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Microelectrode determination of the intracellular chloride concentration in nerve cells
Life Sciences Yol . 5, pp . 453-456, 1966 . Printed in Great Britain.
.Pergamon Press Ltd.
MIGROELECTRODE DEPEFd~iÁTION OF THE INZRACdllJLAR QII.ARIIIE CONCEt1TRAlZON IN NERVE (:ELLS G . A. Kerlait and R. W. Meech Departrnent of Physiology and Biochemistry, The University of Southampton, Southampton, England. (Received 3 January 1966) THERE has been corLSidereble interest in the values of thé chloride ooa~centration within nerve cellel 2 3, the det~++~;motion being carried out on the large axons of the squid or cx~yfish .
The chloride levels within the nerves
of the snail are of considerable ürterest since Oomura4 has suggested that the depolarising action of acetylcholine on some cells ( D cells) and the hyperpolarising action of acetylcholine on other cells (H cells) owld be due to differences in the chloride concentration within those cells . We have developed a microelects+ode that is sensitive to chloride concentrations and our experiments using these el.e~odes show that the H cells in the snail bn3in have a laser chloride ooncentr+ation than the D cells . Preparation of the chloride electrodes Two different methods for the prepart3tion of the microelectrodes have been used . (1) Electrolytic deposition of silver within the electrode . (2) chemical deposition of silver .
Since the second method was the mire
successful, this will be the one described here . (1) Glass microelectrodes of approximately five megohms resistance were filled w2th distilled water.
Most of the water was ranoved from the shank
and the electrodes were filled with amnoniacal silver nitrate solution ( Solution A - see next page) .
This solution was again removed from the 453
454
INTRACELLULAR CHLORIDE
shank and the electrodes refilled with A .
Yol . 5, No . 5
This double filling is to overcame
any dilution due to the presence of distilled water remains ~ in the electrode . (2) The electrode was washed and dried and placed with its tip in a solution of formaldehyde ( Solution B ) . The electrodes were left for about ten hrxu s . (3) The electrode was washed and mounted for observation in a bath of KC1 . We used a ooncentr~atipn of 118 mM KC1 ; this being the value of the chlcmide in the snail ringer . The mirrnsoopé .
tip of the electrode was e7~nxi order the
Those electrodes that had blobs of silves on the tip or which
were of blunt profile were discarded .
It is possible to reduce the percentage
of electrodes that had such a blob by malting solution A more dilute or by standing the tips in solution B for less than ten hoiu~s . (4) The electrode was washed and its tip placed in a testing bath which was connected through an agar bridge to a silver-silver chloride electrode . The potential between the two electrodes was measured by means of a Vibron IIecá~eter (Electronic Instrtments Ltd, Richmond, Surrey, U .K.) .
The
chloride concemrt~tion in the bath was altered a~ the relationship between the chloride concenrtretion and the potential detenained .
The higher the
chloride concentration, the more negative the potential between the two electrodes .
A 'good electrode gave a linear response between the concern-
retionsfrom 300 mM C1' and 1 mM C1' . (5) The electrodes were tested to see if they were specific for C1' . The electrode was first placed in a solution containing 118 mM K+1 mM C1' and then in a solution containing 1 mM K+1 mM C1' ; the ionic strength was adjusted with sodium acetate .
If the electrodes were i~letely blocked by the
silver then they proved to be sensitive to this change .
If the electrodes
were blocked with silver then the potentials in the two solutions were within 5-10 mV of each other . (6) Only those electrodes that were insensitive to changes in external K+ and which gave a linear potential response to C1' were used .
vol . 5, No . 5
INTRACELLIILAR cHLOaIDE
Solution A (~niar~al silver nitnate) 0.85g of silver nitrete was dissolved in a small (1-2m1) vole of 33$ ammonium hydroxide .
One drop of 1 M NaOH was added and the colorless
solution ra3de up to 6m1 .
This will be appmnci~tely 0 .8M E~gN03.
The ammonium hydzrnáde was made by taking 33m1 of 0 .880 ammon%a and adding 67m1 of water . Solution B ( Fommaldehyde ) 5ml of stock formaldehyde solution (containing 40 volumes of formaldehyde) is added to Sml of water. Measurement of chloride
ntxation in snail nerve cells .
The brc+ain óf the snail Helix aspexsa was removed and the cells in the suboesophageal gar°~ exposed by x+~mval of the connective tissue . A 1M potassium acetate filled microelectr~ode was inserted into a knoam cell 5 and the response to 10 g/ml acetylcholine tested . In this way one could check if the cell was a H or a D cell . A selected chloride electrode w~s inserted into the céll, this making two electrodes in the one cell ; a potassium acetate electrode and a chloride electrode.
The potential on the chloride electrode was measiu~ed .
The validity of the intracellular measurement was checked in two ways . (1) Hy injecting chloride ions into the cell and finding has the internal chloride concentx~tion changed both with regard to the chloride electrode and with regard to the acetylchol ;+~ reversal potential
(E~) .
(2) By inserting the electrode into the large algal cell Nitella, whose chloride concentration was already known. The chloride level for the snail D cells was 27 .5 + 1 .5 mM (n=25) . The chloride level for the snail H cells was
8.7 + 0 .4 mM (n=25) .
Though the D cells do have a higher internal C1' there are certain differences between the E~ and the ECl,. which indicate that other ions
X55
INTRACELLULAR'('HhORIDE
456
Vol . 5, No . 5
may play a role in bringing about thé depolarisation . From the values of C1' described here and also other experiments it would seem that the action of acetylcholine on the H cells is to increase the permeability to C1' and this agrees with the previous suggestion that the 5 E~ is near the value for ECl, . Canolusion (1) By using silver treated microelectrndes one can measure the chloride concentration within snail nerve cells . (2) There is a marked difference between the concentration of chloride in the H cells ( 8 .7mM) and the D cells ( 27 .5mM) . (3) This indicates that there is an intracellular chemical heterogeneity between nerve cells and this should be considered in addition to the well known transmitter heterogeneity in the b7lain when evaluating the cca~iplexities of the Central Nervous S~rstem. This work was supported by a grant DA-91-591-EUC-3081 fnxn the European Research Office of the U.S . Departrnent of the Army . References 1. R.D .Keynes . J. Physiol . 169, 690 (1963) 2 . A. Muaro. Fed. ~. 13, 96 (1954) 3. A. Strickholm and B.G .Wallin . Nature 208, 790 (1965) 4. Y. Oo~t3 (personal commmication and also quoted in The Physiology of S
ses by J.C .Eccles . Springer Verlag, 1953 . page 200) .
,5 . G.A .Kerkut and R:C .Than3s . Ccenp. Biodran. Physiol. 11, 199 (1964)
.Pergamon Press Ltd.
MIGROELECTRODE DEPEFd~iÁTION OF THE INZRACdllJLAR QII.ARIIIE CONCEt1TRAlZON IN NERVE (:ELLS G . A. Kerlait and R. W. Meech Departrnent of Physiology and Biochemistry, The University of Southampton, Southampton, England. (Received 3 January 1966) THERE has been corLSidereble interest in the values of thé chloride ooa~centration within nerve cellel 2 3, the det~++~;motion being carried out on the large axons of the squid or cx~yfish .
The chloride levels within the nerves
of the snail are of considerable ürterest since Oomura4 has suggested that the depolarising action of acetylcholine on some cells ( D cells) and the hyperpolarising action of acetylcholine on other cells (H cells) owld be due to differences in the chloride concentration within those cells . We have developed a microelects+ode that is sensitive to chloride concentrations and our experiments using these el.e~odes show that the H cells in the snail bn3in have a laser chloride ooncentr+ation than the D cells . Preparation of the chloride electrodes Two different methods for the prepart3tion of the microelectrodes have been used . (1) Electrolytic deposition of silver within the electrode . (2) chemical deposition of silver .
Since the second method was the mire
successful, this will be the one described here . (1) Glass microelectrodes of approximately five megohms resistance were filled w2th distilled water.
Most of the water was ranoved from the shank
and the electrodes were filled with amnoniacal silver nitrate solution ( Solution A - see next page) .
This solution was again removed from the 453
454
INTRACELLULAR CHLORIDE
shank and the electrodes refilled with A .
Yol . 5, No . 5
This double filling is to overcame
any dilution due to the presence of distilled water remains ~ in the electrode . (2) The electrode was washed and dried and placed with its tip in a solution of formaldehyde ( Solution B ) . The electrodes were left for about ten hrxu s . (3) The electrode was washed and mounted for observation in a bath of KC1 . We used a ooncentr~atipn of 118 mM KC1 ; this being the value of the chlcmide in the snail ringer . The mirrnsoopé .
tip of the electrode was e7~nxi order the
Those electrodes that had blobs of silves on the tip or which
were of blunt profile were discarded .
It is possible to reduce the percentage
of electrodes that had such a blob by malting solution A more dilute or by standing the tips in solution B for less than ten hoiu~s . (4) The electrode was washed and its tip placed in a testing bath which was connected through an agar bridge to a silver-silver chloride electrode . The potential between the two electrodes was measured by means of a Vibron IIecá~eter (Electronic Instrtments Ltd, Richmond, Surrey, U .K.) .
The
chloride concemrt~tion in the bath was altered a~ the relationship between the chloride concenrtretion and the potential detenained .
The higher the
chloride concentration, the more negative the potential between the two electrodes .
A 'good electrode gave a linear response between the concern-
retionsfrom 300 mM C1' and 1 mM C1' . (5) The electrodes were tested to see if they were specific for C1' . The electrode was first placed in a solution containing 118 mM K+1 mM C1' and then in a solution containing 1 mM K+1 mM C1' ; the ionic strength was adjusted with sodium acetate .
If the electrodes were i~letely blocked by the
silver then they proved to be sensitive to this change .
If the electrodes
were blocked with silver then the potentials in the two solutions were within 5-10 mV of each other . (6) Only those electrodes that were insensitive to changes in external K+ and which gave a linear potential response to C1' were used .
vol . 5, No . 5
INTRACELLIILAR cHLOaIDE
Solution A (~niar~al silver nitnate) 0.85g of silver nitrete was dissolved in a small (1-2m1) vole of 33$ ammonium hydroxide .
One drop of 1 M NaOH was added and the colorless
solution ra3de up to 6m1 .
This will be appmnci~tely 0 .8M E~gN03.
The ammonium hydzrnáde was made by taking 33m1 of 0 .880 ammon%a and adding 67m1 of water . Solution B ( Fommaldehyde ) 5ml of stock formaldehyde solution (containing 40 volumes of formaldehyde) is added to Sml of water. Measurement of chloride
ntxation in snail nerve cells .
The brc+ain óf the snail Helix aspexsa was removed and the cells in the suboesophageal gar°~ exposed by x+~mval of the connective tissue . A 1M potassium acetate filled microelectr~ode was inserted into a knoam cell 5 and the response to 10 g/ml acetylcholine tested . In this way one could check if the cell was a H or a D cell . A selected chloride electrode w~s inserted into the céll, this making two electrodes in the one cell ; a potassium acetate electrode and a chloride electrode.
The potential on the chloride electrode was measiu~ed .
The validity of the intracellular measurement was checked in two ways . (1) Hy injecting chloride ions into the cell and finding has the internal chloride concentx~tion changed both with regard to the chloride electrode and with regard to the acetylchol ;+~ reversal potential
(E~) .
(2) By inserting the electrode into the large algal cell Nitella, whose chloride concentration was already known. The chloride level for the snail D cells was 27 .5 + 1 .5 mM (n=25) . The chloride level for the snail H cells was
8.7 + 0 .4 mM (n=25) .
Though the D cells do have a higher internal C1' there are certain differences between the E~ and the ECl,. which indicate that other ions
X55
INTRACELLULAR'('HhORIDE
456
Vol . 5, No . 5
may play a role in bringing about thé depolarisation . From the values of C1' described here and also other experiments it would seem that the action of acetylcholine on the H cells is to increase the permeability to C1' and this agrees with the previous suggestion that the 5 E~ is near the value for ECl, . Canolusion (1) By using silver treated microelectrndes one can measure the chloride concentration within snail nerve cells . (2) There is a marked difference between the concentration of chloride in the H cells ( 8 .7mM) and the D cells ( 27 .5mM) . (3) This indicates that there is an intracellular chemical heterogeneity between nerve cells and this should be considered in addition to the well known transmitter heterogeneity in the b7lain when evaluating the cca~iplexities of the Central Nervous S~rstem. This work was supported by a grant DA-91-591-EUC-3081 fnxn the European Research Office of the U.S . Departrnent of the Army . References 1. R.D .Keynes . J. Physiol . 169, 690 (1963) 2 . A. Muaro. Fed. ~. 13, 96 (1954) 3. A. Strickholm and B.G .Wallin . Nature 208, 790 (1965) 4. Y. Oo~t3 (personal commmication and also quoted in The Physiology of S
ses by J.C .Eccles . Springer Verlag, 1953 . page 200) .
,5 . G.A .Kerkut and R:C .Than3s . Ccenp. Biodran. Physiol. 11, 199 (1964)