- Email: [email protected]
Transient calcium current of retinal bipolar cells of the mouse
Neuroscience Research, Suppl. 10 (1989) $67-$76
$67
Elsevier ScientificPublishersIreland Ltd. TRANSIENT CALCIUM CURRENT OF RETINAL BIPOLAR CELLS OF THE MOUSE
A. KANEKO, M. TACHIBANA and L. H. PINTO* National Institute for Physiological Sciences, Okazaki 444 Japan, and *Department of Neurobiology and Physiology, Northwestern University, Evanston, IL 60201 USA
INTRODUCTION Motion detection is one of the important functions of the visual system. Motion sensitive retinal ganglion cells have been found in various species of vertebrates. Well known examples are frogs (1) and rabbits (2). A characteristic feature of motion sensitive neurons is that their responses to a light flash have prominent phasic component. Yet it is well known that cells in the distal retina (photoreceptors, horizontal and bipolar cells) all show sustained light responses. It is still a puzzle how the sustained signals in the distal retina are shaped into phasic responses in cells of the proximal retina, i.e., amacrine and ganglion cells. The shaping of sustained signals into transient ones may involve various mechanisms: activation of specific membrane conductances of amacrine and ganglion cells, interaction between excitatory and inhibitory signals that arrive at a postsynaptic cell with a time delay, or a transient release of transmitter substance from presynaptic (bipolar) cells. It is important, in this sense, to study the nature of calcium current in bipolar cells, because if the calcium current flows transiently, it will produce a transient increase in intracellular calcium concentration, which in turn will trigger a transient transmitter release from bipolar cells. A few years ago we studied ionic currents of bipolar cells isolated from the goldfish retina, and identified a Ca current and three other types of cationic currents (3). The Ca current in goldfish bipolar cells was sustained, showing no decay during the 2 s depolarization. More recently we analyzed membrane currents of solitary bipolar cells dissociated from the mouse retina (4). The reason that we selected mouse is two-fold. First, we wanted to fill a gap in our knowledge of mammalian retinal interneurons. Despite a vast amount of work on retinal interneurons in lower vertebrates, reports on those of mammals are very limited (5-7). Second, there is a large variety of mouse mutations which affect the visual system without causing gross degeneration (8).
Comparison of the retinae of such mutants with
those of normal animals will allow certain inferences to be made about the function of the affected gene products; a first requirement for this comparison is a thorough study of the retinal neuron in the wild type animal. We found that the Ca current of mouse bipolar cells has a transient character and thus differs from the Ca current of goldfish bipolar cells. Here, we summarize our findings.
Presented at the 11th TaniguchiInternationalSymposiumon Visual Science,November28-December 2, 1988 0168-0102/89/$03.50 © 1989 ElsevierScientificPublishersIreland Ltd.
$68
Figure 1: Solitary bipolar cells dissociated from the mouse retina. Scale bar 20 #m.
A
B
C 120
6~406,~,4,ore
]
:8"o
50pA 24mV 50msec
-6mV
v
-60]
-46 -86
isec
IIOpA
-86
Figure 2: M e m b r a n e currents recorded from a bipolar cell of the mouse. A. C u r r e n t s evoked by 2 s c o m m a n d pulses. Vh = -46 mV. Note an inward current tail at the cessation of the hyperpolarizing c o m m a n d . B. Currents evoked by 100 ms depolarization to 24 m V {upper trace} a n d to -36 m V {lower trace}. Records are displayed on a much faster time scale t h a n in A. The vertical broken line indicates the time at which the current a m p l i t u d e was measured a n d plotted in the current-voltage relation curve of C. V h = -86 mV.
$69
MEMBRANE CURRENTS OF SOLITARY BIPOLAR CELLS OF THE MOUSE We obtained solitary bipolar cells from adult mouse (C57BL/6J and C57BL/6crSIc) retinae by enzymatic (papain) dissociation (Fig. 1). Membrane currents of these cells were measured by a patch clamp technique in the whole-cell configuration. As shown in Fig. 2A, hyperpolarization from the holding voltage, Vh, of-46 mV evoked a slowly-activated inward current (identified as an h-current on the criterion that this current component was blocked by 10 mM Cs + applied to the superfusate). Depolarization evoked a TEA-sensitive, outward current (a combination of voltage-sensitive and calcium-dependent K currents). Since a 2 s depolarization to around -10 mV evoked a sustained Ca current in goldfish bipolar cells (3; also see Fig. 9 of this article), we expected to observe a sustained Ca current also in mouse bipolar cells and used initially the same voltage-clamp protocol as we had used in the experiment on goldfish bipolar cells. However, no such Ca current was seen even after the counteracting K currents were suppressed by an application of Cs + and/or TEA to the external medium or by replacing K + of the pipette solution with Cs +. In response to a 2 s hyperpolarizing command pulses, we found a transient inward tail current when a command more negative than -70 mV was terminated (Fig. 2A, lower trace). This tail current was identified as a Ca current by the experiments to be described. The reason that the Ca current was detected as an inward tail current was that the Ca conductance had been inactivated while the cell was held at -46 mV and was reactivated during the long (>2 s) hyperpolarizing command.
THE TRANSIENT INWARD CURRENT IS IDENTIFIED AS A Ca CURRENT A prominent inward current was generated when the cell was depolarized from a more hyperpolarized Vh (Fig. 2B, lower trace). The current was identified as a Ca current (Ica) on the basis of the following observations. First, the transient inward current was resistant to 5 #M TTX. (The same concentration of TTX blocked action potentials of ganglion cells in the same dish. Ganglion cells were identified by a large cell size, 4-5 long dendrites radially extending from the perikaryon.) Second, the transient inward current became undetectable in 0 Ca ~+ medium (Fig. 3A), and became larger in high Ca medium (Fig.
3B).
Third, the
transient inward current was blocked completely by 4 mM Co 2+ (Fig. 4). Thus, Ica was isolated by computer subtraction of current recorded in the presence of 4 mM Co 2+ from that recorded in the absence of Co 2+ (K currents had been suppressed by using Cs + for the pipette solution). Ica, thus isolated, was detectable at membrane potentials between about -60 mV and +40 mV with the peak at about -30 mV. The decrease in amplitude with strong depolarization of the membrane is probably due to the reduction of driving force, because over this depolarized potential range calcium conductance (gCa) is fully activated (see Fig. 7).
$70
B
A
~ l
2Ca
2Ca "
2Ca A
5Omsec -86
-86
Figure 3: Effects of 0 Ca 2+ (A, middle trace) and of elevation of [Ca2+],, (B, middle trace) on the transient inward current of mouse bipolar cells. The inward current was activated by depolarization to -36 (A) or to -26 (B) mV from the Vh of -86 mV. Top traces, control; bottom traces, recovery. Pipette solution contained 120 mM-CsOl. (Reproduced from ref. 4)
C
B
A
recovery
Co
control
-86
E
D difference
:
'
'
VT'o
'
r-:~-n__ -86
J2OpA 50msec
1-40
Figure 4: Effect of Co ~+. The transient inward current of mouse bipolar cell (A, control) is completely blocked by an application of 4 mM Co2+ to the superfusate (B). D, the Co2+-sensitive current obtained by computer subtraction of trace B from trace A. E, current-voltage relation of the Co2+-sensitive current. Pipette solution contained 120 mM-CsCI. (Reproduced from ref. 4)
S71
(
50pA 10 pm
20msec
Figure 5: Spatial distribution of Ic(,. The upper trace illustrates L;, flow at the soma.
The lower trace
illustrates L;., flow at the axon terminal. See the text for the method to obtained these records. Currents were evoked by a 20 ms depolarizing c o m m a n d to -36 m V from a holding voltage of -96 m V . Pipette solution contained 120 rnM-OsCl. (Reproduced from ref. 4)
DISTRIBUTION OF Ica ON THE CELL SURFACE To examine the possibility that Ica may participate in transmitter release from bipolar cells, we studied whether Ica flow at the axon terminal. Ica flowed at the axon terminal was obtained by subtraction of whole-cell current recorded when 4 mM Co 2+ was applied only to the axon terminal (from ca. 1 /zm tip pipette placed at the axon terminal) from total whole-cell Ica. A similar procedure was used to determine Ica flow at the soma (this time the Co2+-containing pipette was placed at the soma). We found that I(Ta flowed both at the soma and at the axon terminal. At the axon terminal the amplitude Of Ica was small (Fig. 5), but its time course was similar to the Ica flowing at the soma. These observations suggestthe possibility that this current may participate in transmitter release from the axon terminal.
VOLTAGE DEPENDENCE OF Ica ACTIVATION AND INACTIVATION
Activation
As shown in Fig. 4, Ice was detected as an inward current at membrane potentials between about -60 mV and +40 mV. Since the current amplitude is the function of both the driving force (the potential difference between the membrane potential and the equilibrium potential of Ca s+, Eta) and the conductance, it is important to know the voltage dependence of gCa. By using tail current, we measured gCa directly. Tail current analysis was performed in the following way. First, gCa was activated by
$72
~r
50pA 20msec
- 6 mV I
I
1
1
L
l
-96 Figure 6: Six superimposed current traces obtained from a mouse bipolar cell evoked by depolarizing commands (to -6 mV from -96 mV Vh) of various duration ranging from 20 ms to 140 ms. Note large tail currents evoked at the end of the command pulses. Pipette solution contained 120 mM-CsCl. membrane depolarization to a specific potential.
When gCa was fully activated (for that
membrane potential), the depolarizing c o m m a n d was terminated by driving the membrane potential back to Vh.
Calcium channels remained open at this moment, and a Ca (tail)
current flowed. As seen in Fig. 6, the amplitude of the tail current was proportional to the amplitude of Ic~ that flowed immediately before the termination of the depolarizing command. Since the driving force during the tail current (the difference between Ev~ and Vh) remained constant, independent of c o m m a n d voltages, the amplitude of the tail current was directly proportional to gCa. As summarized in Fig. 7, gCa showed a sigmoidal increase with depolarization to potentials more positive than -65 mV. The half maximal activation occurred at about -25 mV, and the full activation at around +30 m V (in 10 m M [Ca2+]o).
Inactivation Inactivation of gCa was also voltage-dependent. Data illustrated in Fig. 7 were obtained from a series of experiment in which cells were held for 1 s at conditioning voltages prior to activating pulses (to -6 mV). No suppression of gCa was seen so long as the cell was held at potentials more negative than -80 mV. gCa was almost completely suppressed by a 1 s conditioning at -30 mV or more. Between -80 mV and -30 m V the degree of inactivation of gCa was sigmoidally related to the m e m b r a n e potential (half inactivation at about -50 mV). These observations indicate that I c , can be activated at m e m b r a n e potentials between -65 and -10 m V (in 10 m M [Ca2+]o). Under physiological conditions, (2 m M [Ca~+]o), both the activation and the inactivation curve must be moved by approximately 20 mV in the
$73
B
A
I sec conditioning
10-40 msec
10msec activating pulse
~ - ~
T
-6mV -96mV
-96mV
measurement
messurement
C 1.0 o c t~ (J -s "O
0.5 o
i
-100
i
-80
i
-60
i
-40
i
-20
i
i
i
0
20
40
i
60mV
membrane potential
Figure 7: Activation and inactivation curves, e: relative conductance (mean + S.D., n = 4), measured using tail currents (pulse protocol shown in A), plotted against test pulse voltage, o: relative conductance (mean + S.D., n = 4), measured using the tail current to a -6 mV activating pulse that followed a 1 s conditioning pulse of each of many voltages (pulse protocol shown in B}, plotted against voltage of the conditioning pulse. Vn = -96 mV. [Ca2+}o = 10 raM. Pipette solution contained 120 mM-CsCl. (Reproduced from ref. 4)
$74
Mouse
Goldfish
Nifedipine 00pA
5pA
Bay K 8644 --'~ ~I I 20msec
---I
lOOpA
~
I
lOOpA
.J 2secI
Figure 8: Effects of dihydropyridine compounds on Ca2+-currents of bipolar cells of the mouse and of the goldfish. Each panel consists of two superimposed traces, one obtained under control condition (&) and another taken during application of test substances (10 #M, each; marked with /x). Note identical traces of the mouse records, indicating the absence of any effect by dihydropyridine. Mouse bipolar cells were held at -96 mV and depolarized to -6 mV for 20 ms. Goldfish bipolar cells were held at -46 mV and depolarized to -6 mV for 2 s. Pipette solution contained 120 mM-CsCl. (Reproduced from ref. 4) h y p e r p o l a r i z i n g direction along the voltage axis to c o m p e n s a t e for the difference in surface charge. T h u s , I w would be activated at m e m b r a n e p o t e n t i a l s between -80 a n d -25 mV. Two different m e c h a n i s m s for Iea inactivation are known; v o l t a g e - d e p e n d e n t inactivation a n d inactivation by elevated [Ca2+]~. I n a c t i v a t i o n of Iv~ in m o u s e b i p o l a r cells is likely to be C a - i n d e p e n d e n t , because it did not parallel Ca z+ influx. Ira was i n a c t i v a t e d at m e m b r a n e voltages at which no Ie~ was detected (e.g., more positive t h a n + 4 0 m V or below -65 mV).
E F F E C T S O F D I H Y D R O P Y R I D I N E S ON Ic~ O F M O U S E B I P O L A R CELLS It has been s h o w n so far t h a t Ira in mouse bipolar cells exhibits a s t r o n g v o l t a g e - d e p e n d e n t inactivation. In t h e light of recent classifications of Ca 2+ c h a n n e l s (9, 10), Ic~ of m o u s e bipolar cells was identified as t h e t r a n s i e n t (T) type. In c o n t r a s t to Ic~ in m o u s e bipolar cells, the calcium c u r r e n t of goldfish bipolar cells was of the longlasting (L) type, showing no decay d u r i n g the 2 s depolarization. It has also been r e p o r t e d t h a t d i h y d r o p y r i d i n e c o m p o u n d s modify t h e T - t y p e a n d L-type C a c h a n n e l s differentially. As in o t h e r p r e p a r a t i o n s (11), Ica in t h e m o u s e bipolar cells was unaffected either by 10 # M nifedipine or 10 /~M Bay K 8644 while t h e calcium c u r r e n t of
$75
Mouse
Goldfish lOpA
-86
I -36mY t
_I -60
lOOmsec
-80
Mouse~
lOmV
L
2sec
-40
I0
40
mV
""\ ~ • /~(.o/ Goldfish
Figure 9: Comparison of Ic,~ in mouse and goldfish bipolar cells. IG. in mouse bipolar cell was activated by
100 ms depolarising command (from Vh of -86 mV to -36 mV), while L;'. in goldfish bipolar cell was activated by 2 s depolarizing command (from Vh of-60 mV to 10 mV). Current-voltage relations of I(~.,,in mouse (.) and goldfish (o) bipolar cells are shown for comparison after normalization of the peak amplitude. goldfish bipolar cells was either blocked or enhanced to a remarkable extent (Fig. 8). T-type Iea of mouse bipolar cells was also resistant to 50/~M Cd 2+ that blocked Ira in goldfish.
P H Y S I O L O G I C A L R O L E O F Ic~ IN M O U S E B I P O L A R CELLS We demonstrated that the transient inward current of mouse b i p o l a r ceils is carried by Ca 2+. The current is blocked by Co ~+, but was resistant to T T X . Activation and inactivation analysis shows that Ica can be activated at m e m b r a n e potentials between -80 and -25 m'V under physiological conditions, ([Ca2+]o ~, 2 raM). It is unfortunate that reports of intracellular recordings from mouse bipolar cells have yet to be published. If, however, the light responses of mouse bipolar cells lie within a potential range similar to that of light responses in lower vertebrates (12), they will also lie well within the activation range of Ica. The finding t h a t Ic~ flows b o t h at the perikaryon and at the axon terminal of mouse bipolar cells raises the possibility t h a t this current may participate in synaptic transmission, and in shaping the transient responses of mouse ganglion and amacrine cells. Presence of the transient Ic~ strongly suggests that the resulting increase in intracellular calcium concentration must also be transient.
Since the increase of [Ca2+]o is prerequisite to the transmitter re-
lease, it may be reasonable to believe that transmitter release from mouse bipolar cells is also transient. It has long been a puzzle how the transient ON- or OFF-responses of amacrine or
$76
ganglion cells are formed. The present finding offers a possible explanation of these transient responses, at least in the mouse. ACKNOWLEDGEMENT We thank Michi Hosono for her excellent technical assistance in preparing isolated photoreceptors. This research was supported in part by the Grants in Aid for Scientific Research from the Ministry of Education, Science and Culture (Nos. 61304032, 63638517, 63112007 to AK and 63480111, 63641537 to MT), NEI R01EY01221 (to LHP), NSF INT8613447 and Japan Society for the Promotion of Science. REFERENCES 1. Maturana HR, Lettvin JY, McCulloch WS, Pitts WH (1960) Anatomy and physiology of vision in the frog (Rana pipiens). J. Gen. Physiol. 43 (suppl 2):129-171. 2. Barlow HB, Levick WR (1965) The mechanism of directionally selective units in rabbit's retina. J Physiol (Load) 178:477-504. 3. Kaneko A, Tachibana M (1985) A voltage-clamp analysis of membrane currents in solitary bipolar cells dissociated from Carassius auratus. J Physiol (Load) 358:131-152 4. Kaneko A, Pinto LH, Tachibana M (1989) Transient calcium current of retinal bipolar cells of the mouse. J Physiol (Lond) 410:613-629. 5. Baylor DA, Nunn B J, Schnapf JL (1984) The photocurrent, noise and spectral sensitivity of rods of the monkey Maeaea fascilularis. J Physiol (Lond) 357:575-607 6. Kolb H, Nelson R (1984) Neural architecture of the cat retina. Prog Retinal Res 3:21-60 7. Suzuki H, Pinto LH (1986) Response properties of horizontal cells in the isolated retina of wild-type and pearl mutant mice. J Neurosei 6:1122-1128 8. Balkema GW, Mangini N J, Pinto LH, Vanable JW Jr (1984) Visually evoked eye movements in mouse mutants and inbred strains. Invest Ophthalmol Visual Sci 25:795-800 9. Nilius B, Hess P, Lansman JB, Tsien RW (1985) A novel type of cardiac calcium channel in ventricular cells. Nature 291:497-500 10. Hagiwara N, Irisawa H, Kameyama M (1988) Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells. J Physiol (Load) 395:233-253 11. Nowycky MC, Fox AP, Tsien RW (1985) Three types of neuronal calcium channels with different calcium agonist sensitivity. Nature 316:440-443 12. Saito T, Kondo H, Toyoda JI (1979) Ionic mechanisms of two types of on-center bipolar cells lin the carp retina. I. The responses to central illumination. J. Gen. Physiol. '/3:73-90.
$67
Elsevier ScientificPublishersIreland Ltd. TRANSIENT CALCIUM CURRENT OF RETINAL BIPOLAR CELLS OF THE MOUSE
A. KANEKO, M. TACHIBANA and L. H. PINTO* National Institute for Physiological Sciences, Okazaki 444 Japan, and *Department of Neurobiology and Physiology, Northwestern University, Evanston, IL 60201 USA
INTRODUCTION Motion detection is one of the important functions of the visual system. Motion sensitive retinal ganglion cells have been found in various species of vertebrates. Well known examples are frogs (1) and rabbits (2). A characteristic feature of motion sensitive neurons is that their responses to a light flash have prominent phasic component. Yet it is well known that cells in the distal retina (photoreceptors, horizontal and bipolar cells) all show sustained light responses. It is still a puzzle how the sustained signals in the distal retina are shaped into phasic responses in cells of the proximal retina, i.e., amacrine and ganglion cells. The shaping of sustained signals into transient ones may involve various mechanisms: activation of specific membrane conductances of amacrine and ganglion cells, interaction between excitatory and inhibitory signals that arrive at a postsynaptic cell with a time delay, or a transient release of transmitter substance from presynaptic (bipolar) cells. It is important, in this sense, to study the nature of calcium current in bipolar cells, because if the calcium current flows transiently, it will produce a transient increase in intracellular calcium concentration, which in turn will trigger a transient transmitter release from bipolar cells. A few years ago we studied ionic currents of bipolar cells isolated from the goldfish retina, and identified a Ca current and three other types of cationic currents (3). The Ca current in goldfish bipolar cells was sustained, showing no decay during the 2 s depolarization. More recently we analyzed membrane currents of solitary bipolar cells dissociated from the mouse retina (4). The reason that we selected mouse is two-fold. First, we wanted to fill a gap in our knowledge of mammalian retinal interneurons. Despite a vast amount of work on retinal interneurons in lower vertebrates, reports on those of mammals are very limited (5-7). Second, there is a large variety of mouse mutations which affect the visual system without causing gross degeneration (8).
Comparison of the retinae of such mutants with
those of normal animals will allow certain inferences to be made about the function of the affected gene products; a first requirement for this comparison is a thorough study of the retinal neuron in the wild type animal. We found that the Ca current of mouse bipolar cells has a transient character and thus differs from the Ca current of goldfish bipolar cells. Here, we summarize our findings.
Presented at the 11th TaniguchiInternationalSymposiumon Visual Science,November28-December 2, 1988 0168-0102/89/$03.50 © 1989 ElsevierScientificPublishersIreland Ltd.
$68
Figure 1: Solitary bipolar cells dissociated from the mouse retina. Scale bar 20 #m.
A
B
C 120
6~406,~,4,ore
]
:8"o
50pA 24mV 50msec
-6mV
v
-60]
-46 -86
isec
IIOpA
-86
Figure 2: M e m b r a n e currents recorded from a bipolar cell of the mouse. A. C u r r e n t s evoked by 2 s c o m m a n d pulses. Vh = -46 mV. Note an inward current tail at the cessation of the hyperpolarizing c o m m a n d . B. Currents evoked by 100 ms depolarization to 24 m V {upper trace} a n d to -36 m V {lower trace}. Records are displayed on a much faster time scale t h a n in A. The vertical broken line indicates the time at which the current a m p l i t u d e was measured a n d plotted in the current-voltage relation curve of C. V h = -86 mV.
$69
MEMBRANE CURRENTS OF SOLITARY BIPOLAR CELLS OF THE MOUSE We obtained solitary bipolar cells from adult mouse (C57BL/6J and C57BL/6crSIc) retinae by enzymatic (papain) dissociation (Fig. 1). Membrane currents of these cells were measured by a patch clamp technique in the whole-cell configuration. As shown in Fig. 2A, hyperpolarization from the holding voltage, Vh, of-46 mV evoked a slowly-activated inward current (identified as an h-current on the criterion that this current component was blocked by 10 mM Cs + applied to the superfusate). Depolarization evoked a TEA-sensitive, outward current (a combination of voltage-sensitive and calcium-dependent K currents). Since a 2 s depolarization to around -10 mV evoked a sustained Ca current in goldfish bipolar cells (3; also see Fig. 9 of this article), we expected to observe a sustained Ca current also in mouse bipolar cells and used initially the same voltage-clamp protocol as we had used in the experiment on goldfish bipolar cells. However, no such Ca current was seen even after the counteracting K currents were suppressed by an application of Cs + and/or TEA to the external medium or by replacing K + of the pipette solution with Cs +. In response to a 2 s hyperpolarizing command pulses, we found a transient inward tail current when a command more negative than -70 mV was terminated (Fig. 2A, lower trace). This tail current was identified as a Ca current by the experiments to be described. The reason that the Ca current was detected as an inward tail current was that the Ca conductance had been inactivated while the cell was held at -46 mV and was reactivated during the long (>2 s) hyperpolarizing command.
THE TRANSIENT INWARD CURRENT IS IDENTIFIED AS A Ca CURRENT A prominent inward current was generated when the cell was depolarized from a more hyperpolarized Vh (Fig. 2B, lower trace). The current was identified as a Ca current (Ica) on the basis of the following observations. First, the transient inward current was resistant to 5 #M TTX. (The same concentration of TTX blocked action potentials of ganglion cells in the same dish. Ganglion cells were identified by a large cell size, 4-5 long dendrites radially extending from the perikaryon.) Second, the transient inward current became undetectable in 0 Ca ~+ medium (Fig. 3A), and became larger in high Ca medium (Fig.
3B).
Third, the
transient inward current was blocked completely by 4 mM Co 2+ (Fig. 4). Thus, Ica was isolated by computer subtraction of current recorded in the presence of 4 mM Co 2+ from that recorded in the absence of Co 2+ (K currents had been suppressed by using Cs + for the pipette solution). Ica, thus isolated, was detectable at membrane potentials between about -60 mV and +40 mV with the peak at about -30 mV. The decrease in amplitude with strong depolarization of the membrane is probably due to the reduction of driving force, because over this depolarized potential range calcium conductance (gCa) is fully activated (see Fig. 7).
$70
B
A
~ l
2Ca
2Ca "
2Ca A
5Omsec -86
-86
Figure 3: Effects of 0 Ca 2+ (A, middle trace) and of elevation of [Ca2+],, (B, middle trace) on the transient inward current of mouse bipolar cells. The inward current was activated by depolarization to -36 (A) or to -26 (B) mV from the Vh of -86 mV. Top traces, control; bottom traces, recovery. Pipette solution contained 120 mM-CsOl. (Reproduced from ref. 4)
C
B
A
recovery
Co
control
-86
E
D difference
:
'
'
VT'o
'
r-:~-n__ -86
J2OpA 50msec
1-40
Figure 4: Effect of Co ~+. The transient inward current of mouse bipolar cell (A, control) is completely blocked by an application of 4 mM Co2+ to the superfusate (B). D, the Co2+-sensitive current obtained by computer subtraction of trace B from trace A. E, current-voltage relation of the Co2+-sensitive current. Pipette solution contained 120 mM-CsCI. (Reproduced from ref. 4)
S71
(
50pA 10 pm
20msec
Figure 5: Spatial distribution of Ic(,. The upper trace illustrates L;, flow at the soma.
The lower trace
illustrates L;., flow at the axon terminal. See the text for the method to obtained these records. Currents were evoked by a 20 ms depolarizing c o m m a n d to -36 m V from a holding voltage of -96 m V . Pipette solution contained 120 rnM-OsCl. (Reproduced from ref. 4)
DISTRIBUTION OF Ica ON THE CELL SURFACE To examine the possibility that Ica may participate in transmitter release from bipolar cells, we studied whether Ica flow at the axon terminal. Ica flowed at the axon terminal was obtained by subtraction of whole-cell current recorded when 4 mM Co 2+ was applied only to the axon terminal (from ca. 1 /zm tip pipette placed at the axon terminal) from total whole-cell Ica. A similar procedure was used to determine Ica flow at the soma (this time the Co2+-containing pipette was placed at the soma). We found that I(Ta flowed both at the soma and at the axon terminal. At the axon terminal the amplitude Of Ica was small (Fig. 5), but its time course was similar to the Ica flowing at the soma. These observations suggestthe possibility that this current may participate in transmitter release from the axon terminal.
VOLTAGE DEPENDENCE OF Ica ACTIVATION AND INACTIVATION
Activation
As shown in Fig. 4, Ice was detected as an inward current at membrane potentials between about -60 mV and +40 mV. Since the current amplitude is the function of both the driving force (the potential difference between the membrane potential and the equilibrium potential of Ca s+, Eta) and the conductance, it is important to know the voltage dependence of gCa. By using tail current, we measured gCa directly. Tail current analysis was performed in the following way. First, gCa was activated by
$72
~r
50pA 20msec
- 6 mV I
I
1
1
L
l
-96 Figure 6: Six superimposed current traces obtained from a mouse bipolar cell evoked by depolarizing commands (to -6 mV from -96 mV Vh) of various duration ranging from 20 ms to 140 ms. Note large tail currents evoked at the end of the command pulses. Pipette solution contained 120 mM-CsCl. membrane depolarization to a specific potential.
When gCa was fully activated (for that
membrane potential), the depolarizing c o m m a n d was terminated by driving the membrane potential back to Vh.
Calcium channels remained open at this moment, and a Ca (tail)
current flowed. As seen in Fig. 6, the amplitude of the tail current was proportional to the amplitude of Ic~ that flowed immediately before the termination of the depolarizing command. Since the driving force during the tail current (the difference between Ev~ and Vh) remained constant, independent of c o m m a n d voltages, the amplitude of the tail current was directly proportional to gCa. As summarized in Fig. 7, gCa showed a sigmoidal increase with depolarization to potentials more positive than -65 mV. The half maximal activation occurred at about -25 mV, and the full activation at around +30 m V (in 10 m M [Ca2+]o).
Inactivation Inactivation of gCa was also voltage-dependent. Data illustrated in Fig. 7 were obtained from a series of experiment in which cells were held for 1 s at conditioning voltages prior to activating pulses (to -6 mV). No suppression of gCa was seen so long as the cell was held at potentials more negative than -80 mV. gCa was almost completely suppressed by a 1 s conditioning at -30 mV or more. Between -80 mV and -30 m V the degree of inactivation of gCa was sigmoidally related to the m e m b r a n e potential (half inactivation at about -50 mV). These observations indicate that I c , can be activated at m e m b r a n e potentials between -65 and -10 m V (in 10 m M [Ca2+]o). Under physiological conditions, (2 m M [Ca~+]o), both the activation and the inactivation curve must be moved by approximately 20 mV in the
$73
B
A
I sec conditioning
10-40 msec
10msec activating pulse
~ - ~
T
-6mV -96mV
-96mV
measurement
messurement
C 1.0 o c t~ (J -s "O
0.5 o
i
-100
i
-80
i
-60
i
-40
i
-20
i
i
i
0
20
40
i
60mV
membrane potential
Figure 7: Activation and inactivation curves, e: relative conductance (mean + S.D., n = 4), measured using tail currents (pulse protocol shown in A), plotted against test pulse voltage, o: relative conductance (mean + S.D., n = 4), measured using the tail current to a -6 mV activating pulse that followed a 1 s conditioning pulse of each of many voltages (pulse protocol shown in B}, plotted against voltage of the conditioning pulse. Vn = -96 mV. [Ca2+}o = 10 raM. Pipette solution contained 120 mM-CsCl. (Reproduced from ref. 4)
$74
Mouse
Goldfish
Nifedipine 00pA
5pA
Bay K 8644 --'~ ~I I 20msec
---I
lOOpA
~
I
lOOpA
.J 2secI
Figure 8: Effects of dihydropyridine compounds on Ca2+-currents of bipolar cells of the mouse and of the goldfish. Each panel consists of two superimposed traces, one obtained under control condition (&) and another taken during application of test substances (10 #M, each; marked with /x). Note identical traces of the mouse records, indicating the absence of any effect by dihydropyridine. Mouse bipolar cells were held at -96 mV and depolarized to -6 mV for 20 ms. Goldfish bipolar cells were held at -46 mV and depolarized to -6 mV for 2 s. Pipette solution contained 120 mM-CsCl. (Reproduced from ref. 4) h y p e r p o l a r i z i n g direction along the voltage axis to c o m p e n s a t e for the difference in surface charge. T h u s , I w would be activated at m e m b r a n e p o t e n t i a l s between -80 a n d -25 mV. Two different m e c h a n i s m s for Iea inactivation are known; v o l t a g e - d e p e n d e n t inactivation a n d inactivation by elevated [Ca2+]~. I n a c t i v a t i o n of Iv~ in m o u s e b i p o l a r cells is likely to be C a - i n d e p e n d e n t , because it did not parallel Ca z+ influx. Ira was i n a c t i v a t e d at m e m b r a n e voltages at which no Ie~ was detected (e.g., more positive t h a n + 4 0 m V or below -65 mV).
E F F E C T S O F D I H Y D R O P Y R I D I N E S ON Ic~ O F M O U S E B I P O L A R CELLS It has been s h o w n so far t h a t Ira in mouse bipolar cells exhibits a s t r o n g v o l t a g e - d e p e n d e n t inactivation. In t h e light of recent classifications of Ca 2+ c h a n n e l s (9, 10), Ic~ of m o u s e bipolar cells was identified as t h e t r a n s i e n t (T) type. In c o n t r a s t to Ic~ in m o u s e bipolar cells, the calcium c u r r e n t of goldfish bipolar cells was of the longlasting (L) type, showing no decay d u r i n g the 2 s depolarization. It has also been r e p o r t e d t h a t d i h y d r o p y r i d i n e c o m p o u n d s modify t h e T - t y p e a n d L-type C a c h a n n e l s differentially. As in o t h e r p r e p a r a t i o n s (11), Ica in t h e m o u s e bipolar cells was unaffected either by 10 # M nifedipine or 10 /~M Bay K 8644 while t h e calcium c u r r e n t of
$75
Mouse
Goldfish lOpA
-86
I -36mY t
_I -60
lOOmsec
-80
Mouse~
lOmV
L
2sec
-40
I0
40
mV
""\ ~ • /~(.o/ Goldfish
Figure 9: Comparison of Ic,~ in mouse and goldfish bipolar cells. IG. in mouse bipolar cell was activated by
100 ms depolarising command (from Vh of -86 mV to -36 mV), while L;'. in goldfish bipolar cell was activated by 2 s depolarizing command (from Vh of-60 mV to 10 mV). Current-voltage relations of I(~.,,in mouse (.) and goldfish (o) bipolar cells are shown for comparison after normalization of the peak amplitude. goldfish bipolar cells was either blocked or enhanced to a remarkable extent (Fig. 8). T-type Iea of mouse bipolar cells was also resistant to 50/~M Cd 2+ that blocked Ira in goldfish.
P H Y S I O L O G I C A L R O L E O F Ic~ IN M O U S E B I P O L A R CELLS We demonstrated that the transient inward current of mouse b i p o l a r ceils is carried by Ca 2+. The current is blocked by Co ~+, but was resistant to T T X . Activation and inactivation analysis shows that Ica can be activated at m e m b r a n e potentials between -80 and -25 m'V under physiological conditions, ([Ca2+]o ~, 2 raM). It is unfortunate that reports of intracellular recordings from mouse bipolar cells have yet to be published. If, however, the light responses of mouse bipolar cells lie within a potential range similar to that of light responses in lower vertebrates (12), they will also lie well within the activation range of Ica. The finding t h a t Ic~ flows b o t h at the perikaryon and at the axon terminal of mouse bipolar cells raises the possibility t h a t this current may participate in synaptic transmission, and in shaping the transient responses of mouse ganglion and amacrine cells. Presence of the transient Ic~ strongly suggests that the resulting increase in intracellular calcium concentration must also be transient.
Since the increase of [Ca2+]o is prerequisite to the transmitter re-
lease, it may be reasonable to believe that transmitter release from mouse bipolar cells is also transient. It has long been a puzzle how the transient ON- or OFF-responses of amacrine or
$76
ganglion cells are formed. The present finding offers a possible explanation of these transient responses, at least in the mouse. ACKNOWLEDGEMENT We thank Michi Hosono for her excellent technical assistance in preparing isolated photoreceptors. This research was supported in part by the Grants in Aid for Scientific Research from the Ministry of Education, Science and Culture (Nos. 61304032, 63638517, 63112007 to AK and 63480111, 63641537 to MT), NEI R01EY01221 (to LHP), NSF INT8613447 and Japan Society for the Promotion of Science. REFERENCES 1. Maturana HR, Lettvin JY, McCulloch WS, Pitts WH (1960) Anatomy and physiology of vision in the frog (Rana pipiens). J. Gen. Physiol. 43 (suppl 2):129-171. 2. Barlow HB, Levick WR (1965) The mechanism of directionally selective units in rabbit's retina. J Physiol (Load) 178:477-504. 3. Kaneko A, Tachibana M (1985) A voltage-clamp analysis of membrane currents in solitary bipolar cells dissociated from Carassius auratus. J Physiol (Load) 358:131-152 4. Kaneko A, Pinto LH, Tachibana M (1989) Transient calcium current of retinal bipolar cells of the mouse. J Physiol (Lond) 410:613-629. 5. Baylor DA, Nunn B J, Schnapf JL (1984) The photocurrent, noise and spectral sensitivity of rods of the monkey Maeaea fascilularis. J Physiol (Lond) 357:575-607 6. Kolb H, Nelson R (1984) Neural architecture of the cat retina. Prog Retinal Res 3:21-60 7. Suzuki H, Pinto LH (1986) Response properties of horizontal cells in the isolated retina of wild-type and pearl mutant mice. J Neurosei 6:1122-1128 8. Balkema GW, Mangini N J, Pinto LH, Vanable JW Jr (1984) Visually evoked eye movements in mouse mutants and inbred strains. Invest Ophthalmol Visual Sci 25:795-800 9. Nilius B, Hess P, Lansman JB, Tsien RW (1985) A novel type of cardiac calcium channel in ventricular cells. Nature 291:497-500 10. Hagiwara N, Irisawa H, Kameyama M (1988) Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells. J Physiol (Load) 395:233-253 11. Nowycky MC, Fox AP, Tsien RW (1985) Three types of neuronal calcium channels with different calcium agonist sensitivity. Nature 316:440-443 12. Saito T, Kondo H, Toyoda JI (1979) Ionic mechanisms of two types of on-center bipolar cells lin the carp retina. I. The responses to central illumination. J. Gen. Physiol. '/3:73-90.