CABI SILWOOD LIBRARY
24 0066171 8
THE EFFECTS OF BARBITURATES AND OTHER SEDATIVES
ON FISH RETINA NEURONES : AN ELECTROPHYSIOLOGICAL STUDY
by
LINDA JANE GREGORY
Thesis submitted for the Degree of Doctor of Philosophy
University of London nd for the Diploma of Membership of the Imperial College (DIC)
1988
Imperial College of Science and Technology Department of Pure & Applied Biology Silwood Park Ascot, Berkshire, SL5 7PY 2
ABSTRACT
This thesis describes research into the effects of a number of ‘sedative' type drugs on electrophysiological activity of horizontal cells from the isolated retina of the roach (Rutilus rutilus). Chapter 1 reviews literature concerning the structure, function and pharmacology of the vertebrate retina, relevant to this thesis. The drugs investigated, primarily the barbiturates and the benzodi azepines are considered briefly in Chapter 2, following which a short outline of the aims of the study is given.
Chapter 3 describes the isolation of the retina and the intracellular recording techniques employed. Chapter 4 deals with an investigation of the effects of the barbiturate pentobarbital and the nature of the underlying activity. It is shown that pentobarbital has a persistent hyperpolarizing action on the dark resting membrane potential of the cells and reduces the size of the light
induced S-potentials. The hyperpolarization is not GABA- ergic as it is not blocked by bicuculline, but is due to some non-specific action on the cell membrane. In Chapter
5 the ionic mechanisms underlying the barbiturate effect are consdired and the results are consistent with a non
specific action which appears to increase potassium, but not the sodium ion conductance. In Chapter 6 a comparison
is made between the effects and relative potencies of a number of 'sedative' type drugs, all of which hyperpolarize 3
the horizontal cell dark resting membrane potential to varying degrees. Their relative efficiences in hyperpolar- izing the cells essentially correspond to their order of clinical potency. Chapter 7, describes a brief study of the action of the benzodiazepine flurazepam. Flurazepam hyperpolarizes the horizontal cell dark resting membrane potential and reduces the light induced S-potential, but the effect is less persitent than that seen for the barbiturate. The effect is GABAergic, due to its sensitivity to the GABA antagonist bicuculline, but does not appear to be a direct action on the horizontal cell membrane. No interaction occurs between flurazepam and excitatory neurotransmission. Chapter 8 summarizes the main conclusions of the thesis. 4
ACKNOWLEDGEMENTS
Firstly I would like to thank Dr. Keith Ruddock for his patient and friendly supervision throughout these studies and for his guidance in the completion of the thesis. Secondly, I would like to thank Dr. Mustafa
Djamgoz who first introduced me to neurobiology and electrophysiology, and also Dr. Mark Hankins for the help he gave in the first few months of tackling intracellular r e cording.
As a biologist, problems with the recording equip ment have sometimes baffled me, so I am grateful to both
Golly Righton and Stephen Dankin, for their assistance in both electrical and technical difficulties.
I am grateful to Frank Wright at Silwood Park and the IC Physics photography department for all the photo graphic work. Also, thanks should go to Fisher's Pond who supplied fish throughout the year in all weathers.
Lastly, I would like to acknowledge family and friends who have supported me throughout these studies, especially Philippa for the many discussions we had on the advantages and disadvantages of doing a Ph.D and to David for the endless support and patience he has shown over the last few years.
This project was funded by a grant from the Science and Engineering Research Council. 5
TABLE OF CONTENTS
Page No ABSTRACT 2 ACKNOWLEDGEMENTS 4 CONTENTS 5
CHAPTER 1: INTRODUCTION 16
PART A. THE GENERAL STRUCTURE OF THE VERTEBRATE RETINA
1.1. The Structure of the Vertebrate Retina 16 1.2. The Structure Characteristics of the Retina 20 Cells 1 .2 .1 . The Photoreceptors 20 1 .2 .2 . The Horizontal Cells 23 1.2.3. The Bipolar Cells 27 1.2.4. The Amacrine Cells 29 1.2.5. The Ganglion Cells 31 1 .2 .6 . The Interplexiform Cells 32 1.3 The Inter-Relationships and Connectivity of 34 the Retinal Neurones 1.4. Electrophysiological Responses and Function 37 of the Retina and Individual Retinal Neurones 1.4.1. Introduction 37 1.4.2. The Photoreceptor Response 38 1.4.3. The Horizontal Cell Response 46 1.4.4. The Bipolar Cell Response 51 1.4.5. The Amacrine Cell Response 52 1.4.6. The Ganglion Cell Response 53
PART B. THE NEUROPHARMACOLOGY OF THE VERTEBRATE 54 RETINA
1.5. Neurotransmission in the Vertebrate Central 54 Nervous System 1.6. The Neuropharmacology of Retinal Neurones 55 Within the Outer Plexiform Layer of Teleost Fish 1.6.1. Introduction 55 1.6.2. Amino-acid Neurotransmitters 57 1.6.3. Excitatory Amino-acid in the Retina 58 1.6.4. Inhibitory Amino-acids in the Retina 64 A. Introduction 64 B. GABA-ergic Activity in the Retina 67 1.6.5. Dopamine in the Retina 71 A. Introduction 71 B. Dopaminergic Activity in the Retina 73 6
Page. No CHAPTER 2: THE ACTIONS OF ANAESTHETIC AND 76 SEDATIVE DRUGS USED IN THIS STUDY
2.1. Introduction 76 2.2. The Barbiturates 77 2.2.1. Potentiation of Inhibitory Activity 80 2.2.2. Depression of Excitatory Activity 83 2.3. The Benzodiazepines 87 2.4. Interactions between Barbiturates and 91 Benzodiazepines 2.5. Other Sedative-Type Drugs Studied 93 2.6. Ionic Aspects of Sedative-Type Drug Action 97
CHAPTER 3: MATERIALS AND METHODS 102
3.1. Introduction 182 3.2. Preparation of the Retina 182 3.3. The Intracellular RecordingSystem 183 3.3.1. General 183 3.3.2. The Perfusion Chamber andPerfusion 184 System 3.3.3. The Micromanipulator andMicroscope 187 System 3.3.4. The Stimulating System '88 3.3.5. The Recording System H2 3.3.6. The Microelectrodes 113 3.3.7. The Ringer Solution 1*6 3.4. Procedure for Recording 119 3.5. Transreceptor Recordings 121
AIMS OF THE PRESENT STUDY 123
CHAPTER 4: THE ACTION OF PENTOBARBITAL ON 124 HORIZONTAL CELLS OF THE ISOLATED FISH RETINA
4.1. Introduction 124 4.2. The Effect of Pentobarbital on Retinal 125 Horizontal Cells 4.3. Investigation into the Nature of the 129 Depression of Neuronal Excitability by Pentobarbital 4.3.1. Effect of Inhibitory Neuro- 131 transmission 4.3.2. Effect on Excitatory Receptor Sites 134 4.4. Pentobarbital Interaction with Dopaminergic 145 Activity 4.5. Discussion 147
CHAPTER 5: THE IONIC BASIS FOR THE ACTION OF l57 PENTOBARBITAL ON THE HORIZONTAL CELLS OF THE ISOLATED FISH RETINA
5.1. Introduction _ 167 5.2. The Role of Extracellular Chloride (Cl ) 167 Ions in Pentobarbital Evoked Responses 7
+ Page No 5.3. Sodium (Na ) Substitution and Pentobarbital 162 Hyperpolarizations 5.4. Extracellular Potassium (K ) Concentration 164 and Pentobarbital Activity 5.4.1. The Effect on Pentobarbital Action 164 of Increasing Extracellular K Concentration 5.4.2. The Effects on Pentobarbital 170 Activity of Potassium Ion Channel Blockers 5.5 Discussion 175
CHAPTER 6 i AN INVESTIGATION OF THE EFFECTS OF A 186 NUMBER OF 'SEDATIVE' TYPE DRUGS ON THE HORIZONTAL CELLS OF THE VERTEBRATE RETINA
6.1. Introduction 186 6.2. Comparison of the Effects of 2 mM 'Sedative' 187 with those Induced by 2 mM Pentobarbital 6.3. Estimation of the Concentration of 197 Pentobarbital Required to Produce a Similar Degree of Hyperpolarization as 2 mM ' Sedative' 6.4. Discussion 202
CHAPTER 7: THE ACTION OF THE BENZODIAZEPINE, 206 FLURAZEPAM ON HORIZONTAL CELLS OF THE ISOLATED FISH RETINA
7.1. Introduction 206 7.2. The Effect of Flurazepam on Retinal 207 Horizontal Cells 7.3. Investigation of the Nature of the 214 Depression of Neuronal Activity by Flurazepam 7.3.1. Effect on Excitatory Activity 214 7.3.2. Effect on Inhibitory Activity 215 7.4. Discussion 220
CHAPTER 8 : GENERAL DISCUSSION AND CONCLUSIONS 228
8.1. The Effect of Pentobarbital onRetinal 228 Neuronal Activity. 8.2. Ionic Mechanisms Mediating the 231 Hyperpolarization of Horizontal Cells by Pentobarbital. 8.3. The Effects of a Number of 'Sedative' Type 234 Drugs on the Activity of Horizontal Cells, in Comparison with the Effects of Pentobarbital. 8.4. The Effect of Flurazepam on RetinalNeuronal 235 Activity.
REFERENCES 238 APPENDIX 266 8
TABLE LEGENDS Page No
Table 1.1 Excitatory receptor classification based on sensitivity to antagonists. L-GLU, L-glutamate; L-ASP, L-aspartate? tf-DGG, 2f-D-glutamylglycine; PDA, CIS-2, 3-piperidine dicarboxylic acid; GDEE, glutamate diethylester? APV, 2-amino-5- phosphonovalerate; D ek - AA, D- <*- amino ad ipate.
Table 1.2 Pharmacological differences and 68 properties of vertebrate GABA* and GABA- receptor sites. (based on Simmonds, 1983).
Table 1.3 Classification of dopamine receptor into 72 D-l and D-2 in the vertebrate CNS. (based on Kebabian and Caine, 1979).
Table 3.1. A Comparison of Cyprinid Fish Ringer 117 Formulations.
Table 4.1. Comparison of experimental results using 130 the Student's t-test. (Appendix 1).
Table 5.1. Comparison of experimental results using 161 the Student's t-test. (Appendix 1).
Table 5.2. Extracellular and intracellular concen- 179 trations and Nerijist equilibrium potentials for K , Cl and Na .
Table 6.1. Comparison of the hyperpolarization 193 induced by 2 mM of drug 'x' with that for 2 mM pentobarbital.
Table 6.2. Anaesthetic concentrations of the 196 'sedative' drugs investigated calculated from their anaesthetic doses.
Table 6.3. Concentrations of pentobarbital which 201 induce similar effects to 2 mM of drug 'x' .
Table 7.1. Comparison of experimental results using 209 the Student's t-test (Appendix 1).
Table 7.2. Measurement of the photoreceptor response 213 using transretinal recordings. 9
FIGURE LEGENDS Page No 18 F ig .1.1. Vertical sections of the teleost retina, (stained by the Golgi technique).
Fig . 1.2. Schematic representation of the neurones 18 and neuronal connectivity of the vertebrate retina. 21 F i g .1.3. The structure of the vertebrate photo receptor.
F i g . 1.4. Camera lucida drawings to show Hi, H2, 26 and H3 type goldfish cone horizontal c e l l s .
F i g . 1.5. Summary showing colour coding of 28 connections between bipolar cells and different receptors in the rudd retina (cyprinidae retina).
F i g . 1.6. Amacrine cells from Golgi stained 30 preparations of the retina of goldfish.
Fig. 1.7. Connections made by amine containing 33 interplexiform cells (I) of the goldfish retina.
Fig. 1.8. Cell interconnectivity within the verte- 36 brate retina.
Fig. 1.9. Na+ currents associated with the rod 41 photoreceptor.
Fig. 1.10. Diagram showing the process involved with 43 the "calcium Hypothesis".
Fig. 1.11. The mechanistic pathways proposed for 45 cyclic nucleotides acting as the internal transmitter of photoreceptors.
Fig. 1.12. Diagram to show the characteristic S- 50 potential recorded from horizontal cells.
Fig. 1.13. Intracellular recordings from a bipolar 56 cell in the goldfish retina.
Fig. 1.14. The proposed transmitters for 56 conventional chemical synapses within the vertebrate retina and their location. 10
Page No
Fig. 1.15. The structures of the excitatory amino 61 acids, L-glutamate and L-aspartate and also of the agonists, kainic acid quisqualic acid and N-methyl D-aspartate.
Fig. 1.16. Diagram to show the structure of the GABA 66 receptor complex.
Fig. 2.1. The chemical structure of barbituric acid 79 and its active barbiturate derivatives, phenobarbital, pentobarbital and hexobarbital.
Fig. 2.2. The chemical structure of the 88 benzodiazepine, flurazepam (and the benzodiazepine antagonist R015-1788 (an imidazodiazepine)).
Fig. 2.3. The chemical structure of other drugs 94 used in this study.
Fig. 3.1. Schematic diagram showing the various 105 systems involved in making intracellular recordings from the perfused fish retina, whilst stimulating in the physiological w a y .
Fig. 3.2. The recording /perfusion chamber. 106
Fig. 3.3. The recording platform with microscope 109 and micro- manipulator.
Fig. 3.4. The optical system. 110
Fig. 3.5. The recording system. 114
Fig. 3.6. The microelectrode and electrode holder 115 shown in half section.
Fig. 3.7. The recording system for measuring 122 transreceptor potentials.
Fig. 4.1. The degree of hyperpolarization and the 126 time taken for it to occur.
Fig. 4.2. The effect of pentobarbital (5 mM) on the 127 responses of horizontal cells.
Fig. 4.3. The direct action of 5 mM pentobarbital 128 (PENT) on horizontal cells,^synaptically isolated by 2 mM cobalt (Co^+ ). 11
Page No Fig. 4.4. The accumulative effect of pentobarbital 128 (PENT).
Fig. 4.5. Pentobarbital (PENT) hyperpolarization at 132 5 mM, is not affected by the GABA antagonist picrotoxin (Pi) at 50 juM.
F i g . 4.6. The effect of the GABA antagonists 132 picrotoxin (Pi) and bicuculline (Bi) on the hyperpolarizing response to 5 mM GABA.
F i g . 4.7. The GABA antagonist bicuculline (Bi) does 133 not reverse the hyperpolarization produced by 5 mM pentobarbital (PENT).
F i g . 4.8. No potentiation of the GABA (15 mM) 133 induced hyperpolarization by 5 mM pentobarbital (PENT) is apparent.
Fig. 4.9. Excitatory agonist and putative 135 excitatory transmitter depolarizations.
Fig. 4.10. The resting membrane potential of a 136 horizontal cell is hyperpolarized by the application of 5 mM pentobarbital (PENT).
F i g . 4.11. The depolarizing action of kainic acid 136 (KA), for comparison with that seen in the presence of 5mM pentobarbital (PENT).
F i g . 4.12. The resting membrane potential of a 138 horizontal cell is depolarized in the presence of 5 mM pentobarbital (PENT), and the excitatory amino-acid agonist quisqualic acid (QA), at 50 juM.
Fig. 4.13. The depolarizing action of quisqualic 138 acid (QA) at 50 juM, for comparison with that produced by 50 juM QA in the presence of 5mM pentobarbital (PENT).
Fig. 4.14. The excitatory amino-acid agonist kainic 139 acid (KA) at 50 JUM was applied to the horizontal cell and depolarization occurs. Pentobarbital (PENT) at 5 mM applied to the depolarized cell, causes a relaxation of the depolarization.
F i g . 4.15. The relaxation of the excitatory 140 agonist/transmitter induced depolarization. 12
Page No F ig . 4.16. The depolarization induced by kainic acid 140 (KA) at 50 juM in the presence of 2 mM cobalt ions (Co^ ).
F ig . 4.17. The excitatory amino-acid 142 neurotransmitter L-glutamate (L-GLU) at 50juM potentiated by D-aspartate (D-ASP) at 3 mM depolarizes the horizontal cell membrane potential.
F ig . 4.18. The excitatory amino-acid 142 neurotransmitter L-glutamate (L-GLU).
F ig. 4.19 The GABA antagonist bicuculline (Bi) at 144 250 juM, failed to block the relaxation of the kainic acid (KA) induced depolarization by 5 mM pentobarbital (PENT). 146 Fig. 4.20 Kainic acid (KA) at 50 juM and the antagonist kynurenic acid (KYN), at a concentration which does not induce antagonism of the KA depolarization (250 juM) .
F ig. 4.21 Kynurenic acid (KYN) acts as an effective 146 antagonist of depolarization of the horizontal cell membrane potential by 50 uM kainate (KA).
Fig. 4.22 40 uM dopamine (DA) depolarizes the 148 horizontal cell membrane potential, and this depolarization is reversed by the addition of 5 mM pentobarbital (PENT), with the loss of the light evoked S-potentials.
F i g . 5.1. The effects of chloride-free (Cl~-free) 159 perfusate on the 5 mM pentobarbital (PENT) evoked hyperpolarization.
F ig . 5.2. The depolarization of the horizontal cell 159 dark membrane potential induced in Cl~-free Ringer.
Fig. 5.3 The effects of a Cl -free perfusate 60 containing 5 mM PENT applied to the preparation.
F ig . 5.4 The effect of doubling the perfusate 160 chloride ion concentration on 5 mM pentobarbital (PENT). 13
+ Page No Fig. 5.5. The effect of a sodium-free (Na -free) 163 perfusate on 5 mM pentobarbital activity.
F i g . 5.6. The effect of a high potassium ion (K+) 165 concentration in the perfusate on the effect of 5 mM pentobarbital.
F i g . 5.7. The effect of a high potassium ion (K ) 165 concentration in the perfusate on the effect of 5 mM pentobarbital using a potassium salt other than potassium chloride.
F i g . 5.8. The excitatory amino-acid antagonist 167 kynurenic acid (KYN) at a concentration of 2 m M .
F i g . 5.9. All three traces show the effects of 75 167 mM potassium chloride (KC1) on the hyperpolarization induced by2J mM PENT and 2 mM cobalt chloride (C(j .
F i g . 5.10. The effect of 100 mM and 150 mM potassium i68 chloride (KC1) on the hyperpolarization induced by 5 mM pentobarbital (PENT) and 2 mM cobalt chloride (Coz ).
F i g . 5.11. The effect of 150 mM potassium chloride 169 (KCl) on the activity of 5 mM pentobarbital (PENT^+in the absence of cobalt chloride (Co ).
F i g . 5.12. 75 mM potassium chloride (KCl) prevents 169 the relaxation effect of 5 mM pentobarbital (PENT) on depolarizations induced by 50 uM excitatory amino-acid agonist kainic acid (KA).
F i g . 5.13. The effect of the potassium ion (K+) 171 channel blocker, 4-amino-pyridine (4-AP) on the effects induced by 5 mM pentobarbital (PENT).
F i g . 5.14. The effect of 5 mM 4-amino-pyridine 173 (4-AP) on the hyperpolarizai^on induced by 2 mM cobalt chloride (Co ).
F i g . 5.15. The effect of 5 mM tetraethyl^mmonium 174 chloride (TEA) a potassium (K ) channel blocker, on the activity induced by 5 mM pentobarbital (PENT). 14
Page No Fig. 5.16. Hyperpolarization of the horizontal cell 174 membrane potential, induced by 5 mM pentobarbital (PENT).
Fig. 5.17. Depolarization of the horizontal cell 176 membrane potential induced by 5 mM tetraethylammonium chloride (TEA).
Fig. 5.18. Relaxation by pentobarbital (PENT) of the 176 depolarization induced by the excitatory amino-acid agonist kainic acid (KA).
Fig. 5.19. No relaxation of the kainic acid (KA) 177 induced depolarization of the horizontal cell membrane potential by pentobarbital (PENT) in synaptically isolated cells (isolated by cobalt ions) occurs in the presence of tetraethylammonium chloride (TEA).
Fig. 6.1. Diagram to show measurements to assess 188 hyperpolarization of the horizontal cell dark resting membrane potential by 'sedative' and pentobarbital.
Fig. 6.2 Comparison of ethanol (ETH, 2 mM) with 188 pentobarbital (PENT 2 mM).
Fig. 6.3. Comparison of hexobarbital (HEXO, 2 mM) 189 with pentobarbital (PENT, 2 mM).
Fig. 6.4. Comparison of chloral hydrate (CHL HYD, 2 189 mM) with pentobarbital (PENT, 2 mM).
Fig. 6.5. Comparison of urethane (2 mM) with 190 pentobarbital (PENT 2 mM).
Fig. 6.6. Comparison of phenobarbital (PHENO, 2 mM) 190 with pentobarbital (PENT, 2 mM).
Fig. 6.7. Comparison of oC -chloralose ( -CHLOR, 2 191 mM) with pentobarbital (PENT, 2 mM).
GRAPH 6.1. Hyperpolarization induced by sedative 'x' 194 as % of the hyperpolarization induced by pentobarbital (PENT) vs. log anaesthetic concentrations (mM).
Fig. 6.8. -chloralose ( c* -CHLOR) compared 198 pentobarbital (PENT).
Fig. 6.9. Phenobarbital (PHENO) compared with 199 pentobarbital (PENT). 15
Page No F i g . 6.10. Hexobarbital (HEXO) compared with 199 pentobarbital (PENT).
Fig. 6.11. Chloral hydrate (CHL HYD) compared with 200 pentobarbital (PENT).
F i g . 6.12. Ethanol (ETH) compared with pentobarbital 200 (PENT).
F i g . 7.1. 2 mM flurazepam (FLUR) hyperpolarizes* the 208 horizontal cell dark resting potential and reduces the size of the light induced S-potentials.
2 + Fig. 7.2. The synaptic blocker cobalt (Co ) at 2 210 mM, hyperpolarizes the horizontal cell dark resting potential with a reduction in the size of the light induced S-potentials.
Fig. 7.3. Transreceptor recordings made across the 212 photoreceptors, using the method outlined in Chapter 3 (section 3.5.).
Fig. 7.4. Depolarization induced by 50 uM kainic 216 acid (KA).
Fig. 7.5. Depolarization induced by 50 uM kaini<£ 216 acid (KA) in the presence of 2 mM co^ .
Fig. 7.6. Hyperpolarization induced by 2 mM 218 flurazepam (FLUR) is not reversed by the GABA antagonist picrotoxin (Pi) at a concentration of 50 uM.
F i g . 7.7. a and b. Hyperpolarization induced by 2 mM 219 flurazepam (FLUR) is reversed by the GABA antagonist bicuculline (Bi) at a concentration of 50 uM. 16
CHAPTER 1
INTRODUCTION
PART A: THE GENERAL STRUCTURE OF THE VERTEBRATE RETINA/
THE CHARACTERISTICS OF THE CELLS MAKING UP THE
STRUCTURE AND THEIR CONNECTIVITY
1.1. The Structure of the Vertebrate Retina
All vertebrate retinae have a common basic structure. The retina is the light sensitive tissue of the eye and is ontogenetically derived from the forebrain.
It extends across the back of the eye-cup in a layer approximately 300 Aim in thickness/ and has a well defined laminar structure consisting of six types of retinal neurone.
It was Cajal (1893) who first developed an understanding of the structure and circuitry of the retina. By using the Golgi staining technique/ Cajal was able to reveal the cellular structure of the retina (fig.
1 .1.) and note how as a part of the central nervous system/ it shares the same cellular complexity as the brain. However/ the retina is more accessible and the cells are more precisely orientated allowing ideas on possible function to be easily deduced. 17
The main types of retinal neurone have been classified on the basis of function and histochemical characteristics. The six types are as follows:
1. Photoreceptor cells - these are the transducer elements, absorbing and converting light to electrical signals. Two types of photoreceptor are known/ rods and cones/ and in the fish retina to be considered/ the latter are subdivided into three spectral groups which are red/ green and blue sensitive. In the roach/ receptors sensitive to light in the ultra-violet region of the spectrum have been demonstrated (Avery et al./ 1982)/
2. Horizontal cells - second order interneurones/ which connect in a lateral fashion across the outer plexiform layer/
3. Bipolar cells - second order neurones which transmit electrical signals to cells deeper within the retina/
4. Amacrine cells - interneurones with no axons/ laterally connecting across the inner plexiform layer/
5. Interplexiform cells - these interneurones are found with the amacrine cells and feed signals from the inner plexiform layer to the outer plexiform layer/ 18
F i g ,1.1 Vertical sections of the teleost retina stained by the Golgi technique. Vertical elements (left section) and horizontal elements (right section) are shown separately, c/ cones: cb / small bipolar cells; rb, large bipolar cells; eh/ external horizontal cells; mh/ intermediate horizontal cells; a, amacrine cells; q, ganglion cells. (Reproduced from Ramon y Cayal/ 1893).
Schematic representation of the neurones and neuronal connectivity of the vertebrate retina. Synapses between neurones are confined to two well defined plexiform layers (from Dowling & Boycott/ 1966). 19
6. Ganglion cells - the output neurones of the retina. These are the only spike generating neurones and have axons which combine to form the optic nerve/ carrying various signals to the brain.
All six classes of neurone synapse in one of two layers. At the outer plexiform layer/ photoreceptors/ horizontal and bipolar cells interconnect whereas at the
inner plexiform layer/ connections between bipolar/ amacrine and ganglion cells are made. These connections have been observed by Witkovsky and Dowling (1969) and
Stell (1972) using light and electron microscopy. The interplexiform cells are known to synapse post- synaptically at the inner and presynaptically at the outer
layer/ thus they act as feed-back interneurones. The general structure of the retina is shown diagramatically
in fig. 1.2. Other retinal components include glial cells/ which are thought to provide physical and chemical support for the retinal structure.
The retina is attached to a layer at the back of
the eye cup known as the pigment epithelium. In the
light adapted state there are apical processes from this epithelium which surround the photoreceptor outer segments whereas in the dark adapted state these processes are withdrawn. There are interactions between the neural
retina and the pigment epithelium/ such as the phagocytosis of discarded outer segments of the photoreceptors. 20
1.2. The Structural Characteristics of the Retinal Cells
1.2.1. The Photoreceptors
The photoreceptors lie in the distal region of the retina, attached to the back of the eye cup and projecting to the outer plexiform layer. They are the transducer elements of the retina, receiving, absorbing and converting the light energy falling on the back of the eye chamber to electrical signals.
Their classification into two classes, rods and cones is based on both structural and functional differences (fig. 1.3.). The basic structure of the photoreceptor is similar in both classes, there being two main regions, an outer and an inner segment. The outer segment is structured as a lamellar membrane, which in the rods has numerous free floating saccules, and in the cones consists of multiple invaginations of the plasma membrane,
in both cases, these structures provide the matrix for the
light-sensitive photopigment molecules. The inner segment can be sub-divided into four parts, the distally situated ellipsoid region containing densely packed mitochondria,
the myoid region containing granular endoplasmic
reticulum, the nuclear region forming the outer nuclear
layer of the retina separated from the synaptic terminal by a neck like region. The synaptic terminals are located
in the outer plexiform layer. Functionally, the rods and OUTER SEGMENT
SPHERULE PEDICLE
Fig. 1.3 The structure of the vertebrate photoreceptor. Both rods and cones have similar basic structures, an outer segment, an inner segment, in which the nucleus is situated, and a synaptic terminal. The lamellar structure of the outer segments of rods (R) is made up of free floating discs, whilst the structure of cones (C) is formed from invagination of the plasma membrane. 22
cones can be classified by their spectral response characteristics and light sensitivities. Studies made on various species of animal/ from diurnal and nocturnal groups/ have suggested that rods serve dim light (scotopic vision)/ whereas cones are involved with visual stimulation at higher light intensities (phototopic vision)/ and colour discrimination. Microspectro- photometric studies of single goldfish cones have revealed that there are three classes of cone photoreceptor responding to light in the visible part of the spectrum and one class responding to ultra-violet. The three main classes are classified by their spectral sensitivities.
One group is sensitive to light in the red region (X max =
625 nm)/ one sensitive in the green region (X max = 530 nm) and one sensitive in the blue region (Xmax = 455 nm) of the visible light spectrum (Marks/ 1965). In the roach/
Avery et al. (1982) found photoreceptors which respond maximally to light of wavelengths shorter than 370 nm which are in the ultra-violet region of the spectrum. Thus/ the roach is believed to be tetrachromatic/ with one of the widest spectral sensitivities known in vertebrates.
The three classes of cone in the visible part of the spectrum have been shown to correspond to three structurally distinct cone types/ in cyprinid fish
(Scholes/ 1975; Stell and Harosi/ 1976). In the roach/ 23
Avery et al. (1982) showed the following relationship between structural type and wavelength sensitivity:
1. Double cones (composed of two outer segments),
a. Principle (long) cones -
with maximum absorbance at 619nm,
b. Accessory (short) cones -
with maximum absorbance at 526nm
2. Single cones,
a. Short single cones -
with maximum absorbance at 447nm,
b. Miniature' single cones -
with maximum absorbance between 355nm and 360nm
1.2.2. The Horizontal Cells
The horizontal cells, the largest of the vertebrate
retinal neurones, are interneurones which modify signals at the level of the outer plexiform layer. Grouped into distinct rows or layers, the horizontal cells occupy the distal region of the inner nuclear layer (fig. 1 .2.).
Cajal (1893) classified horizontal cells of the fish
retina into three classes, which have subsequently been
shown to give rise to specific classes of responses 24
(Ruddock and Svaetichin, 1975). The three classes are as follows:
1. External - sending dendrites to the cone
synaptic terminal of the outer plexiform layer/
2. Intermediate - sending dendrites to the rod
synaptic terminal of the outer plexiform layer/
3. Internal - sending no dendrites to the outer
plexiform layer and having no nuclei.
The first two classes were found in goldfish (Stell/
1967)/ and later/ using electron microscopy/ the
'internal* class of horizontal cells were identified as fusiform terminal expansions of the external cells
(Stell/ 1975). Parthe (1972) found similar classes of horizontal cells in marine teleosts. True 'internal' horizontal cells/ with a nucleus/ were seen in the carp by
Weiler and Zettler (1976).
Stell (1975)/ using flat mount Golgi staining techniques/ observed cone driven horizontal cells and identified the following groups of horizontal cells in the goldfish retina based on/ decreasing size of perikaryon/ increasing dendritic spread/ increasing distance from the 25
distal regions of the outer plexiform layer and increasing density of cone synapses:-
H1 - associated with red, green and blue cones,
H2 - associated with green and blue cones
H3 - associated with blue cones.
All have axons with fusiform expansions, forming Cajal's
'internal* horizontal cells in the innermost nuclear layer
(fig. 1.4.). Weiler and Zettler (1976) observed similar cells in the carp, but also found a fourth type, H4, which was an axonless horizontal cell lying below the cell bodies of those cells connected to cone photoreceptors.
Equivalent H4 cells lying below cell bodies of cells associated with rod photoreceptors have also been seen.
Teranishi, Negishi and Kato (1985) have observed, in flat mounts of carp retina using lucifer yellow under dopamine treatment, horizontal cells with bifurate axons possibly forming a fifth type.
In mammals, horizontal cells are less elaborate in organization than those in the lower vertebrates, for example, in the cat there appears to be just two classes of horizontal cell:
Type A - axonless cells, with dendrites contacting cone
synaptic terminals,
Type B - with axons, with dendrites contacting cone
terminals and with terminal aborizations
contacting only rod terminals. 2 6
ONL ...... T f f i ------— ...... INL
HI
Fig, 1.4 Camera lucida drawings to show HI/ H2/ and H3 type goldfish cone horizontal cells. Shown in 90uM vertical sections and flat whole mounts. Horizontal lines indicate the limits of the outer nuclear later (ONL) and the inner nuclear layer (INL) (from Stell & Lightfoot/ 1975). 27
1.2.3. The Bipolar Cells
The bipolar cells are second order neurones/ receiving input from the photoreceptors and horizontal cells (Stell/ 1972) and from interplexiform cells in the outer plexiform layer (Dowling et al./ 1976) (fig. 1.2.).
They relay visual information from cells which synapse in the outer plexiform layer to cells which synapse in the inner plexiform layer.
Cajal (1893) classified cyprinid bipolar cells into two groups:
1. 'Giant' bipolar cells with connections to rod
photoreceptors
2. 'Small' bipolar cells with connections to cone
photoreceptors.
Stell (1967) observed/ in the goldfish/ that there were mixed input 'giant* bipolar cells with connections to both rod and cone terminals and 'small' bipolar cells with connections to only cone terminals. Scholes (1976) stated that there might be as many as 15 types of bipolar cell in the fish retina/ but described 3 main classes as follows:
1. Rod Bipolar Cells - these are equivalent to
Cajal's 'giant' bipolar cells/ receiving rod and red cone input. Terminations of the cells reach the innermost 28
n n rods
cones (red) principal
(green) accessory
(blue) single
1 2 3 4 5 G
Fig. 1.5 Summary showing colour coding of connections between bipolar cells and different receptors in the rudd retina (cyprinidae retina). Three classes of bipolar cells are shown, 1. Rod; 2. Cone; 3. Mixed/ as described in the text (from Scholes, 1976). 29
layers of the inner plexiform layer. There appear to be two distinct morphological sub-types/
2. Selective Cone Bipolar Cells - equivalent to
Cajal's 'small' bipolar cells, these cells receive inputs from blue or green cones. The terminations of the cells reach the mid-region of the inner plexiform layer,
3. Mixed Cone Bipolars - these cells receive input from cone and rod photoreceptors. Cell terminations reach to the outer and mid regions of the inner plexiform layer.
The classifications are illustrated in fig. 1.5.
1.2.4. The Amacrine Cells
These neurones were first described and named by
Cajal (1893) 'amacrine', meaning axonless. The amacrine cells are interneurones whose perikarya occupy the inner region of the inner plexiform layer, with dendrites terminating in the same layer. Cajal (1893) divided the cells into three morphological types depending on the relationship with particular levels of the inner plexiform layer. Stell (1972) showed that in the teleost retina there were obvious stratified layers. The classes of cells, according to Cajal (1893) are as follows (fig
1 .6.):
1. Diffuse amacrine cells - these have processes which extend and terminate throughout the whole of the inner plexiform layer, Fig. 1,6 Amacrine cells from Golgi stained preparations of the retina of goldfish. Cells are classified by their dendritic branches/ whether diffuse throughout the inner nuclear layer (L/M/N) or stratified/ branching to particular levels as follows: A/B first level; C/J/ second level; D/E third level; F/H/O fourth level; GtI fifth level (from Cayal/ 1893). 31
2. Stratified araacrine cells - a, Bistratified or multistratified, with dendrites branching to two or more
levels of the inner plexiform layer.
b. Monostratified with dendrites confined to only one level of the inner plexiform layer.
There are also many sub-types of amacrine cell.
1.2.5. The Ganglion Cells
The current classification based on morphological
features for ganglion cells originated from the
classification by Cajal (1893). The neurones have cell
bodies within the innermost layers of the retina, directly
beneath the inner plexiform layer. The axons of the ganglion cells run radially across the retinal plane
forming an optic fibre layer which combines at the optic
disc to form the myelinated optic nerve. Cajal used a
classification system based on the branching patterns of
the ganglionic dendrites within the inner plexiform layer
as follows:
1. Diffuse Ganglion Cells - these have dendrites
ramifying throughout the inner plexiform layer, 32
2. Stratified Ganglion Cells - these can be divided into three sub-divisions/
a. monostratified; b. bistratified; c. polystratified.
Ganglion cells have also been classified by their complex neuronal responses. These will not be considered in this present study.
1.2.6. The Interplexiform Cells
The interplexiform cells were first described morphologically in Golgi stained sections of the cat retina (Gallego/ 1971). With the development of fluorescent histochemical techniques/ cells containing monaomines (noradrenaline/ dopamine/ serotonin/ etc) could be stained. Dowling and Ehinger (1975) used this technique to look at monoamine containing neurones in the retina of the goldfish and cebus monkey. They found that certain cells showed up which had their perikarya in the inner nuclear layer/ with extensions to the plexiform layers (fig. 1.7.). They were identified as interplexiform cells/ and showed dopamine fluorescence.
Subsequently/ Dowling et al. (1976) showed dopaminergic interplexiforra cells in the retinae of many different species and stated that they could be found in all retinae. 33
Fig. 1.7 Connections made by amine containing interplexiform cells (I) of the goldfish retina. Amacrine cells (A) synapse via conventional chemical synapses and form the input to the interplexiform cells, in the inner plexiform layer (IPL). In the outer plexiform layer (OPL), interplexiform cells synapse on the cell bodies of external horizontal cells (Hi) and onto bipolar cell dendrites (redrawn from Dowling & Ehinger, 1975). 34
In the cat/ Nakamura et al. (1980) observed that there were populations of interplexiform cells which had a 3 high affinity for the uptake of [ H]—GABA/ and using permanganate fixation/ showed dopaminergic terminals in electronmicrographs.
1.3. The Inter-Relationships and Connectivity of the
Retinal Neurones
The physiological function of the retina is to extract information from the optical image presented to the eye/ by transducing the distribution of light energy into neural electrical signals. These signals are then modified and encoded by neuronal interactions resulting in an output signal transmitted to the higher visual centres along the optic nerve pathway. The retinal network therefore operates to modulate and encode transduced visual signals.
As discussed previously/ there are two synaptic layers where cell interaction occurs/ the outer plexiform
layer (OPL) where photoreceptors/ horizontal and bipolar cells synapse and the inner plexiform layer (IPL) where bipolar/ amacrine and ganglion cell synapse/ with the
interplexiform cells synapsing in both the inner and outer plexiform layers (fig. 1 .2.). 35
Two functional pathways are present within the retinal structure as follows, shown schematically in fig.
1 .8 .
1. Direct Vertical Propagation - As already discussed, photoreceptors synapse chemically with particular classes of bipolar cells at the rod or cone synaptic terminals. Likewise in the inner plexiform layer, the bipolar cells synapse to ganglion cells using chemically mediated neurotransmission (Witkovsky and
Dowling, 1969; Stell, 1972). The direct vertical transmission pathway is under the influence of,
2. Modulation by lateral transmission and between plexiform layers by interneurones. The three
interneurones, horizontal, amacrine and interplexiform cells modulate the direct vertical pathways and so enable
information processing to occur within the retina. The neurones involved with the present study, the horizontal
cells, as already discussed (section 1 .2.2.), can be
either cone or rod driven with chemical synapses between
these cells and the photoreceptors. Likewise, the
horizontal cells synapse conventionally with bipolar cell
dendrites within the outer plexiform layer, and also with
other horizontal cells (Dowling, 1968; Dowling and
Werblin, 1969). Interplexiform cells synapse with
external horizontal cells only (section 1 .2.2.). 36
Fig. 1.8 Cell interconnectivity within the vertebrate retina. S denotes conventional synapses, E denotes electrical synapses. PR - photoreceptors; H - horizontal cells; B - bipolar cells; A - amacrine cells; G - ganglion cells; IPL - inner plexiform layer; OPL - outer plexiform layer. 37
Horizontal cells also interact with each other by gap
(electrical) junctions (Witkovsky and Dowling/ 1969).
The functional aspects of the inter-relationships and cell connectivity discussed here/ will be further dealt with in the next section when considering the electro-physiology of retinal neurones.
1.4. Electrophysiological Responses and Function of the
Retina and Individual Retinal Neurones
1.4.1. Introduction
Compared with other neuronal networks in the vertebrate CNS/ the retina is relatively simple/ with well defined cells occupying specific positions within the cellular network. Many studies/ including that of
Witkovsky et al. (1975)/ have investigated the overall electrical activity of the retina/ the electro- retinogram (E.R.G.). These studies have shown that the
E.R.G. is composed of activity from both neuronal and non neuronal cells/ such as glial and retinal epithelial cells. In the fish retina/ much work has been done on the intracellularly recorded cell responses of all the retinal cell types and these will now be considered. 38
1.4.2. The Photoreceptor Response
The photoreceptors are responsible for absorbing light energy and converting it to electrical signals.
Intracellular recordings made from mudpuppy (Bortoff,
1964)/ carp (Kaneko and Hashimoto/ 1967) and turtle retina
(Baylor and Fuortes/ 1970), have shown that both rods and cones hyperpolarize in response to light, from a 'dark' resting potential in the range -lOmV to -40mV. The hyperpolarization is a slow potential maintained for the duration of the light stimulus. The response is graded with respect to the illumination level (intensity) in a way which has been represented by the following equation:
V\ = I where, Vi = peak recorded V I + 0 potential change, max
I = light intensity,
V potential change max recorded when the light is very bright, and 0 the light intensity for which V. 1/2 V l max
(Baylor and Fuortes, 1970). This is a similar function to that used by Naka and Rushton (1966) to describe the relationship between horizontal cell S-potentials and light intensity in the fish retina (see section 1.4.3.). 39
Toyoda et al• (1969) showed/ in the mudpuppy and gecko/ that light stimulation is associated with an increase in photoreceptor cell membrane resistance/ which causes the observed hyperpolarization.
Using ionic substitution techniques in the frog retina/ it was shown that the photoreceptor response changed/ except at very low ionic concentrations/ in a direct linear proportion to the logarithm of the external
Na concentration and inversely to the external K concentration (Sillman et al./ 1969 a, b).
In the dark/ the receptor extracellular space is typically at a potential which is 5mV negative to the basal end of the receptor. This difference was found to be reduced by light stimulation (Hagins et al./ 1970). It was proposed that the current observed under no light stimulation/ the dark-current/ originated from a local Na+ current in the outer segment. In the dark/ the outer segment membrane is permeable to Na+ ions/ the inward movement of which maintains the cell in a depolarized state. With light stimulation/ the outer segment membrane
. + resistance increases/ decreasing the Na permeability across the membrane. The inner segment of the photoreceptor has metabolic pumps/ which expel Na+ ions out of the cell/ against the Na+ concentration gradient.
These pumps function both during dark and light/ thus during light stimulation/ as Na+ ions are expelled via the
inner segment but movement into the photoreceptor via the 40
outer segment membrane is blocked/ the cell assumes a hyperpolarized state. Hagins (1972) was able to abolish the photoreceptor response by applying the metabolic inhibitor ouabain in combination with normal extracellular ionic concentrations. The proposed mechanism is shown in fig. 1.9.
The light incident on the retina is absorbed by photopigments present in internal membranes within the outer segment of the photoreceptor (section 1.2.1.). The absorption of light energy/ the activation of the photopigment and the conversion of information contained within the incident light to electrical signals
(transduction) is linked by a mechanism within the photoreceptor/ which is believed to involve an internal transmitter. Many studies have been carried out to reveal the nature of the internal transmitter and to identify a possible candidate for the role.
Yoshikami and Hagins (1973)/ raised the external 2+ calcium (Ca ) concentration around the photoreceptors and found that this produced an effect similar to that of light/ by suppressing the Na dark current. Likewise/ 2+ reducing the Ca concentration of the extracellular environment reduced the light sensitivity of the photoreceptors. It was found that there was a transient 2+ increase m the intracellular Ca concentration/ recorded 2 + during light stimulation and the concentration of Ca was found to be directly related to the amount of photopigment 41
DARK LIGHT Fi^. 1.9 Na currents associated with the rod photoreceptor. Ir^ the dark/ Na flows into the outer segment through open Na channels and+is+extruded from the inner segment by a metabolic Na /K pump/ giving the voltage gradient over the cell as described in the Jext. In light the oujer segment becomes impermeable to Na due to closing of Na channels. However/ Na is still extruded from the inner segment/ thus hyperpolarizing the photoreceptor. 42
bleached (Mason et al./ 1974). It was thus proposed that 2+ Ca ions were involved with tranduction/ and could possibly be the internal transmitter. The 'Calcium
Hypothesis' proposed that the discs of the outer photoreceptor segments use metabolic pumps to accumulate
Ca ions during darkness (Neufield/ Miller and Bitensky,
1972; Mason et al./ 1974). Light induced changes in the photopigment cause the release of calcium from the discs/ the calcium then diffuses through the extrasaccular space/ to the sites of Na+ channels within the plasma membrane/ and blocks these channels (fig. 1 .10.).
The 'Calcium Hypothesis' has been developed further with subsequent studies. It has been suggested that calcium acts as a primary internal transmitter. There are a number of light activated enzymes located in the outer segments of rod photoreceptors. Bitensky et al. (1975)/ located a light activated photophodiesterase (P D E ), within the internal membrane of the rod outer segments.
Cyclic guanosine monophosphate (cGMP) is the substrate most readily hydrolysed by this enzyme. Phosphorylation of membrane proteins occurs under the influence of the kinase/ for which cGMP is a co-factor. Thus the phosphorylation of the membrane proteins within the photoreceptor plasma membrane was proposed as being the
. + ... final link in the mechanism by which Na permeability is governed. Woodruff et al. (1977) proposed a model of transduction involving the cyclic nucleotides and 43
Fig. 1.10 Diagram showing the process involved with the "calcium Hypothesis". Light interacts with the photopigment situated within the di sc^rpembranes of the receptor. This evokes the release of from the disc into the extracellular space, where it then diffuses to the plasma membrane and acts to reduce the sodium conductance into the photoreceptor. Extracellular Ca is continually accumulated in the discs by metabolic pumps in the disc membrane. 44
phosphorylation, which is shown diagramatically in fig.
1.11. Kawamura and Murakami (1983), produced further evidence to support the involvement of cGMP in phototransduction. They iontophoretically injected cGMP into gecko photoreceptors and produced, under normal extracellular sodium concentrations, a prolonged depolarization, accompanied by a marked increase in membrane conductance. However, with a sodium-free bathing solution, injected cGMP produced no depolarization.
Similar results were obtained using other nucleotides such as cyclic adenosine monophosphate (cAMP).
How calcium and cGMP might interact, or whether they interact at all, is still unclear. Yoshikami et al. 2+ (1980) looked at the kinetics of light induced Ca effluxes within the photoreceptor outer segment. They found that there were calcium fluxes which were fast enough, of sufficient magnitude and distribution to operate as the internal transmitter. It was later found 2+ that cGMP stimulates Ca uptake and storage m rod disc membranes (George and Hagins, 1983). More recently
Fesenko et al. (1985) have proposed a more direct role of cGMP. Using frog retinae and patch clamp techniques, they studied the effects of cGMP on membrane conductance on excised 'inside-out' patches from the plasma membrane of rod outer segments. cGMP caused a reversible increase in cationic conductance when applied to the 'inner' side of the membrane. Calcium applied in a similar way was 45
Fig 1.11 The mechanistic pathways proposed for cyclic nucleotides acting as the internal transmitter of photoreceptors (from Ernst, 1979). 46
ineffective, as were other cyclic nucleotides such as cyclic adenosine monophosphate (cAMP) and 2'3' cyclic GMP.
The effect occurred in the absence of nucleoside triphosphates, e.g. adenosine triphosphate (ATP) and guanosine triphosphate (GTP), which are required for phosphorylation of membrane proteins, thus this process appeared not to be involved with phototransduction, contrary to the model put forward by Woodruff et al.
(1977). Fesenko et al. (1985) proposed that cGMP and not calcium was the messenger modulating the conductance of the plasma membrane. The direct action of the cyclic nucleotide on the conductance of the plasma membrane has the advantage of having a fast response time, essential for the functioning of photoreceptors, where the response to intense illumination develops within milliseconds. The 2+ effect of Ca on the rod photoresponse might then be mediated by the action on cGMP metabolism in the 2+ photoreceptor. A decrease m Ca concentration is known to produce a marked increase in intracellular cyclic GMP concentration (Polans et al., 1981), explained by the
2 -4- effects of Ca on the cyclic GMP phosphodiesterase
(Robinson et al., 1980).
1.4.3. The Horizontal Cell Response
Svaetichin (1953) was the first to intracellularly record from horizontal cells The response recorded became known as the S-potential. S-potentials are slow hyperpolarizations (fig. 1.12.) having the following relationship to light intensity:
V KI where V - response amplitude, I - illumination level, K - constant which is the maximum level of V, I, - level of illumination at which V is the maximum value
(Naka and Rushton, 1966).
Svaetichin (1956) divided the S-potentials into two spectrally different types:-
1. Luminosity or L-type - these hyperpolarize to
all spectral stimulation,
2. Colour or C type - these hyperpolarize to
certain stimulating wavelengths, but depolarize
to others.
It was suggested by Svaetichin and MacNicol (1958) that the function of the L-type potentials was to encode luminosity information, whereas the C-type potentials coded colour. The C-type potentials were differentiated as follows:
1. Biphasic or C^ type - these hyperpolarize to short wavelength stimulation and depolarize to long wavelengths, 48
2. Triphasic or Cfc type - these hyperpolarize to both short and long wavelengths, whilst depolarizing to any wavelengths between.
In extensive studies of S-potential responses of cyprinid retinae, Naka and Rushton (1966 a, b, c) identified five different classes of S-potential. In the roach, Djamgoz and Ruddock (1978b) described 4 classes of cone connected S-potentials and 1 rod connected potential
(shown in fig. 1.12.):
Ll-type - hyperpolarizes to red more than green stimuli (connected to red cones only),
1.2-type - hyperpolarizes to green/blue more than red stimuli (connected to blue and green cones only),
C^-type - biphasic cells depolarizing to red and hyperpolarizing to green stimuli,
Cfc-type - triphasic cells depolarizing to green and hyperpolarizing to red and blue light. These cells are rarely seen in the dark adapted retina,
L^-type - these cells hyperpolarize to all wavelengths at low illumination levels.
The type cell was described in fish retinae by Kaneko and Yamada (1972). Their spectral sensitivity follows that of the rod photopigment absorption spectrum. It was observed that these cells received no cone input (Kaneko and Yamada, 1972; Ruddock and Svaetichin, 1973) and so were exclusively rod driven. 49
Horizontal cells have large receptive fields, within which an increase in the area of illumination produces an increase in response amplitude (fig. 1.12.).
The area of summation is much greater than the dendritic
field (Norton et al., 1968). This ability to respond over such a wide area is attributed to gap junctions (section
1.3.) between the cells, which effectively produce a continuous 'space' across the retina. Naka and Rushton
(1967) defined this space ('s-space'), as a laminar
conducting medium bound by two high resistance membranes.
As already stated, the S-potential response is a slow graded hyperpolarization. The cells receive direct
input from photoreceptors, which also hyperpolarize in
light conditions, thus, Trifonov (1968) proposed that when photoreceptors are hyperpolarized there is a suppression
of the release of excitatory transmitter from the photoreceptor synaptic terminals, which in turn leads to
hyperpolarization of the horizontal cells. Various
studies have provided evidence for the 'Trifonov
Hypothesis':
1. When photoreceptors are depolarized by a
transretinal current, horizontal cells also depolarize in
a graded way (Byzor & Trifonov, 1968; Kaneko & Shimazaki,
1976),
2. Blocking synaptic transmission between the . 2+ 2+ photoreceptors and horizontal cells with Co , Ca or 2+ Mg , produces a horizontal cell hyperpolarization and 5U
LI type L2 type
iQmV CmV
stim J TL__J 1 ___ ” jjreerV" red green
Cb type Ct type
IOmV lOmV
““greeh”
2 A B J------— SmV 5 mV
stim I------i stim__i------l
Fig. 1.12 Diagram to show the characteristic S-potential recorded from horizontal cells. 1. S-potentials originating from cone driven cells are classified by their spectral characteristics as shown and described in the text. (Hyperpolarizations are shown as downward deflections). 2. L-type S-potentials recorded from the cyprinid retina to show A. the response to a spot stimulus of 0.2 mm diameter; B. the response to a diffuse stimulus illumination. This illustrates the ability of horizontal cells to summate responses over a broad receptive field.
Fig 1.13 Intracellular recordings from a bipolar cell in the goldfish retina showing depolarizations as upward deflections. Cell are hyperpolarized by a spot (A) and depolarized by an annulus (B) (from Kaneko, 1970) 51
suppression of the S-potential, but no change in the photoreceptor potential (Cervetto & MacNichol, 1972;
Kaneko & Shimazaki/ 1975, 1976),
3. L-type S-potentials can be reversed in polarity by extracellular depolarization of the cell membrane to the zero level, implying that the postsynaptic event is an excitatory postsynaptic potential (EPSP), developed in darkness (Trifonov et al., 1974),
4. Putative excitatory neurotransmitters depolarize the horizontal cell membrane (Murakami et al.,
1972).
Depolarizing responses of horizontal cells have been explained by considering feed-back pathways. Several schemes have been put forward which describe the connections which must exist to produce the responses seen
(Stell and Lightfoot, 1975; Djamgoz and Ruddock, 1980).
1.4.4. The Bipolar Cell Response
Bipolar cells have small cell bodies, so intracellular recordings are difficult to achieve. Werblin and Dowling (1969) recorded two types of bipolar cell in the retinae of mudpuppy, as did Kaneko (1970) in the goldfish:
1. ON-Centre Bipolar cells - these depolarize to a small central light spot centred on the cell and hyperpolarize to an annular surround, 52
2. OFF-Centre Bipolar cells - these hyperpolarize to a central stimulation and depolarize to an annulus, as illustrated in fig. 1.13. The classification was later reviewed by Kaneko (1973) to incorporate spectral sensitivities of the cells.
The size of the bipolar cell receptive field in the central region is close to that of the dendritic field in the outer plexiform layer (0 P L ). The larger surround area is probably mediated by the horizontal cells via intercellular connections (Werblin and Dowling, 1969;
Piccolino and Gerschenfeld, 1977).
1.4.5. The Amacrine Cell Response
Recordings have been made from goldfish (Kaneko,
1973), carp (Murakami and Shimoda, 1977) and roach
(Djamgoz and Ruddock, 1978a). The studies produced evidence for two functional classes of amacrine cell:
1. Sustained amacrine cell - these cells respond to light stimulation with a maintained slow depolarizing or hyperpolarizing change in membrane potential, which is colour coded in respect to the polarity of the response.
These correspond to monostratified amacrine cells (section
1.2.4.),
2. Transient amacrine cells - these cells respond to light stimulation with transient depolarizations at the light ON point and light OFF point, and have a maintained 53
depolarization or hyperpolarization for the duration of
the stimulus. These cells correspond to bistratified amacrine cells (section 1.2.4.). Both these amacrine cell
types have relatively large receptive fields and have not been shown to have any marked centre surround organization
(Kaneko/ 1973).
1.4.6. The Ganglion Cell Response
The ganglion cells are the only retinal neurones which produce the true 'Hodgkin and Huxley' type action potential. Using extracellular recording techniques/ it has been shown that ganglion cells respond to light by changing the frequency at which they fire/ in either a sustained or transient way with respect to the stimulus
(Daw, 1968). As the receptive field organization of the cells is very complex, reflecting the degree of complex processing which takes place in the retina, this will not be considered in the present discussion. 54
PART B: THE NEUROPHARMACOLOGY OF THE VERTEBRATE RETINA
1.5. Neurotransmission in the Vertebrate Central Nervous
System
Communication between neurones in the vertebrate central nervous system (CNS)/ takes place via two types of interneuronal junction:
1. Electrotonic or electrical gap junctions. The cell membranes of adjacent cells are very close together and linked at specialized regions known as gap junctions.
Here clusters of protein molecules form 'connexons' which act as channels connecting the interiors of the cells.
The gap junctions allow the passage of small molecules and
ions from cell to cell/ in either direction/ so that cells connected in this way are raetabolically and also electrically coupled by ionic activity/
2. Chemically mediated junctions or synapses. In
these cases/ two neurones are separated by a synaptic cleft which is bridged by the release/ from the presynaptic nerve/ of a chemical/ (the neurotransmitter)/ which diffuses across the cleft and binds to the postsynaptic neuronal membrane/ hence producing changes in postsynaptic neuronal activity. 55
The occurence of either type of junction in a neuronal network/ is closely related to the function of the system- Electrical junctions allow fast/ synchronised relay of information/ whereas chemical synapses are restricted to slower/ unidirectional transmission which may be excitatory or inhibitory. It is well established that interneuronal communication in the vertebrate central nervous system (CNS) is predominantly a chemical phenomenon mediated by neurotransmitters (Krnjevic/ 1974).
1.6. The Neuropharmacology of Retinal Neurones Within
the Outer Plexiform Layer of Teleost Fish
1.6.1. Introduction
The vertebrate retina is well established as a preparation used to study neurotransmission in the vertebrate CNS/ due mainly to the background knowledge which has been acquired on the organisation and responses of individual neurones. Investigations using different types of neuropharmacological techniques have led to a variety of putative transmitters being put forward (fig.
1.14.).
In the fish retina/ the second order neurones/ the horizontal cells (section 1 .2 .2 .) are large and numerous and so are the common cells from which intracellular recordings are made. 56
Fig. 1.14 The proposed transmitters for conventional chemical synapses within the vertebrate retina and their location 57
Using specific antagonists and agonists/ much information has been produced regarding the types of receptor sites on retinal neurones/ in particular the horizontal cells and on the probable putative transmitters. The following sections will deal with the transmitters identified so far in the outer plexiform layer of the teleost fish which are relevant to this study:
1. Amino-acids a. Excitatory (Aspartate & Glutamate)/
b. Inhibitory (^-amino-butyric acid-GABA)/
2. Dopamine
1.6.2. Amino-acid Neurotransmitters
Until the late 1940's/ it was generally considered that amino-acids had no significant neuropharmacological properties. However/ in 1949/ Brooks et al./ showed that dicarboxylic amino acids and some other related compounds could evoke excitatory activity in isolated frog brains.
There was much confusion about whether the amino-acids were putative neurotransmitters due to the fact that physiologically/ when in the free state/ they play various roles/ such as precursors for the formation of protein molecules/ or as sources of metabolic energy. However/ extensive studies/ as described in the following sections/ have shown that the amino-acids do play an important part in neurotransmission within the CNS. 58
Acidic amino-acids such as L-glutamate/ L-aspartate
and L-cysteic acid have been shown to have excitatory
effects on neurones/ whereas the neutral amino-acids
Y-amino-butyric acid (GABA) and glycine have inhibitory
effects.
1.6.3. Excitatory Amino-acids in the Retina
Extensive studies have provided evidence that two
amino-acids/ L-glutamate and L-aspartate/ which occur
abundantly as free amino acids in the brain/ may be
excitatory neurotransmitters in the vertebrate central
nervous system and in the vertebrate retina. Furakawa and
Hanawa (1955) were the first to report that L-glutamate
and L-aspartate at mM concentrations could have an effect
on the electroretinogram (E.R.G.)/ and subsequently it was
found/ at these concentrations/ that the amino acids
induced a reversible depolarization of the horizontal
cells with the abolition of the light evoked S-potential
(Murakami et al./ 1972). It was believed that the
excitatory amino-acids bind to sites on the horizontal
cell membrane and produce effects equivalent to those of
the putative excitatory transmitters released by the
photoreceptors/ described in the 'Trifonov Hypothesis'
(section 1.4.3.). Similar effects occur at the other
second order retinal neurone membrane/ that of the bipolar
cel l . 59
Using perfused goldfish retinae preparations/
Ishida and Fain (1981) reported that horizontal cells were
depolarized by L-glutamate potentiated by 3mM D-aspartate
(this may block the uptake mechanisms for exogenous amino-acids)/ but not by L-aspartate. This phenomena was also found by Rowe and Ruddock (1982b) in the perfused
roach retina preparation. Slaughter and Miller (1983/
1985) found that both second order retinal neurones/
horizontal cells and the two types of bipolar cells (ON and OFF/ section 1.4.4.)/ were sensitive to glutamate in
the mudpuppy preparation. As the amino-acid had variable
effects/ they decided that there must be multiple postsynatic receptor sites. Blocking the glutamate sites with Cis - 2/3-piperidinedicarboxylic acid (PDA) and
D-O-phosphoserine/ Slaughter and Miller (1985) were able
to differentiate three glutamate-like receptor sites on
second order retinal neurones. It was therefore decided
that the likely excitatory amino-acid neurotransmitter
released according to the 'Trifonov Hypothesis'/ by the
photoreceptors (1968)/ and acting on the second order
retinal neurones/ was L-glutamate or a closely related
analogue.
Using selective agonists and antagonists at
receptor sites has helped to further identify the putative
transmitter. Selective agonists are/ kainic acid (KA) and
quisqualic acid (QA), both of which have structures
similar to the putative transmitter amino-acid/ 60
L-glutamate and N-methyl-D-aspartate (NMDA) which is an analogue of L-aspartate (fig 1.15.). All three of these compounds are potent agonists at excitatory amino-acid receptor sites, NMDA being the most potent.
Davies and Watkins (1981) and McLennan and Lodge
(1979) classified three receptor sites based on the sensitivity of the three agonists, KA, QA and NMDA at frog and cat spinal motorneurone membranes. The endogenous transmitters, L-glutamate and L-aspartate are regarded as mixed agonists at the three classified sites. Using the perfused roach retina preparation, Rowe and Ruddock (1981a, 2+ 1982b) investigated synaptically isolated (using 2mM Co ) horizontal cells, looking at the responses to the excitatory agonists, KA, QA and NMDA. They reported that rod and cone driven horizontal cells depolarized in solutions containing KA and QA at concentrations of 10 urn or more, whilst NMDA at concentrations up to 250 Aim, had no effect. Slaughter and Miller (1983) reported similar results in the mudpuppy retina. Using isolated retinal horizontal cells of the goldfish and carp, Dowling et al.
(1983) and Ishida et al. (1983) found that there was a common sensitivity to the agonists KA and QA at concentrations of 10 jum or more, inducing depolarizations.
Hankins and Ruddock (1984b) found that KA and QA at low concentrations (1 to 3 Jum), hyperpolarized horizontal cells of the roach retina. This hyperpolarization was reversed by the GABA antagonist bicuculline,whilst the 61
c h 2 c h - c o o h CH-COOH
CH-COOH CH-COOH
Glutamate Aspartate
CH,
Hjl-C - CH-COOH 2
-COOH
“N‘
Kainic acid
Quisqualic acid N methyl D aspartate
CH-COOH / ° \ 2 o - N-CH-CH-COOH 2. I NH \. / 2 .CH-COOH \\ o Hj: NH
Fig, 1.15 The structures of the excitatory amino acids, L-glutamate and L-aspartate and also of the agonists, kainic acid quisqualic acid and N-methyl D-aspartate. 62
antagonist picrotoxin reversed the KA induced hyperpolarization/ but not that induced by QA. The GABA antagonists did not affect the depolarization observed when the agonists were applied at 50 urn. It was concluded that the hyperpolarizing and depolarizing effects originated from two independent mechanisms.
The excitatory amino-acid receptors are susceptible to agents which specifically block the receptor sites and hence the actions of putative transmitters or agonists.
Folic acid (pteroyl-L-glutamic acid/ FA) was proposed as an endogenous 'kainate-like' compound (Olney et al./
1981). At concentrations of 5mM it was shown to antagonise the actions of 50 juM KA and QA in goldfish retinae/ but was less effective at blocking the action of
L-glutamate. This was also found in the roach preparation
(Rowe and Ruddock/ 1981b).
Other antagonists of the excitatory amino-acids have been observed in various neuronal preparations.
These are outlined in Table 1.1. Rowe and Ruddock (1981a,
1982a) found that neither the antagonist at KA sites,
H -D-glutamylglycine ( -DGG) or the antagonist at QA sites/ glutamate diethyl ester (GDEE) were effective at blocking the depolarizing effects of the agonists at the excitatory receptors sites of the roach horizontal cells.
Ishida et al. (1983) and Dowling et al. (1983) observed the same results in isolated goldfish horizontal cells. 63
Receptor Classification Antagonists Proposed putative agonists
KAINATE 1. -DGG 2. Folic acid L-GLU 3. Kynurenic acid (insensitive to KA 4. PDA an tagonists)
QUISQUALATE 1. GDEE 2. Folic acid L-GLU 3. Kynurenic acid 4. PDA
N-methyl-D 1. APV -Aspartate 2. Doi-AA L-ASP 3. PDA 4. # - DGG 5. Quinolinic acid
Table 1.1 Excitatory receptor classification based on sensitivity to antagonists. L-GLU/ L-glutamate; L-ASP/ L-asparotate; 8 -DGG/ -D-glutamylglycine; PDA/ CIS-2/3-piperidine dicarboxylic acid; GDEE/ glutamate diethylester; APV/ 2-amino-5-phosphonovalerate; Do(- AA, D-oC- amino adipate. 64
Kynurenic acid/ a naturally occuring metabolite of tryptophan, is a non-specific antagonist of excitatory responses in the rat cortex (Perkins and Stone/ 1982), It was found that at 1 mM or above/ it antagonises the depolarizing actions of excitatory amino-acids or the agonists kainic and quisqualic acids/ whilst hyperpolarizing horizontal cell membranes in the roach retina to their potassium Nernst equilibrium potential
(-70mV). The effect occurs in cells synaptically isolated 2+ by the application of 2 mM Co . A related compound/ quinolinic acid/ hyperpolarized the horizontal cells/ reducing the light evoked responses (S-potentials) in a way similar to that of NMDA/ and it was therefore proposed that quinolinic acid acted at sites sensitive or specific to NMDA (Hankins and Ruddock/ 1986)/ as previously argued by Perkins and Stone (1982/ 1983).
1.6.4. Inhibitory Amino-Acids in the Retina
A. Introduction
y-amino-butyric acid (GABA) is a naturally occurring amino-acid and functions as an inhibitory transmitter in many species. Invertebrate studies have shown that at the Crustacean stretch receptor neurones/ low concentrations of GABA (4 10 Aim) block afferent discharges evoked by the stretch stimuli. GABA was shown 65
to evoke a hyperpolarization of the neuronal membrane potential similar to that seen with naturally evoked inhibitory postsynaptic potentials (IPSP's) (Kuffler and
Edwards, 1958). Since this early study, it has been shown that GABA acts as an inhibitory transmitter at a variety of invertebrate and vertebrate synapses, both in the peripheral and central nervous systems.
Much data has been collected concerning the pharmacological properties of the GABA receptor and its associated ionic channels. The present idea of the GABA receptor is a complex group of binding sites with an associated ion channel, shown in fig 1.16. and as follows:
1. A receptor site where GABA or a GABA agonist binds,
2. An ion channel, whose opening or closing is controlled by the binding of GABA or a GABA agonist at the receptor site. The ion has been identified as chloride
( C l - ) .
3. A regulatory site or regulatory sites where specific substances act, influencing 1. and 2. above.
These substances have been identified as benzodiazepines and barbiturates, both of which will be considered in
Chapter 2, and picrotoxin which acts as an antagonist influencing the Cl ionophore. 66
Fig. 1.16 Diagram to show the structure of the GABA receptor complex. (See text for details; from Davidoff/ 1983). 67
In vertebrates there is evidence for two distinct types of GABA receptor, GABA, and GABA_ (Bowery, Hill and
Hudson, 1983). Their classification is based on sensitivity to agonists, antagonists or potentiators as shown in Table 1.2.
Antagonism of the GABA-receptor complex is by two methods (Simmonds, 1980):
1. Bicuculline inhibits Na+ independent GABA b i n d i n g ,
2. Picrotoxin acts at the Cl” ion channel, regulating the opening of the chloride ionophore and hence regulating the degree of hyperpolarization produced by a given concentration of GABA.
Regulatory sites potentiating the GABA effect classified as benzodiazepine or barbiturate sites, will be dealt with in Chapter 2.
B. GABA-ergic Activity in the Retina
Noell (1959) was the first to show that GABA had a general inhibitory effect on the electroretinogram
(E.R.G.). The uptake and metabolism of GABA was shown to be influenced by light, accumulation being in external and internal horizontal cells (Lam and Steinman, 1971, 1972).
Marc et al. (1978), using the goldfish retina, localised 68
VERTEBRATE GABA. VERTEBRATE GABAn A B
AGONISTS
Muscimol Potent Weak GABA Potent Potent Baclofen No action Potent
ANTAGONISTS
Bicuculline Potent (competitive) No action Pierotoxin Potent (non-competitive) No action
POTENTIATORS
Benzodiazepines Potent No action Barbiturates Mod. Potent No action
Table 1.2 Pharmacological differences and properties of vertebrate GABA^ and GABAg receptor sites (based on Simmonds/ 1983). 69
GABA exclusively to HI type horizontal cells (section 3 1.2.2.) sensitive to red light/ and showed that [ H] GABA
uptake increases under red light stimulation of the
retina. This evidence suggested that GABA was the
neurotransmitter for HI type horizontal cells of the outer plexiform layer.
Electrophysiological evidence for GABA activity in
the retina was demonstrated by Murakami et al. (1972).
GABA appeared to have no effect on transreceptor potential
in the carp preparation. However/ all cone horizontal
cells were hyperpolarized and light evoked activity
(S-potentials) were suppressed. In the turtle retina
(Laufer/ 1982) it was found that exogenous GABA
depolarized the cells and suppressed the S-potentials. In
the roach GABA appears to have two actions. At low
concentrations (0.1 mM to 5 mM) hyperpolarization occurred
whereas at high concentrations (> 5 mM) depolarization of
the horizontal cell membrane potential was seen (Hankins
and Ruddock/ 1984a). The depolarization was reversed by bicuculline but not by picrotoxin. Also nipecotic acid/ which is a GABA uptake blocker/ hyperpolarized horizontal
cells and reduced the S-potentials/ this action could be
reversed by bicuculline but not by picrotoxin/ indicating
that GABA acted in a Cl" independent manner. Further
Gregory/ Hankins and Ruddock (1985)/ showed that GABA polarizations induced in the horizontal cells of the
isolated roach retina/ persisted in Cl" free Ringer. 70
It was proposed that the depolarizing components of
C-type S-potentials in the retina (section 1.4.3.) originated in feedback networks involving inhibitory inputs from horizontal cells to photoreceptors (Stell and
Lightfoot, 1975; Djamgoz and Ruddock, 1980). The depolarization was blocked by picrotoxin (Djamgoz and
Ruddock, 1990; Murakami et al., 1982b) and suppressed by exogenous GABA (Murakami et al., 1972). In the carp retina, the HI horizontal cells were shown to be
GABA-ergic neurones forming inhibitory feedback connections to red cone receptors (Murakami et al.,
198 2 a.b).
Experiments using labelled GABA looking at release of the transmitter from isolated cells and cells from the intact retina, have produced supportive evidence for
GABA-ergic pathways in the outer plexiform layer. Ayoub and Lam (1982) and Yazulla and Kleinschmidt (1983) showed that the excitatory amino-acid, L-glutamate, could evoke 3 the release of labelled [ H] GABA from horizontal cells of . . 2+ the goldfish retina, by a Ca independent mechanism. The likely sites of action of the excitatory amino-acids are
KA or QA agonist sensitive sites (section 1.6.3.) and these in some way are related to mechanisms releasing GABA
(Yazulla, 1983). Cunningham and Neal (1985) showed that excitatory amino-acid induced release of GABA from frog horizontal cells was 40x that recorded for the spontaneous resting release, and they concluded that the effect 71
appeared to be mediated via KA or QA receptor sites, consistent with the suggestions of other workers. It can be deduced from these studies that the likely mechanism for GABA release is indirectly, via excitatory amino-acids acting at their receptor sites.
Kaneko and Tachibana (1986) used isolated turtle cones to show that GABA is continuously released from monophasic horizontal cells in the dark, producing tonic hyperpolarization in red and green cones. Light suppresses the GABA release, depolarizing the cones by disinhibition.
1.6.5. Dopamine in the Retina
A. Introduction
The amines, dopamine (DA) and noradrenaline (NA) are synthesised from the amino-acid tyrosine, and occur naturally within the vertebrate body. They are both strong neurotransmitter candidates and are believed to be implicated in many mental disorders of the central nervous system.
The DA receptors have been divided into two classes, termed D1 and D2 (Kebabian and Caine, 1979) on the basis of their sensitivities to agonists and antagonists (Table 1.3.). 72
DOPAMINE RECEPTORS
D-l D-2
AGONISTS
Dopamine Full agonist Full agonist Apomorphine partial agonist at juM Full agonist at nM
ANTAGONISTS
Phenothiazines nM po te nc y nM potency Thioxanthenes nM po te nc y nM potency Butyrophenones juM potency nM potency Substi tuted be nz am id es inactive juM potency
Adenylate Stimulatory Inhibitory or Cyclase unlinked linkage
Table 1,3 Classification of dopamine receptor into D-l and D-2 in the vertebrate CNS (based on Kebabian and Caine/ 1979). 73
B. Dopaminergic Activity in the Retina
Using the perfused fish retina, Negishi and Drujan
(1978), applied 100-200 jum DA and observed that the centre-surround properties of the horizontal cells were altered. The response recorded at the centre of the stimulated area was enhanced, but there was a marked inhibition of responses around the central area. DA appeared to affect the spatial properties of the horizontal cell layer, originating from the lateral spread of S-potentials mediated via gap junctions between adjacent horizontal cells (section 1.4.3.) (Negishi and
Drujan, 1978; Teranishi, Negishi and Kato, 1983). Similar results were found in the carp retina (Mangel and Dowling,
1985). Application of DA reduced the responsiveness and receptive field size of the cone horizontal cells, as did a prolonged period in complete darkness, suggesting that prolonged darkness induced a release of DA from the DA - containing interplexiform cells (section 1.2.6.). In another study of the action of DA on retinal neurones,
Hedden and Dowling (1978) evoked horizontal cell depolarization and S-potential inhibition. The actions were assumed to be mediated via postsynaptic receptors in the interplexiform cell synaptic pathways of the outer plexiform layer, which had a direct presynaptic influence on horizontal cells. It was later suggested (Dowling et al., 1983) that horizontal cells (in goldfish) have receptors for DA. 74
Ikeda et al. (1986) iontophoretically applied 500 mM DA to the intact cat retina and observed that there was a general inhibitory effect on all ganglion cells. This effect was weak compared to GABA or glycine at similar concentrations. They also used the Dl and D2 antagonist haloperidol/ the Dl antagonist/ oc -flupenthixol dihydrochloride and the D2 antagonist L-sulpiride on the preparation/ and from these studies concluded that the ganglion cell receptors were D2 type rather then Dl type.
3 2+ Radiolabelled [ H] GABA can be released in a Ca independent fashion from horizontal cells by the excitatory amino acids and their agonists acting at kainate (KA) or quisqualate (QA) receptor sites (section
1.6.4.). Kato/ Negishi and Teranishi (1985) showed that DA from DA-containing interplexiform cells in the carp retina/ inhibited release of [ H] GABA induced by KA and
•j* also high K / m a dose dependent manner. They also showed that serotonin/ bicuculline and picrotoxin could mimic this effect of DA at similar concentrations and the
DA antagonist haloperidol blocked the inhibitory effect on 3 [ H] GABA release/ induced by DA.
Using whole cell voltage clamp methods/ Knapp and
Dowling (1987)/ studied isolated perch horizontal cells to show whether DA could alter light responsiveness of the horizontal cells by changing their sensitivity to excitatory photoreceptor transmitters. Dopamine and cyclic adenosine/ monophosphate (cAMP) enhanced ionic 75
conductances gated by the excitatory neurotransraitter
L-glutamate and its agonist KA. The results indicated that there was evidence for DAergic regulation of excitatory amino-acid neurotransmitter action and indicated a possible mechanism to explain the reduction of responsiveness of horizontal cells when the retina was treated with dopamine. 76
CHAPTER 2
THE ACTIONS OF ANAESTHETIC AND SEDATIVE DRUGS USED
IN THIS STUDY
2.1. Introduction
The drugs and chemicals used in this study belong to a wide range of compounds which have sedative-like actions.
There are varying degrees of sedation which can be defined in clinical terms in the following ways:
1. Anaesthetics - drugs which induce a reversible absence of all feeling and sensation/ either in local areas or generally throughout the whole body/
2. Hypnotics - drugs inducing drowsiness and sleep/
3. Sedatives - drugs inducing a 'calming1 effect.
It is generally found that drugs producing sedation at low concentrations/ can have hypnotic effects at slightly higher concentrations and anaesthetic effects at high concentrations. Most are considered as non-specific depressants of the central nervous system (CNS).
Theories of anaesthsia and sedation have been divided into those which account for the depression of synaptic transmission in terms of a blockade of axonal conduction/ and those which explain the effects observed by 77
considering the disturbance of the mechanism of synaptic transmission. As synaptic transmission is depressed at concentrations of drugs which do not block axonal conduction (Richards/ 1972)/ it appears more likely that the depression originates at the synaptic site.
Disturbances of synaptic functioning have been proposed as follows:
1. A decrease in the presynaptic release of excitatory transmitter/
2. An increase in the presynaptic release of
inhibitory transmitter/
3. A decrease in postsynaptic chemosensitivity to excitatory transmitter/
4. Stabilization of the postsynaptic membrane/ which thus inhibits the activity induced by
transmitter-receptor interactions at the postsynaptic
membrane.
Two main classes of drugs were used in this study
the barbiturates and the benzodiazepines/ whilst other
sedatives/anaesthetics were briefly examined. Each of the
classes will be considered in the following sections.
2.2. The Barbiturates
The barbiturates are used clinically as hypnotics
and sedatives at low concentrations/ but can produce
anaesthesia at higher concentrations. All are related 78
structurally to barbituric acid/ which is itself without depressant activity. Active derivatives are produced by substitution of alkyl and aryl radicals on carbon 5 or by substitution at a nitrogen (N)atom (fig. 2.1.).
Barbiturates are general depressants affecting the central nervous system (CNS). They are classified into three groups/ based on their duration of activity:
Group I - Long lasting with low solubility. The drugs in this group are generally used as sedatives and
include phenobarbital/
Group II - Intermediate duration of activity/ with
lipid solubility greater than that of Group I. The group
includes pentobarbital and other drugs which are used both as sedatives and hypnotics/
Group III - Short acting with high lipid solubility.
Drugs of Group III are used as intravenous anaesthetics and
include hexobarbital.
Barbiturates are thought to depress neuronal
activity in either of two ways:
1. By potentiating -amino-butyric acid (GABA)
mediated inhibition/
or 2. Directly depressing excitatory synaptic
transmission
These appear to be the two main ways in which the
barbiturates act on the nervous system. Each will be
considered in turn. 79
O -- H C-- fsj \ > 0 H
C—N II O
Barbituric acid
? V
c ^ - CxH 2 / C _ N \ C-OH CHj CHjC ^ - C H \ / A Pentobarbital CH. I ■ n O. \ ^ O H
CHz
o Hexobarbital
F i g . 2.1. The chemical structure of barbituric acid and its active barbiturate derivatives, phenobarbital, pentobarbital and hexobarbital, all of which are used in this present study. 80
2.2.1. Potentiation of Inhibitory Activity
Several studies have provided evidence to suggest that pentobarbital enhances GABA-ergic synaptic inhibition in the vertebrate central nervous system. These include,
Ransom and Barker (1976) using cultured mouse spinal neurones, Nicoll (1978) using frog sympathetic ganglion cells and Scholfield (1978) working on guinea-pig olfactory cortex neurones. There is also evidence that potentiation of both exogenous and synaptically released GABA occurs
(Macdonald and Barker, 1979; Nicoll and Wojtowicz, 1980;
Study and Barker, 1981).
Bowery and Dray (1976, 1978) have reported that the antagonism by bicuculline of GABA induced depolarization of isolated sympathetic ganglia and GABA induced depression of brain stem neurones in the rat are 'reversed' by pentobarbital. GABA enhancement was shown in experiments where low doses of pentobarbital were applied with the GABA antagonist bicuculline. Evans (1977), found that a prolonged GABA enhancement was produced by low concentrations of pentobarbital acting with a direct GABA mimetic action, without the addition of a GABA antagonist.
It is believed that pentobarbital remains in the tissue for a longer period of time than GABA and to explain Bowery and
Dray's finding (1976), Evans put forward the idea that bicuculline is displaced from the GABA receptor by the GABA mimetic (eg. pentobarbital), which binds more firmly to the 81
GABA receptor site. Curtis and Lodge (1977) investigated the action of pentobarbital on cat dorsal horn interneurones and found that there was a partial reversal of bicuculline antagonism of GABA. However/ they stated that barbiturate anaesthesia could then only be explained in terms of interference with some unknown endogenous GABA antagonist. Bowery and Dray (1976) also suggested that the depressant action of pentobarbital might involve the reversal of the action of an endogenous bicuculline like substance present in the central nervous system. Evans
(1979) found that pentobarbital did not 'reverse' the effects of bicuculline in antagonizing the depolarization of primary afferent fibres induced by GABA. It was also found in the study by Evans (1979)/ that bicuculline did not block the potentiation of GABA-induced responses produced by pentobarbitone/ even though GABA antagonists block the GABA like actions of the drug (Nicoll/ 1975a/ b).
Two independent sites of action for pentobarbital were suggested/ one which is GABA potentiating/ but unaffected by GABA antagonists and one where the mimetic action of pentobarbital is antagonised by bicuculline (Evans/ 1979).
Binding studies have provided more evidence for
interactions between pentobarbital and GABA at GABA receptor sites. Willow and Johnston (1980) using crude
synaptosomal membrane from rat brain/ studied the binding activity of GABA. They found that in the presence of pentobarbital GABA binding was enhanced by 30-50%/ but this 82
increase was abolished by picrotoxin. Similar results were observed in bovine cerebral cortex membrane preparations by
Asano and Ogasawara (1981)/ where enhancement occurred in the presence of Cl" ions. Radiolabelled GABA has also been used to show enhanced binding under pentobarbital treatment
(Olsen and Snowman, 1982; Skerritt and Johnston, 1983). 3 Barbiturate inhibition of [ H] bicuculline binding to GABA receptor sites, apparently involves a decrease in affinity which at higher barbiturate concentrations, results in the effective loss of any detectable binding
(Wong et al., 1984). This effect is reversed by picrotoxin, which leads to the conclusion that there is an indirect action at the previously defined picrotoxin/barbiturate modulatory site on the GABA- benzodiazepine receptor/chloride ion channel complex.
Barbiturates can have both anticonvulsant and anaesthetic properties (phenobarbital is predominantly anticonvulsant, whereas pentobarbital is used as an anaesthetic). Macdonald and Barker (1978a) studied both types of drug and found that:
1. Anaesthetic, but not anticonvulsant barbiturates, abolish all spontaneous synaptic activity, and anticonvulsants abolish picrotoxin induced activity,
2. Anaesthetics, but not anticonvulsants, directly
increase the membrane conductance, an action which is antagonized by the antagonist picrotoxin,
3. Anaesthetics, but not anticonvulsants, substantially prolong GABA responses, 83
4. Both classes enhance postsynaptic GABA responses and antagonize postsynaptic glutamate responses/ although anaesthetics are 2 to 3 times more potent. The results obtained indicate both qualitative and quantitative differences in the pharmacological actions of the two classes of barbiturates. Pentobarbital has been shown to be more potent than phenobarbital at displacing picrotoxin from the GABA receptor (Olsen et al./ 1978) and in addition/ GABA binding is enhanced more effectively by pentobarbital (Willow and Johnston/ 1981; Olsen/ 1981).
Iadorola et al. (1985) using both electrophysiological and biochemical techniques/ on pyramidal cells in the hippocampal slice preparation/ compared phenobarbital/ as a clinically used anticonvulsant drug/ to pentobarbital and observed that phenobarbital did not enhance GABA. They proposed that the anticonvulsant actions of barbiturates do not depend on their ability to enhance GABA-ergic neuronal activity/ and must therefore be due to some other regulatory effect/ such as inhibition of excitatory mechanisms.
2.2.2. Depression of Excitatory Activity
Brooks and Eccles (1947) were the first to demonstrate that general anaesthetics could depress excitatory postsynaptic potentials and subsequent ionophoretic studies have shown that barbiturates cause a 84
general reduction in chemically evoked excitation (Johnson,
Roberts and Straughan, 1969; Crawford, 1970).
From observations on guinea-pig olfactory cortex slices, Richards (1972) suggested that the depression of excitatory synaptic transmission could account for the effects seen during anaesthesia. Barker and Gainer (1973) looked at the effects of pentobarbital on synaptic transmission and postsynaptic potentials, using several invertebrate preparations including crayfish and lobster, and found that there was a reversible depression of both mechanisms, but no effect on inhibitory postsynaptic - + potentials or Cl and K dependent postsynaptic responses.
Thus it was proposed that the peripheral and central depression observed during general anaesthesia may be due to a selective depression of excitatory synaptic events.
If this were true then anaesthetics and also sedatives may either;
1. Decrease the release of transmitter by nerve impulses,
2. Depress the sensitivity of postsynaptic membranes to transmitter substances.
More specifically, neuronal excitability produced by
the excitatory amino-acid, L-glutamate, was reduced by the barbiturates. Richards and Smaje (1976) using neurones of
the prepiriform cortex, excited by applications of pulses of L-glutamate, before, during and after exposure to 85
anaesthetic levels of pentobarbital, observed that there was a reduction of the postsynaptic membrane sensitivity to
L-glutamate. They concluded that this was probably the main action of pentobarbital and showed a similar action with other anaesthetics in the guinea-pig olfactory cortex.
D-L-homocysteic acid (an excitatory amino-acid) produces stable increases in membrane conductance (Gm), whilst large long-lasting applications of L-glutamate produce slowly increasing changes in membrane conductance.
Barbiturates (pentobarbital and thiopentone) greatly reduce the change in conductance brought about by L-glutamate, but although they also reduce the response to D-L-homocysteic acid, no effect on the conductance change was observed
(Lambert and Flatman, 1981). The high Gm is manifested by a low affinity L-glutamate uptake. If this uptake is therefore inhibited by barbiturates, then the reduction of the high Gm state can be explained. It was proposed by
Lambert and Flatman (1981), that in the case of
D-L-homocysteic acid, the barbiturates probably interact with receptor complex associated conductance changes, rather than directly with the receptors. 22 + Using radiolabelled sodium ( Na ) efflux assay experiments, it was shown that the barbiturates depress the action of excitatory substances, such as glutamate analogues, acting at sites separate from those for glutamate (Teichberg et al., 1984). Four main classes of receptor are known in the rat striatum (Luini et al., 86
1981). These are N-methyl D-aspartate (NMDA)t
L-aspartate/L-glutamate, kainate (KA) and quisqualate (QA).
These receptor sites were discussed in Chapter 1 .
Teichberg et al. (1984)/ examined the effects of barbiturates and some alcohols on excitatory amino-acid stimulated labelled anion efflux from pre-loaded rat striatal slices. This type of assay permitted the authors to study barbiturate interactions with the excitatory amino-acids whilst/ at the same time monitoring the primary membrane permeability changes produced by receptor activation. The barbiturates partially antagonised the KA . 22 + and QA induced Na efflux from the striatal slices/ but the effluxes due to NMDA/ L-aspartate or L-glutamate were less affected. In the case of KA and QA and in contrast to that for the other excitatory amino-acids/ antagonism was even more pronounced when aliphatic alcohols/ such as ethanol were used. It was proposed therefore/ that there are hydrophobic domains associated with KA and QA receptor sites/ at which barbiturates and alcohols can interact.
Similar results were obtained by Harrison (1985) using rat cerebral cortex slices. Depolarizing response amplitudes to various excitants were reduced in the following way by application of pentobarbital:
NMDA 8 + 5%
L-aspartate 15 + 6%
L-glutamate 3 + 5% + KA 37 9%
QA 57 - 4%. 87
These reductions were not antagonized by bicuculline or picrotoxin, and were consistent with Teichberg et al.
(1984) who provided evidence that the barbiturates appear to have a specific effect on responses to KA and QA.
2.3. The Benzodiazepines
Like the barbiturates/ the benzodiazepines are used clinically as broad spectrum general anaesthetics/ sedatives/ hypnotics and anticonvulsants. The drug group includes/ chlordiazepoxide , diazepam/ nitrazepam and flurazepam. All possess a similar range of activity/ but differ in the degree to which they produce their various effects. Flurazepam (fig. 2.2.)/ the drug used in this study is a long-lasting hypnotic which like other benzodiazepines appears to interact at the GABA-receptor complex. The structure of the complex and the possible modulatory site for benzodiazepines has already been discussed in Chapter 1 (section 1.6.4.).
Neurotransmitters such as serotonin (Stein et al./
1975)/ catecholamines (Fuxe et al./ 1975) and glycine
(Snyder and Enna/ 1975)/ have been proposed as interacting with the benzodiazepines/ however/ many studies have confirmed that the likely neurotransmitter candidate with which the benzodiazepines interact is GABA. It was demonstrated that diazepam could potentiate the presynaptic inhibitory pathway in the cat spinal cord/ where the 88
Fig, 2.2. The chemical structure of the benzodiazepine/ flurazepam 89
neurotransmitter is belived to be GABA (Schmidt et al. ,
1967), Specificity for GABA inhibitory pathways was demonstrated by the lack of effect on postsynaptic inhibition in the same region/ which is thought to be mediated by glycine (Schmidt et al.; 1967). Further studies have shown similar effects in other areas of the
CNS/ for example in the sympathetic ganglia (Suria and
Costa/ 1973)/ in fetal rat spinal cord cell cultures
(Macdonald and Barker/ 1978b)/ in the hippocampus (Biscoe and Duchen/ 1985) and the retina (Ikeda and Robbins/
1986b). Costa et al. (1979) reported the specific effects of Cl“ in enhancing benzodiazepine binding to GABA receptor sites. As GABA receptors are associated with chloride ionophores/ it was inferred that the receptor sites at which the benzodiazepines were acting were GABA receptor sites. Also/ purification of the benzodiazepine receptor has shown that the benzodiazepine receptor protein is linked to the GABA receptor protein (Stephenson and Olsen/
1982; Sigel et al./ 1985; Kirkness and Turner/ 1986).
Benzodiazepines do not however always enhance inhibitory mechanisms. There appears to be a degree of dose dependency/ such that at low doses benzodiazepines enhance GABA mediated inhibition, whereas at higher doses, antagonism of the GABA response occurs (Macdonald and
Barker, 1978b). Whichever occurs, the underlying mechanism is not clear. Bormann and Sakmann (1984), were able to show in spinal cord and neuronal cultures, that diazepam 90
increased either the affinity of GABA binding to its receptor site or the number of available GABA receptor sites for binding. Studying the GABA activated membrane currents using patch-clamp recording techniques, benzodiazepines, such as diazepam, were found to enhance
GABA mediated synaptic transmission and increase the peak current by a factor of 3-4 times, whilst the time constant for desensitization was decreased. Single channel recordings showed that diazepam does not change the conductance or the gating properties of the GABA-activated c h a n n e l s .
GABA has been proposed as an inhibitory transmitter at mammalian retinal ganglion cells (Ikeda, 1985) and it also contributes, through its effects on neuronal activity to the electroretinogram (E.R.G.). Thus it should be expected that benzodiazepines might also influence the
E.R.G. However, Ikeda and Robbins (1986a) showed that in the rat, benzodiazepines such as midazolam malate and flurazepam and the benzodiazepine antagonist R015-1788 (An imidazodiazepine) have no effect on the E.R.G. It was concluded from the data collected by Ikeda and Robbins
(1986b), that the benzodiazepines do not affect preganglionic retinal activity, but at the ganglion cell level they found that benzodiazepines had an inhibitory effect, reducing the spontaneous activity by 30-90%.
Further, only the sub-population of ganglion cells sensitive to GABA were depressed, and not those sensitive 91
to the inhibitory amino-acid transmitter glycine, providing further evidence for GABA-benzodiazepine interaction-
2.4. Interactions between Barbiturates and
Benzodiazepines
Interactions between benzodiazepines and sedative-type drugs such as pentobarbital have been suggested by several studies. Pentobarbital reversibly increases Na+ independent, chloride dependent GABA binding and flurnitrazepam binding in bovine cerebral cortex membranes (Asano and Ogasawara, 1981) and similar results were seen in washed rat cerebral cortex membranes (Skolnick et al., 1981). Skerritt et al. (1983) applied a range of sedative and anticonvulsant drugs, all related derivatives of pentobarbital, to rat brain synaptosomal membranes, and studied their effects on GABA and benzodiazepine binding.
The study showed that benzodiazepines and barbiturates act at different loci of the GABA receptor complex. It was also demonstrated by Skerritt and Johnston (1983) that 3 there was a modulation of benzodiazepine ([ H]diazepam) and 3 GABA ([ H]GABA) binding in the presence of sedatives and anticonvulsant type drugs. They concluded, however, that not all sedatives and anticonvulsants have the same ability to enhance GABA activity. A more recent study by Iadarola et al. (1985) used biochemical as well as electrophysiological techniques, to compare the effects of 92
anaesthetic and anticonvulsant barbiturates on GABA and
GABA related responses. Biochemical methods were applied to examine the barbiturate regulation of binding of radiolabelled benzodiazepine to the benzodiazepine modulatory site on the GABA-receptor/ whereas the electrophysiological method involved studying the regulation of somatic recurrent inhibition of CAl pyramidal cells in hippocampal slice preparations. Results indicated that anaesthetic barbiturates such as secobarbital and pentobarbital/ enhance benzodiazepine binding and inhibition/ whereas phenobarbital and diphenyl barbituric acid/ which are anticonvulsants/ do not. This suggests that the anticonvulsant action of the barbiturates may not depend on the ability to enhance GABA-ergic neuronal activity.
The barbiturate interaction with benzodiazepine binding at GABA receptor sites is susceptible to GABA antagonists/ such as picrotoxin (TickU/ 1981; Leeb-Lundberg and Olsen/ 1982) and bicuculline (TickU/ 1981;
Leeb—Lundberg et al./ 1981; Leeb—Lundberg and Olsen/ 1983).
Wong et al. (1984) also found that the barbiturates could 3 inhibit the binding of [ H] bicuculline to GABA sites and the binding of a benzodiazepine 'inverse agonist' ft -carboline-3-carboxylate methyl ester (ft CCM) to benzodiazepine binding sites in the mammalian brain. The effects were concentration dependent and chemically specific in a way which correlated with the activity of 93
barbiturates to enhance GABA responses in neurones and GABA and benzodiazepine binding iri vitro. Both inhibitory effects involved a decrease in affinity of binding to receptor sites/ inhibition being greater for bicuculline than /3 CCM. Reversal of the inhibition could be achieved by picrotoxin/ suggesting an indirect action at picrotoxin/barbiturate modulatory sites on the GABA-C1" ionophore complex.
2.5. Other Sedative-Type Drugs Studied
In addition to the barbiturates and benzodiazepines/ several other drugs classed as sedatives/ hypnotics or anaesthetics were included in the study to be described.
These were ethanol/ urethane/ chloral hydrate and cS -chloralose (fig. 2.3.).
Negishi and Svaetichin (1966) measured the relative effects of alcohols (ethanol and methanol) and volatile anaesthetics (ether/ flurothane and chloroform)/ on plasma membrane potentials. Their experimental preparations were
"controller cells" (S-potential producing horizontal cells)/ from the isolated fish retina and "conductor cells"
(spike producing cells)/ from the isolated frog dorsal root ganglion. The results obtained demonstrated that at concentrations of 0 .02% to 0.08% (in dry air)/ both alcohols and volatile anaesthetics suppressed S-potential producing cells/ whereas other cells of the retina/ such as 94
H H B I I K f H - C — C — O H N-C-O-C-C-H I I H 6 * ^ H H
C h ^ O H D. C- H-C-OH C L H I I ,o CL-C-C-OH i l C L O H \°n / o o \ M XC CL 3
Fig. 2.3. The chemical structure of other drugs used in this study. A - ethanol; B - urethane (ethyl carbamate); C - chloral hydrate and D - ©< -chloralose. 95
photoreceptors/ the spike producing cells of the frog dorsal root ganglion and synaptic transmission were more resistant to the effects of the drugs. The depolarizing component of the C-type S-potential (Chapter 1/ section
1.4.3.) was selectively depressed by ethanol.
Alcohols were examined to show their effects on the excitatory amino-acid stimulated radioactive cation efflux from preloaded rat striatal slices (Teichberg et al./
1984). It was revealed that the alcohols (such as ethanol) could selectively antagonise kainate (KA) and quisqualate 22 + (QA) induced radiolabelled sodium ( Na ) efflux from striatal slices/ but affected the efflux produced by
N-methyl D-aspartate (NMDA)/ L-glutamate and L-aspartate much less. It was proposed that the hypnotic effects of the alcohols could be attributed partially to their interference with excitatory neurotransmission. If ethanol interferred with NMDA binding and response/ than lack of
NMDA response would cause sedation. JEn vivo experiments have shown that NMDA antagonists cause loss of muscle tone/ righting reflex and produce general sedation (Teichberg et al./ 1984)/ which lead to the idea that the depressant action of the alcohols was to disturb the activation of excitatory amino acid receptors.
It has been reported that ethanol/ like barbiturates/ enhances the affinity for benzodiazepine binding both in membrane and solubilized membrane preparations (Birch and Ticku/ 1980; Ticku and Davis/ 96
1981). Isopropylbicyclophosphate (IPPO) binds to the
GABA-receptor complex near or at the chloride ionophore/ greatly reducing the activity of drugs such as pentobarbital, at the receptor complex (see section 2.6 .).
Mendelson et al. (1985) demonstrated that there was a significantly reduced duration of the loss of righting reflex induced by ethanol in the presence of IPPO and it may be the case therefore, that ethanol, in common with some other sedatives eg. barbiturates and benzodiazepines, interacts at the GABA receptor complex, specifically at the
Cl" ionophore.
Urethane (ethyl carbamate) (fig. 2.3.), a laboratory anaesthetic, was also investigated in this study. The influence of urethane on electrical properties of giant neurones of gastropods was studied by Gierasimov and
Janiszewski (1967). In general, urethane reduces the amplitude of spontaneous activity spikes, whilst increasing the duration of activity, and prolonging the rise time of the spikes. Voltage-current relationships after application of urethane were non-linear. Similar effects were seen in Na+-free solutions (no urethane added) and these were reversible. Gierasimov and Janiszewski (1967) concluded from the results that urethane and possibly other narcotics influence Na+-permeability. Urethane selectively depresses postsynaptic excitatory mechanisms (Barker and
Gainer, 1973) and it also hyperpolarizes neurones by + increasing the potassium (K ) conductance, thus it might be 97
deduced that this drug's action is not due to interactions with inhibitory mechanisms (Nicoll and Madison, 1982).
Two other drugs used in this study were chloral hydrate, an hypnotic drug which is formed by crystalisation of chloral, and metabolised to the active trichloroethanol within the body (fig. 2.3.) and oC -chloralose, a laboratory animal anaesthetic, formed from anhydrous glucose and chloral (fig. 2.3.). Both depress postsynaptic excitatory events (Barker and Gainer, 1973) and the hyperpolarization • “f* produced is possibly due to increased K conductance
(Nicoll and Madison, 1982).
2.6. Ionic Aspects of Sedative-Type Drug Action
The work previously described has associated various ions with the action of the sedative/anaesthetic type drugs. It has been demonstrated that the predominant action of benzodiazepines is on GABA activity, which is mediated by Cl” ionophores, whilst the barbiturates also enhance inhibitory mechanisms. Consequently, a number of attempts have been made to correlate barbiturate effects with activity at Cl" ionophores (Willow and Johnston, 1980;
Asano and Ogasawara, 1981; Skolnick et al., 1981).
Mendelson et al. (1985) administered a series of drugs which bind at various sites on the GABA complex, and attempted to antagonise lethal doses of pentobarbital and ethanol in mice. Isopropylbicyclophosphate (IPPO), binds 98
near or at the chloride ionophore and was shown to reduce the overall mortality of the experimental animals treated with lethal doses of pentobarbital/ whilst increasing the latency to death. Likewise/ picrotoxin decreased pentobarbital lethality/ but only at doses lethal when given alone/ but in those animals dying/ the latency to death was decreased. These results suggested that pentobarbital was acting at the Cl" ionophore.
Previously/ Huang and Barker (1980) carried out a study which looked at the effects of stereoisomers of the barbiturates on neuronal activity. Barbiturates exist in
(+) and (-) isomeric forms/ which have slightly different physiological effects as follows:
(+) - causes a transient period of extreme hyperexcitability/ before having a depressing effect/
(-) - produces a relatively smooth and progressively deeper hypnotic state.
Huang and Barker (1980) studied the cellular mechanisms underlying stimulant and depressant effects of the barbiturate isomers , by applying them to cultured mouse spinal neurones/ and found evidence of cellular activity which correlated well with the clinical effects of the isomers. The depressant effect on cultured mouse spinal neurones of the (-) isomer was shown by Mathers and Barker
(1980)/ to involve an increase in Cl" conductance and this effect was blocked by picrotoxin/ which acts specifically 99
at the Cl" ionophore. When compared with GABA/ (-) pentobarbital activated Cl” ionophores were kept open five times longer than those activated by GABA.
Barbiturates have also been shown to interact with
the GABA receptor to enhance the binding of benzodiazepines
(section 2.4.) and this interaction is believed to involve
the Cl”ionophore (Skolnick et al., 1981; Asano and
Ogasawara/ 1981; Olsen/ 1982). Compounds which bind either at the Cl" ionophore or some other component of the complex may block some or all of the pharmacological actions of the barbiturates. Thus/ bicuculline/ reverses barbiturate
induced increases in benzodiazepine affinity (Skolnick et al./ 1981; Leeb-Lundberg/ et al./ 1980).
Other ions have also been investigated. Evidence
from studies on invertebrate preparations (Zbicz et al./
1981)/ myelinated nerves (Arhem and Kristbjarnarson/ 1981) and artificial membranes (Johnston and Miller/ 1970) suggests that general anaesthetics and sedatives may
increase the permeability of membranes to potassium ions
(K+). Nicoll and Madison (1982) studied vertebrate systems
(frog motor neurones and rat hippocampal pyramidal cells) and showed that a number of general anaesthetic drugs/
including pentobarbital/ phenobarbital/ ether/
ot-chloralose and many others hyperpolarize central
neurones. The responses to barbiturates and o(-chloralose
were blocked by GABA antagonists and thus appear to be
dependent on Cl* activity. The responses to other 100
anaesthetics/ such as halothane/ ether/ chloral hydrate/
urethane and chloroform were generated by a separate
mechanism/ possibly an increase in potassium ion (K )
conductance. It was suggested that barbiturates and
, 4- -chloralose also increase K conductance and such a
non-synaptic action could contribute both to the decreased
neuronal responses produced by the compounds studied by
Nicoll and Madison (1982) and their anaesthetic actions.
Sodium ions (Na ) has also been studied for any possible involvement which they may have with the pentobarbital depressing effect. Barker and Gainer (1973)
using molluscan and crustacean preparations and looking at
synaptic transmission and resulting post synaptic
potentials/ found that pentobarbital selectively and
reversibly depressed excitatory postsynaptic potentials
.f. (EPSP's) and Na dependent postsynaptic activity induced by
putative excitatory transmitters/ whilst inhibitory
postsynaptic potentials and Cl* or K+ dependent
postsynaptic activity to putative transmitters were not
affected by pentobarbital. It therefore appeared from this
study that pentobarbital selectively depressed transmitter
coupled Na+ conductances/ with the preservation of
transmitter coupled Cl* and K+ conductances. Similar
results were found by Nicoll (1978)/ using frog sympathetic
ganglion to study fast excitatory postsynaptic potentials.
It was found that pentobarbital blocked central excitatory 101
synapses by a postsynaptic mechanism involving an increased
Na+ conductance, coupled with a postsynaptic enhancement of
GABA - mediated synaptic inhibition. Previously it had been shown that excitatory responses to glutamate, which involve increases in sodium conductance were also blocked by barbiturates (Nicoll, 1975; Barker, 1975; Ransom and
Barker, 1975).
•mm . , Chloride (Cl ), potassium K ) and sodium (Na ) ions have all been shown to be involved in the pentobarbital effect, however which ion is involved would appear to depend on the nature of the pentobarbital activity, whether an enhancement of inhibition or a depression of excitation. 102
CHAPTER 3
MATERIALS AND METHODS
3.1. Introduction
Isolated retinae of the roach (Rutilus rutilus ), a common freshwater fish of the Cyprinidae family/ were used
for all experiments. Electrophysiological equipment was set up to record intracellularly from the retina/ whilst stimulating in the normal physiological way with light
incident from the vitreal side. During experiments the
retina was perfused with the various drugs under study/
using a system similar to that described by Rowe and
Ruddock (1982a).
3.2. Preparation of the Retina
Roach were kept in a large outdoor tank/ through which there was a continuous flow of water/ and only brought inside to a small holding tank when required for
experimen ts.
The roach has a particularly large eye size to body
length ratio/ so that dissection was possible from fish as
small as 10 cm in length.
The fish were first dark adapted for a period of
10-30 minutes prior to removal of the eye. This was 103
necessary to induce the separation of the pigment epithelium (choroid), from the neural tissue of the retina, which in the light adapted state penetrates between the photoreceptor cells. The fish were killed and the eyeballs removed, in dim red light (A> 650 nm) , by cutting the connective tissue surrounding the eye-cup, and the optic nerve where it leaves the optic cavity.
The eyeball, supported on a pad of tissue which had been moistened with Ringer solution was cut equatorially and cornea and lens were lifted away and removed. The retina was then made accessible and was carefully peeled away from the back of the eye-cup, using fine forceps, and isolated by cutting the optic nerve.
Once removed, the retina (S 250 jum in thickness) was supported by the vitreous humour of the eye.
3.3. The Intracellular Recording System
3.3.1. General
All electrophysiological experiments were performed inside a 'walk-in' Faraday cage, which shielded against any external sources of electrical interference and noise.
Four main systems made up the intracellular recording system, as follows:
1. The perfusion/recording chamber and perfusion
system, 104
2. The micromanipulator and microscope system,
3. The stimulating system,
4. The recording system.
These are shown in fig 3.1.
The four main systems all interact when used as the
'complete' intracellular recording system, however for simplicity, each will be dealt with according to the order in which they come into use when recording.
3.3.2. The Perfusion Chamber and Perfusion System
The recording or perfusion chamber (fig. 3.2.) was constructed from a block of clear Perspex, from which a well of 0.35 ml had been machined. The well had a clear, optically smooth base to enable the preparation to be stimulated in a normal physiological way, with light entering the recording chamber from below. A stainless steel ring (fig 3.2.) fitted tightly into the recording chamber holding the retina in place from above. Four human hairs cemented as a mesh across the ring, helped to keep the retina stable during recording without causing any mechanical damage or restricting manipulation of the electrodes onto the retinal surface.
Inlets and outlets were positioned on opposite sides of the chamber. The inlet was a tight fitting stainless steel tube, attached to a silicone rubber inlet 105
M icroscope and Manipulator System
B—
Perfusion System
Stimulating System
Fig. 3,1. Schematic diagram showing the various systems involved in making intracellular recordings from the perfused fish retina., whilst stimulating in the physiological way. A. Perfusion system; B. Micromanipulator and Microscope system; C. Stimulating system; D. Recording system. 106
HAIR MESH
RETINA
Fig. 3.2. The recording /perfusion chamber was manufactured from a clear block of perspex/ having an optically smooth bottom which allowed light to enter the chamber from below. The retina, photoreceptor side facing upwards/ was clamped in the recording chamber by a tight fitting steel ring/ over which a hair mesh had been cemented. This aided retina stability. Ringer solution entered via an inlet which was positioned below the working fluid level of the chamber/ and left via a shallow dam/ as shown/ feeding to an outlet pipe. 107
tube positioned so as to be just below the normal working fluid level of the chamber- The outlet was a rubber tube leading to a waste vessel/ via a shallow dam/ which maintained the fluid level of the chamber and also reduced flow turbulence.
The inlet tube was supplied from one of six 100 ml teflon conical separating funnels (Scientific Supplies) arranged above the recording chamber. The perfusion solutions/ whether Ringer or a drug solution/ were aerated with medical gas (95% 0^/ 5% CO^) throughout experiments using scintered glass gas distribution tubes (Corning
Glass/ porosity 1). A fluid switch (Rheodyne/ type 50) divided the separating funnels from the recording chamber inlet. The switch could connect any one of the six funnels to the single inlet to the recording chamber. A clamp on the silicone rubber tube adjusted the flow rate/ which was typically 1-4 ml/min. This combination of gravity feed/ precision switching and a recording chamber designed so as to minimise turbulence/ allowed recordings to be made from single retinal cells for as long as 20-30 minutes/ whilst studying the effects of a variety of chemical or drug solutions/ applied continuously to the retina.
3.3.3. The Micromanipulator and Microscope System
The recording chamber/ micromanipulator and microscope were supported on a recording platform 108
constructed from a 3 cm thick slate sheet on a 1 metre high concrete pillar. The micromanipulator was mounted on a heavy steel base plate (2 cm thick) by a 20 cm high steel rod, fixed to one side of the base plate. The micromanipulator was used to hold and position the amplifier probe or head amplifier into which glass microelectrodes were inserted, using half-cell electrode holders (WPI Instruments). The recording chamber was positioned with respect to the stimulating light path by the use of an 'XY' control microscope stage, which was mounted onto a stainless steel platform supported above the base plate by 4 20 cm high steel rods. A 1.5 cm, diameter hole cut into the stainless steel platform, allowed the light stimulus to fall onto the surface of the retina. A zoom stereo-microscope (Zeiss) mounted above the recording platform allowed the preparation and manipulation of the microelectrodes to be viewed under high magnification. This system is illustrated in fig. 3.3.
3.3.4. The Stimulating System
An optical stimulating system (fig. 3.4.) was constructed outside the Faraday cage, on a slate table.
The light source with which the retina was stimulated during experiments, originated from a D.C. driven 250
Watts quartz-iodine lamp. The filament of the lamp was 109
MICROSCOPE
Fig. 3.3. The recording platform with microscope and micro manipulator. The micromanipulator/ carrying the amplifier probe/ electrode holder and microelectrode/ was mounted on one side of a stainless steel recording platform. This platform/ carrying the recording chamber/ had a central aperture through which the light stimulus was directed. Both micromanipulator and recording platform were supported by 20cm high steel supports/ all bolted to a heavy steel base plate. The recording platform was used to mount the 'XY' position microscope stage/ which allowed the recording chamber and preparation movement relative to the stimulus. A dissecting microscope was used above the recording platform to observe electrode manipulations. tI
The optical system. The light source (a D.C. driven 250 Watt quartz-iodine lamp) was focussed onto two pinhole diagrams (PH^ and Pt^) by a Fresnel lens and semi-silvered mirror combination (SR). Each beam produced then passed through a combination of lenses ( and L^) interference filters (IPj and Prisms (P^ and P 2 ) and stops (S). The two beams were then recombined with the beam splitter (BS) and directed to the retinal preparation by prism P , situated beneath the recording platform. Electromagnetic shutters (EMS, and EMS2 ) determined which beam was presented to the retina. m
focused by a 'Fresnel lens' onto two diaphragm pinholes.
Using achromatic lenses/ two collimated beams were obtained which were recombined with a beam splitter cube/ having passed through filters/ stops and shutters. This combined beam then entered the Faraday cage through an opening behind the microscope and micromanipulator system.
A prism directed the light beam upwards through the optically smooth bottom of the recording chamber which was then focused onto the retina by a projection lens combination.
Two light beams of different wavelengths were obtained by placing interference filters (Balzers B40/ interference filters/ 5 mm half intensity bandwidths) in each of the two beams. The two wavelengths used for most experiments were 618 nm and 518 nm (appearing respectively red and green to a human observer). The intensity of the stimulus could be controlled by placing neutral density filters in the light paths.
The two stimulating light beams were presented alternately by the use of two electromagnetic shutters placed in the paths of the beams/ driven by Grass stimulators with inter-stimulus delay. The duration and frequency of stimulation could be controlled by these stimulators/ the former being typically 400 ms/ and the latter typically 0.5 pps. 112
3.3.5. The Recording System
The electrical equipment used to record the cell responses was mounted on a rack within the Faraday cage.
All components were centrally bound to earth via a copper bar, attached to the outside of the Faraday cage.
Earthing in this way helped to keep electrical interference to a minimum and also prevented earth loops from forming.
The microelectrode (see section 3.3.6.) used to record cell responses/ was held in a half cell electrode holder (WPI)/ having a solid Ag - AgCl contact (fig 3.6.).
This half cell was prefilled with 2.5M KC1 and fitted into the head amplifier of the recording system/ which was in the form of a probe/ mounted directly onto the micro manipulator system. The head amplifier fed straight into the main recording amplifier (WPI Instruments - M701).
The unity gain output of the impedance matching amplifier was displayed on one channel of a storage type oscilloscope (Tektronix - 5103N). The trace originating from the main amplifier was used to monitor cell penetrations and the electrical responses of the impaled neurones.
Due to the fact that the typical horizontal cell response has a relatively slow rise time/ permanent recordings could be made directly from the main amplifier output to a chart recorder (W + W -314). The chart 113
recorder had two input channels, one was used as described to record cell responses, the other was used to mark where the perfusate was changed via the switching system. In this way, correlation between cell responses and applied chemicals could be seen directly. The system is shown in fig 3.5.
3.3.6. The Microelectrodes
The microelectrodes employed for intracellular recording were pulled on a modified manual, horizontal,
Livingstone-type electrode puller (Hockman), from 1 mm outer diameter borosilicate glass capillaries fitted with a fine inner fibre for ease of filling (Clark Electro medical GC-100F). The electrode puller had a variable heating control in the form of a power supply giving variable current output. Electrodes of the desired shape and resistance, were pulled at a specific temperature and for a particular time period, (Typically times of 11 secs and currents of 0.4 - 0.45 milliamps were used). The tip shape was checked visually under a compound microscope, and then electrodes were back filled with 2.5M KC1, using a 1 ml syringe fitted with an Acrodisc filter (0.22 jum) and 28 gauge needle (Hamilton). Electrodes were filled to ensure no air bubbles blocked the electrode interior, and then held in a half cell electrode holder (WPI), as used for recording (fig 3.6.). The half cell was prefilled 114 AMPLIFIERS PROBE M701xl
Fig. 3.5. The recording system. The electrode holder and microelectrode were connected to the amplifier probe which was then in connection with the main amplifier (M701; xl). (The preparation and equipment were earthed via this main amplifier). The output from the amplifier was monitored directly on the oscilloscope (Range 20mV/division) and recorded permanently by the chart recorder (Speed 8cm/min; Range lOOmV). An event recording system/ was used to mark/ on the chart recordings/ where changes in solution were made. 115
F i g . 3-6. The microelectrode and electrode holder are shown in half section. The microelectrode and holder were back-filled with 2.5M KC1. The Ag-AgCl block provided a direct connection to the amplifier probe in which the holder was mounted by the connector pin. 116
with 2.5M KC1 and fitted directly into the head amplifier probe. The resistance of the electrode was measured by passing a small calibrated current (by pressing the
'electrode test' button on the amplifier and employing a part of the amplifier circuit), through the electrode tip to earth, via a small petri-dish of fish Ringer. The resulting voltage deflection shown on the oscilloscope screen indicated the voltage change at the amplifier input. By Ohms Law, the resistance of the electrode could be determined. The standard injection current was 1 nA, so a voltage change or deflection of 1 mV corresponded to a DC tip resistance of 1 M n . Good electrodes had a tip resistance of 50 - 150 Mfl, as cells were difficult to hold with electrodes of less than 50 Mil, whereas above
150 M the electrode tended to block.
3.3.7. The Ringer Solution
The Ringer used to maintain the preparation and in which the various chemicals under study were dissolved, was that originally described by Rowe and Ruddock (1982a).
The composition of the Ringer is similar to a variety of other Cyprinid fish Ringer solutions (Table 3.1.). The composition of the Ringer according to Rowe and Ruddock
(1982a) is as follows:
NaCl H O m M ,
KC1 2 .5 m M , 117
Table 3.1. A Comparison of Cyprinid Fish Ringer
Formulations.
Chemical (mM) A B c D E
NaCl 110.00 80.00 119.50 120.00 110.00
NaHC03 - 22.70 22.60 - 20.00
KC1 2.50 3.50 3.60 2.50 2.50
MgS04 .7H20 - 2.40 1.04 1.20 -
CaCl 2 2H20 2. 20 2.30 1.15 2. 20 -
N a H 2P04 - - 0.40 - -
Na2HP04 - - 0.10 - -
Dextrose 10.00 10.00 10.00 10.00 20.00
Hepes 5.00 10.00 — 3.00 —
pH 7.70 7.35 8.00-8.10 7.80 7.70
Authors and preparations:
A - Kaneko and Shimazaki (1975) - carp
B - Wu and Dowling (1978) - carp
C - Kato and Negishi (1978) - carp
D - Ishida and Fain (1981) - goldfish
E - Rowe and Ruddock (1982) - roach 118
NaHC03 20mM
Glucose 20mM
Calcium 30uM
corrected to a pH 7.7 with 0.1 M NaOH or HC1.
It should be noted that there is a very low 2+ . concentration of Ca m the Ringer/ due to the fact that 2+ it was found that Ca m high concentrations/ such as 2 2+ mM/ hyperpolanzed the cells in a similar fashion to Co
(Rowe and Ruddock/ 1981; Schwartz/ 1982; Yau et al./
1981).
The Ringer was prepared from Analar grade chemicals
(B.D.H.) and distilled - deionized water (to minimise the 2+ occurence of high concentrations of free Ca ), and buffered by bicarbonate/ so that the pH was dependent on dissolved C02« Before experiments were started/ solutions were aerated with 95% 02 and 5% C0 2 via scintered glass gas distribution tubes (Corning Glass/ porosity 1). The pH was adjusted to 7.7 with 0.1 M NaOH or HC1/ and this was maintained throughout experiments by continuous aeration with 95% 02 and 5% C02 -
Ionic composition of the Ringer could be altered/ and the various drugs and chemicals under study could be dissolved directly in the Ringer solution/ these occasionally required sonication or magnetic stirring.
Drugs were obtained from Sigma Chemical Company or B.D.H. 119
The benzodiazepine, flurazepam, was supplied by Roche
Products Ltd. Solutions containing the chemical agents were prepared immediately prior to each experiment, in order to minimize breakdown and chemical change. The drug/chemical solutions were continually aerated with 95%
Q 2 and 5% CC>2 and the pH corrected and maintained at 7.7.
3.4. Procedure for Recording
Solutions were freshly prepared, checked for pH, then loaded into the separating funnels of the perfusion system. Air bubbles were tapped out of the system. The retina of a dark adapted fish was isolated as already described (section 3.2.) and placed photoreceptor side up in the recording chamber. The retina was clamped down with the metal ring and left for a few minutes to settle, with a continuous flow of Ringer solution maintained over the retinal surface. The preparation was earthed by a
Ag - AgCl wire and wick placed on one side of the retina
(for most experiments using Ringer with normal ionic composition). Indirect earthing was used for experiments involving ionic changes, achieved by connecting the perfusion chamber to a separate chamber via a small tube filled with Ringer-Agar gel. This separate chamber was then earthed with a wick earth.
The recording electrode was positioned by use of the micromanipulator and the dissecting microscope, in the 120
centre of the light stimulus falling on the retinal surface. The electrode was advanced using the coarse control of the micromanipulator, until the retinal surface was touched. This could be monitored by observing a small positive or negative deflection on the oscilloscope screen. The two light stimuli (618 nm and 518 nm) were then presented alternately and the electrode advanced through the retina using the fine control of the micromanipulator. The application of excessive capacity compensation through the amplifier was used to facilitate the penetration of cells, which could be observed as a hyperpolarizing change in the electrode tip potential, the cell should then show the characteristic response to the light stimulus depending on the type of cell encountered
(Chapter 1). A permanent record of the cell responses was made, as described, using the chart recorder. The normal experimental procedure was to record initially in Ringer solution until a stable cell response was achieved, then to switch to the various chemical/drug solutions, in each case recording until a stable state was seen. Finally, the various solutions were flushed out with Ringer solution and recording continued to observe whether the response of the cell had changed from its initial state at the beginning of the experiment. At the end of a successful recording, the electrode was withdrawn from the cell to record the zero membrane potential at the retinal surface, thus the cell potential could be measured against a standard zero giving a reference level for all cell responses. Cellular activity could be recorded in this way from a single retina for anything up to 3 hours.
3.5. Transreceptor Recordings.
Due to the difficulty associated with intracellular recording from photoreceptors, transrecptor potentials
(i.e. the potentials between the basal and free ends of the photoreceptors, see section 1.4.2., chapter 1) were isolated from light evoked transretinal potentials. the isolated retina was clamped in a perfusion chamber which had been modified by the introduction of a silver chloride pellet electrode embedded in the base of the chamber, as shown in fig. 3.7. A silver chloride wire electrode was positioned in such a way as to just touch the surface of the retina when clamped in the perfusion chamber and the transretinal potential was recorded between the two electrodes whilst perfusing the pre paration as for intracellular recording. Photoreceptor responses were isolated by adding 50 juM kainic acid (KA) to the solution in order to block all post-receptor activity. The differential signal between the two electrodes was recorded via the D.C. amplifier, and this differential potential was observed on the oscilloscope and the chart recorder with typical range settings of 10 cm/sec. and 10 mV/cm. 122
F i g . 3.7. The recording system for measuring transreceptor potentials. The recording chamber was similar to that used for intracellular recordings, although a silver chloride pellet electrode was embedded in the base of the chamber. A silver chloride wire electrode placed over the retinal surface recorded the transretinal potential with reference to the silver chloride pellet electrode, by feeding both signals into a D.C. differential amplifier. 123
AIMS OF THE PRESENT STUDY
The following chapters describe an investigation of the electrophysiological effects of sedative/anaesthetic drugs on the retinal horizontal cells in the cyprinid fish, the roach (Rutilus rutilus). Experiments were performed on the isolated, perfused retina and were designed to investigate:
1. The electrophysiological effects of the barbiturate pentobarbital, on the membrane potential and light evoked responses (S-potentials) of the horizontal cells,
2. Whether the effects seen with the barbiturates are mediated by interaction with inhibitory or excitatory transmitter binding sites on the horizontal cell membrame,
3. The ionic mechanisms underlying the actions of the barbiturates,
4. A comparison of the effects of a variety of other sedative/anaesthetic type drugs with those of the barbiturate pentobarbital,
5. The actions of the benzodiazepine, flurazepam, on the membrane potential and light evoked responses (S- potentials) of the horizontal cells,
6 . Whether the effects of the benzodiazepine are mediated by interaction with inhibitory or excitatory transmitter binding sites on the horizontal cell membrane. 124
CHAPTER 4
THE ACTION OF PENTOBARBITAL ON HORIZONTAL CELLS
OF THE ISOLATED FISH RETINA.
4.1. Introduction
In Chapter 2, the actions of the anaesthetic and sedative barbiturates, such as pentobarbital, on neuronal responses were discussed. The aim of experiments described in this chapter is to show.the effects of pentobarbital on the retinal horizontal cell membrane potential and light evoked responses.
It has already been explained in Chapter 1, that the results of experiments with the agonists, kainic acid (KA) and quisqualic acid (QA), indicate that the likely excita tory neurotransmitter acting on retinal horizontal cells is
L-glutamate (L-GLU), and that the inhibitory neurotrans mitter ^-amino-butyric acid (GABA) is also effective on these cells. In this chapter, the interactions of pento barbital with the excitatory and inhibitory neurotrans mitter systems of the horizontal cell layer of the fish retinal preparation, are examined. 125
4.2. The Effect of Pentobarbital on Retinal Horizontal
Cells
The barbiturate pentobarbital (PENT) was initially investigated for electrophysiological activity on neuronal responses of roach horizontal cells in the perfused fish retina. The responses of pentobarbital were measured from the chart recordings as shown and described in fig.4.1.
At concentrations of 0.1 mM to 5.0 mM, PENT evokes a hyperpolarization of the horizontal cell dark resting potential and in many cases suppresses the light evoked
S-potentials (average hyperpolarization, 11.0 + 1.1 mV? average rate of hyperpolarization, 0.19 + 0.05 mV/sec: n =
20). These effects persist following the removal of pento barbital from the perfusing solution. Fig.4.2 illustrates the effect seen when 5mM PENT is added to the perfusate.
Pentobarbital is also effective in the presence of 2 mM 2+ cobalt chloride (Co , which acts to isolate the cell by blocking synaptic input from other cells). In fig. 4.3 2 + Co is seen to hyperpolarize the cell initially, with further hyperpolarization by 5 mM PENT (12 cells from 11 retinae). Average values for pentobarbital hyperpolari- 2 + zation in the presence of 2 mM Co were, hyperpolanza- tion, 9.39 + 1.53 mV? average rate of hyperpolarization
0.15 + 0.02 mV sec? where n = 10. Using the Student's t-test (Table 4.1.), it is shown that the average degree of hyperpolarization and rate of hyperpolarization for PENT 126
F i g . 4 .1. The degree of hyperpolarization and the time taken for it to occur, were measured as shown above, A - the membrane potential change (mV), taken from a steady resting level (in Ringer) to a steady hyperpolarized level. B - the time course (seconds), taken from the point where the membrane potential starts to change (a), until a steady hyperpolarized level is reached (b). The criteria of measuring responses from steady state conditions were used for all experiments, where possible. 127
F i g . 4.2. The effect of pentobarbital (5 mM) on the responses of horizontal cells. The membrane potential, V(mV), is displayed against time (seconds) with depolarizing responses corresponding to upward and hyperpolarizing corresponding to downward displacements. (Light responses were elicited by periodic flashes o^light, 400 m.sec. in duration and of 618 nm, 0.04 juW. mm with a 3 mm spot diameter). 5mM pentobarbital (PENT) evokes a hyperpolari zation of the dark resting potential of the horizontal cell, which is usually accompanied by loss of light evoked S-potentials. Often there is no recovery from this hyperpolarization when the cell is returned to normal Ringer solution. The horizontal bar denotes the period of drug application. G, 128
5 m M P F N T
[ 1 min____| F i g . 4.3. The direct action of 5 mM pentobarbital (PENT) on horj^ontal cells^synaptically isolated by 2 mM cobalt (Coz ). 2 mM Coz hyperpolarizes the cell from a steady resting potentia^.,. When no further hyperpolarization occurs in 2 mM Coz , 5 mM PENT is applied and this further hyperpolarizes the cell. (Stimulus as before from fig. 4.2. ) .
F i g . 4.4. The accumulative effect of pentobarbital (PENT) ImM pentobarbital was applied in short bursts at A, B, C and D. When the cell is returned to the normal Ringer solution after successive applications of pentobarbital, the resting membrane potential does not return to its original value, but one which is increasingly more negative. Also, there is a reduction in the light evoked S-potential size and a reduction in the sensitivity to successive bursts of pentobarbital (the degree of hyperpolarization achieved with ImM pentobarbital is reduced). The record is continuous (indicated by the arrow). (Stimulus as for fig. 4.2. ) . 129
2 + (5 mM) in the presence of Co (2 mM) does not differ
significantly from those for 5 mM PENT alone (Table 4.1.).
Pentobarbital appears to have an accumulatory effect
when applied in short bursts over a period of time, com
bined with intervals of normal Ringer solution. Fig. 4.4.
shows the effect of applying bursts of pentobarbital at 1 mM. When the cell is returned to normal Ringer solution,
the light evoked S-potentials are reduced in size and the
resting membrane potential to which the cell returns is of
an increasingly negative value (The cell shown in fig. 4.4.
depolarized to -27.0 mV after one burst of pentobarbital
(A), to -28.0 mV after two (B), and -32.0 mV with three
bursts (C)). More significant is the reduction in sensit
ivity to successive bursts of pentobarbital. The hyper
polarization from the 'depolarized' state in Ringer
solution being reduced from -14.0 mV to -5.0 mV after 4
applications with rates of hyperpolarization, 0.31 mV/sec
(A); 0.22 mV/sec (B)? 0.15 mV/sec (C)? 0.07 mV/sec (D).
4.3. Investigation into the Nature of the Depression of
Neuronal Excitability by Pentobarbital
As already discussed in Chapter 2, pentobarbital and
other sedatives act either to disturb excitatory synaptic
transmission or potentiate inhibitory activity by potentia
ting ^-amino-butyric acid (GABA). The next sections deal
with results from experiments to determine the interaction 130
Table 4.1. Comparison of experimental results using the Student1s t-test. (Appendix 1 ) .
Comparison Degrees of Calculated Tabulated freedom t t (P=0.05)
A. a . 28 0.86 1.70 b. 28 0.94 1.70
B . a. 8 1.00 1.86 b. 8 0.66 1.86
C . a . 8 0.26 1.86 b. 8 0.98 1.86
D . a . 8 1.12 1.86 b. 8 0.30 1.86
Key to comparisons:
2+ A. = PENT (5 mM) vs PENT (5 mM) and Co (2 mM) t B. = KA (50 AIM) and PENT (5 m M ) vs KA (50 AIM) , PENT (5 mM) and Co^+ (2 m M ), C. = KA (50 AiM) and PENT (5 mM) vs KA (50 AIM) , KYN ( 250 juM) and PENT (5 mM), D. = KA (50 AiM) and PENT (5 mM) vs KA (50 AIM) , Bi (50 aiM) and PENT (5 mM). a. = membrane potential change (mV), b. = rate of membrane potential change (mV/sec). 131
between pentobarbital and excitatory/inhibitory neurotrans mission within the fish retina preparation.
4.3.1. Effect on Inhibitory Neurotransmission
The hyperpolarizing response of pentobarbital was tested for its sensitivity to the GABA antagonists picro- toxin (Pi) and bicuculline (Bi). Picrotoxin at a concen tration of 50 juM is found to be ineffective in reversing the response to 5 mM PENT (fig.4.5., 7 cells from 7 retinae) as is also found when picrotoxin (50 a iM) is used against 5 mM GABA, shown in fig. 4.6. Likewise, bicuculline at a concentration of 50 juM is found to be ineffective in reversing hyperpolarizations induced by 5 mM PENT (7 cells from 7 retinae; fig. 4.7.). However, it was found that bicuculline (50 juM) reverses the hyperpolarizing effect of
5 mM GABA (fig. 4.6.). These results suggest that pento barbital does not involve GABA-ergic activity.
A number of experiments were performed in which GABA was applied in combination with pentobarbital. These were in the main inconclusive, as the effects of GABA itself were often negligible, and when it was effective, were sometimes hyperpolarizing and sometimes depolarizing
(Hankins and Ruddock, 1984a). An example of a record in which there is a hyperpolarizing effect with 5 mM GABA is given in fig. 4.8. Addition of pentobarbital causes
increased membrane hyperpolarization, but this could be due 132
0
I 5mM PENT , I 50liM i Pi I 1 min
F i g . 4.5. Pentobarbital (PENT) hyperpolarization at 5 mM, is not affected by the GABA antagonist picrotoxin (Pi) at 50uM. (Stimulus as for fig. 4.2.).
Of
■ 20 -
% 0
|50uM i iSO juM i ^Pi | 50juM Bi | "P i , 50uM Bi | _____ 5mM GABA
min
F i g . 4.6. The effect of the GABA antagonists picrotoxin (Pi) and bicuculline (Bi) on the hyperpolarizing response to 5 mM GABA. The response is blocked by bicuculline (50 uM) but not by picrotoxin (50 juM). Bicuculline repolarizes the cell potential and evokes S-potential recovery. (Stimulus as for fig. 4.2.). 133
F i g . 4.7. (Bi) does not reverse the The GABA antagonist bicuculline pentobarbital (PENT). hyperpolarization produced by 5 (Stimulus as for fig. 4.2.)*
Or-
tttff 1 Hull
15mM GARA .SmM PFNT
1 min
F i g . 4.8. No potentiation of the GABA (15 mM) induced hyperpolari zation by 5 mM pentobarbital (PENT) is apparent. However, it is difficult to determine whether the hyperpolarization seen when both pentobarbital and GABA are applied to the retina, is due to a potentiation of GABA or the action of pentobarbital. (Stimulus as for fig. 4.2.). 134
either to independent action of the barbiturate, or to its potentiation of GABA action. In view of this ambiguity and the extreme variability of GABA activity, such experiments were not pursued.
4.3.2. Effect on Excitatory Receptor Sites
As discussed in Chapter 1, the excitatory amino-acid
L-glutamate depolarizes retinal horizontal cells with a reduction or total loss of S-potentials, whilst the gluta mate agonists, kainic acid.(KA) and quisqualic acid (QA) and L-glutamate (L-GLU) when potentiated by D-aspartate
(D-ASP), have similar effects on the dark membrane potential of the horizontal cells, but act at lower concentrations. It is important to emphasize that these depolarizing actions are maintained as long as the drug is applied to the retina, without desensitization (Ishida et al., 1984).
Investigations were made to look at the effects of pentobarbital (5 mM) on the response to KA and QA at concentrations of 50 juM. Measurements of the voltage changes seen with these drugs are explained in fig. 4.9.
When KA (50 juM) is applied to cells hyperpolarized by 5mM PENT, depolarization occurs (fig. 4.10.). The depolarization induced by KA was of an average value of
10.0 + 1.4 mV (12 cells from 12 retinae). In previous studies, the depolarization induced by KA (50 juM), in 135
Fig. 4.9. Excitatory agonist and putative excitatory transmitter depolarizations were measured as shown. Both agonist and putative transmitters produce transient hyperpolarizations before depolarizing the membrane potential. The depolarization (A) of the membrane potential (mV) was measured from the maximum hyperpolarized level reached, to a level where a steady depolarization is attained. The time course (B) in seconds for the change in membrane potential to occur, was measured from the point where hyperpolarization is at its maximum (a), to the point at which steady depolarization occurs (b). The criteria here also apply to cells first hyperpolarized by pentobarbital then depolarized by excitatory amino-acid transmitters or ag o n i s t s . 136
0 fttnttmmm > -z-
5mM PENT 50 uM KA 1M!N
F i g . 4 » 1 0 1 t -i -i i | g The resting membrane potential of a h°rizonta , , hyperpolarized by the application of 5 niM pen (PENT). The subsequent addition of the excitatory ,f_ amino-acid agonist kainic acid (KA)/ at 50 JiM is t to depolarize the cell with a reduction in size or t e light evoked S-potentials, whilst removal of kainic acid returns the cell to a hyperpolarized level. (Stimulus as for fig. 4.2.).
I------1 50 pM KA
Fig. 4.11. The depolarizing action of kainic acid (KA), for comparison with that seen in the presence of 5mM pentobarbital (PENT). At 50 JUM the excitatory amino-acid agonist causes a reversible depolarization associated with a reduction in the size of the light evoked S-potentials. The effect is preceded by a transient hyperpolarization. (Stimulus as for fig. 4 . 2 .) .From Hankins. 1985. 137
normal Ringer solution was of an average value of 14.0 -
15.0 mV (Hankins, 1985). An example of a KA (50 juM) depolarization without the addition of 5mM PENT is shown in fig. 4.11. Fig. 4.12 shows that QA (50 juM) applied in the presence of 5 mM PENT, also depolarizes the horizontal cell membrane potential. Depolarization was observed in 6 cells from 6 retinae with an average depolarization of 11.7 +1.6 mV. Previous experiments (Hankins, 1985) showed QA depolar izing the membrane potential by an average value of 11.0 mV
(fig. 4.13).
Thus pentobarbital does not completely block the depolarizing action of the glutamate agonists on horizontal cells. If however, 5 mM PENT is applied to cells already in a state of depolarization induced by 50 juM KA, or in com bination with the agonist, then relaxation of the membrane potential is observed (fig. 4.14.) with an average value of
4.20 + 1.67 mV, and average rate of hyperpolarization of
0.06 + 0.02 mV/sec, n = 5. (The measurements taken from chart recordings showing pentobarbital induced relaxation of the kainate depolarization are illustrated and described in fig. 4.15. The measurements were also used in other cases where ‘relaxation* of the depolarizations are considered).
If the horizontal cells are synaptically isolated by 2 + the addition of 2 mM cobalt chloride (Co ) to the Ringer solution, application of KA (50juM) depolarizes the hori zontal cells and further addition of PENT (5 mM) evokes a 138
o >e > -20
1 min
F i g . 4 ♦12. The resting membrane potential of a horizontal cell is depolarized in the presence of 5 mM pentobarbital (PENT), and the excitatory amino-acid agonist quisqualic acid (QA), at 50 juM. The depolarization is accompanied by loss of S-potentials. Removal of the agonist results in hyper polarization of the membrane potential. (Stimulus as for fig. 4.2.).
50 mM QA |
Fig. 4.13. The depolarizing action of quisqualic acid (QA) at 50 juM, for comparison with that produced by 50 juM QA in the presence of 5mM pentobarbital (PENT). The excitatory amino-acid agonist causes a reversible depolarization associated with a reduction in the size of light evoked S-potentials. The effect is preceded by a transient hyperpolarization. (Stimulus as for fig. 4 • 2.) .From Hankins, 1985......
50juM KA ______I
I 1 min , 5mM PENT______,
F i g . 4.14. The excitatory amino-acid agonist kainic acid (KA) at 50 nM was applied to the horizontal cell and depolarization occurs. Pentobarbital (PENT) at 5 mM applied to the de polarized cell, causes a relaxation of the depolarization. (Stimulus as for fig. 4.2.). 140
F i g ♦ 4.15. The relaxation of the excitatory agonist/transmitter induced depolarization was measured from traces as shown above. The excitant was applied to the cell, with pentobarbital applied either in combination or at a point where the cell is in a depolarized state. Relaxation (a ) was measured from the highest level of depolarization attained in the presence of the excitant and pentobarbital, to a point where a steady relaxed level is attained. The time course for the relaxation (B) was taken from a. where the membrane potential begins to change, to b. where a steady relaxed level is reached.
F i g . 4.16. The depolarization induced by kainic2^cid (KA) at 50 ;uM in the presence of 2 mM cobalt ions (Coz ), is relaxed by 5 mM pentobarbital (PENT) when applied in combination with KA and Coz . (Stimulus as for fig. 4.2.). 141
relaxation of this depolarization (fig. 4.16.). The
average relaxation value was 3.10 + 0.64 mV, with an
average rate of hyperpolarization of 0.05 + 0.01 mV/sec, n
= 5. Table 4.1 shows that comparing these average values with those for KA (50 juM) and PENT (5 mM), without the 2 + additon of Co (2 mM), gives no significant difference
between the two experimental conditions. This confirms
that the effects of pentobarbital, like those of the
excitatory amino-acids, are localised at the horizontal
cell membrane.
Pentobarbital is also effective against the depolar
izations induced by the putative transmitter, L-glutamate
(L-GLU) at 50 juM, potentiated by 3 mM D-aspartate (D-ASP)
(D-aspartate blocks the high affinity uptake mechanism for
acidic amino-acids, which was discussed in Chapter 1,
section 1.6.3.), as is illustrated by fig. 4.17. The
average relaxation observed (5 cells from 5 retinae), is
5.02 + 0.78 mV, whilst the average rate of hyperpolari
zation is 0.06 + 0.01 mV/sec. L-glutamate applied at > 1 mM
concentrations, produces a depolarizing effect without the
addition of a potentiating agent, and 5 mM PENT relaxes
this depolarization in a manner similar to that observed
with the other excitatory amino-acids. The average values
for the relaxation and rate of relaxation are 4.40 + 1.75mV
and 0.04 + 0.01 mV/sec, n = 5, respectively (fig. 4.18.).
The interaction between inhibitory and excitatory
neurotransmitter mechanisms at the horizontal cell layer of 142 0 V (mV)
Fig. 4.17. The excitatory amino-acid neurotransmitter L-glutamate (L-GLU) at 25 juM potentiated by D-aspartate (D-ASP) at 3 mM depolarizes the horizontal cell membrane potential. The subsequent addition of 5 mM pentobarbital (PENT) relaxes this depolarization, and continues to do so even when removed from the perfusate. L-GLU, D-ASP and PENT applied together also show depolarization, followed by a relaxation of the membrane potential. The data represents a continuous recordinig (denoted by the arrow). (Stimulus as for fig. 4.2.).
JD > E > -20
“AO
F i g . 4.18. The excitatory amino-acid neurotransmitter L-glutamate (L-GLU), applied to the cell at 5 mM, depolarizes the horizontal cell membrane potential. The addition of 5 mM pentobarbital (PENT) causes a relaxation of this depolarization. (Stimulus as for fig. 4.2.). 143
the retina, was discussed in Chapter 1 (section 1.6.4.).
It has been established that GABA is accumulated by the Hi type horizontal cells (Lam and Steinman, 1971) and then released by excitatory trasmitters such as L-GLU (Ayoub and
Lam, 1982? Yazulla and Kleinschmidt, 1983), acting at KA and/or QA receptors. If this is the case then the relaxation by pentobarbital of the depolarization induced by KA (fig. 4.14.) may be GABA-ergic, even though the direct action of pentobarbital does not appear to be GABA mediated (figs. 4.5. and 4.7.). This possibility was investigated by adding 50 juM KA, the GABA antagonist, bicuculline (Bi) at 250 aiM and 5 mM PENT to the perfusate.
It was observed in 6 cells from 6 retinae that the relaxation effect of pentobarbital on KA induced depolari zation of the membrane potential is not affected by bicuculline. Relaxation of the KA induced depolarization occurs (fig. 4.19.) with average values of 5.80 + 1.62 mV for the degree of relaxation and 0.06 + 0.08 mV/sec for the rate of relaxation, where n = 5. The Student's t-test applied to compare these results with those obtained when bicuculline was not included in the perfusate, indicates that the average values for the two perfusate compositions are not significantly different from each other (Table
4.1.).
Kynurenic acid (KYN) applied at > 1 mM concen trations, is an effective antagonist of excitatory amino- acid type depolarizations and produces marked hyper- 144
F i g . 4.19. The GABA antagonist bicuculline (Bi) at 250 juM, failed to block the relaxation of the kainic acid (KA) induced depolarization by 5 mM pentobarbital (PENT), when applied simultaneously. The record is continuous, denoted by the arrow. (Stimulus as for fig. 4.2.). 145
polarization of the membrane potential (see fig. 4.21.).
At concentrations below 1 mM, KYN does not produce this antagonistic action. Pentobarbital (5mM) was applied with
250 yuM KYN in order to test whether the antagonistic effect of kynurenic acid could be potentiated by pentobarbital.
KA (50 juM) and KYN (250 juM) were applied to the retinal preparation and depolarization of the membrane potential was observed. PENT (5 mM) was then added and the typical relaxation of the membrane potential was seen (fig. 4.20., average values from 5 cells, 5 retinae, were 3.90 + 0.84 mV for relaxation; rate of relaxation 0.07 + 0.01 mV/sec).
Again, the Student's t-test was applied to compare these values with those from previous experiments using kainic acid and pentobarbital alone, and the results indicated that there is no significant difference between the response produced by the two types of perfusate (Table
4.1.). This implies that there is no interaction between the antagonistic effect of kynurenic acid and the pento barbital induced relaxation of the kainate depolarization.
Fig. 4.21 shows kynurenic acid (1 mM) against 50 juM KA induced depolarization in the absence of pentobarbital for comparison.
4.4. Pentobarbital Interaction with Dopaminergic Activity
Evidence that dopamine (DA) is a neurotransmitter in the fish retina was reviewed in Chapter 1 (section 1.6.5.) 146
Fig. 4.20. Kainic acid (KA) at 50 juM and the antagonist kynurenic acid (KYN), at a concentration which does not induce antagonism of the KA depolarization (250 juM) / were applied to the retina and depolarization occurs. 5 mM pentobarbital when added to the perfusate causes a relaxation of the depolarized membrane potential. No apparent potentiation of the antagonist occurs, as might be expected if pent obarbital and kynurenic acid acted by the same mechanism. (Stimulus as for fig. 4.2.).
Fig. 4.21. Kynurenic acid (KYN) acts as an effective antagonist of depolarization of the horizontal cell membrane potential by 5 0 ;uM kainate (KA). The addition of 1 mM KYN results in the repolarization of the dark resting potential and recovery of the light evoked S-potentials. This antagonism is comparatively different from the relaxation of the KA induced depolarization seen in the presence of 250 uM KYN and 5 mM PENT. (Stimulus as for fig. 4 . 2 .) .From Hankins, 1985, 147
and electrophysiologically dopamine has a varied effect on
the horizontal cells. Dowling et al. (1983) showed that 20 juM to 50 juM dopamine depolarizes some horizontal cells and
hyperpolarizes others, but the former action is more
common, and this was regularly observed in the present
s t u d y .
The possible interaction between pentobarbital and
dopamine, was examined in a way similar to that used to
study the interactions of pentobarbital with excitatory
amino acids. The results show that the depolarization of
horizontal cells induced by 40 ajM DA is reversed by PENT (5
mM) and that the effect is more dramatic than in the case
of the excitatory amino acids (4 cells from 3 retinae,
illustrated by fig. 4.22.). In addition to its hyperpolar-
izing action, pentobarbital also produces a marked increase
in the electrical noise of the membrane potential. In fig.
4.22., the electrode is withdrawn from the cell at point A,
showing that the noise arises in the neuronal membrane
potential and not in the electrode, as a consequence of
blockage in the electrode tip.
4.5. Discussion
It has been shown in this chapter that pentobarbital
at concentrations of 0.5 - 5.0 mM hyperpolarizes the hori
zontal cell membrane potential (fig. 4.2.). This depressant
action on neuronal activity is in general agreement with 148
0 V(mV)
- 2 0
Or V(mV)
F i g . 4.22. 40 juM dopamine (DA) depolarizes the horizontal cell mem brane potential, and this depolarization is reversed by the addition of 5 mM pentobarbital (PENT), with the loss of the light evoked S-potentials. The resulting membrane potential is very noisy, illustrated at point A where the electrode is withdrawn from the cell and the noise is lost, indi cating that the noise is associated with neuronal activity and not electrode blockage. (Stimulus as for fig. 4.2.). 149
results obtained by workers using other CNS preparations, as already discussed in Chapter 2, including Barker and
Gainer, (1973), Ransom and Barker (1976), Nicoll (1978),
Macdonald and Barker (1979) and Study and Barker (1981).
Hyperpolarization also occurs in the presence of 2mM 2+ 2 + cobalt chloride (Co ; fig. 4.3.). The action of 2 mM Co is to block synaptic input to individual cells and so effectively isolate them, thus any effects seen in this situation are due to actions at the isolated cell membrane.
The average values for the degree of hyperpolarization and rates of hyperpolarization for pentobarbital (5 mM) and 2 + Co (2 mM) and pentobarbital (5 mM) alone, are not signi
ficantly different, suggesting that pentobarbital acts directly on sites on the horizontal cell membrane and not by interacting with synaptic transmission.
Pentobarbital at 1 mM, is also shown to have an accumulatory effect with some reduction in membrane
sensitivity to the drug (fig. 4.4.). Pentobarbital has a
long lasting effect which persists after its removal from
the perfusate and is often irreversible. Therefore, it
appears that pentobarbital is binding at some site on the horizontal cell membrane and that the cell then remains in
some degree of hyperpolarization, whilst returning to a
less depolarized state after removal of pentobarbital from
the perfusing solution. This would explain the accumulatory effect and also the reduction in the sensitivity of the cells to successive bursts of 1 mM pentobarbital. 150
In Chapter 2, two possible actions of sedatives and anaesthetics on neuronal activity were discussed, namely:
1. enhancement of GABA-ergic activity,
2. reduction in neuronal excitability.
Pentobarbital's interactions with both inhibitory and excitatory activity were studied to determine which was the predominant action of pentobarbital in the retinal preparation.
GABA is active within the outer plexiform layer of the vertebrate retina (Murakami et al., 1972) and is localised in the horizontal cell layer, where it is accumulated (Lam and Steinman, 1971? Marc et al., 1978).
In fish its effects are usually hyperpolarizing, but at higher concentrations (> 5 mM) there can be depolarization
(Hankins and Ruddock, 1984a). Picrotoxin is not an effective GABA antagonist at horizontal cells in the roach retina, but bicuculline (50 ajM) blocks both hyperpolarizing and depolarizing effects of GABA (Hankins and Ruddock,
1984a). There have been many reports of barbiturate/GABA interactions in the vertebrate CNS, as was discussed in
Chapter 2 (section 2.2.1.), for example, the enhancement of
GABA-ergicactivity in cultured mouse spinal neurones
(Ransom and Barker, 1976) and the enhancement of GABA binding to membrane receptor sites (Willow and Johnston,
1980; Asano and Ogasawara, 1981? Skerritt and Johnston,
1983). If the hyperpolarizing effects of pentobarbital are due to its action at GABA sites, then they should be 151
reversed at least by the application of bicuculline. In practice, neither antagonist was found to be effective at reversing or reducing the pentobarbital induced hyper polarization (figs. 4.5. and 4.7.) and it would appear therefore, that the action of pentobarbital in this vertebrate retinal preparation is not GABA-ergic. Bowery and Dray (1976) found that pentobarbital could reverse the antagonistic effects of bicuculline against GABA in the vertebrate CNS and Evans (1977) proposed that bicuculline is displaced from the GABA receptor by pentobarbital, which then binds with greater affinity to the GABA receptor site.
It has been reported that the barbiturates allo- 3 sterically inhibit GABA antagonist ([ H] bicuculline) binding to GABA receptor sites (Wong et al., 1984), there being a decrease in the affinity of the antagonist for the binding sites. Wong et al. (1984) proposed that the barbiturates may stabilize the GABA/benzodiazepine receptor complex in a conformation which has a high affinity for the agonist, eg. GABA, and a low affinity for the antagonist, eg. bicuculline. Thus the failure of bicuculline to induce reversal of pentobarbital effects may not be an effective monitor of GABA-ergic involvement. None the less, certain studies including that of Nicoll (1975) have revealed such reversal of the pentobarbital effect, and clearly the mechanisms of retinal action are different from those cases. 152
Picrotoxin's action is specific to chloride ion
channels and there have been several reports of it blocking
the potentiation of GABA by pentobarbital (Willow and
Johnston, 1980; Asano and Ogasawara, 1981). In the retina,
however, it has been found that GABA can hyperpolarize
horizontal cells in the absence of Cl ions (Gregory et
al., 1985) and in the present study, GABA actions do not
appear to involve chloride ion channels as picrotoxin is
not an effective blocker of GABA action on the horizontal
cells (Hankins and Ruddock, 1984a). The absence of picro
toxin inhibition of pentobarbital induced hyperpolarization
is therefore not a reliable indication of possible GABA
involvement. Overall therefore, the results of these
experiments provide no evidence of GABA involvement in the
pentobarbital induced hyperpolarization of horizontal
c e l l s .
The remaining results presented in this chapter
relate to the problem of pentobarbital interaction with
excitatory neurotransmitter activity at the horizontal cell
layer. In Chapter 2, reports of the depression of excita
tory responses by the barbiturates and other sedatives/
anaesthetics were discussed. These include loss of
sensitivity of the post-synaptic membrane to the putative
transmitter L-glutamate (Richards and Smaje, 1976) and
interaction at the quisqualate (QA) and kainate (KA)
binding sites (Teichberg et al., 1984). 153
The initial experiments involved first hyperpola- rizing the cell with pentobarbital (5 mM), then applying either KA or QA (50 ajM) to see whether depolarization occurred. The results shown in figs. 4.10. and 4.12. establish that pentobarbital fails to block depolarization by either of the excitatory amino acids, although kainate induced depolarization in the presence of pentobarbital is somewhat reduced when compared to that found in previous studies (Hankins, 1985), when kainate is applied alone
(average depolarization of KA (50 aiM) and PENT (5 mM) is
10.0 + 1.4 mV; average depolarization of KA (50 pM) is 14.0 to 15.0 mV).
If however, the cells are in a depolarized state induced by the agonist and then subjected to 5 mM pento barbital (fig. 4.14.), or both agonist and pentobarbital are applied simultaneously, a relaxation of the depolar ization is observed. Both KA and QA appear to act directly on horizontal cells (Rowe and Ruddock, 1981a, 1982a,b), so the action of pentobarbital must also be generated from a direct action on the horizontal cell membrane. Pento barbital partially reverses KA induced depolarizations in 2 + the presence of 2 mM Co (fig. 3.16.) and there is no significant difference in either degree of relaxation (mV) 2 + or rate of relaxation (mV/sec) in the presence of 2 mM Co or with PENT (5 mM) alone (Table 4.1.). As cobalt (2 mM) blocks synaptic transmission at chemical synapses these last results imply that pentobarbital does not hyper- 154
polarize horizontal cells through transmission via chemical synapses, thus its hyperpolarizing actions appear to occur at the neuronal membrane itself.
The depolarizations induced by the putative trans mitter, L-glutamate at a concentration of 25 juM when poten tiated by 3 mM D-aspartate, and also at a concentration of
5 mM, which requires no potentiation, are also relaxed by the application of 5 mM pentobarbital (figs. 4.17. and
4.18. ) .
Interactions between excitatory and inhibitory
GABA-ergic processes have been observed, as was reviewed in
Chapter 1 (section 1.6.4.). The GABA antagonist bicuculline does not however affect the action of pentobarbital in any way (fig. 4.19.), there being no significant difference between degree of relaxation or the rate of relaxation for retinae depolarized by excitatory amino acids, with or without bicuculline (Table 4.1.). Therefore, there appears to be no GABA-ergic influence on the relaxation of the kainate depolarization and this is consistent with the observation that the hyperpolarizing action of pento barbital on the horizontal cells is not sensitive to GABA antagonists. Furthermore, it has been shown previously that GABA antagonists do not reverse the depolarization of horizontal cells by 50 uM KA or QA (Hankins and Ruddock,
1984b).
Kynurenic acid applied at concentrations of < 1 mM is not effective as an antagonist against excitatory amino- 155
acids, although it does cause hyperpolarization of the horizontal cells. If pentobarbital acted in a similar manner to kynurenic acid, it would be expected to potentiate the action of the antagonist when this is applied at low concentrations. The experiment with 250 juM kynurenic acid shows, however, that this does not occur
(fig. 4.20.) and the effects do not differ significantly from those where pentobarbital is applied alone. Also, when compared with experiments where KA (50 /uM) and KYN
(1 mM) are applied without pentobarbital (fig. 4.21.), an obvious difference between the pentobarbital relaxation effect and the antagonistic action of kynurenic acid is observed. The specificities of the two drugs in their antagonistic effects also differ, as kynurenic acid fails to antagonise depolarization by 5 mM L-glutamate (Hankins and Ruddock, 1986), whereas pentobarbital causes a relaxation of the glutamate induced depolarization.
5 mM pentobarbital reverses 40 aim dopamine (DA) induced depolarizations and causes the membrane potential to become very noisy as is illustrated by the traces in fig. 4.22. The effects of dopamine are not antagonised by kynurenic acid (Hankins and Ruddock, 1986), thus the actions of pentobarbital and kynurenic acid are further differentiated by this observation.
Dopamine affects the spatial properties of the horizontal cell response by changing the lateral spread of
S-potentials mediated through gap junctions between 156
adjacent horizontal cells (Negishi and Drujan, 1979). The resulting noisy membrane potential indicates that the resistance between adjacent cells has increased, an action which may involve either dopamine or pentobarbital alone or the potentiation of the dopamine effect by pentobarbital.
In summary, this chapter has described and discussed experiments which have shown that pentobarbital has a hyperpolarizing action on the horizontal cell membrane in the vertebrate retina. On closer inspection, it was shown that pentobarbital causes a relaxation of the membrane depolarization induced by excitatory amino-acids and their agonists. The mechanisms mediating this and also the reversal of the dopamine induced depolarization of the horizontal cell membrane, will be discussed in a later chapter. Richards and Smaje (1976) reported that pentobarbital reduces post-synaptic membrane sensitivity to
L-glutamate, and this could provide an explanation for the observed effects. However, pentobarbital is far less specific than kynurenic acid regarding its action against kainic acid and dopamine, and there is a lack of synergism between the two agents, suggesting that the effects of pentobarbital are not mediated by any interaction with specific binding sites. 157
CHAPTER 5
THE IONIC BASIS FOR THE ACTION OF PENTOBARBITAL
ON THE HORIZONTAL CELLS OF THE ISOLATED FISH RETINA.
5.1. Introduction
In general, the barbiturates depress neuronal excitability and enhance GABA activity and there is evidence that this action involves the chloride (Cl ) ion channel, which is part of the GABA binding sit complex
(Chapter 2, section 2.6.). Likewise, the possible contributions of ions such as potassium (K+ ) and sodium
Na+) have also been studied (Chapter 2, section 2.6.) and it has been found that anaesthetic and sedative type drugs may increase the permeability of the neuronal cell membrane to either of these ions. In the previous chapter it was shown that pentobarbital has a hyperpolarizing effect on the horizontal cell membrane potential, in the fish retina preparation. In this chapter the ionic mechanisms mediating this action of pentobarbital are examined.
5.2. The Role of Extracellular Chloride (Cl ) Ions in
Pentobarbital Evoked Responses
The importance of the extracellular chloride (Cl ) ion concentration on pentobarbital hyperpolarization of the 158
horizontal cell membrane potential, was investigated by
2 - — substituting sulphate ions (SO^ ) for chloride ions (Cl ) in the perfusate (55 mM N a 2S04 was substituted for 110 mM
NaCl, and 1.25 mM K 2SC>4 for 2.5 mM KC1, to maintain the cation concentration of the perfusate). The omission of
Cl" ions from the Ringer solution, induces a slight hyper polarization followed by depolarization. When 5 mM pento barbital is added to the chloride-free Ringer, hyperpolari zation of the depolarized cell is observed (fig. 5.1.).
Cells which are depolarized in Cl -free Ringer by an average of 12.6 +4.8 mV (measured from the initial steady hyperpolarized level, as shown in fig. 5.2.), were subse quently hyperpolarized by an average of 5.1 + 1.1 mV, at an average rate of 0.2 + 0.1 mV/sec. (3 cells from 3 retinae) in 5 mM PENT (fig. 5.1.). When the preparation is subjected to a Cl -free perfusate and 5 mM PENT simultaneously, hyperpolarization is observed (fig. 5.3.) with an average value of 9.2 + 2.8 mV, and an average rate of 0.3 + 0.1 mV/sec. (5 cells from 5 retinae).
Although the hyperpolarization induced by 5 mM PENT in cells depolarized by Cl -free Ringer is significantly different from that induced by 5 mM PENT in normal Ringer, it was found that in experiemnts where 5 mM PENT was applied simultaneously with a Cl -free Ringer solution, the hyperpolarization induced by the barbiturate was not significantly different from that induced by 5 mM PENT in normal Ringer (Table 5.1. using the Student's t-test). Or 159
I 1 min |
Fig. 5.1» The effects of chloride-free (Cl -free) perfusate on the 5 mM pentobarbital (PENT) evoked hyperpolarization. Chloride ions were substituted by SO. so that the Ringer contained 55 mM N a 2SO^ and 1.25 mM K 2SO.. This Cl -free Ringer gives an initial hyperpolarizing effeet with suppression of the S-potentials, followed by a depolarizing response. The subsequent addition of 5 mM PENT hyperpolarizes the horizontal cell dark membrane potential, but to a level which is less than that obtained in a normal Ringer solution. When PENT is then removed from the perfusate the cell potential returns to the level seen in Cl -free Ringej. (Stimulus: 400 ms in duration, of 618 nm and 0.04 wW/min and spot diameter of 3 mm) .
> T
F i g . 5 . 2 . The depolarization of the horizontal cell dark membrane potential induced in Cl”-free Ringer was measured as shown above. A- is the membrane potential change (mV) taken from the point of maximum steady hyperpolarization (a) to the point of maximum steady depolarization (b). These measurements were used in other cases where hyperpolarization preceded depolarization (see text). 160
Y" WTTK
'' imTrrmTtrrrr ,Cf- free ,
5m M PENT
■ 1 min
F i g . 5.3. The effects of a Cl -free perfusate containing 5 mM PENT applied to the preparation (The Cl+-free ginger solution contained the concentrations of Na and K+ salts as stated for fig. 5.1.). Hyperpolarization occurs with a Cl”-free perfusate containing 5 mM PENT, with further hyperpolariz ation when pentobarbital is added in a normal Ringer solution. The degree of hyperpolarization induced by pento barbital in Cl”-free Ringer is not different from that induced in normal Ringer solution. (Stimulus as for fig. 5.1. ) .
Oi—
F i g . 5.4. The effect of doubling the perfusate chloride ion concentration on 5 mM pentobarbital (PENT) evoked hyperpolarization of the horizontal cell dark resting membrane potential. Depolarization occurs in 2 x Cl with a reduction in light evoked S-potential size, whilst the subseguent addition of 5 mM PENT evokes a relaxation of the horizontal cell membrane potential, to a level close to the resting membrane potential. (Stimulus as for fig. 5.1.). 161
Table 5.1. Comparison of experimental results using the Student's t-test. (Appendix 1 ).
Comparison Degrees of Calculated Tabulated freedom t t (P = 0 .05)
A . a . 23 2.95 1.714 b . 23 0.83 1.714
B . a . 23 0.70 1.714 b. 23 1.40 1.714
C . a . 23 1.27 1.714 b . 23 0.89 1.714
Key to comparisons:
A. = PENT (5 mM) vs PENT (5 mM) applied to cells depolarized in Cl -free Ringer, B. = PENT (5 mM) vs PENT (5 mM) in Cl~-free Ringer, C. = PENT (5 mM) vs PENT (5 mM) in Na+-free Ringer,
a. = membrane potential change (mV), b. = rate of membrane potential change (mV/sec). Thus pentobarbital has some hyperpolarizing action in
Cl -free Ringer, indicating that the effects seen are not mediated solely by changes in chloride ion conductance across the cell membrane.
Experiments were also carried out where the extra cellular Cl level was doubled by the addition of choline chloride to the perfusate. The effect of increasing Cl by a factor of 2 is a depolarization of the horizontal cell dark membrane potential by an average of 8.4 + 1.2 mV (6 cells from 6 retinae). This effect was previously observed in the roach by Hankins et al. (1985). The subsequent addition of 5 mM PENT relaxes this depolarization as illustrated in fig. 5.4. (relaxation of 5.8 + 2.9 mV).
5.3. Sodium (Na+) Substitution and Pentobarbital
Hyperpolarizations
When choline is substituted for sodium (Na+ ) ions in the Ringer perfusate, hyperpolarization of the horizontal cell membrane potential occurs (10 cells from 9 retinae).
When 5 mM pentobarbital is applied to cells already hyper- polarized by Na+-free Ringer, it further hyperpolarizes the horizontal cell membrane potential (5 cells from 5 retinae) with an average hyperpolarization of 8.1 + 1.6 mV, and average rate of 0.24 + 0.04 mV/sec. (fig. 5.5.). Table
5.1. shows that this hyperpolarization is not significantly different from that produced by pentobarbital in a normal 163
1 min
Or > J- > rfrrrrrrtTy " 2 0 - \ V
% 0-
Na+ FREE______. , 5mM PENT | 1 1 min
Fig. 5.5. The effect of a sodium-free (Na -free) perfusate on 5 mM pentobarbital activity. Horizontal cells perfused by Na -free Ringer in which Na salts were replaced by choline salts, show hyperpolarization of the horizontal cell dark membrane potential with loss of light evoked S-potentials, whilst addition of 5 mM PENT induces further hyper polarization of the membrane potential. (Stimulus as for f ig. 5.1.). 164
Ringer perfusate. From these results it could be concluded that as pentobarbital further hyperpolarizes cells already hyperpolarized by Na+-free Ringer, then its action is + independent of the Na ion concentration gradient.
5.4. Extracellular Potassium (K ) Concentration and
Pentobarbital Activity
5.4.1. The Effect on Pentobarbital Action of Increasing
Extracellular K+ Concentration
In order to investigate the possible relationship between the action of pentobarbital (5 mM) and extra cellular K+ concentration in the fish retina, recordings were made from horizontal cells in perfusates containing various concentrations of potassium (K+ ) ions, made by adding K+ salts to the normal Ringer solution, containing
2•5mM K+ .
Potassium chloride (KC1) at 75mM evokes a depola rization of the membrane potential with an average value of
11.1 + 2.2 mV (7 cells from 7 retinae). There is no hyperpolarization or relaxation of the depolarized level when 5 mM PENT is added to the perfusate (5 cells from 5 retinae). This is illustrated in fig. 5.6. which also shows the irreversibility of the effect produced by a high
K+ concentration, that is the cells do not return to their resting potential and no recovery of the S-potential is 165 O r -
> E > - 3 0 75 mM KCL
u5mM , P E N T 1 [ 1 min
F i g . 5.6, The effect of a high potassium ion (K+ ) concentration in the perfusate on the effect of 5 mM pentobarbital. 75 mM potassium chloride (KC1) evokes a depolarization of the horizontal cell dark membrane potential with loss of light evoked S-potentials. This depolarization is not altered by the subsequent addition of 5+mM PENT. The effect induced by a high concentration of K ions is usually irreversible. (Stimulus as for fig. 5.1.).
I 1 min
F i g . 5.7. The effect of a high potassium ion (K ) concentration in the perfusate on the effect of 5 mM pentobarbital using a potassium salt other than potassium chloride. 75 mM potassium carbonate (KHCO^) induces a depolarization of the horizontal cell dark membrane potential, after an initial hyperpolarization, with loss of the light evoked S-potentials. As with 75 mM potassium chloride (fig. 5.6.), no change in the membrane potential is
seen. Other potassium salts, including bicarbonate
(KHCOg) and sulphate (K2SC>4 ) were also added at a con
centration of 75 mM, and as with the chloride, these
potassium salts evoke depolarizations of the membrane
potential which are not reversed by 5 mM PENT (KHCO^/ shown
in fig. 5.7., 5 cells from 4 retinae; K 2SC>4 , 3 cells from 3
retinae). The depolarization of the membrane potential by
75 mM KC1, is also not reversed by the excitatory amino-
acid antagonist kynurenic acid at a concentration of 2 mM
(fig. 5.8., 3 cells from 3 retinae).
As already discussed in Chapter 4 (section 4.2.),
PENT (5 mM) hyperpolarizes the horizontal cell membrane in 2+ the presence of 2 mM Co , but with the addition of 75 mM
KCl (fig. 5.9.) this hyperpolarization is reversed, with an
average depolarization of 13.3 + 2.1 mV (6 cells from 6
retinae; the depolarization was measured from the steady hyperpolarized state in 5 mM pentobarbital, as shown in
fig. 5.2.). Reversal of the hyperpolarization induced by 2 2+ mM Co and 5mM PENT was also observed for higher concen
trations of KCl at 100 mM (fig. 5.10.a; 3 cells from 3
retinae) and 150 mM (fig. 5.10.b; 5 cells from 5 retinae). . . 2 + Cells hyperpolarized by 5 mM PENT in the absence of Co ,
are subsequently depolarized by 150 mM K+ (fig. 5.11.). -f* | Thus changing the K gradient across the cell membrane, by
increasing the extracellular concentration, consistently
suppresses the hyperpolarizing action of pentobarbital.
The fact that this is observed in the presence of 2 mM "30 depolarization induced by a perfusate containing 75 mM mM 75 containing perfusate a by induced depolarization The excitatory amino-acid antagonist kynurenic acid (KYN) (KYN) acid kynurenic antagonist amino-acid excitatory The oasu clrd (C) (tmls s o fg 5.1.). fig. for as (Stimulus (KC1). chloride potassium at a concentration of of concentration a at 5. . g i F All three traces show the effects of 75 mM potassium potassium mM 75 of effects the show traces three All chloride (KC1) on the h y p e r p o l a r i ^ t i o n induced by 5 mM mM 5 by induced n o i t ^ i r a l o p r e p y h the on (KC1) chloride PENT and 2 mM cobalt chloride (CO , used to synaptically synaptically to used , (CO chloride cobalt mM 2 and PENT 5.9. . g i F potassium ions (K ) which evoke a depolarization of the the of depolarization a evoke which ) (K ions potassium horizontal cell membrane potential. (Stimulus as for for as (Stimulus potential. membrane cell horizontal isolate the horizontal cells). Jtjie hyperpolarization Jtjie hyperpolarization cells). horizontal the isolate i. 5.1.). fig. nue b 5 M jlT n 2 M O s eesd y 7 mM by 5 reversed is CO mM PjjlNT 2 mM 5 and by induced V(m V! V( mV) VimV) -20 -30 301— 0 -3 8 . 0r- i r 0 *— m PN , PENT mM5 m PENT mM5 7 M K+ mM 75 . 2 mM is shown not to reverse the the reverse to not shown is mM m Co** mM2 M Co** mM2 167 5m K* mM75 1min | 1 min I min 1 168 A
I 1 min
-20 L 1 min. c 0r-
> E > -30L
F i g » 5.10. The effect of 100 mM and 150 mM potassium chloride (KC1) on the hyperpolarization induced by 5 mM pentobarbital (PENT) and 2 mM cobalt ^hloride (Co^+). A. 100 mM, B. 150 mM potassium ion (K ) concentration, both show that the hyperpolarization induced by 5 mM PENT ^nd 2 mM CoZ is reversed by the high concentration of K which evokes a depolarization of the horizontal cell membrane potential, as is seen with 75 mM K (fig. 5.9.). C. shows a similar effect on an amacrine cell. (Stimulus as for fig. 5.1. ) . 169
jl min | 1,50 mM K + r-^- 1
F i g . 5.11. The effect of 150 mM potassium chloride (KC1) on the activity of 5 mM pentobarbital (PENT) in the absence of cobalt chloride (Co ). The hyperpolarization of the horizontal dark membrane potential which is induced by 5 mM PENT in the absence of 2 mM Co , is reversed by 150 mM potassium ion (K ) concentration. (Stimulus as for fig. 5.1. ).
1 min
F i g . 5.12. 75 mM potassium chloride (KC1) prevents the relaxation effect of 5 mM pentobarbital (PENT) on depolarizations induced by 50 juM excitatory amino-acid agonist kainic acid (KA), an effect which ^as shown in Chapter 4 (fig. 4.14.). 75 mM potassium ion (K ) concentration and 50 urn KA added simultaneously to the perfusate induce a depolarization of the horizontal cell membrane potential with loss of light evoked S-potentials. This depolarization is not relaxed by the addition of 5 mM PENT to the perfusate. (Stimulus as for fig. 5.1.). 170
cobalt shows that no chemical synapses are involved. It 2 + could also be noted here that in the presence of Co / some cell recovery occurs when cells are returned to normal
Ringer solutions (eg. fig. 5.9.). It was shown in Chapter
4 (section 4.3.2.) that at 5 mM, pentobarbital relaxes the depolarization induced by 50 juM kainic acid (KA). However, in the presence of 75 mM KC1, this relaxation does not occur (fig. 5.12., 5 cells from 4 retinae), which provides further evidence that the hyperpolarizing action of pento- • ■}* t barbital is sensitive to changes in K concentration.
5.4.2 The Effects on Pentobarbital Activity of Potassium
Ion Channel Blockers
The experiments described in section 5.4.1 establish that changes in the K+ concentration are effective in modifying the hyperpolarizing actions of pentobarbital and attempts were made to modify this action by using the potassium ion channel blockers, 4-aminopyridine (Yeh et al., 1976) and tetraethylammonium chloride (Tasaki and
Hagiwara, 1975? Armstrong and Binstock, 1965? Hermann and
Gorman, 1981).
Experiments with 4-aminopyridine (4-AP) did not however yield significant modifications of the previous observations. Thus, in the presence of 4-AP (5 mM), the hyperpolarizing action of 5 mM PENT is still clearly observed (fig. 5.13., 4 cells from 4 retinae). The 171
-AO
Fig. 5*13. The effect of the potassium ion (K ) channel blocker, 4-amino-pyridine (4-AP) on the effects induced by 5 mM pentobarbital (PENT). A. 5 mM PENT in combination with 5 mM 4-AP produce a strong hyperpolarization of the horizontal cell membrane potential, with a reduction in the size of the light evoked S-potentials. B. The hyper polarization induced by 5 mM PENT is not affected by the addition of 5 mM 4-AP, although further hyperpolarization occurs. Results from both A. and B. appear to sh^w that the pentobarbital effect is not associated with K activity. (Stimulus for A and B as for fig. 5.1.). 172
efficiency of 4-AP in modifying the electrophysiological activity of the horizontal cells was therefore examined
independently of the pentobarbital action. 2 mM cobalt was added to the perfusate and this hyperpolarizes the horizontal cells towards their K+ equilibrium potential
(Kaneko and Shimazaki, 1975). It is apparent that 4-AP has 2+ little effect on the hyperpolarizing action of Co , causing only a small relaxation of the membrane potential
(fig. 5.14.a). Furthermore, a high extracellular K+ concentration (75 mM), added to the cells hyperpolarized by 2+ . 2 mM Co in the presence of 5 mM 4-AP, produces an immediate and rapid depolarization of the cell membrane
(fig. 5.14.b, observed in 4 cells from 3 retinae). Thus,
4-AP does not appear to be a particularly effective blocker + of K activity at the horizontal cell membrane and experiments employing this blocker were abandoned.
More interesting results were obtained with the second potassium channel blocker, tetraethylammonium chloride (TEA). TEA applied at a concentration of 5 mM 2 + with 2 mM Co produces a hyperpolarization as would be expected, but no further hyperpolarization is seen when 5 mM pentobarbital is added to the perfusate (fig. 5.15., 4 cells from 4 retinae), unlike those results seen in Chapter
4 (section 4.2.). More significantly, the hyperpolarization
induced by the barbiturate is reversed when TEA (5 mM) is added to the perfusate (fig. 5.16., 8 cells from 8 retinae)
and cells depolarized by 5 mM TEA are insensitive to 5 mM 173
B o
1 iflMfcJj-U > 5mM A-AP + 2mM Co' 'AO-
1 min
Fig. 5.14. A. shows the effect of 5 mM 4-amino-pyridine (4-AP) og+the hyperpolarization induced by 2 mM cobalt chloride (Co^ ), which hyperpolar^zes the horizontal cell towards its potassium ion (K ) equilibrium potential (Kaneko and Shimazaki, 1975). 5 mM 4-AP causes a sligh£+repolarization of the hyperpolarization induced by 2 mM Coz , but when 5 mM 4-AP is removed from the perfusate, the cell again hyperpolarizes the cell to the level induced by 2 mM Co^ . B. Both traces show that 75 mM potassium chloride (KC1) depolarize the horizontal cell membrane potential in the prj^ence of 5 mM 4-AP and 2 mM CoZ . 5 mM 4-AP and 2 mM Co hyperpolarize the cell with a large reduction in the light evoked S-potential, the addition of 75 mM KC1 produces a rapid depolaraization of the horizontal cell membrane potential. (Stimulus for A and B as for fig. 5.1. ). 174
> E
- 3 0
F i g . 5.15. The effect o£ 5 mM tetraethylammonium chloride (TEA) a potassium (K ) channel blocker, on the activity induced by 5 mM pentobarbital (PENT). 5 mM TEA and 2 mM cobalt chloride (Co to synaptically isolated the horizontal cells) produce a hyperpolarization of the horizontal cell membrane potential with a reduction in the light evoked S-potentials. The subsequent addition of 5 mM PENT induces a further hyperpolarization of only a few mV. (Stimulus as for fig. 5.1.).
5 mM TEA
1 min
F i g . 5.16. Hyperpolarization of the horizontal cell membrane potential, induced by 5 mM pentobarbital (PENT) is reversed by the addition of 5 mM tetraethylammonium chloride (TEA) to the perfusate. (Stimulus as for fig. 5.1.). 175
PENT, with no relaxation or reversal of the depolarized state (fig. 5.17., 5 cells from 5 retinae). These results demonstrate that TEA prevents pentobarbital from acting in its usual way, and further confirmation of this is obtained with experiments involving kainic acid (KA). It was observed that pentobarbital causes a relaxation of the depolarization induced by 50 uM KA (Chapter 4, fig. 4.15.), however, it is shown here that 5 mM TEA suppresses the effect (fig. 5.18., 3 cells from 3 retinae). This action 2+ of TEA persists in the presence of 2 mM Co , thus it is not dependent on chemical synaptic activity (fig. 5.19., 6 cells from 6 retinae).
5.5 Discussion
The results presented in this chapter were difficult to obtain, partly because of the instability of retinal electrical potentials, which accompanies prolonged exposure to Ringer solutions containing abnormal ionic concen trations. Consequently, a great many incomplete recordings were obtained and could not be used in the analysis, although almost invariably the results of these were consistent with the complete recordings, some of which were illustrated in the preceeding results sections. It was noted that after exposure to high concentrations of K+ , the retinal neurones rarely recovered their resting potential or responsiveness to light. Sometimes when cobalt was 176
O p
■20 j * y y"j"' * i^r 5mM PENT
1 min
Fig. 5.17. Depolarization of th» u induced bv 5 mM teh>. horizontal cell membrane potential reveled L reLxedre ^ h^ aT ° niUm Shl^ id? Fig. 5.18. Relaxation by pentobarbital (PENT) of the depolarization induced by the excitatory amino-acid agonist kainic acid (KA) as shown in Chapter 4 (fig. 4.14.) is not seen in the presence of tetraethylammonium chloride (TEA). Depolarization of the horizontal cell membrane potential by 50 juM KA in the presence of 5 mM TEA is not affected by the addition of 5 mM PENT. (Stimulus as for fig. 5.1.). 177 rrrtKV .2 mM Co"+ 50 juM . ■2. mM C o ^ 5 0 juM . 'KA + 5mM TEA 1 KA + 5mM TEA 5[mM_PE^T 5,nnM PENT H l m i n 0 > E > 2 mM Co+ + 50 juryl 2TnM Co"+ 50 'Is - 2 0 KA + 5 mM TEA juM KA + 5 mM TEA 5 mM PENT 1 -1 min ’f^ v T Y v rrrY -. > - 20L- + 5 mM ,5mM PENT i 1 min F i g . 5.19. No relaxation of the kainic acid (KA) induced depolari zation of the horizontal cell membrane potential by pentobarbital (PENT) in synaptically isolated cells (isolated by cobalt ions) occurs in the presence of tetraethylammonium chloride+(TEA). All 3 traces show that in the presence of 2 mM Co , the depolarization induced by 50 juM KA applied with 5 mM TEA is not relaxed or reversed by the subsequent addition of 5 mM PENT. (Stimulus as for fig . 5.1.). 178 added to the perfusate recovery was observed (eg. fig. 5.9.), but in most cases it was not. These later recordings were accepted for analysis, only if there was no upward drift in cell potential from the equilibrium value established under a high K+ concentration (eg. fig. 5.6.). A further complication lies in the fact that changes in ionic solution affect both the photoreceptors and the second order horizontal cells. Thus there is some ambiguity in the interpretation of the observed potential changes, although the results of the previous chapter have already established that the observed effects of pento barbital are localized at the membrane of the horizontal cell. Finally, it is assumed throughout the discussion that the ions studied can diffuse freely through the extracellular tissue. This assumption seems justified, as the larger molecules such as the excitatory amino acids appear to diffuse through the retina, at least to their binding sites on horizontal and amacrine cells. However, partitioning of ions can occur in the retinae, as has been shown for calcium ions by Galley et al. (1982). The results in this chapter describe the effects of ionic substitution and concentration changes of the basic Ringer solution, on the actions of pentobarbital which were discussed in Chapter 4. Three ions were investigated, — + + chloride (Cl ), sodium (Na ) and potassium (K ). The reported extracellular and intracellular concentrations of these ions are given in Table 5.2., together with the corresponding Nernst equilibrium potentials. 179 Table 5.2. Extracellular and intracellular concentrations and Nernst equilibrium potentials for K+ , Cl and Na+ . Ion + K+ Cl" Na CQ (mM) 4.6 128.0 145 (mM) 77.0 64.0 5-15 Ex (mV) -70.0 -19.0 + 50 -+6 5 C - extracellular concentration (mM) o - intracellular concentration (mM) Ex - Nernst equilibrium potential (mV) Values for K+ and Cl are mean measurements made with ion sensitive electrodes from type horizontal cells of the roach retina (Laming, 1982). Values for Na are those for a typical vertebrate cell (resting values). 180 The results for each ion will be examined separately, starting with K+ , which shows the clearest results. In every recording, the hyperpolarizing effects of pentobarbital were abolished by raising the extra- cellular K concentration (eg. fig. 5.6.). Under such conditions, the equilibrium potential established for the new concentration gradient is unaffected by the addition of pentobarbital to the perfusate. Thus, it is concluded that the hyperpolarization induced by pentobarbital in normal Ringer reflects a change in the membrane conductances causing a shift in the membrane potential towards the K+ equilibrium level. It has been shown that when the neuro transmitter release from photoreceptors is blocked with 2 + Co , the membrane potential of the horizontal cell shifts towards the K Nernst potential (Kaneko and Shimazaki, 1975), although in a series of measurements on 35 cells, Hankins et al. (1985), found that 2mM cobalt reduced the cell potential to an average value of -58 mV, whereas the theoretical Nernst potential is -70 mV. The results discussed in the previous chapter establish that pento barbital does not interact specifically with the depolari zation induced by the excitatory amino-acids, thus its ability to shift the membrane potential towards the K+ equilibrium potential must be mediated by changes in the ionic conductances of the cell membrane, so that the K+ conductance (g„) dominates. This could be achieved either by an increase in gvf or a decrease in the conductances of K 181 other ions. Experiments using the specific potassium channel blocker, tetraethylammonium chloride (TEA), show that under a variety of conditions it suppresses the hyperpolarizing action of pentobarbital (figs. 5.15., 5.16. and 5.17.). It is concluded, therefore, that a major contribution to the hyperpolarization of the cell membrane by pentobarbital is due to a direct action on the potassium channel, causing an increase in gR. Abolishing the extracellular Na+ concentration by substitution with choline, hyperpolarizes the horizontal cells, as would be expected on two counts, firstly, photo receptors maintain their depolarized state in darkness by allowing Na+ to leak across the cell membrane of the outer segment (Chapter 1, section 1.4.2.) and so removal of extracellular Na+ ions should hyperpolarize the photo receptors and by suppressing release of neurotransmitter, so hyperpolarize the horizontal cells (Trifonov, 1968). Secondly, the action of excitatory amino-acids in depola rizing the post-synaptic membrane is usually attributed to the opening of Na+ channels (Burle et al., 1978), and this appears to be true of retinal horizontal cells, both in the intact retina and in isolated cells (Waloga and Pak, 1975; Ishida et al., 1984). It should be noted, however, that Hankins et al. (1985), concluded that the excitatory action of the excitatory amino-acids on the horizontal cell mem brane in the roach preparation, can only be partially attributed to their actions on Na+ channels and both in the 182 previous and this present study, a reduction in the extra cellular Na+ concentration fails to hyperpolarize the 2+ horizontal cells to the same extent as Co and induces a membrane potential which is more positive than the E... If pentobarbital influences the sodium channels and sodium ion conductance (gNfl), it must do so by blocking or closing the channels and so decreasing gNa, as opening the channels would give rise to a depolarization of the cell membrane. The hyperpolarization by pentobarbital in a Na+-free solution (fig. 5.5.) is consistent with a change in mem brane potential towards the potassium Nernst potential, independent of the activity of Na+. Chloride ions have been shown not to be in equili brium across the horizontal cell membrane in dark adapted retinae (Laming, 1982), as the Nernst potential is some 14mV more positive with respect to the dark equilibrium potential of the cells. Thus it was proposed that Cl ions are pumped into the cell, in order to maintain the observed concentration gradient, which was found to be relatively constant for different cells, although the absolute intra cellular concentration (Ci) and extracellular concentration (Co) for Cl varied greatly from cell to cell. There is however, no evidence as to the value of gc^, thus the level of chloride leakage and the degree of pumping required are unknown. As with Na+ the hyperpolarizing action of pentobarbital cannot, therefore, involve an increase in gc l, which would tend to depolarize the cells. However, 183 blocking the chloride channels would tend to increase the relative efficiency of the potassium conductance and there fore would lead to a hyperpolarization of the horizontal cells. Picrotoxin (Pi) a selective chloride channel blocker, fails to antagonise the effects of pentobarbital (fig. 4.5.), but as any activity of pentobarbital mediated by Cl must be to block the chloride channel, no antagonis tic action should be expected. Hyperpolarization of the cell membrane by pentobarbital persists both in the absence of extracellular Cl ions (figs. 5.1. and 5.3.) and in double the normal extracellular concentration of Cl ions (fig. 5.4.), which is consistent, as before, with the membrane shifting towards the potassium equilibrium poten tial. Some variation in the degree of hyperpolarization is observed, depending on whether the pentobarbital is added to the preparation some time after the switch to Cl -free perfusate (fig. 5.1.), or as soon as the cell is subjected to Cl -free Ringer (fig. 5.3.). In the latter case the hyperpolarization is of a smaller value than that seen when the cell is depolarized initially in Cl -free Ringer, and this suggests that Cl ions may have some influence on the membrane response to pentobarbital and therefore it may be an over-simplification to attribute activity solely to changes in K+ ion activity. In summary, the data obtained from ion substitution experiments suggests that the most significant effect of pentobarbital is to increase gR across the cell membrane 184 relative to other ionic conductances, causing the cell membrane potential to shift towards the ER value. This is in agreement with results obtained by Nicoll and Madison (1982) using a variety of anaesthetics on frog and rat neuronal preparations. The non-specific action of pento barbital in the roach retina preparation is consistent with results obtained in the previous chapter, showing that pentobarbital causes relaxation of the membrane depolari zation induced by a variety of excitatory substances. The possibility that the Cl channels are blocked in some way by pentobarbital cannot be excluded, as this would tend to lead to similar results as those seen, but the results with TEA imply that this cannot be the primary action. It should also be noted here that although the experiments discussedm have treated each ion in isolation, this does not occur physiologically and an increase or decrease in one ionic conductance will influence other ion activities. Therefore, the underlying ionic mechanism for the pentobarbital effect might well involve changes, directly on one ionic conductance and indirectly on another. The conclusions made in this discussion could be further confirmed by measuring changes in the cell membrane resistance during drug application. However, as discussed in Chapter 1 (section 1.3.), there is extensive electrical coupling between horizontal cells, which unlike chemical 2+ synapses cannot be blocked by Co , thus there is always interaction between horizontal cells which influences cell 185 membrane resistance and so unless the cells are isolated, individual cell membrane resistance cannot be measured. As cell isolation methodology is beyond the scope of this present project, experiments to measure membrane resistance were not pursued. 186 CHAPTER 6 AN INVESTIGATION OF THE EFFECTS OF A NUMBER OF 'SEDATIVE1 TYPE DRUGS ON THE HORIZONTAL CELLS OF THE VERTEBRATE RETINA 6.1. Introduction In the preceding chapters, I have demonstrated that pentobarbital causes hyperpolarization of the horizontal cell membrane potential. In this chapter, I describe experiments in which a number of 'sedative' type drugs were applied to the retina, in order to investigate their effects on the horizontal cell membrane potential. Some estimate of the order of potency for these 'sedatives' was obtained and this was then compared to the anaesthetic potencies of the 'sedatives'. (The anaesthetic potencies are measured in vivo, as the concentration of drug required to induce anaesthesia, in many species of animal, see Barnes and Eltherington, 1973). The 'sedative' drugs used were phenobarbital and hexobarbital, barbiturates of long and medium duration respectively, which are used clinically as sedatives? -chloralose and urethane, both used as laboratory animal anaesthetics? chloral hydrate a clinical sedative and ethanol, which in high concentrations can induce anaesthesia. 187 The following sections describe experiments aimed at first looking at the effects of each of the 'sedatives' on the horizontal cell membrane potential, and then measuring the degree of the effects seen by comparing them with those induced by pentobarbital. 6.2. Comparision of the Effects of 2 mM 'Sedative' with those Induced by 2 mM Pentobarbital. The experimental procedure adopted to measure the effect of the 'sedative' against that of pentobarbital, was to apply 2 mM 'sedative' to the preparation, followed by 2 mM pentobarbital. It was always found that at 2 mM, the 'sedative' induced a degree of hyperpolarization which was less than the hyperpolarization seen when 2 mM pento barbital was also applied to the preparation. This method was used to eliminate the problems of measuring the absolute concentration of 'sedative' which just induces a hyperpolarization of the cell membrane potential, and also eliminates variability in cell sensitivity. Fig. 6.1. illustrates the measurements used to calculate the efficiency of the 'sedative* compared to that of pento barbital. The hyperpolarization induced by the 'sedative' at a concentration of 2 mM, is taken as a percentage of the total hyperpolarization, which includes that induced by 2 mM pentobarbital. 188 Fig. 6.1. Diagram to show measurements to assess hyperpolarization of the horizontal cell dark resting membrane potential by 'sedative' and pentobarbital. A. Hyperpolarization (mV) induced by 2 mM 'sedative^. B. Hyperpolarization (mV) induced by 2 mM pentobarbital. C. Total hyperpolarization (mV). TTnTmttTmtttt^ ^ ... "20*- 2mM.„ETH______A I 1 mm _____| I— ’ Fig. 6.2. . oentobarbital (PENT Comparison of ethanol (ETH, 2 mM) wit msec duration, 618 2 mM).. Stimulus^ Light flashes of nm, 0.04 uW. mm and 3 mm spot diamet 189 Two main assumptions are made: 1. That no interaction occurs between the 'sedative' and pentobarbital which could either enhance or inhibit the action of pentobarbital, 2. That the hyperpolarization induced by the 'sedative' and by pentobarbital is a linear effect. How ever this is never the case, because as the membrane potential approaches the new ionic equilibrium potential produced by the 'sedative', the rate of hyperpolarization decreases, thus producing a non-linear effect. Fig. 6.2. to 6.7. show the effects of the various drugs studied at a concentration of 2 mM. Ethanol (fig. 6.2.), hexobarbital (fig. 6.3.), chloral hydrate (fig. 6.4.) and urethane (fig. 6.5.) hyperpolarize the cells by between 20.0 - 30.0% of that induced by pentobarbital (values range from 19.0% for ethanol to 31.0% for urethane). Phenobarbital (fig. 6 .6 .) and o(-chloralose (fig. 6.7.) both have a strong hyperpolarizing effect on the horizontal cell membrane. The hyperpolarizations were 56.0% and 93.0% of the pentobarbital induced effect for phenobarbital and o<. -chloralose respectively, at concen trations of 2 mM. All 'sedatives' have some effect on the light induced S-potentials. Table 6.1. and Graph 6.1. show that the efficiency of the 'sedative* drugs used in comparison with pento barbital in the isolated vertebrate retina partially follows the order in which the drugs are placed according 190 , 2mM PENT , I 1 min F i g . 6.3. Comparison of hexobarbital (HEXO, 2 mM) with pentobarbital (PENT, 2 mM). Stimulus as for fig. 6.2. F i g . 6.4. Comparison of chloral hydrate (CHL HYD, 2 mM) with pento barbital (PENT, 2 mM). Stimulus as for fig. 6.2. 191 0 - TTUnTHT^TTr^s, - 20- ■ i1. i. i: i i!: ’ > i.:!;;: • ’T’f' > ...... I _ i h-Lmio___ | J_mio_ Comparison of urethane (2 mM) with pentobarbital (PENT 2 mM). Stimulus as for fig. 6.2. > £ rfiiiiiiHnimintitiiifl, 0 ! 2 mM PHEfJO | 2 mM PENT| 2 mM PENT 1 min > 0 E • (tttut -20 0 mM PHENO -AO 7 mM PEtyT I 1 min r ly • v • o • 9 x Comparison of phenobarbital (PHENOr 2 mM) with pento- barbital (PENT/ 2 mM). Stimulus as for fig. 6.2. 192 Or- 1 min > E -20 2mM o-CHL 1 min -AO 2mM PENT 0 > E -20 -AO F i g . 6 .7. Comparison of -chloralose («*-CHLOR, 2 mM) with pentobarbital (PENT, 2 mM). Stimulus as for fig. 6.2. 193 Table 6.1. Comparison of the hyperpolarization induced by 2 mM of drug 'x1 with that for 2 mM pentobarbital. Drug 1x ' xX X PENT Total xX expressed SE + (mV) (mV) (Mv) as % of total oi -Chloralose 8.5 0.7 9.2 92.0 3.0 Phenobarbital 6 • 6 5.2 11.8 56.0 4.6 Urethane 5.4 11.8 17.2 31.5 4.0 Chloral Hydrate 2.8 6.9 9.7 29.0 4.8 Hexobarbi tal 1.5 5.5 7.0 21.5 2.0 Ethanol 1.8 7.2 9.0 20.0 5.7 n = 6 xX(mV) = average hyperpolarization induced by 2 mM drug ' x' xPENT(mV) = average hyperpolarization induced by 2 mM PENT. ETH ethanol HEXO hexobarbi tal CHL HYD chloral hydrate 100 •Pent URE urethane PHENO phenobarbi tal [occhlor -CHLOR c* -chloralose % pent hyPer- 80 polarization 60 Pheno 40 2 0 iHexo Eth i------,------1------0 I 2 log anaesthetic cone (mM) GRAPH 6 . 1 . Hyperpolari zation induced by sedative 'x' as % of the hyperpolarization induced by pentobarbital (PENT) vs. log anaesthetic concen brations (mM). 195 to their anaesthetic concentrations. Deviants from this order are phenobarbital and urethane which appear to be more efficient in hyperpolarizing the retinal horizontal cells than the anaesthetic concentration might imply, and hexobarbital which is less effective in the retina than would be expected. The simple calculation used in Table 6.2. converts the dose of anaesthetic in mg/kg (Barnes and Eltherington, 1973) for a laboratory rat, directly to a concentration in mM/kg of tissue. This does not take into consideration a number of parameters which determine that the concentration of anaesthetic required to induce in vivo anaesthesia, is greater than that which would produce an inhibitory effect in vitro. These include: 1. The ability of the anaesthetic to reach target cells via the extravascular space and distribute itself throughout the whole body (Richards, 1972). This would be determined by the lipid solubility of the drug. 2. The uptake of the drug by various tissues (Richards, 1972) eg. fat depots. 3. The breakdown of the drug by various means, within the body of the animal. Therefore, the anaesthetic concentrations calculated in Table 6.2. are greater than would be required to induce inhibition in an in vitro situation, although the order of potency should remain the same. 196 Table 6.2. Anaesthetic concentrations of the *s e d ative' drugs investigated calculated from their anaesthetic doses. Drug Anaesthetic Molecular Anaesthetic Dose Weight Concentration (mg/kg) (g) (mM/kg) Pentobarbital 25 248.3 0.1 o • oC -Chloralose 55 309.5 to Hexobarbital 75 236.3 0.3 Phenobarbital 100 232.2 0.4 -» k . Chloral hydrate 300 165.4 00 . Urethane 780 89.1 00 Ethanol 6000 46.0 130.0 The anaesthetic doses for the above drugs in the laboratory rat were obtained from Barnes and Eltherington, 1973. Doses for o^-chloralose, hexobarbital, chloral hydrate and ure thane were applied via intravenous administration, whilst pentobarbital and phenobarbital were applied by intra- peritoneal administration. These two routes were assumed to be equally efficient. The dose for ethanol was applied via oral administration and so is of a higher value than would be used for intravenous or intraperitoneal admini stration. Bowman and Rand (1980) stated that a 7-7.5% solution (corresponding to a 1.63 M solution), injected intravenously, was effective at inducing anaesthesia in man 197 6.3. Estimation of the Concentration of Pentobarbital Required to Produce a Similar Degree of Hyper polarization as 2 mM 'Sedative'. As in section 6.2. the 'sedative' drug was applied to the retinal preparation at a concentration of 2 mM, and this induced some degree of hyperpolarization. Pento barbital was then applied at various concentrations starting with 2 mM and diluting down until no further hyperpolarization was observed from that induced by 2 mM 'sedative* (Only hyperpolarizations were considered and not changes in the light induced S-potentials). Due to inaccuracy introduced by attempting to dilute the pento barbital to very low concentrations, pentobarbital was applied in 0.1 mM gradations, and so this gives only a quantified measure of the relative order of potency of the 'sedatives' in the retina. Figs. 6 .8 . to 6.12. illustrate the results presented in Table 6.3., which show that there is a trend which follows that seen for the anaesthetic activity of the drugs, with oc -chloralose more potent in its actions than the other 'sedatives' investigated. The drugs can be placed in the following order of decreasing effectiveness: phenobarbital, hexobarbital, chloral hydrate and ethanol. The concentrations of pentobarbital required to match the hyperpolarization induced by 2 mM 'sedative' were : -chloralose, 2 mM (fig. 6 .8 .)? phenobarbital, 0.2-0.5 mM (fig. 6.9.); hexobarbital, 0.1-0.2 mM 198 0 E > -20 mtrtTfrrrtttitntrrrmtT^ 0 |5 m M gx- Qj5 mM,c<- ,0.5 mM cx- - 4 0 CHLOH WiUOR 1 CHLOR ,0.5 mM i ,0.5 mM , , 1 min PENT PEN' Fig. 6 .8 . a. 2 mM oc-chloralose ( oC -CHLOR) compared with 2 mM pentobarbital (PENT). b. 0.5 mM oc-chloralose (<*. -CHLOR) compared with 0.5 mM pentobarbital (PENT). (Stimulus as for fig. 6.2.) > ttltm tlttltlttmti tm tH H tm t -20 I 1min 'PHENO !n.,0-5mM PENT m T 1^ ^ TTTTTITT[Tm TTTmrTTTTTTTiTvrvTrmii-rri-.-n , 2mM FtiENO______| I 1min | [. 0-2mM PENT Fig. 6.9. a. 2 mM phenobarbital (PHENO) compared with 0.5 mM pentobarbital (PENT) b. 2 mM phenobarbital (PHENO) compared with 0.2 mM pentobarbital (PENT) (Stimulus as for fig. 6.2.) a I f P m M F F N T TrmnnmnnnmnmfimffltfflttrattiiittmthhWtrttrm^ ] 2mM HEXO , 1 min H j Q-1 mM PENT F i g . 6.10. a. 2 mM hexobarbital (HEXO) compared with q %2 m pentobarbital (PENT) % mw b. 2 mM hexobarbital (HEXO) compared with o, 1 mM pentobarbital (PENT) (Stimulus as for fig. 6.2.) 1 min D Or- | hiiiTuinnirrTttTi ninmtrttTHiTtttttT ttttnu([trfmf m t > " 20*— i2mM CHL. HYD, 0-1 mMPEMT H 1 min F i g . 6.11. a. 2 mM chloral hydrate (CHL HYD) compared with 0.2 mM pentobarbital (PENT) b. 2 mM chloral hydrate (CHL HYD) compared with 0.1 mM pentobarbital (PENT) (Stimulus as for fig. 6.2.) , OlmM PENT, F i g . 6.12. a. 2 mM ethanol (ETH) compared with 0.2 mM pentobarbital (PENT) b. 2 mM ethanol (ETH) compared with 0.1 mM pentobarbital (PENT) (Stimulus as for fig. 6.2.) 201 Table 6.3. Concentrations of pentobarbital which induce similar effects to 2 mM of drug 'x1. Drug 'x' Concentration of PENT required to (2 mM)______induce a similar effect (mM) o( - Chloralose < 2 o LD Phenobarbital 0.2 - • Hexobarbital 0.1 - 0.2 Chloral hydrate 0.1 E th a n o l 0.1 202 (fig. 6 .10.)? chloral hydrate (fig. 6 .11.) and ethanol (fig. 6.12.), 0.1 mM. Because these experiments do not require comparison of the different degrees of hyperpolarization induced by both 'sedative' and pento barbital, as was the case in section 6 .2., no linearity assumption is required. 6.4. Discussion. The aim of the experiments presented in this chapter was to look at the effects, in the vertebrate retina, of a number of drugs which have 'sedative' activity, and attempt to form some order of potency which could then be compared to the order of anaesthetic potencies or concentrations of these drugs. It was found (figs. 6.2. to 6.12.) that all the 'sedative' drugs investigated induce a hyperpolarizing effect on the horizontal cell membrane at a concentration of 2 mM, but this varies according to the 'sedative' investigated. The order in which the 'sedatives' can be placed depending on the degree of hyperpolarization seen, partially follows their anaesthetic concentrations (Graph 6.1.). The main discrepancy occurs for urethane and phenobarbital which are more effective, and hexobarbital which is less effective, than would be expected from the anaesthetic concentrations. 203 All the •sedatives* investigated are less effective in their actions than a similar concentration of pento barbital (figs. 6.2 to 6.7.). There is little information regarding relative potencies of these drugs, although pentobarbital has been shown to be 2-3 times more potent than phenobarbital in cultured spinal cord neurones (Macdonald and Barker, 1978a) and phenobarbital, unlike pentobarbital, is unable to enhance GABA-ergic activity (Iadarola et al., 1985). Data from the experiments in which pentobarbital concentrations were adjusted (Table 6.3.), gave results similar to those found in the experiments discussed above, with the hyperpolarization induced by 2 mM o4 -chloralose matched by a higher concentration of pentobarbital (fig. 6 .8 ., concentrations up to 2 mM), than the other 'sedatives' investigated (fig. 6.9., phenobarbital, 0.2-0.5 mM; fig. 6.10., hexobarbital, 0.1-0.2 mM? fig. 6.11., chloral hydrate and fig. 6.12., ethanol 0.1 mM). The assumptions made in analysing these experiments were stated in section 6 .2 ., the main one being that no interaction occurs between the 'sedative' and pento barbital. As anaesthetics/sedatives induce similar effects, this might not be true, although as the inhibitory effects can be produced in a number of ways, synergism may not necessarily occur. Ethanol, however, is known to act synergistically with hypnotics and sedatives, including the barbiturates (Bowman and Rand, 1980) and so there may be 204 enhancement of the total hyperpolarization seen which includes both hyperpolarization of the 'sedative' and hyperpolarization induced by the barbiturate. Table 6.1. does not provide evidence for synergistic interaction, however, the total hyperpolarization induced by pento barbital and ethanol is 9.0 mV, which is if anything less than the value of 11.0 mV for pentobarbital alone. In chapters 4 and 5, it was found that the pentobarbital action on horizontal cells does not appear to involve GABA-ergic or associated Cl activity, whereas ethanol has been found to interact at sites on the GABA receptor complex near or at the Cl” ionophore (Mendelson et al., 1985). The lack of synergism between the two drugs in this preparation may therefore reflect the non-GABA-ergic nature of pentobarbital activity. From Table 6.1. the values of the total hyperpolari zation ('sedative' and pentobarbital), which do appear to differ significantly from those expected for pentobarbital alone, are hexobarbital (7.0 mV) and urethane(17.2 mV). There may, therefore, be some interaction between hexo barbital and pentobarbital which reduces the effectiveness of 2 mM pentobarbital and, conversely, an enhancement of pentobarbital activity by the 'sedative', urethane. Urethane has been reported to depress selectivity post- synaptic excitatory mechanisms (Barker and Gainer, 1973) and hyperpolarize neurones by increasing their potassium conductance (Nicoll and Madison, 1982). In chapters 4 and Z05 5 it was shown that these effects also appear to be shared by pentobarbital, thus there may be some enhancement of the barbiturate activity via these mechanisms. In summary, the results discussed in this chapter have shown that a variety of 'sedative' type drugs can produce hyperpolarization of the horizontal cell membrane potential, and the degree to which this occurs for each 'sedative' investigated essentially follows the order in which the 'sedatives' can be ranked according to their anaesthetic concentrations. Z06 CHAPTER 7 THE ACTION OF THE BENZODIAZEPINE, FLURAZEPAM ON HORIZONTAL CELLS OF THE ISOLATED FISH RETINA 7.1. Introduction The benzodiazepines, including flurazepam, were discussed in Chapter 2 (section 2.3.) and in common with the barbiturates they are characterised by sedative type activity which is believed to be mediated primarily by interaction at the GABA binding site complex. In Chapter 1 , it was shown that there is much evidence to suggest that a benzodiazepine receptor site is associated with the GABA receptor complex, which may have a regulatory or modulatory function (section 1.6.4). From electroretinogram studies (erg), made on the rat retina, it was concluded that the benzodiazepines do not interact at the preganglionic cell level, but there is an inhibitory effect on the spike forming ganglion cells (Ikeda and Robbins, 1986a) and recent binding studies have shown, that in the primate retina, benzodiazepine activity is specific to the inner plexiform and inner nuclear layers (Mariani et al., 1987). The experiments described in this chapter investigate the electrophysiological effects of the benzodiazepine, flurazepam, at the horizontal cell layer. 207 7.2. The Effects of Flurazepam on Retinal Horizontal Cells Electrophysiological effects of flurazepam (FLUR), on the neuronal activity of the horizontal cell layer were examined with microelectrodes, using the isolated retina preparation. Responses to flurazepam were measured as for pentobarbital in Chapter 4 (see fig. 4.1.). At a concentration of 2 mM, flurazepam induces a hyperpolarization of the horizontal cell dark resting potential (fig. 7.1.), with an average value of 11.1 + 1.8 mV, and an average rate of 0.17 + 0.03 mV/sec; n = 11. When compared to the average hyperpolarization induced by 5 mM pentobarbital (Chapter 4, section 4.2., where hyper polarization was 11.0 + 1.1 mV and rate of hyperpolariza tion was 0.19 + 0.05 mV/sec.), both degree and rate of hyperpolarization are not significantly different for the two drug solutions (Table 7.1. using the Student's t-test). However, in comparison with pentobarbital, flurazepam produces a smaller reduction of the light evoked S-potential and on removal of the benzodiazepine from the perfusing solution, full recovery from the hyperpolariza tion is usually observed. Flurazepam at a concentration of 2 mM, applied to cells already hyperpolarized by an average of 8.80 + 0.96 7 + mV in 2 mM cobalt (Co ), induced a further hyperpolariza- tion of less than 1 mV (fig. 7.2.; 5 cells from 5 retinae), 208 0 > E > '20 *+**+*+*+mHHUH 2mM FLUR [^2mM-ELUR ljuin On “20 Fig. 7.1. 2 mM flurazepam (FLUR) hyperpolarizes the horizontal cell dark resting potential and reduces the size of the light induced S-potentials. Once flurazepam is removed from the perfusate the potential reverts almost to its resting level and there is some recovery of the light induced S-po£entials. (Stimulus: 400 ms duration; 618 nm, 0.04 ju Wmm , spot diameter 3 mm). 209 Table 7.1. Comparison of experimental results using the Student's t-test (Appendix 1). Comparison Degrees of Calculated Tabulated freedom t t(P=0.05) A . a . 29 0.56 1.699 b . 29 0.390 1.699 Key to Comparisons: A. = Flurazepam (2 mM) vs Pentobarbital (5 mM) a. = membrane potential change (mV) b. = rate of membrane potential change (mV/sec) '20 7 , 0 0 V(mV) - ^ | | ^ i m t f r f r r r r S-potentials and the level of the dark resting potential. potential. induced resting dark light the of of level recovery the some and shows cell the S-potentials perfusate, barbital. When Coz and flurazepam are removed from the the from removed are pento mM 5 flurazepam with and seen that Coz unlike When v ^ i t za i r a l barbital. hyperpo of addition of 2 M flurazepam (FLUR) does not affect the level level the affect not does (FLUR) flurazepam M 2 of addition The synaptic blocker cobalt (Co ) at 2 mM, hyperpolarizes hyperpolarizes mM, 2 at ) (Co cobalt blocker synaptic The the horizontal cell dark resting potential with a reduction reduction a with potential resting dark cell horizontal the in the size of the light induced S-potentials. The The S-potentials. induced light the of size the in 1- 2- ,+ - -2 7 F19- Siuu: s o fg 7.1.). fig. for as (Stimulus: Co' M m 2 FLURi R U L F M m 2 +•*- ^ r r r ^ n r r r r v r ^ 1 min M Co m 2 + + 2mMFLlJR 211 compared to 9.39 + 1.53 mV seen with 5 mM pentobarbital (Chapter 4, fig. 4.3.). This result gives no indication as to whether or not flurazepam acts directly on the horizontal cell membrane, in contrast to the case of pento barbital, where the further hyperpolarization of the mem brane potential in the presence of the synaptic blocker 2+ Co , indicates the direct action of the drug. The effect of 2 mM flurazepam on photoreceptor activity was investigated by recording the trans-retinal potential, where post-photoreceptor cell activity (horizontal and bipolar cells) had been blocked by the application of 50 juM KA. Fig. 7.3. shows the change in photoreceptor response to the application of a. 50 juM KA; b. 5 0 juM KA and 2 mM flurazepam? c. 50 jum KA, 2 mM fluraze pam and 1 mM ouabain, for comparison. Table 7.2. shows the values obtained from 3 experiments. 90 seconds after application of the benzodiazepine the photoreceptor response was reduced by an average of 52% from that seen in 50 juM KA, measured 45 seconds after application, and by an average 82% in 1 mM ouabain and 2 mM flurazepam, 60 seconds after application. Although there may be some general reduction in the photoreceptor response with time, the results indicate that there is some interaction between the benzodiazepine and photoreceptor activity. If this interaction is inhibitory, as has been indicated, it will affect neurotransmitter release from the photoreceptors and hence horizontal cell activity. min F i g . 7.3. Transreceptor recordings made across the photoreceptors, using the method outlined in Chapter 3 (section 3.5.). A- shows photoreceptor response in normal Ringer perfusate; B- shows photoreceptor response in 50 jliM KA, 45 seconds after application; C- shows photoreceptor response in 50 pM KA and 2 mM flurazepam, 90 seconds after application; D- shows photoreceptor response in 50 jjM KA, 2 mM flurazepam and 1 mM ouabain, 60 seconds after application. (Stimulus: as for fig. 7.1., but spot diameter 4 mm). 213 Table 7.2. Measurement of the photoreceptor response using transretinal recordings. Solution Size of Photoreceptor response Average in mV (% reduction from that ______seen in 50 yM KA)______% A 07 Q9 2.0 — B 0.6 Q9 1.4 - C 0.4 (33%) 04 (55%) Q5 (64%) 52% D 0.1 (83%) 02 (78%) 02 ( 8 6 %) 82% Solution A = Ringer? B = 50 pM KA, measurement taken 45 secs after start of application; C = 50 pM KA + 2 mM flurazepam, measurement taken 90 secs after start of application; D = 50 pM KA, 2 mM flurazepam and 1 mM Ouabain, measurement taken 60 secs after start of application. 214 7.3. Investigation of the Nature of the Depression of Neuronal Activity by Flurazepam Evidence from numerous studies made with the benzodiazepines (reviewed in Chapter 2), indicates that the effects produced by these drugs are mediated by interaction with inhibitory neuronal activity. Little evidence has so far been found to indicate that the benzodiazepines disturb excitatory mechanisms. The following sections describe experiments designed to investigate the effects of fluraze pam on the depolarization of the horizontal cell membrane by the excitatory amino-acid agonist kainic acid, and on inhibitory activity induced by the inhibitory neurotran smitter, # -amino-butyric acid (GABA). 7.3.1. Effect on Excitatory Activity As discussed elsewhere in this thesis, the excitatory amino-acids and their agonists, depolarize retinal horizontal cells with a reduction or loss of the light evoked S-potentials. The depolarizing action is maintained for the duration of the drug application without desensitization of the neuronal membrane (Ishida et al., 1984) . Experiments were performed to measure the effects of 2 mM flurazepam on the depolarization induced by the excitatory amino-acid agonist kainic acid (KA) at a concen 215 tration of 50 juM. It was demonstrated in Chapter 4 (section 4.3.2. ) that the barbiturate pentobarbital, at a concen tration of 5 mM, relaxes the KA induced depolarization by an average of 4.20 + 1.67 mV (fig. 4.15.). Fig. 7.4. shows that in contrast, flurazepam at a concentration of 2 mM, does not reverse or relax the kainate induced depolariza tion (4 cells from 4 retinae). This was also seen in 2 + horizontal cells synaptically isolated by 2 mM Co and exposed to 50 juM KA, which causes a depolarization of the horizontal cell membrane potential, whilst the subsequent addition of 2 mM flurazepam, does not relax or reverse this depolarization (fig. 7.5.). This was observed in 7 cells from 7 retinae. If flurazepam is acting by an interaction with excitatory mechanisms then it would be expected that some reversal or relaxation of the depolarization induced by the excitatory amino-acid agonist might be observed, as was the case for pentobarbital. This is not seen, however, so it may be that flurazepam's action on the horizontal cells is mediated by its interaction with an inhibitory mechanism, and this possibility is examined in the next section. 7.3.2. Effect on Inhibitory Activity As already stated, various studies have shown that the benzodiazepines act by interactions at the GABA- receptor complex. To test this in the vertebrate retinal 216 0 1 > •20 ■40 Or- I 1 min Fig. 7.4. Depolarization induced by 50 juM kainic acid (KA) is not relaxed or reversed by the addition of 2 mM flurazepam (FLUR) to the perfusate, unlike that seen for pento barbital. (Stimulus: as for fig. 7.1.). w m ^ / ' , C o * * + 50uM KA______1-1, min i-2nnM..R.UR Or- “rr Trr,l"iiininniitT E > ~20h- Co+* »50jjM KA , C(£%.50jjM KA f-.lm.in l2iiMELU81 I 2mM FLUR 0r- • 201- F i g . 7.5. Depolarization induced by 50 ajM kainic acid (KA) in the presence of 2 mM co 2 , is not relaxed or reversed by adding 2 mM flurazepam (FLUR) to the perfusate. (Stimulus: as for fig. 7.1.). preparation, the hyperpolarization induced by flurazepam was examined for sensitivity to the GABA antagonists picrotoxin and bicuculline. Picrotoxin (Pi) at a concentration of 50 ajM is ineffective in reversing the inhibitory effect of 2 mM flurazepam (fig. 7.6.), which was observed in 6 cells from 6 retinae. The significance of this result is however minimal as hyperpolarizations induced by 5 mM GABA itself are not antagonised by 50 juM picrotoxin (fig. 4.6., also see Hankins and Ruddock, 1984a). In this respect, the sensitivity of flurazepam to the GABA antagonist is similar to that of pentobarbital (fig. 4.5.). However, bicuculline (Bi) at 50 juM was effective in antagonising the 2 mM flura zepam induced hyperpolarization (fig. 7.7., 10 cells from 9 retinae). It should be noted that bicuculline at a concen tration of 50 juM also blocks the action of GABA (fig. 4.6., also see Hankins and Ruddock, 1984a), although it does not influence the action of pentobarbital. From the results described here, it appears that flurazepam is acting by an interaction with GABA-ergic activity, probably at the GABA-receptor complex, but not necessarily in the horizontal cell layer. This involvement with inhibitory mechanisms agrees with results obtained in various preparations using other benzodiazepines, including diazepam, as was discussed in Chapter 2, and which will be further discussed later in this chapter. 218 _2mM FLUR I 50uM Pi_____ j 1 min 2mM FLUR lim a. 50uM Pi F i g . 7.6. Hyperpolarization induced by 2 mM flurazepam (FLUR) is not reversed by the GABA antagonist picrotoxin (Pi) at a concentration of 50 juM. (Stimulus: as for fig. 7.1.). 219 a 50u Bi . I 1min I_ Imin 50uMBi Fig. 7.7.a and b. Hyperpolarization induced by 2 mM flurazepam (FLUR) is reversed by the GABA antagonist bicuculline (Bi) at a concentration of 50 juM. a. Shows full recovery of the light induced S-potential and dark resting membrarne potential when bicuculliine is added to the perfusate. (Stimulus: as for fig. 7.1.). 220 7.4. Discussion The results described in the previous sections of this chapter have shown that in the isolated perfused roach retina, the benzodiazepine, flurazepam, has a hyperpolar- izing action which is consistent with the 'sedation' which benzodiazepines exercise on the vertebrate central nervous system (CNS). Flurazepam at a concentration of 2 mM is as effective at inducing hyperpolarization of the horizontal cell membrane potential (fig. 7.1.) as 5 mM pentobarbital (Chapter 4, fig. 4.2.). The quantitative changes in the membrane potential produced by the two drugs, are not significantly different from each other (Table 7.1.), although flurazepam differs from pentobarbital in that it has less effect on the light evoked S-potentials and there is a greater degree of recovery once the benzodiazepine is removed from the perfusate. The hyperpolarizing action of flurazepam on the horizontal cells of the outer nuclear layer, does not agree with results obtained with retinae of other species, where it has been reported that only cells within the inner nuclear layer are sensitive to the benzo diazepines. Evidence for this comes from binding studies (Skolnick et al., 1980; Mariani et al., 1987), and also the lack of effect induced by these drugs on the electroretino- gram (Ikeda and Robbins, 1986a). However, when benzodiaze pines are applied iontophoretically to the ganglion cell 221 layer, an inhibitory effect is observed (Ikeda and Robbins, 1986b). These observations will be discussed in more detail later in this section. In Chapter 4, it was shown that pentobarbital could induce hyperpolarization of cells synaptically isolated by 2 + 2 mM Co , cells which were already hyperpolarized towards their K+ equilibrium potential of -70 mV (Djamgoz and Laming, 1982). Results from experiments where 2 mM flura- 2+ zepam was applied to cells hyperpolarized in 2 mM Co , suggest that the benzodiazepine is not effective at the isolated horizontal cell membrane, as no further hyper polarization is seen (fig. 7.2.), although an explanation 2 + for this could be that the Co hyperpolanzation masks that produced by 2 mM flurazepam, so there is an action on the individual cell membrane, but the effect is not a p p a r e n t . It could be concluded from this present study that the benzodiazepine does not act by direct interaction with receptor sites on the horizontal cell membrane. This view is supported by the absence of any blocking of the kainic acid (KA) induced depolarization by the benzodiazepine (fig. 7.4.), indicating a lack of activity at the kainate/ quisqualate binding sites on the postsynaptic horizontal cell membrane. This result differentiates the benzodiaze pine effect from those of pentobarbital. Fig. 7.3. and Table 7.2. show that 2 mM flurazepam reduces the size of the photoreceptor response to light (studied by using 222 transretinal recordings and 50 uM KA, to block all post photoreceptor cell activity), and so it might also be con cluded that the effects of the benzodiazepine, in contrast with those of pentobarbital, are related to an interaction with the photoreceptor cell membrane, which affects the synaptic release of excitatory neurotransmitter from the photoreceptor synaptic terminal. There is little evidence that benzodiazepines interact with excitatory receptor sites or excitatory neurotransmission, but it has been reported by Macdonald et al. (1979), that in cultured mouse spinal neurones, flura- zepam produces an excitatory response which is rapidly desensitizing and which suppresses further excitation of the membrane. The consequence of such action on the horizontal cells would be similar to the relaxation effect observed with pentobarbital in Chapter 4, but in practice the depolarization induced by the excitatory amino-acid agonist, kainic acid, is maintained in the presence of flurazepam, for as long as the agonist is in the perfusate (fig. 7.4.). Thus it is concluded that flurazepam has no effect on excitatory activity, although an action on the release of neurotransmitter from the synaptic terminals of the photoreceptors cannot be excluded. In Chapter 1 (section 1.6.4.) it was stated that GABA is active in the outer plexiform layer of the vertebrate retina (Murakami et al., 1972) and is accumulated by the horizontal cells (Lam and 223 Steinman, 1971? Marc et al., 1978). Furthermore, Murakami et al. (1978) showed in the carp that GABA could hyper- polarize a dark adapted cone and reduce its light evoked responses, whilst Kaneko et al. (1985) reported that isolated turtle photoreceptors (cones) are sensitive to GABA, with the sensitivity localised in the synaptic terminals of the cells. Therefore, as there is much evidence for an association between benzodiazepines and the GABA receptor complex (as briefly discussed in Chapter 2), and despite evidence that benzodiazepines bind only in the inner retinal layer, specific antagonists of GABA were used to investigate flurazepam's involvement with GABA-ergic a c t i v i t y . It was shown earlier in this chapter that picr- otoxin, the GABA antagonist which is specific to the Cl ionophore associated with the GABA receptor complex, is ineffective at antagonising 2 mM flurazepam induced hyper polarizations (fig. 7.6.). This disagrees with a number of studies in various vertebrate CNS preparations (eg. Costa et al., 1975; Gallager, 1978; Okamoto and Sakai, 1979) where picrotoxin has been observed to affect the activity of the benzodiazepines, mediated at the GABA-receptor complex. As discussed in Chapter 1 (section 1.6.4.) the model GABA-receptor complex consists of a number of components, including a benzodiazepine receptor and a Cl ionophore, which have been shown to be in close associ ation. However, in the isolated roach retina, picrotoxin is ineffective against the horizontal cell response to GABA 224 at concentrations up to 600 juM, (Hankins and Ruddock, 1984a) and previously, similar findings had been observed in a number of other vertebrate CNS preparations, including the spinal cord (Curtis et al., 1969) and the cerebellum (Kawamura and Provini, 1970). Furthermore, several reports have described a lack of picrotoxin antagonism against benzodiazepine activity. Tallman et al. (1978) showed that 3 picrotoxin was unable to alter the specific [ H] diazepam binding to cortical neurones, and it was specifically shown by Skerritt and Johnston (1983) in rat brain synaptosomal membranes, that picrotoxin at concentrations up to 100 juM 3 failed to alter [ H] diazepam binding. It also did not block the enhancement of GABA binding by benzodiazepines, whilst this GABA enhancement by diazepam was not stimulated by the addition of Cl ions, unlike that found by Costa et al. (1979) in rat brain membranes. Thus it is concluded that in the vertebrate retina, picrotoxin is an ineffective antagonist of the hyperpolarizing actions of the benzo diazepine, furazepam. This could be explained in a number of ways: 1. GABA receptors in the roach retina, differ from those of the model illustrated by Davidoff (1983), there being no Cl ionophore associated with the receptor. The insensitivity of the GABA evoked responses to the antago nist picrotoxin, suggests that the observed activity is not mediated by changes in neuronal chloride conductances and that the benzodiazepine sites on the GABA receptors do not interact with a Cl*~ ionophore. 225 2. Benzodiazepines act at a separate site away from the GABA-activated ionophore, enhancing GABA receptor affinity (Skerritt and Johnston, 1983 ). 3. There is no enhancement or facilitation of GABA-ergic activity by the benzodiazepines. Steiner and Felix (1976) reported that in the cerebellum and lateral vestibular nucleus of the cat brain, GABA-ergic activity of certain cells was antagonised rather than facilitated by benzodiazepines. Macdonald and Barker (1978b) studying the effects of benzodiazepines on amino acid mediated post- synaptic responses in cultured mammalian spinal cord neurones, observed that there was a selective modification of the GABA mediated synaptic inhibition, which was dose dependnent, with antagonism at higher doses and augment ation at low doses. If the hyperpolarizing actions of the benzodiaze pines in the retina are due to interactions with GABA receptor sites or enhancement of GABA-ergic activity, then the effects seen with flurazepam, if not sensitive to picrotoxin, should be reversed by bicuculline. Bicuculline was found to be an effective antagonist of the benzodiaze pine induced hyperpolarization (fig. 7.7.), agreeing with other investigations which have shown that bicuculline can 3 reverse [ H] diazepam binding to cortical neurones (Tallman 3 et al., 1978) and pentobarbital enhanced [ H] diazepam binding in rat cerebral cortex preparations (Skolnick et al. , 1981). The benzodiazepine effects on GABA-ergic activity have also been blocked by bicuculline (see review 226 by Tallman et al. 1980). From the specificity shown by the two GABA antagonists, it is concluded that the benzodiaze pine hyperpolarization of the horizontal cells, which in the roach retina involves an interaction with GABA-ergic activity, does not depend on the activity of Cl iono- p h o r e s . In summary, flurazepam hyperpolarizes the horizontal cell membrane in the isolated roach retina, cells which are situated in the outer plexiform layer of the retina (Chapter 1, section 1.2.2.) but there is no evidence that this effect represents a direct interaction with sites located on the cell membrane. Thus it is concluded that the benzodiazepine action is mediated at sites on cells which affect the horizontal cell membrane itself. These cells could either be photoreceptors or cells deeper within the retinal structure. It has been reported that the benzo diazepines are inactive within the outer plexiform layers of the rat retina, but active at sites within the inner nuclear layer, the inner plexiform layer and the ganglion cell layer (Ikeda and Robbins, 1986b), where benzodiazepine binding sites have also been located (Skolnick et al., 1980; Mariani et al., 1987). Therefore, feedback from these inner layers to the outer plexiform layer may be involved, with effects in the inner layers altering activity in the outer plexiform layer via interplexiform cells. Likewise, it was shown earlier in this chapter that the benzodiaze pine, flurazepam, has some effect on photoreceptor activity 227 and this may therefore affect the release of transmitter from the photoreceptors and thus horizontal cell activity. The action of flurazepam appears to be mediated by GABA-ergic mechanisms because bicuculline reverses the hyperpolarizing effect of the drug, but as picrotoxin is ineffective against benzodiazepine activity it is not associated with Cl ionophores. Although there is GABA activity in the outer plexiform layer of the fish retina (Murakami et al., 1972? Marc et al., 1978; Reynolds, 1981), and at the photoreceptor level in turtle (Kaneko et al., 1985), the benzodiazepines are inactive at the outer retinal layers, but active within the inner nuclear and plexiform layers and in the ganglion cell layer (Ikeda and Robbins, 1986b). In these layers both ganglion cells, which are GABA-ergic (Ikeda, 1985) and amacrine cells which accumulate GABA (Mariani et al., 1987) are sensitive to benzodiazepines. Therefore, in this present study, the observed effect may be one in which: 1. Flurazepam is interacting with GABA-ergic activity of either amacrine or ganglion cells and this, via feedback through interplexiform cells, affects the horizontal cell layer. 2. Interaction with GABA sites on the photoreceptor cells in the outer retina affects the release of trans mitter from the photoreceptors, so affecting the activity of the post-synaptic horizontal cells. 3. A combination of both the mechanisms described in 1 . and 2 228 CHAPTER 8 GENERAL DISCUSSION AND CONCLUSIONS The aim of this thesis was to investigate the effect of sedative/anaesthetic type drugs on the membrane potentials and light induced responses (S-potentials) of horizontal cells in the perfused, isolated roach retina. There is much information about the actions of 'sedative' type drugs in other neuronal preparations, as reviewed in Chapter 2, but very little about the retina, despite it being an ideal preparation for intracellular electrophysio- logical studies of drug action on neurones and neurotrans mission. This chapter summarises the major findings of the present study, already discussed in the preceding chapters. 8.1. The Effect of Pentobarbital on Retinal Neuronal Activity In the first experimental chapter, an investigation into the effects of the sedative barbiturate pentobarbital was presented. Recordings made from single horizontal cells in the isolated intact retina, perfused with drug solutions, show that pentobarbital at 0.5 - 5.0 mM hyper- polarizes the cell membrane potential and abolishes the light evoked S-potentials. This effect is long lasting, 229 persisting after removal of pentobarbital from the per fusate and often irreversible (fig. 4.1.). Furthermore, the effect is seen in the presence of the synaptic blocker 2 + 2 mM cobalt (Co ), so that it can be concluded that the action of pentobarbital originates from a direct inter action with the horizontal cell membrane (fig. 4.2.). Two main mechanisms have been proposed to account for the 'sedative' action induced by the barbiturates: 1. enhancement of GABAergic mechanisms, 2 . reduction in excitatory neurotransmission, both mechanisms were investigated in the retinal pre paration . Although GABA has been localised in the outer plexi- form layer of the retina, no receptor sites have yet been localised on horizontal cells, so that the inhibitory effect of GABA on horizontal cells is probably via photo receptors, where there are sites specific to GABA. Binding at these sites would decrease the excitatory transmitter release from photoreceptors and so hyperpolarize the horizontal cell membrane (in accordance with Trifonov's hypothesis). Experiments involving the GABA antagonists picrotoxin and bicuculline have shown that neither antagonist is effective against pentobarbital (figs. 4 .5 . and 4.7.). As picrotoxin has been shown to be ineffective against GABA activity in the retina, this is not an unexpected result, but the ineffectiveness of bicuculline indicates that there is no GABA involvement with the pentobarbital effect. 230 Horizontal cells possess receptor sites which are sensitive to the excitatory neurotransmitter L-glutamate (L-GLU) and its agonists kainic acid (KA) and quisqualic acid (QA), and as pentobarbital appears to act at the horizontal cell membrane investigations were made to look at any interactions occurring between the barbiturate and agonists at the receptor sites. Two effects were observed. Firstly, when cells were hyperpolarized by pentobarbital and then subjected to the excitatory agonist, depolari zation occurs similar to that seen in the absence of pento barbital (fig. 4.9.). Secondly and more significantly, if the cell is first depolarized by the agonist and then sub jected to pentobarbital, relaxation of this depolarization occurs. The effect was seen for depolarization induced by kainic acid (50 uM),(fig. 4.4.), L-glutamate (25 juM) potentiated by D-aspartate (3 mM),(fig. 4.17.), L-glutamate (5 mM),(fig. 4.18.) and dopamine (20 ;uM),(fig. 4.22.). As the effect of pentobarbital is a direct one on the hori zontal cell membrane, it can be concluded that the barbit urate affects neither release of excitatory transmitter from the photoreceptors nor synaptic functioning. Although there are interactions between inhibitory and excitatory transmitter systems in the retina, depolar ization by the excitatory amino-acids is not affected by antagonists of GABA. Likewise, the GABA antagonist bicuculline was ineffective in blocking the relaxation of the kainate induced depolarization, indicating that the observed effect is not via some GABA feedback mechanism. The possibility that pentobarbital is acting as an antagonist of excitatory activity was also studied, by investigating whether pentobarbital could potentiate low concentrations (<1 mM) of the excitatory amino acid antagonist, kynurenic acid (KYN). It was found that no potentiation occurred (fig. 4.20.) and this coupled with the fact that unlike pentobarbital, kynurenic acid is ineffective against dopamine or 5 mM L-glutamate, indicates that pentobarbital is not necessarily an antagonist of the excitatory effect. Thus it appears that although pento barbital interacts with excitatory mechanisms in the retina, it does so with some general, non-specific effect on the horizontal cell membrane rather than by interaction at specific binding sites. 8.2. Ionic Mechanisms Mediating the Hyperpolarization of Horizontal Cells by Pentobarbital To investigate the roles of potassium (K+ ), sodium (Na+ ) and chloride (Cl ) in mediating the pentobarbital hyperpolarizing effect, ion substitution of the perfusate, applied to the retina was employed. Changes made in the potassium concentration of the retinal perfusate produced the most interesting results regarding ionic mechanisms underlying the pentobarbital 232 inhibitory effect. When the extracellular K+ concentration was raised, the pentobarbital hyperpolarization was eliminated, whilst the depolarized membrane potential attained in high extracellular potassium ion concentrations was unaffected by the addition of pentobarbital (fig. 5.6.). Application of the K+ channel blocker tetraethyl- ammonium (TEA) suppressed pentobarbital activity (fig. 5.16.). Therefore, pentobarbital may be altering ionic conductances, so that an increased outward conductance of K+ ions from the cell dominates, either by increasing potassium conductance or by decreasing the conductances of other ions, and the hyperpolarization seen is due to increased efflux of potassium ions taking the cell towards the potassium equilibrium potential of -70 mV. Results involving sodium and chloride ions were less clear than for potassium. However, pentobarbital was •j* effective in a perfusate where Na ions had been substitu ted by choline (fig. 5.5.). As any effect pentobarbital 4* # 4* has regarding Na ions must be to block Na channels, the observation that pentobarbital is effective in Na+-free perfusates, indicates that the barbiturate's action is independent of Na activity. Although many investigations have linked pento barbital with chloride ions, due to its association with GABAergic mechanisms, in this study pentobarbital was - 2 - effective both in Cl -free Ringer (SO^ substituted) and a perfusate where Cl” ions had been doubled by the addition 233 of choline chloride (figs. 5.1. and 5.4.). However, if pentobarbital was applied at the same point as Cl -free Ringer, the hyperpolarization induced by the barbiturate is of a lesser value than that seen when the cell is initially depolarized in Cl -free Ringer, and this suggests that there may be some Cl involvement with the pentobarbital effect, possibly coupled with K+ activity. From the results discussed in this and the preceding section, it is apparent that pentobarbital affects excitatory neuronal activity in some way other than by directly reducing the effectiveness of the excitatory transmitter. If it were acting in a direct way, then it might be expected that pentobarbital would be more specific in its action on the depolarization induced by excitatory substances, and its activity might be similar to the antagonist kynurenic acid. Furthermore, depolarizations induced by the excitatory agonist primarily involve Na+ ions and not K+ ions, whereas it is apparent that pento barbital has no effect on Na+ ion conductance, but probably increases K+ ion conductance. Hence pentobarbital has an effect on the cell membrane which involves K+ ions and this may be superimposed on the depolarization induced by the excitatory transmitter or agonists, causing a relaxation of the depolarized level. 234 8.3. The Effects of a Number of Sedative' Type Drugs on the Activity of Horizontal Cells, in Comparison with the Effects of Pentobarbital In Chapter 6 , experiments were described in which a variety of drugs with 'sedative* properties were investigated for: 1. Their action on the horizontal cell membrane, 2. The degree of activity which each drug induces compared to that of pentobarbital. The drugs studied were the barbiturates, pheno- barbital and hexobarbital, eC - chloralose, urethane, chloral hydrate and ethanol, all of which induce a hyperpolarizing effect at concentrations of 2 mM (figs. 6.2. to 6.7.). This varies according to the 'sedative' investigated, with phenobarbital inducing the greatest effect and ethanol the least. These drugs have been ranked according to their anaesthetic potencies (the concentration of drug required to produce anaesthesia) by Barnes and Etherington (1973). The order shown in the vertebrate retina essentially follows that found in the rat, although slight discrepancies occur with urethane, phenobarbital and hexobarbital, but all the drugs studied are less effective than pentobarbital at 2 mM. Similar results were obtained in experiments where pentobarbital concentrations were adjusted to produce the same effect as 2 mM 'sedative'. 235 The results discussed in Chapter 6 , show that a variety of 'sedative' type drugs can produce hyperpolari zation of the horizontal cell membrane potential, similar to that seen with pentobarbital, whilst the degree to which this occurs for each 'sedative' investigated essentially follows the order in which the sedatives are ranked according to their anaesthetic concentrations (Tables 6.1. and 6.2.; Graph 6.1.). 8.4. The Effect of Flurazepam on Retinal Neuronal Activity The last experimental chapter deals with the benzo diazepine flurazepam, a drug which has similar clinical actions to the barbiturate pentobarbital. Intracellular recordings from horizontal cells, showed that flurazepam at 2 mM has a hyperpolarizing action which is as effective as 2 mM pentobarbital, although there is less effect on the light evoked S-potentials and it is reversible (fig. 7.1.). The 'sedatory' action seen here is consistent with reports from investigations in other preparations as discussed in Chapter 2, but not consistent with activity seen in other investigations on the retina. Binding studies (Skolnick et al., 1980; Mariani et al., 1987) have shown that flurazepam is active within the inner nuclear layer and electrophysio- logical studies have indicated that there is no benzodiaze pine activity in the outer layers of the retina, but acti vity at ganglion cell layers (Ikeda and Robbins, 1986a,b.). 236 Flurazepam, unlike pentobarbital, does not appear to act through a direct action on the horizontal cell membrane, as no hyperpolarization of the horizontal cell membrane is seen in cells synaptically isolated by cobalt ions, and there is no apparent interaction at KA or QA binding sites present on the horizontal cell membrane (figs. 7.4. and 7.5.). However, in transphotoreceptor recordings, 2 mM flurazepam reduces the size of the photoreceptor response to light (fig. 7.3.) so that there may be an interaction at the photoreceptor level possibly involving the release of excitatory neurotransmitter from the photoreceptor synaptic terminals. As benzodiazepines have been closely associated with the GABA receptor complex and no interaction appears to occur with excitatory neurotransmission, the benzodiaze pines were investigated for their interaction with GABA mediated activity. GABA has been localised in the outer plexiform layer, with photoreceptors possessing specific sites and horizontal cells accumulating the inhibitory amino-acid, thus GABA antagonists were used against benzodiazepine activity. Picrotoxin, as expected due to its lack of GABA antagonism in the retina, was not effective against the flurazepam hyperpolarization but bicuculline has an effect similar to its action against GABA in the retina (figs. 7.6. and 7.7.). 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