h i n g was c o m p l e t e d , a n y l o n p e r f u s i o n c a n n u l a was p l a c e d so t h a t it was a b o v e t h e g a n g h a b u t a v o i d e d t h e o p e n e n d s of t h e tracheal system, a n d R i n g e r solution was p e r f u s e d over t h e p r e p a r a t i o n . T h e fifth n e r v e was d r a w n into t h e b a r r e l of a suct:on e l e c t r o d e a n d t h e p r e p a r a t i o n allowed 10 rain to settle down. T h e suction electrode led d i r e c t to a 502A T e k t r o n i x oscilloscope a n d t h e o u t p u t of this was c o n n e c t e d to a n E K C O N 522c rate m e t e r a n d a S e r v o s c r l b e recorder. T h e rate m e t e r was set at a c o u n t i n g rate of 100/sec a n d t h e t i m e c o n s t a n t of 80 sec to s m o o t h o u t a n y fluctuations in t h e firing rate m the fifth nerve.
Intracellular recor&ng : micro-electrodes I n general it p r o v e d easier to p e n e t r a t e t h e ceils of t h e s i x t h a b d o m i n a l g a n g h o n t h a n t h e cells m t h e m e t a t h o r a c l c g a n g h o n . T h e isolated g a n g l i o n a n d n e r v e c o r d was p l a c e d o n a glass slide a n d t h e c o n n e c t i v e s fixed b y m e a n s of elastic b a n d s . T h e n e r v e s h e a t h over t h e dorsal surface was r e m o v e d w i t h fine forceps (this has to b e done very carefully o t h e r w i s e t h e n e r v e cells w d l b e damaged). T h e p r e p a r a t i o n was p l a c e d in a b a t h a n d o b s e r v e d w l t h a b i n o c u l a r m i c r o s c o p e at m a g n i f i c a t i o n s × 20 to × 80. I t was usually possible to see t h e large n e r v e cell bodies in t h e d e s h e a t h e d area. T h e m i c r o - e l e c t r o d e s were p u l l e d o n a vertical N a r a s h i g e electrode puller. T h e y were n o r m a l l y filled w i t h 1 M p o t a s s i u m acetate s o l u t i o n b y b o d i n g u n d e r r e d u c e d pressure. T h e electrodes h a d a resistance of b e t w e e n 20 a n d 80 M ~ . T h e electrode led to a M e d i s t o r c a t h o d e follower a n d t h e n to a T e k t r o n i x 502A oscdloscope. T h e results were r e c o r d e d o n a G r a s s P7 P o l y g r a p h or were filmed f r o m t h e oscilloscope. A b r i d g e circuit was u s e d at t h e i n p u t of t h e c a t h o d e follower to allow t h e cell to b e h y p e r p o l a r l z e d or s t i m u l a t e d t h r o u g h t h e electrode. T h e i o n t o p h o r e t i c electrodes were filled w i t h m o l a r solutions of a c e t y l c h o h n e or G A B A a n d h a d a resistance b e t w e e n 5 a n d 14 M ~ . T h e l o n t o p h o r e t i c electrodes were b r o u g h t close to t h e r e c o r d i n g electrode. A b a c k i n g c u r r e n t was u s e d to stop t h e d r u g diffusing o u t of t h e electrode. D u r i n g s t l m u l a t i o n , a pulse of k n o w n c u r r e n t s t r e n g t h a n d
FIG. 1. Cell body m cockroach ganglion penetrated with a micro-electrode and marked with Procion yellow. The cell fluoresces (white m photograph) m u.v. hght and shows the position of the micro-electrode
ACH AND G A B A ON INSECT C N S NEURONES
613
duration was passed through the iontophoretic electrode to carry the drug out onto the cell membrane. T h e experimental bath was made of perspex with a front wall of glass. T h e volume of the hath was reduced to 10 ml by filling the space not required with wax. T h e solutions were changed by means of glass tubes entering the bath. T h e solutions were washed through the bath at a rate of 6 ml/min. T h i r t y mlllilitres of Ringer was used to wash away applied drugs. Tests with coloured solutions indicated that 15 ml would wash out the bath. T h e following agonists of acetylcholine were used: acetylcholine, carbachol, pilocarpme, nicotine. Antmhohne esterases: eserine, neostigmine, edrophonium. Antagonists: tubocurarine, atropine. T h e chemicals were made up in wt/vol, except for nicotine and neostigrmne which were made up vol/vol. T h e solutions were made in Ringer solution. T h e Ringer solution was similar to that used by Yamasakl & Narahashi (1958, 1959, 1960) but m place of the 2'0 m M phosphate buffer which tends to precipitate the calcium, we used 1"0 Tris-HC1 buffer. T h e composition of the Ringer solutions is given in Table 1. T h e CaC12 concentration was also increased to 9'0 m M from 1"8 raM. TABLE 1--THE COMPOSITION OF THE DIFFERENT RINGER SOLUTIONSUSED
A NaC1 KC1 CaC18 Tris-HCl NaAc KAc CaAc2 Trls-Ac
214 3"1 9"0 1"0
B -3"1 9"0 284"4
C
D
44"3 40 9"0 1"0 214 3"1 9"0 1"0
Values are expressed in mM/l. A. Normal Ringer solution. B. Sodium-free solution. C. Ringer solution in which the potassium concentration has been increased to 40 m M by replacement of sodium. D. Chloride-free Ringer solution. RESULTS
1. Micro-electrode recording T h e n e r v e cells on t h e m i d l i n e of t h e d o r s a l surface o f t h e g a n g l i a p r o v e d to b e easy to p e n e t r a t e w i t h m i c r o - e l e c t r o d e s . I t was p o s s i b l e t o see t h e e l e c t r o d e a p p r o a c h i n g t h e surface o f t h e cell b o d y , a n d s o m e t i m e s to see t h e n e u r o n e d i m p l e as t h e e l e c t r o d e p e n e t r a t e d . I n s o m e e x p e r i m e n t s t h e e l e c t r o d e was filled w i t h P r o c i o n Y e l l o w a n d l a t e r p u l s e d so t h a t t h e n e r v e cell filled w i t h t h e dye. T h e y e l l o w cell b o d y o f t h e n e u r o n e c o u l d b e seen in t h e f r e s h p r e p a r a t i o n . H i s t o l o g i c a l s e c t i o n s o f t h e s e g a n g l i a u n d e r u.v. l i g h t s h o w e d t h e d y e in t h e cell b o d y (Fig. 1) a n d i n d i c a t e d t h a t t h e e l e c t r o d e t i p h a d b e e n in t h e cell b o d y . U s i n g t h i s t e c h n i q u e a n d l o n g e r d y e infusion, o n e can m a p o u t t h e p a t h w a y s o f t h e axons f r o m t h e s e cells ( S t r e t t o n & K r a v i t z , 1968).
614
G. A. I~RKUT, R. M. PXTMANAND R. J. WALiC_eR
T h e cell bodies had a resting potential of 54.5 + 1.9 mV (n = 10). M a n y of the cells showed action potentials of 81.0+ 1 . 9 m V ( n = 10) and a positive after potential of 14.9 + 1.5 mV. Figure 2 shows the shape of a single action potential at a fast time sweep whilst Fig. 3 shows a series of action potentials. T h e duration of the action potential was between 2 and 3 msec. Preparations could show 30
--
0
-40
j
-80 5 m sec
FIo. 2. Action potentials from a nerve cell body in the cockroach CNS.
50mY
3
sec
FIG. 3. Series of action potentials from a cell body in the cockroach CNS. spontaneous activity for 3 hr or more. T h e use of the word "spontaneous" only implies that the activity was not directly attributable to any specific or known input excitation. Approximately 15 per cent of the penetrated cells showed spontaneous action potentials. Over 500 cells have been penetrated. T h e silent
ACH AND GABA ON INSECTCNS NEURONES
615
cells could be stimulated to activity either through the bridge circuit or by applying drugs. The nerve cell bodies thus appear to be both chemically and electrically excitable.
2. Action of acetylcholine (ACh) An iontophoretic electrode filled with 1 M ACh was brought up close to the penetrated cell. A backing current prevented the ACh from diffusing out and stimulating the cell. Figure 4 shows the results of an experiment where different
D
80nA
I10 nA
IH_ 150 nA
220 nA
50 mV I 500
nA
3 sec
FIG. 4. I o n t o p h o r e t i c application of acetylcholine onto the nerve cell body. As the iontophoretic current increased, so the a m o u n t of acetylcholine applied to the nerve cell b o d y increased and the depolarization increased. T h e n u m b e r refers to the current applied. T h e retaining current = 100 nA. T h e A C h electrode resistance was 14 M f ] .
amounts of ACh (as shown by the increasing iontophoretic current) were applied to the surface of the impaled nerve cell. There was a depolarization when the ACh was applied and the amount of depolarization and the number of action potentials increased as the amount of acetylcholine was increased. Figure 5 shows
G. A. KERKUT,R. M. PITMAN AND R. J. WALKER
616
the relationship between the amount of A C h applied (in t e r m s of the iontophoretic current) and the depolarization of the nerve cell. It is possible to calculate the amount of A C h that is liberated f r o m the iontophoretic electrode. Such calculations show that assuming the transport n u m b e r of A C h to be 0.5, the threshold amount 32 - -
I0--
8 --
6
-
4
-
2
--
/
d o
0
jrJl
2"r5
3o
Ionfophoref~c
/
/
,
40
50 current,
o
f
I
I
1
60
70
80
nA
FIG. 5. Dose-response curve showing the relationship between the amount of acetylcholine applied to the nerve cell (iontophoretic current) and the depolarization of the cell. Retaining current = 60 nA. ACh electrode resistance = 12 M ~ . of A C h was 1.31 x 10 -13 mole. T h i s value is of the same order as that for neurones in other animals and tests using the same iontophoretic electrode on cockroach nerve cells and nerve cells in the brain of the snail Helix aspersa, showed that there was little difference in the sensitivity of the neurones of these two animals to ACh. T h e s e experiments show that the sensitivity of the cockroach neurones to iontophoretically applied A C h is m u c h the same as neurones in other animals and that insect neurones are not less sensitive to ACh.
3. Iontophoretic application of GABA G A B A hyperpolarizes the nerve cells. W h e n an iontophoretic pipette filled with 1 M G A B A was brought up close to an impaled nerve cell and current passed t h r o u g h the pipette, G A B A hyperpolarized the nerve cell and could stop action potentials. T h e degree of hyperpolarization is proportional to the amount of G A B A applied (Fig. 6) and a dose-response curve for the application of G A B A is shown in Fig. 7.
ACH ANDGABA ON INSECTCNS NEURONES
617
The threshold to GABA, assuming a transport number of 0.5, was 1.05 × 10-13 mole.
!~,._.[/~. 90 nA
150 nA
lOOmY 5 sec
250 nA
450 nA
700 nA FIG. 6. Iontophoretic application of G A B A onto the nerve cell body. As the a m o u n t of G A B A applied increased so the hyperpolarization increased. It also stopped the action potentials. Retaining current = 100 nA. G A B A electrode resistance = 12 M ~ .
4. Ionic basis of GABA hyperpolarization Alteration in potassium concentration. The addition of GABA hyperpolarizes the neurone. This could be brought about either by increasing the membrane permeability to potassium ions or to chloride ions or to both. Figure 8 shows the effect of increasing the external potassium ion concentration from 3 mM to 40 mM. When GABA was applied to the bath of standard Ringer solution there was a hyperpolarization of the nerve cell by 6 mV. In the high potassium solution there was still a hyperpolarization following addition of GABA but it was only 4 mV. It should be noted that increasing the external potassium concentration to 40 mM had depolarized the membrane potential from - 5 3 mV to - 4 5 mV. Replacing
G. A. KERKUT,R. M. PITMANAND R. J. WALKER
618
normal Ringer around the nerve restored the membrane potential to - 50 mV and the GABA hyperpolarization was once more 6 mV. Increasing the potassium ion concentration by about ten times has a small effect on the GABA potential.
12
/
I0
/
/
_q
/ -
/
je 0
2-5
o 50
I00
Ionfophoret,c currenf,
2O0
300
I
600
nA
FIG. 7. Dose response curve showing the relationship between the amount of GABA applied to the nerve cell and the hyperpolarization. Retaining current = 60 hA. GABA electrode resistance = 9 Mr2.
Alteration in chloride concentration. Figure 9 shows the effect of changing the external Ringer to one that is chloride free, the chloride ions being replaced by acetate. In normal Ringer which contains 236 m M chloride ions, addition of GABA to the preparation hyperpolarizes the cell by 6 mV. When the Ringer is replaced by chloride-free Ringer, addition of GABA now brings about a 10 mV depolarization, i.e. the effect of GABA has been reversed. I f the chloride-free Ringer is replaced by normal Ringer then GABA once more brings about a hyperpolarization of 6 mV. T h e effect of removing the chloride ions in the external solution was most marked and reversible. I f the micro-electrodes are filled with potassium chloride solution, the chloride ions diffuse into the nerve cell and increase the internal chloride ion concentration. In such cases the response to GABA changes. Figure 10 shows such a response; a 1 M KC1 filled 30-Mf~ micro-electrode was inserted into the cell. Application of
ACH AND GABA ON INSECTCNS NEtrRONF~
619
50/~g of GABA within 30 sec of insertion of the electrode caused the cell to hyperpolarize by 2.5 mV (the normal hyperpolarization in cells penetrated with a potassium acetate filled electrode was 6 mV). Three minutes after impalement, application of GABA caused the neurone to depolarize by 2.5 mV and after 50 sec A "L
....
"
H
.,
,
R.R - 5 3 50 ~g
GABA
,I,
I
I
R P--45 20 mm 40mM K+
50/zg GABA
I0 rnV]
4sec RR - 5 0 20 rain Wash
k._ 50Fg GABA
FIG. 8. Effect of changing external potassium ion concentration on the GABA hyperpolarization. Changing the external K from 3 mM to 40 mM slightly reduced the effect of GABA. to fire one action potential. After 6 min GABA depolarized tile membrane potentials by 4 mV and an action potential fired after 33 sac. When GABA was added 9 min after impalement, the membrane depolarized by 6.5 mV and after 10 sec fired an action potential. Injection of chloride ions thus rapidly reversed the effect of GABA from a hyperpolarization to a depolarization. During the experiment the membrane potential remained at - 55 mV. These experiments show that the effect of GABA is greatly affected by alteration in the chloride concentration and slightly affected by alteration in the potassium concentration.
G. A. KERKUT, R. M. PITMAN AND R. J. WALKER
620 A
r ,filter !tf r
ll
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i
50 ,u.g GABA
IOmV [ _ _ 5sec
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~-5o ~ ' I 50~g GABA IO rnin Wash Fro. 9. Effect of reducing the external chloride ion concentration of the response to GABA. Reducing the external chloride ion concentration to zero changed the response to G A B A from a hyperpolarlzation to a depolarization. On restoring the normal chloride level (C) the effect of G A B A was partially restored.
5. Ionic basis of ACh depolarization The depolarization following addition of acetylcholine could be due to acetylcholine increasing the membrane permeability to sodium ions. The effect of removing sodium ions on this response is shown in Fig. 11. The normal Ringer solution contains 214 mM sodium ions and this was replaced by a Ringer solution in which all the sodium ions had been replaced by Tris (Table 1).
ACH AND GABA ON INSECT CNS NEURONES
621
I/2 rain after inser'hon
R. P -- 55
/~'50Fg
GABA
3 mln affer inserhon
RP--52
l 50/zg GABA
6 mln affer inset,ion
_,~"~
/
..... "~L'
R.P-55 ~ 50~xg GABA
!
D
9 rnli3 after in msertmn R P--55
I0 mV I
5sec
i
l
50 Fg GAB
FIG. 10. Effect of increasing internal chloride ion concentration on the response to GABA. Inserting the KCl-filled micro-electrode into the cell allowed chloride ions to diffuse into the cell and changed the response to GABA from a hyperpolarization (A) to a depolarization (B-D) as the internal chloride concentration increased.
Figure l l A shows the response of the neurone in normal Ringer when 500 Fg of ACh is applied to the bath. The membrane potential was - 62 inV. On adding ACh there was a depolarization of 12 mV and the cell fired a series of action potentials at the rate of 2/sec. When the Ringer solution was changed to a sodiumfree solution, there was a hyperpolarization of the membrane potential by 16 mV over 10 min. [This would indicate that the membrane potential is not at the potassium equilibrium potential but is at a value less negative than this due to a relatively high permeability to sodium ions: Hodgkin & Horowicz (1959).]
622
G. A. KERKUT, R. M. PITMAN AND R. J. WALKER
Addition of ACh brought about a depolarization of 4 mV and it took much longer to reach this level (25 sec instead of the 15 sec as in Fig. llA). When the normal Ringer was replaced, the response to ACh was once more a quick depolarization leading to many action potentials (Fig. 11C). A ,Jr
'
i
,. ,
F j
f
,
, , ,
,
,
,
R P -62
I 500jag ACh I0 mV 5 sec
R P--78
IO
mM
No +
Free
|
500/zcj
ACh.
I
C
rI
I0
mtn
500
H-g
r
ACh
Wash
FIG. 11. Effect of reducing the external s o d i u m ion concentration on the response to acetylcholine. Removal of external s o d i u m reduced the effect of acetylcholine.
The observed effect of the sodium-free solution was not due to the hyperpolarization of the cell since if the cell in normal Ringer was artificially hyperpolarized by 16 mV through a bridge circuit, addition of 500/zg ACh brought about a fast depolarization. The slow depolarization seen in sodium-free solution could be due to the presence of some residual sodium ions still present around the preparation (Chamberlain & Kerkut, 1969). 6. Post synaptic activity in insect central neurones In many of the penetrated cells it was possible to see indications of postsynaptic activity. The positions of the interneurones that were acting presynaptically have not as yet been determined.
ACH ANDGABA ON INSEC'TCNS NEURONm
623
Figure 12A shows a cell with action potentials and also base-line activity indicative of EPSP. The EPSP are more clearly shown in Fig. 12B which shows the depolarizing potentials as a series of upward spikes. Figure 12C and D show a similar series of IPSP recorded in the cell body.
A
I0 mV
[ 2 see
5mV 3 sec
I0 mV
I sec
----¢--,-----'-v---D
I0 mV
200 msec
Fzc. 12. EPSP and IPSP recorded from insect nerve cell body in the ganglion. A, recording showing action potentials and background EPSP; B, EPSP; C, IPSP; D, IPSP on faster time-scale. The postsynaptic potentials indicate that the site of the synapse is relatively close to the penetrated nerve cell body. Such preparations allow study of the action of drugs and ions on the synaptic systems of the insect CNS and work is in progress on the determination of known pathways that will allow stimulation of either the EPSP or the IPSP.
624
G . A . KERKUT, R. M. PITMAN AND R. J. WALKER
7. Action of acetylcholine agonists T h e relative sensitivity of the cockroach preparation to acetylcholine and related compounds can be tested by seeing how the activity of the preparation changes following application of the drug. In the following series of experiments the drug was applied to the ganglion and the activity was measured in the fifth thoracic nerve by means of a suction electrode. Figure 13 shows the activity in the fifth nerve and the effect of adding 1 mg/ml ACh on this activity. The preparation shows a single unit firing (Fig. 13A); D
AP's/sec ~ _ 5 mm B A
I0
mg/ml ACh
L
100/~g/mL
I00/~g/mL
CCh
IOp.g/mL
PI LO.
NIC.
FIG. 14. Rate meter curves showing the activity m the fifth nerve following apphcation of A, acetylchohne; B, carbachol; C, pilocarpme or D, nicotine. 5 AP's/sec
] 5mm
A
B
IO/.zg/ml ES
C
IOO tzg/ml
IO /~g/mL
NEOSTIG
EDROPH
FIG. 15. Rate meter curves showing the activity in the fifth nerve following application of anticholine esterases; A, eserine; B, neostlgmine and C, edrophonmm.
Normal
/ mln Img/ml
ACh
6
3 mln
IO mm
15 mln
20
mln 0 5mV
L I set
FIG. 13. Actlvlty of nerves m the fifth nerve from the metathoraclc ganghon. When acetylcholme was added to the ganglion there was an mcrease of actnlty The time mdlcates mmutes after the addltmn of the recorded m the fifth nerve acetylcholme.
625
ACI{ AND GABA ON INSECT C N S N~URONES
addition of ACh brought about an increase in activity, the peak being reached after 20 min. In this case ACh was continuously perfused over the ganglion. In the next example the drug was applied once and the response noted. Figure 14 shows the responses of the preparations to the application of ACh, carbachol, pilocarpine A
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.
.
.
.
.
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FIO. 16. The effect of tubocurarine on the electrical activity in the fifth nerve. B - F are the activity 1, 3, 6, 10 and 15 rain after the application of tubocurarine.
626
G . A. KERKUT, R. M . PITMAN AND R. J. WALKER '/ t
,I
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5 ~g CCh
0 5/zg NIC
50 ,u.g MCh
L
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,
!
10 mV ~/. 4 sec
Fro. 17. The effect of A, 500 ~g acetylchol~ne; B, 5 Fg carbachol; C, 0"5 ~g n i c o t i n e ; D , 50 ~ g m e c h o l i n e ; E, 50 ~ g pilocarpLue; o n t h e m t r a c e l l u l a r activity of t h e n e r v e cell.
ACR AND GABA ON INSECTCNS NEURONF.S
627
and nicotine. The responses are shown in terms of rate of activity over time. The records show the response to 10 rag ACh, 100 Fg carbachol, 100 Fg pilocarpine and 10 Fg nicotine. The preparation was most sensitive to nicotine > carbachol > pilocarpine > acetylcholine. Figure 15 shows the response of the preparation to addition of anticholine esterases. The preparation responded to 10 Fg eserine, 100 Fg neostigmine and 100/zg edrophonium. The preparation was most sensitive to eserine>edrophonium > neostigmine. Tubocurarine inhibited the activity of the preparation. Addition of 100 Fg tubocurarine stopped all activity in the preparation (Fig. 16) the effect being seen 10 min after the addition of the drug. The response was not reversible. Similar results were obtained from intracellular studies. Figure 17 shows the effect of adding 500 Fg ACh, 5 tzg carbachol and 0.5 Fg nicotine to the preparation. The cell was most senskive to nicotine. When nicotine was applied at low concentrations (0.05 tzg/ml) the cell showed a brief exckation and the response could
A
~---~500/xg
ADREN
I !!ilfl
!il li '
i ! 'i'J'''''
500#g NORADREN'
C
f
f I I rrl
/!'f!Ifr~!l!!fflf ,,~
500 p.g DOPAMINE
lOmV[~ 4 sec
FIG. 18. The effect of A, adrenaline; B, noradrenaline; and C, dopamine on the intraceUular activity of the nerve cell
628
G. A.
KERKUT,
R. M. PITMANANDR. J. WALKER
be repeated with further application of nicotine. The preparation was excited by adrenaline, noradrenaline and dopamine (Fig. 18). Glycine inhibited activity. Table 2 shows the relative concentrations of the drugs required to be added to the bath in order to get an effect. The values are not those acting on the membrane surface and are merely given to indicate the relative effectiveness of the drugs. TABLE 2--THE
RELATIVE EFFECTIVE CONCENTRATIONS OF DRUGS TESTED ON THE NEURONES OF THE SIXTH ABDOMINAL GANGLION
Compounds Acetylcholine Carbachol Nicotine Mecholine Atropine Tubocurarine Eserine Adrenaline Noradrenaline Dopamme Glycine GABA
Action Depolarizes and excites Depolarizes and excites Depolarizes and e x c i t e s Depolarizes and e x c i t e s Antagonises acetylchohne and pilocarpine Antagonises nicotine Potentmtes acetylchohne Depolarizes and excites Depolarizes and excites Depolarizes and excites Hyperpolarizes and inhibits Hyperpolarizes and inhibits
Threshold 50/zg-5 mg 5/zg 0"05-0'5/~g 50-500/zg 10/zg/ml 100/zg/ml 1"0/xg/ml 500/zg 500/zg 500/zg 50-500/zg 5 ttg
The drugs are not apphed lontophoretlcally and the amounts are added to the bath and only indicate relative effecuveness. DISCUSSION Intracellular recordings from nerve cell bodies in insect C N S The electrophysiological investigations with micro-electrodes of the insect CNS has been rather limited and unrewarding compared with those on the Molluscan CNS neurones. In theory the insect central neurones should have been better experimental material since it is easier to set up and study reflex activity in insects, and insects have more interesting behaviour than that shown by gastropod molluscs. An explanation of the backwardness of the studies on insects is partly given by the initial experimental difficulties in setting up the preparation and partly due to a belief or rumour that the insect cell bodies were silent and a long way away from the site of synaptic activity. Thus the insect CNS would not apparently repay the time and effort that might be put into a study of the electrical activity of the neurones. Examination of the literature shows that some workers have been able to obtain recordings from insect central neurones. Thus Hagiwara & Watanabe (1956) have recorded from cicada central neurones and obtained good action potentials, whilst Rowe (1964, 1969), Callec & Boistel (1967) and Kerkut et al. (1968) obtained activity from cockroach central neurones.
ACH
AND
GABA
ON
INSECT
CNS
NEURONES
629
The main drawback is the initial difficulty in seeing the insect cell bodies and obtaining good penetration with micro-electrodes. We have found that we get much better activity and longer-lasting preparations when the micro-electrodes are filled with potassium acetate instead of potassium chloride and this may be part of the previous difficulty, in that many of the previous workers did use KCl-filled micro-electrodes. The present study on the use of micro-electrodes on neurones in the insect CNS indicates that many such neurones are electrically active and that it should be possible to develop preparations that show synaptic activity. Action of acetylcholine and G A B A on insect neurones
Previous studies on insect central nerve cells have indicated that the neurones were relatively insensitive to ACh. In our experiments we have shown that iontophoretic application of ACh directly onto the neurone gives a good quick response and that the sensitivity of the neurone is of the order of 10 -13 mole. This compares very favourably with estimations of the sensitivity of other preparations; the mollusc neurone was sensitive to 10 -14 mole (Gerschenfeld & Stefani, 1966). The crayfish motor end-plate was senskive to 10 -14 mole GABA (Takeuchi & Takeuchi, 1964). The frog nerve muscle junction was sensitive to addition of 10 -15 mole (del Castillo & Katz, 1955). We have used the same iontophoretic pipette for adding ACh onto cockroach neurones and snail neurones and have found little difference in the sensitivity of the two preparations. On the other hand, when we apply the ACh onto the whole ganglia and record from single neurone or from peripheral axon, we find that a much higher concentration of acetylcholine is required to bring about an effect, the threshold level being about 10 -5 g/ml. Callec & Boistel (1967) applied acetylcholine by means of a pipette placed close to the recording electrode and found that the nerve cell responded to application of 10 -8 g/ml. The most feasible explanation of the high concentration of ACh that has to be applied to the whole ganglion to get an effect is that provided by Treherne & Smith (1965a, b) and Smith & Treherne (1965). It is likely that the high concentration of choline esterase in the insect nerve cord breaks down most of the applied ACh so that the final concentration at the nerve cell membrane is low. Treherne & Smith (1965a) using labelled acetylcholine found that penetration into the intact cockroach ventral nerve cord was quite rapid. They calculated that when the cord was soaked in 10 -2 M ACh solution, the extracellular concentration was approximately 8.1 × 10 -5 M. The low potency of ACh is thus due to a biochemical barrier: the high concentration of choline esterase. This is supported by comparative pharmacological studies. Carbachol which is not destroyed by the choline esterase, is more effective than acetylcholine when applied to the whole ganglion. Thus one of the drawbacks to the acceptance of acetylcholine as a natural excitatory transmitter in the cockroach, namely the high experimental concentration
630
G . A . KERKUT, R. M. PITMAN AND R. J. WALKER
that had to be applied in order to get an effect, now no longer holds. The insect neurone is as sensitive as other neurones (mollusc or vertebrate) to applied acetylcholine provided that the ACh is applied directly and iontophoretically onto the cell membrane. The other evidence in favour of ACh as an insect CNS transmitter is as follows. The cockroach nerve cord has a high ACh content. Colhoun (1958a, b, c) found that the brain and suboesophageal ganglia contained 135.2/~g/g ACh. The ventral nerve cord contained 63.2/zg/g. The thoracic ganglia contained 95.4/zg/g and the terminal abdominal ganglia contained 63.0/zg/g. The ACh content in the thoracic ganglia was more than three times that in the connective. It is in the ganglia that most of the synaptic connections are made and this supports the idea that ACh is the excitatory transmitter. The ACh in the insect CNS is more than twice the level obtained by Brown & Feldberg (1936) for the vertebrate sympathetic ganglion. Tobias et al. (1946), Smallman & Pal (1957) and Colhoun (1958b) have demonstrated the presence of the enzyme choline acetyl transferase in the insect nerve cord. The level in the insect ganglia was eight times higher than that present in the guinea-pig cervical ganglia. There is similar evidence that GABA could be the main inhibitory transmitter in the insect CNS. Ray (1964) showed that the nerve cord of Periplaneta americana contained 2.5 mM/kg of GABA. This is the same order of activity as that in the mammalian brain. We have evidence that the nerve cord can convert labelled glutamate to labelled GABA, thus there is a glutamate decarboxylase present in the nerve cord (Huggins et al., 1967). Usherwood & Grundfest (1965) found that GABA mimics the effect of stimulating the inhibitory axon at the neuro-muscular junction of Romalea microptera and Schistocerca gregaria. Suga & Katsuki (1961) and Gahery & Boistel (1965) showed that GABA would inhibit the electrical activity in the insect CNS. We have found that cockroach central neurones are sensitive to iontophoretic application of GABA. The effect of GABA is to increase the postsynaptic membrane permeability to chloride ions. Glycine can also bring about an inhibition of the cockroach neurones but the level required is some hundred times more than that for GABA. The evidence at present is reasonably good that acetylcholine is an excitatory transmitter at insect central neurones and that GABA is an inhibitory transmitter. There is some evidence that the catecholamines could play a part in normal transmitter systems but this requires more experimental evidence. SUMMARY 1. Intracelhilar recordings from nerve cells in the isolated cockroach nerve cord showed that the cells had a resting potential of 54 _+1.9 mV; action potential of 81 + 1.9 mV and a positive after potential of 14.9 _+ 1.5 mV. The duration of the action potential was 2-3 msec. The position of the penetrated cell has been marked with Procion yellow.
A C H AND G A B A ON INSECT C N S NEURONES
631
2. U p to 15 per cent o f the nerve cells studied s h o w e d s p o n t a n e o u s action potentials and the activity can last for 3 h r in an impaled neurone. 3. T h e preparations can s h o w b o t h E P S P and I P S P . 4. I o n t o p h o r e t i c application of acetylcholine depolarized the nerve cell. T h e threshold to acetylcholine was 1.31 × 10 -13 mole. 5. I o n t o p h o r e t i c application of G A B A hyperpolarized the nerve cells. T h e threshold was 1.05 × 10 -13 mole. 6. G A B A increased the permeability of the m e m b r a n e to chloride ions and slightly to potassium ions. Acetylcholine increased the m e m b r a n e permeability to s o d i u m ions. 7. T h e effectiveness of acetylcholine agonists is n i c o t i n e > c a r b a c h o l > p i l o carpine > acetylcholine. 8. T h e effectiveness of anticholine esterases was e s e r i n e > e d r o p h o n i u m > neostigmine. 9. D o p a m i n e , noradrenaline and adrenaline excite the cells whilst glycine inhibits activity. Acknowledgements--Dr. Robert Pitman is a Research Fellow of the Agricultural Research Council. We are indebted to I.C.I. (Dyestuff Division) for a gift of Procion yellow and to Dr. Niall Horn for his advice and assistance throughout this investigation.
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Key Word Index--Insect CN S ; nerve pharmacology; cockroach; Periplaneta americana; iontophoretlc application of drugs; acetylcholine GABA; mcotme; mecholine; carbachol; eserme; edrophonium; tubocurarine; adrenaline; noradrenaline; dopamlne; ionic basis of drug action in insects.