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Voltage-sensitive dyes measure potential changes in axons and glia of the frog optic nerve
Neuroscience Letters, 66 (1986) 49-54 Elsevier Scientific Publishers Ireland Ltd.
49
NSL 03893 VOLTAGE-SENSITIVE DYES MEASURE POTENTIAL CHANGES IN AXONS AND GLIA OF THE FROG OPTIC NERVE
ARTHUR KONNERTH* and RICHARD K. ORKAND** Department of Physiology & Pharmacology, School of Dental Medicine and Mahoney Institute of Neurological Sciences, University of Pennsylvania, Philadelphia, PA 19104-6002 (U.S.A.) (Received January 13th, 1986; Revised version received and accepted January 29th, 1986)
Key words: neuroglia - voltage-sensitive dye - frog - optic nerve - action potential - afterpotential
Changes in dye absorption and fluorescence produced by electrical stimulation were measured in frog optic nerves stained with voltage-sensitive dyes. Following a single maximal stimulus applied through a suction electrode, the change in transmitted light intensity consisted of two components: one representing an axonal compound action potential and the second a slow depolarizing afterpotential which appeared to arise from the glial cells. The following results support this interpretation: (1) during a train of stimuli the depolarizing potentials sum and can exceed 80% of the initial spike amplitude while the spike amplitude itself remains essentially constant. Thus, the axons cannot have undergone significant depolarization during the train. (2) Optical recordings with simultaneous microelectrode recordings from the glial cells indicate that the change in glial membrane potential during the train has a time-course similar to that of the slow optical response. We conclude that voltage-sensitive dyes can monitor potential changes in both neurons and glia.
Voltage-sensitive dyes for m o n i t o r i n g c h a n g e s in m e m b r a n e p o t e n t i a l are finding wide a p p l i c a b i l i t y in the s t u d y o f the n e r v o u s system [5, 13]. This m e t h o d is b a s e d on the o b s e r v a t i o n t h a t certain m e m b r a n e dyes c h a n g e their a b s o r p t i o n o r their fluorescence linearly with changes in t r a n s m e m b r a n e v o l t a g e [3]. In a d d i t i o n to the c h a n g e in p o t e n t i a l , the m a g n i t u d e o f the optical signal d e p e n d s u p o n the a m o u n t o f dye b o u n d to the m e m b r a n e , the m e m b r a n e a r e a f r o m which it is r e c o r d e d , the n a t u r e o f the dye b i n d i n g sites a n d the sensitivity o f the dye to o t h e r c h a n g e s which m a y a c c o m p a n y the p o t e n t i a l change, as well as o n the optical r e c o r d i n g a p p a r a t u s itself. In a tissue like the n e r v o u s system, consisting o f m o r e t h a n one cell type a n d h a v i n g a c o m p l e x g e o m e t r y , one m i g h t expect s o m e difficulties in the i n t e r p r e t a t i o n o f optical signals arising f r o m voltage-sensitive dyes. In fact, p r e v i o u s studies have suggested the possibility t h a t r e c o r d i n g s f r o m n e u r o n s are c o n t a m i n a t e d b y a contrib u t i o n f r o m the glial cells [6, 8, 10, 15, 16]. In this study, we have c o m p a r e d the optical signals f r o m the whole o p t i c nerve o f the frog (Rana pipiens) with those o b t a i n e d *Present address: Max-Planck Institute for Psychiatry, D-8033 Planegg, F.R.G. **Author for correspondence at: Department of Physiology & Pharmacology, University of Pennsylvania SDM, 4001 Spruce St., Philadelphia, PA 19104-6002, U.S.A. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
50
A
B
lJ
LAMP
LENS
0
HEATFILTER
I
I
FILTER
I
I
1
570 nm
A
I
LENS a
PREPARATION
b
~
.........................
............................
OBJECTIVE FILTER 2
J
[
PHOTODIODE
T
I l/V-CONVERTER I
o--@
f SAMPLE & HOLD~
20 mV 10 ms
Fig. 1. A: schematic diagram of the experimental set-up for stimulation and recording. The optic nerve is mounted on the stage of a Nikon MS inverted microscope which was modified so that the stage remains stationery during focusing. It is held by two suction electrodes used for extracellular stimulation and recording, respectively. The bottom of the experimental chamber was a replaceable glass coverslip. When the experiment did not involve intracellular recording the air-water interface was eliminated with a microscope coverslip. For intracellular recording from glial cells we used a conventional 3 M KCI (20-40 M(~) electrode driven by a Narishige hydraulic manipulator. Light from a tungsten-halogen lamp (Osram Bellaphot) was focused on the nerve with a long-working distance condenser. The light was rendered quasimonochromatic by passing it through a heat filter (KG-1) and an interference filter. For absorption measurements, a × 63 objective formed a real image on a photodiode located in the trinocular tube at the image plane. The nerve filled the optical field (200/~m diameter) o f the objective. For fluorescence measurements a second interference filter (filter 2) was placed in front of the photodiode as a barrier filter. The photocurrent from the photodiode was passed to a current to voltage (I/V) converter whose output was recorded with a sample-and-hold circuit (Morad Autobucker) which subtracted the background light and allowed the recording of DC potentials (0-1 kHz). Signals were displayed on a Tektronix DI5 oscilloscope and photographed with a Grass camera. B: these traces were recorded during a single supramaximal electrical stimulation of an optic nerve stained with NK2367 at 0.2 mg/ml for 25 rain. The top trace (a) is the record of transmitted light intensity at 570_+15 nm. Following the stimulus there is an increase in transmitted light intensity consisting of a large spike and a long-lasting afterpotential of the same sign as the spike. At 660_+ 15 n m (b), there is a decrease in light intensity with the same time-course and relative amplitude of the spike and the afterpotential, respectively. This result is expected as a consequence of the changes
51
B
A
a
I lO-3
20 mY
b
o_f
[ 5x10-4
C
20 mV
I 3x10-4
6r~J nm
ls
20 mV
0.5 s Fig. 2. Responses to trains of stimuli. The optic nerve was stimulated at 30 Hz for 0.8 s. A: the nerve was stained with NK2367 (0.2 mg/ml for 25 min) and the changes in light absorption were recorded; 570+ 15 nm (a); 660+ 15 nm (c). The depolarizing afterpotentials sum and reach 80% of the initial spike height. The spikes do not decrease in amplitude during the train. The relative amplitudes of the spikes and slow potentials are the same at both wavelengths. Trace b shows the spikes as recorded by the suction electrode. B: this nerve was stained with WW781 (0.3 mg/ml for 20 min). Similar train of stimuli recorded at a slower timebase. The suction electrode record is shown in a. The middle trace (b) is a record of the increase in fluorescence of the nerve (exciting filter 630___15 nm; barrier filter 660_ 15 nm) during the train of stimuli. Because the fluorescence intensity is very low, the noise in the measurement is dominated by the noise contributed by the sample-and-hold amplifier. Trace c is an intracellular recording from a glial cell (Vm = - 8 8 mV) obtained simultaneously with the optical trace in b. The time-course of the glial depolarization is similar to that of the slow optical depolarization.
b y i n t r a c e l l u l a r m i c r o e l e c t r o d e r e c o r d i n g s f r o m glial cells i n t h e s a m e p r e p a r a t i o n [12], in o r d e r t o a s s e s s t h e c o n t r i b u t i o n o f t h e g l i a l cells t o t h e o p t i c a l s i g n a l . T h e o p t i c n e r v e , a n a t u r a l l y i s o l a t e d t r a c t o f t h e c e n t r a l n e r v o u s s y s t e m (ca. 4 0 0 # m d i a m e t e r ) , w a s d i s s e c t e d f r o m s m a l l (5 c m ) a d u l t f r o g s a f t e r d e c a p i t a t i o n . I t w a s held by both ends in suction electrodes for stimulation and recording in a 0.5-ml chamber
mounted
on the stage of a Nikon
inverted microscope. The nerves were
in absorption of this dye as a function of wavelength [17] and indicates that the signal results from a change in the properties of the voltage-sensitive dye and is not due to changes in tissue opacity. The bottom trace (c) is the potential recorded by the suction electrode and shows the usual unmyelinated axonal population spike. The peak is delayed relative to the optical spike because the photodiode monitors changes at the middle of the nerve and the suction electrode is located at the distal end. The spike of the myelinated axons (about 3% of total) is obscured by the stimulus artifact and in this preparation did not give rise to an optical response.
52 stained for 15-25 min in a 0.1-0.4 mg/ml solution of the voltage-sensitive dyes NK2367 (merocyanine-oxazolone) [14] or WW781 (merocyanine-rhodanine) [7] in normal Ringer's solution of the following composition (in mM): NaCI 112, KCI 2, CaCl2 2, glucose 33, HEPES 15 (adjusted with N a O H to pH 7.4). A diagram of the set-up for optical and electrical recording is shown in Fig. l A. Fig. 1B shows optical records obtained from a stained optic nerve, at two wavelengths, in response to a single supramaximal electrical stimulus, together with an electrical recording made with a suction electrode. The optical signal consists of a large compound action potential, arising primarily from the unmyelinated axons [2, I l] followed by a long-lasting depolarizing afterpotential. The inversion of both components of the signal with wavelength suggests that they are dye-specific and related to membrane potential and not due to changes in tissue opacity or light scattering [3, 13]. In the absence of dye we did not record optical signals of this magnitude during stimulation. Optical and electrical recordings during repetitive stimulation are illustrated in Fig. 2. The striking feature of the record in Fig. 2A, is that the long-lasting depolarizing afterpotentials sum until they reach 80% of the initial spike amplitude, while the spike amplitude itself does not decline. If the axons themselves were depolarizing slowly during the train, the spike amplitude would have decreased. Thus, it appears that the depolarization must arise not from the axons but from the glial cells. Repetitive stimulation of the frog optic nerve is known to produce a slow glial depolarization due to the transient accumulation of potassium, released from the active axons, in the narrow extracellular clefts separating the axons and glial cells [12, 18]. By contrast, the membrane potentials of axons are relatively insensitive to low levels of potassium and they depolarize little during repetitive activity and may even hyperpolarize. Because of their small diameter, 0.1-2 #m, there have not been any intracellular recordings from unmyelinated axons in the optic nerve. In Necturus optic nerve an analysis of extracellular records indicates that these axons depolarize by up to 2 mV during a brief train and hyperpolarize due to electrogenic sodium pumping immediately after stimulation stops [4]. In invertebrate neurons [1] there is hyperpolarization during and after a train of impulses. Fig. 2B shows a direct comparison of an optical signal from the whole nerve with an intracellular microelectrode recording from a glial cell in the same nerve. The time-course of the slow optical response closely resembles the glial depolarization recorded with a microelectrode, consistent with the assumption that the slow component of the optical signal arises from the glial cells. These results indicate that some voltage-sensitive dyes can respond to potential changes in glial cells as well as in neurons. If the responses were the same in both cell types and the membrane areas of the axons and glial cells were equal, the optical signals should be proportional to the voltage change in each cell type. However, preliminary naorphometric estimates (P.M. Orkand, unpublished), suggest that there is about 5 times as much unmyelinated axon membrane in this nerve as glial membrane. If we assume that the axonal spike is 100 mV and the glial depolarization to a brief train (30 Hz for 0.8 s) is 25 mV (Fig. 2Be), then the amplitude of the optical recording
53
of the spike should be 20 times larger than that of the slow depolarization. The optical signals corresponding to the spike and to the slow depolarization differed by a factor less than two, suggesting either that not all the axons were active in response to a supramaximal stimulus or that the dyes are more sensitive to glial depolarization. This latter possibility could arise if the dyes show greater affinity for glial membranes or if the changes in dye properties were more pronounced in the glial membrane environment. A third possibility is that dye penetration is limited and that the best staining occurs in an outer shell where the glial membrane predominates. Given the wide variety of substances useful as voltage-sensitive dyes [3, 5, 13] it seems reasonable to search for dyes with great specificity for recording from either neurons or glia. Some of these results were presented at a meeting of the Deutsche Physiologische Gesellschaft [9]. We thank Dr. Brian Salzberg for constant advice and the loan of some apparatus and Drs. A.L. Obaid and A. Grinvald for comments on the typescript. Supported by NIH Grant NS-12253 and a Feodor Lynen Fellowship from the Alexander von Humboldt Foundation (A.K.). 1 Baylor, D.A. and Nicholls, J.G., Changes in extracellular potassium concentration produced by neuronal activity in the central nervous system of the leech, J. Physiol. (London), 203 (1969) 555-569. 2 Bishop, G.H., Fiber groups in the optic nerve, Am. J. Physiol., 106 (1933) 460-474. 3 Cohen, L.B. and Salzberg, B.M., Optical measurement of membrane potential, Rev. Physiol. Biochem. Pharmacol., 83 (1978) 35-88. 4 Cohen, M.W., Glial potentials and their contribution to extracellular recording. In A. Remond (Ed.), Handbook of EEG and Clinical Neurophysiology, Vol. 2 Part B, Elsevier, Amsterdam, 1974, pp. 2B43-2B60. 5 Grinvald, A., Real-time optical mapping of neuronal activity, Annu. Rev. Neurosci., 8 (1985) 263-305. 6 Grinvald, A., Manker, A. and Segal, M., Visualization of the spread of electrical activity in rat hippocampal slices by voltage sensitive optical probes, J. Physiol. (London), 333 (1982) 269-291. 7 Gupta, R., Salzberg, B.M., Grinvald, A., Cohen, L.B., Kamino, K., Lesher, S., Boyle, M.B., Waggoner, A.S. and Wang, C.H., Improvements in optical methods for measuring rapid changes in membrane potential, J. Membr. Biol., 58 (1981) 123-138. 8 Konnerth, A., Obaid, A.L. and Salzberg, B.M., Elasmobranch cerebellar slices in vitro: selective binding of potentiometric probes allows optical recording of electrical activity from different cells types, Biol. Bull., 169 (1985) 553. 9 Konnerth, A. and Orkand, R.K., Optical recording from axons and glial cells of frog optic nerve stained with voltage-sensitive dyes, Pflugers Arch., 405, Suppl. 2, (1985) R39. 10 Lev-Ram, V. and Grinvald, A., Is there a potassium-dependent depolarization of the paranodal region of myelin sheath? Soc. Neurosci. Abstr., 10 (1984) 948. 11 Maturana, H., The fine anatomy of the optic nerve of anurans - an electron microscope study, J. Biophys. Biochem. Cytol., 7 (1960) 107-119. 12 Orkand, R.K., Nicholls, J.G. and Kuffter, S.W., Effect of nerve impulses on the membrane potential of glial cells in the central nervous system of amphibia, J. Neurophysiol., 29 (1966) 788-806. 13 Salzberg, B.M., Optical recording of electrical activity in neurons using molecular probes. In J.L. Barker (Ed.), Current Methods in Cellular Neurobiology, Vol. 3, John Wiley & Sons, New York, 1983, pp. 139-187. 14 Salzberg, B.M., Grinvald, A., Cohen, L.B., Davila, H.V. and Ross, W.N., Optical recording of neuronal activity in an invertebrate central nervous system: simultaneous monitoring of several neurons, J. Neurophysiol., 40 (1977) 1281-!_29!,
54 15 Salzberg, B.M., Obaid, A.L., Senseman, D.M. and Gainer, H., Optical recording of action potentials from vertebrate nerve terminals using potentiometric probes provides evidence for sodium and calcium components, Nature (London), 306 (1983) 36-40. 16 Salzberg, B.M., Obaid, A.L., Shimizu, H., Orkand, R.K. and Senseman, D.M., Does the Schwann cell of Loligo act as a potassium electrode? Optical studies using potentiometric probes, Biol. Bull., 163 (1982) 390. 17 Senseman, D.M. and Salzberg, B.M., Electrical activity in an exocrine gland: optical recording with a potentiometric dye, Science, 208 (1980) 1269-1271. 18 Sykova, E., Extracellular K + accumulation in the central nervous system, Prog. Biophys. Mol. Biol., 42 (1983) 135-189.
49
NSL 03893 VOLTAGE-SENSITIVE DYES MEASURE POTENTIAL CHANGES IN AXONS AND GLIA OF THE FROG OPTIC NERVE
ARTHUR KONNERTH* and RICHARD K. ORKAND** Department of Physiology & Pharmacology, School of Dental Medicine and Mahoney Institute of Neurological Sciences, University of Pennsylvania, Philadelphia, PA 19104-6002 (U.S.A.) (Received January 13th, 1986; Revised version received and accepted January 29th, 1986)
Key words: neuroglia - voltage-sensitive dye - frog - optic nerve - action potential - afterpotential
Changes in dye absorption and fluorescence produced by electrical stimulation were measured in frog optic nerves stained with voltage-sensitive dyes. Following a single maximal stimulus applied through a suction electrode, the change in transmitted light intensity consisted of two components: one representing an axonal compound action potential and the second a slow depolarizing afterpotential which appeared to arise from the glial cells. The following results support this interpretation: (1) during a train of stimuli the depolarizing potentials sum and can exceed 80% of the initial spike amplitude while the spike amplitude itself remains essentially constant. Thus, the axons cannot have undergone significant depolarization during the train. (2) Optical recordings with simultaneous microelectrode recordings from the glial cells indicate that the change in glial membrane potential during the train has a time-course similar to that of the slow optical response. We conclude that voltage-sensitive dyes can monitor potential changes in both neurons and glia.
Voltage-sensitive dyes for m o n i t o r i n g c h a n g e s in m e m b r a n e p o t e n t i a l are finding wide a p p l i c a b i l i t y in the s t u d y o f the n e r v o u s system [5, 13]. This m e t h o d is b a s e d on the o b s e r v a t i o n t h a t certain m e m b r a n e dyes c h a n g e their a b s o r p t i o n o r their fluorescence linearly with changes in t r a n s m e m b r a n e v o l t a g e [3]. In a d d i t i o n to the c h a n g e in p o t e n t i a l , the m a g n i t u d e o f the optical signal d e p e n d s u p o n the a m o u n t o f dye b o u n d to the m e m b r a n e , the m e m b r a n e a r e a f r o m which it is r e c o r d e d , the n a t u r e o f the dye b i n d i n g sites a n d the sensitivity o f the dye to o t h e r c h a n g e s which m a y a c c o m p a n y the p o t e n t i a l change, as well as o n the optical r e c o r d i n g a p p a r a t u s itself. In a tissue like the n e r v o u s system, consisting o f m o r e t h a n one cell type a n d h a v i n g a c o m p l e x g e o m e t r y , one m i g h t expect s o m e difficulties in the i n t e r p r e t a t i o n o f optical signals arising f r o m voltage-sensitive dyes. In fact, p r e v i o u s studies have suggested the possibility t h a t r e c o r d i n g s f r o m n e u r o n s are c o n t a m i n a t e d b y a contrib u t i o n f r o m the glial cells [6, 8, 10, 15, 16]. In this study, we have c o m p a r e d the optical signals f r o m the whole o p t i c nerve o f the frog (Rana pipiens) with those o b t a i n e d *Present address: Max-Planck Institute for Psychiatry, D-8033 Planegg, F.R.G. **Author for correspondence at: Department of Physiology & Pharmacology, University of Pennsylvania SDM, 4001 Spruce St., Philadelphia, PA 19104-6002, U.S.A. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
50
A
B
lJ
LAMP
LENS
0
HEATFILTER
I
I
FILTER
I
I
1
570 nm
A
I
LENS a
PREPARATION
b
~
.........................
............................
OBJECTIVE FILTER 2
J
[
PHOTODIODE
T
I l/V-CONVERTER I
o--@
f SAMPLE & HOLD~
20 mV 10 ms
Fig. 1. A: schematic diagram of the experimental set-up for stimulation and recording. The optic nerve is mounted on the stage of a Nikon MS inverted microscope which was modified so that the stage remains stationery during focusing. It is held by two suction electrodes used for extracellular stimulation and recording, respectively. The bottom of the experimental chamber was a replaceable glass coverslip. When the experiment did not involve intracellular recording the air-water interface was eliminated with a microscope coverslip. For intracellular recording from glial cells we used a conventional 3 M KCI (20-40 M(~) electrode driven by a Narishige hydraulic manipulator. Light from a tungsten-halogen lamp (Osram Bellaphot) was focused on the nerve with a long-working distance condenser. The light was rendered quasimonochromatic by passing it through a heat filter (KG-1) and an interference filter. For absorption measurements, a × 63 objective formed a real image on a photodiode located in the trinocular tube at the image plane. The nerve filled the optical field (200/~m diameter) o f the objective. For fluorescence measurements a second interference filter (filter 2) was placed in front of the photodiode as a barrier filter. The photocurrent from the photodiode was passed to a current to voltage (I/V) converter whose output was recorded with a sample-and-hold circuit (Morad Autobucker) which subtracted the background light and allowed the recording of DC potentials (0-1 kHz). Signals were displayed on a Tektronix DI5 oscilloscope and photographed with a Grass camera. B: these traces were recorded during a single supramaximal electrical stimulation of an optic nerve stained with NK2367 at 0.2 mg/ml for 25 rain. The top trace (a) is the record of transmitted light intensity at 570_+15 nm. Following the stimulus there is an increase in transmitted light intensity consisting of a large spike and a long-lasting afterpotential of the same sign as the spike. At 660_+ 15 n m (b), there is a decrease in light intensity with the same time-course and relative amplitude of the spike and the afterpotential, respectively. This result is expected as a consequence of the changes
51
B
A
a
I lO-3
20 mY
b
o_f
[ 5x10-4
C
20 mV
I 3x10-4
6r~J nm
ls
20 mV
0.5 s Fig. 2. Responses to trains of stimuli. The optic nerve was stimulated at 30 Hz for 0.8 s. A: the nerve was stained with NK2367 (0.2 mg/ml for 25 min) and the changes in light absorption were recorded; 570+ 15 nm (a); 660+ 15 nm (c). The depolarizing afterpotentials sum and reach 80% of the initial spike height. The spikes do not decrease in amplitude during the train. The relative amplitudes of the spikes and slow potentials are the same at both wavelengths. Trace b shows the spikes as recorded by the suction electrode. B: this nerve was stained with WW781 (0.3 mg/ml for 20 min). Similar train of stimuli recorded at a slower timebase. The suction electrode record is shown in a. The middle trace (b) is a record of the increase in fluorescence of the nerve (exciting filter 630___15 nm; barrier filter 660_ 15 nm) during the train of stimuli. Because the fluorescence intensity is very low, the noise in the measurement is dominated by the noise contributed by the sample-and-hold amplifier. Trace c is an intracellular recording from a glial cell (Vm = - 8 8 mV) obtained simultaneously with the optical trace in b. The time-course of the glial depolarization is similar to that of the slow optical depolarization.
b y i n t r a c e l l u l a r m i c r o e l e c t r o d e r e c o r d i n g s f r o m glial cells i n t h e s a m e p r e p a r a t i o n [12], in o r d e r t o a s s e s s t h e c o n t r i b u t i o n o f t h e g l i a l cells t o t h e o p t i c a l s i g n a l . T h e o p t i c n e r v e , a n a t u r a l l y i s o l a t e d t r a c t o f t h e c e n t r a l n e r v o u s s y s t e m (ca. 4 0 0 # m d i a m e t e r ) , w a s d i s s e c t e d f r o m s m a l l (5 c m ) a d u l t f r o g s a f t e r d e c a p i t a t i o n . I t w a s held by both ends in suction electrodes for stimulation and recording in a 0.5-ml chamber
mounted
on the stage of a Nikon
inverted microscope. The nerves were
in absorption of this dye as a function of wavelength [17] and indicates that the signal results from a change in the properties of the voltage-sensitive dye and is not due to changes in tissue opacity. The bottom trace (c) is the potential recorded by the suction electrode and shows the usual unmyelinated axonal population spike. The peak is delayed relative to the optical spike because the photodiode monitors changes at the middle of the nerve and the suction electrode is located at the distal end. The spike of the myelinated axons (about 3% of total) is obscured by the stimulus artifact and in this preparation did not give rise to an optical response.
52 stained for 15-25 min in a 0.1-0.4 mg/ml solution of the voltage-sensitive dyes NK2367 (merocyanine-oxazolone) [14] or WW781 (merocyanine-rhodanine) [7] in normal Ringer's solution of the following composition (in mM): NaCI 112, KCI 2, CaCl2 2, glucose 33, HEPES 15 (adjusted with N a O H to pH 7.4). A diagram of the set-up for optical and electrical recording is shown in Fig. l A. Fig. 1B shows optical records obtained from a stained optic nerve, at two wavelengths, in response to a single supramaximal electrical stimulus, together with an electrical recording made with a suction electrode. The optical signal consists of a large compound action potential, arising primarily from the unmyelinated axons [2, I l] followed by a long-lasting depolarizing afterpotential. The inversion of both components of the signal with wavelength suggests that they are dye-specific and related to membrane potential and not due to changes in tissue opacity or light scattering [3, 13]. In the absence of dye we did not record optical signals of this magnitude during stimulation. Optical and electrical recordings during repetitive stimulation are illustrated in Fig. 2. The striking feature of the record in Fig. 2A, is that the long-lasting depolarizing afterpotentials sum until they reach 80% of the initial spike amplitude, while the spike amplitude itself does not decline. If the axons themselves were depolarizing slowly during the train, the spike amplitude would have decreased. Thus, it appears that the depolarization must arise not from the axons but from the glial cells. Repetitive stimulation of the frog optic nerve is known to produce a slow glial depolarization due to the transient accumulation of potassium, released from the active axons, in the narrow extracellular clefts separating the axons and glial cells [12, 18]. By contrast, the membrane potentials of axons are relatively insensitive to low levels of potassium and they depolarize little during repetitive activity and may even hyperpolarize. Because of their small diameter, 0.1-2 #m, there have not been any intracellular recordings from unmyelinated axons in the optic nerve. In Necturus optic nerve an analysis of extracellular records indicates that these axons depolarize by up to 2 mV during a brief train and hyperpolarize due to electrogenic sodium pumping immediately after stimulation stops [4]. In invertebrate neurons [1] there is hyperpolarization during and after a train of impulses. Fig. 2B shows a direct comparison of an optical signal from the whole nerve with an intracellular microelectrode recording from a glial cell in the same nerve. The time-course of the slow optical response closely resembles the glial depolarization recorded with a microelectrode, consistent with the assumption that the slow component of the optical signal arises from the glial cells. These results indicate that some voltage-sensitive dyes can respond to potential changes in glial cells as well as in neurons. If the responses were the same in both cell types and the membrane areas of the axons and glial cells were equal, the optical signals should be proportional to the voltage change in each cell type. However, preliminary naorphometric estimates (P.M. Orkand, unpublished), suggest that there is about 5 times as much unmyelinated axon membrane in this nerve as glial membrane. If we assume that the axonal spike is 100 mV and the glial depolarization to a brief train (30 Hz for 0.8 s) is 25 mV (Fig. 2Be), then the amplitude of the optical recording
53
of the spike should be 20 times larger than that of the slow depolarization. The optical signals corresponding to the spike and to the slow depolarization differed by a factor less than two, suggesting either that not all the axons were active in response to a supramaximal stimulus or that the dyes are more sensitive to glial depolarization. This latter possibility could arise if the dyes show greater affinity for glial membranes or if the changes in dye properties were more pronounced in the glial membrane environment. A third possibility is that dye penetration is limited and that the best staining occurs in an outer shell where the glial membrane predominates. Given the wide variety of substances useful as voltage-sensitive dyes [3, 5, 13] it seems reasonable to search for dyes with great specificity for recording from either neurons or glia. Some of these results were presented at a meeting of the Deutsche Physiologische Gesellschaft [9]. We thank Dr. Brian Salzberg for constant advice and the loan of some apparatus and Drs. A.L. Obaid and A. Grinvald for comments on the typescript. Supported by NIH Grant NS-12253 and a Feodor Lynen Fellowship from the Alexander von Humboldt Foundation (A.K.). 1 Baylor, D.A. and Nicholls, J.G., Changes in extracellular potassium concentration produced by neuronal activity in the central nervous system of the leech, J. Physiol. (London), 203 (1969) 555-569. 2 Bishop, G.H., Fiber groups in the optic nerve, Am. J. Physiol., 106 (1933) 460-474. 3 Cohen, L.B. and Salzberg, B.M., Optical measurement of membrane potential, Rev. Physiol. Biochem. Pharmacol., 83 (1978) 35-88. 4 Cohen, M.W., Glial potentials and their contribution to extracellular recording. In A. Remond (Ed.), Handbook of EEG and Clinical Neurophysiology, Vol. 2 Part B, Elsevier, Amsterdam, 1974, pp. 2B43-2B60. 5 Grinvald, A., Real-time optical mapping of neuronal activity, Annu. Rev. Neurosci., 8 (1985) 263-305. 6 Grinvald, A., Manker, A. and Segal, M., Visualization of the spread of electrical activity in rat hippocampal slices by voltage sensitive optical probes, J. Physiol. (London), 333 (1982) 269-291. 7 Gupta, R., Salzberg, B.M., Grinvald, A., Cohen, L.B., Kamino, K., Lesher, S., Boyle, M.B., Waggoner, A.S. and Wang, C.H., Improvements in optical methods for measuring rapid changes in membrane potential, J. Membr. Biol., 58 (1981) 123-138. 8 Konnerth, A., Obaid, A.L. and Salzberg, B.M., Elasmobranch cerebellar slices in vitro: selective binding of potentiometric probes allows optical recording of electrical activity from different cells types, Biol. Bull., 169 (1985) 553. 9 Konnerth, A. and Orkand, R.K., Optical recording from axons and glial cells of frog optic nerve stained with voltage-sensitive dyes, Pflugers Arch., 405, Suppl. 2, (1985) R39. 10 Lev-Ram, V. and Grinvald, A., Is there a potassium-dependent depolarization of the paranodal region of myelin sheath? Soc. Neurosci. Abstr., 10 (1984) 948. 11 Maturana, H., The fine anatomy of the optic nerve of anurans - an electron microscope study, J. Biophys. Biochem. Cytol., 7 (1960) 107-119. 12 Orkand, R.K., Nicholls, J.G. and Kuffter, S.W., Effect of nerve impulses on the membrane potential of glial cells in the central nervous system of amphibia, J. Neurophysiol., 29 (1966) 788-806. 13 Salzberg, B.M., Optical recording of electrical activity in neurons using molecular probes. In J.L. Barker (Ed.), Current Methods in Cellular Neurobiology, Vol. 3, John Wiley & Sons, New York, 1983, pp. 139-187. 14 Salzberg, B.M., Grinvald, A., Cohen, L.B., Davila, H.V. and Ross, W.N., Optical recording of neuronal activity in an invertebrate central nervous system: simultaneous monitoring of several neurons, J. Neurophysiol., 40 (1977) 1281-!_29!,
54 15 Salzberg, B.M., Obaid, A.L., Senseman, D.M. and Gainer, H., Optical recording of action potentials from vertebrate nerve terminals using potentiometric probes provides evidence for sodium and calcium components, Nature (London), 306 (1983) 36-40. 16 Salzberg, B.M., Obaid, A.L., Shimizu, H., Orkand, R.K. and Senseman, D.M., Does the Schwann cell of Loligo act as a potassium electrode? Optical studies using potentiometric probes, Biol. Bull., 163 (1982) 390. 17 Senseman, D.M. and Salzberg, B.M., Electrical activity in an exocrine gland: optical recording with a potentiometric dye, Science, 208 (1980) 1269-1271. 18 Sykova, E., Extracellular K + accumulation in the central nervous system, Prog. Biophys. Mol. Biol., 42 (1983) 135-189.