Increased Intracellular Sodium Mimics
Some but not All Aspects of Photoreceptor
Adaptation in the Ventral Eye of Limulus
A L A N F E I N and J. S H E R W O O D
CHARLTON
From the Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole,
Massachusetts 02543
INTRODUCTION
I n t r a c e l l u l a r r e c o r d i n g s f r o m a wide variety o f i n v e r t e b r a t e p h o t o r e c e p t o r s
have s h o w n that m o s t o f these r e c e p t o r s d e p o l a r i z e w h e n i l l u m i n a t e d (see
review by F u o r t e s a n d O ' B r y a n , 1972). V o l t a g e c l a m p studies o f Limulus v e n t r a l
p h o t o r e c e p t o r s (Millecchia a n d M a u r o , 1969b) a n d b a r n a c l e p h o t o r e c e p t o r s
( B r o w n et al., 1970) have s h o w n that in these r e c e p t o r s , which d e p o l a r i z e u p o n
i l l u m i n a t i o n , t h e r e is an i n c r e a s e d N a + c o n d u c t a n c e in light. I n these cells,
p h o t o i s o m e r i z a t i o n o f r h o d o p s i n leads to an increase in p e r m e a b i l i t y to N a +
ions ( a n d to o t h e r ions as well) which in t u r n b r i n g s a b o u t an i n f l u x o f N a + a n d
m e m b r a n e d e p o l a r i z a t i o n . T h e l i g h t - i n d u c e d s o d i u m influx causes t h e intracellular s o d i u m c o n c e n t r a t i o n to rise ( B r o w n , 1976) a n d metabolically activated
processes (the s o d i u m p u m p ) t h e n r e s t o r e t h e intracellular s o d i u m c o n c e n t r a tion to its d a r k (resting) level ( B r o w n a n d L i s m a n , 1972; Koike et al., 1971).
E x p e r i m e n t s o n the d e p o l a r i z i n g p h o t o r e c e p t o r s o f Limulus ventral eye
T H E JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 7 0 ,
1977 •
pages 601-620
601
Downloaded from jgp.rupress.org on July 31, 2017
A B S T R A C T The effects of the intracellular iontophoretic injection of Na + ions
have been quantitatively compared with adaptation in ventral photoreceptors of
Limulus. We find that: (a) both light adaptation and sodium injection are associated
with a decrease in the variability of the threshold response amplitude; (b) both
light adaptation and sodium injection are associated with a decrease in the absolute
value of the temporal dispersion of the threshold response time delay; (c) the
same template curve adequately fits the intensity response relationships measured
under light adaptation and Na + injection; (d) both light adaptation and Na +
injection produce a fourfold decrease in response time delay for a desensitization
of 3 log units; (e) the time course of light adaptation and dark adaptation is
significantly faster than the onset of and recover)' from desensitization produced
by Na + injection; Or) unlike local illumination, Na + injection does not produce
localized desensitization of the photoreceptor. These findings suggest that a rise in
intracellular Na + concentration makes at most only a minor contribution (probably
less than 5%) to the total adaptation of these receptors in the intensity range we
have examined (up to 3 log units above absolute threshold). However, changes in
intracellular Na + concentration may contribute to certain components of light and
dark adaptation in these receptors.
602
T H E J O U R N A L OF GENERAL PHYSIOLOGY " VOLUME
70
• 1977
MATERIALS
AND
METHODS
The technique for preparing and the method of stimulating the ventral photoreceptors
of Limulus have all been described previously (Fein and DeVoe, 1973, Fein and Lisman,
1975, Fein and Charlton, 1975b). When we began this study we planned to monitor the
photoresponse by measuring the light-induced membrane depolarization. We had done
this in a similar study where we examined the effects of the intracellular iontophoretic
injection of Ca ++ (Fein and Charlton, 1977b). We soon found that for Na ÷ injections
light-induced changes in membrane potential were not an appropriate measure of the
photoresponse. This was because the injection of Na ÷ into a ventral photoreceptor
causes the cell to hyperpolarize (Brown and Lisman, 1972; Lisman and Brown, 1972b).
This hyperpolarization has a number of effects on the photoresponse. First, the
hyperpolarization causes the driving force for the light-induced current to increase
(Millecchia and Mauro, 1969b). The photocurrent produced by a fixed light induced
conductance change would thereby increase. Second, the hyperpolarization causes the
input resistance to rise (due to membrane rectification [Millecchia and Mauro, 1969b])
and the membrane time constant thereby increases. Thus for a given light-induced
current, the potential change would be larger and possibly slower. And finally, these
photoreceptors have a spike-like potential which is potentiated by membrane hyperpolarization (Millecchia and Mauro, 1969a). All these factors, which are secondary to the
membrane hyperpolarization, tend to confound the comparison of threshold responses.
Therefore we decided to measure the photoresponse (light-induced current) with the
cell voltage clamped to its resting (dark) potential. This procedure eliminates all the
problems associated with the sodium-induced hyperpolarization.
Under visual control, single photoreceptors were impaled with two pipettes, one
containing KC1, the other NaCI. Both pipettes had resistances in the range of 15-20 MI~
measured in the artificial seawater (Fein and Charlton, 1975b) that bathed the preparation. These two pipettes were used for both injecting sodium into the photoreceptor
and voltage clamping the photoreceptor. The amplifiers used for both injecting current
and voltage clamping (Fig. 1 A) were of conventional design. The photoreceptor was
voltage clamped when the switch in Fig. 1 A was in the voltage clamp position. The
procedures followed for establishing that the photoreceptor was isopotential and that
Downloaded from jgp.rupress.org on July 31, 2017
(Lisman and Brown, 1972 b) and the hone), bee d r o n e c o m p o u n d eye (Bader et
al., 1976) have shown that, in these receptors, both light adaptation and
intracellular iontophoretic'injection o f Na + ions p r o d u c e a reversible decrease
in the sensitivity o f the p h o t o r e c e p t o r . T h e s e findings raise the possibility that
in these depolarizing p h o t o r e c e p t o r s , and possibly others as well, a lighti n d u c e d rise in intracellular Na + concentration may contribute to light adaptation (Lisman and Brown, 1972b; B a d e r et al., 1976). However, when ventral
p h o t o r e c e p t o r s are voltage clamped b e y o n d the reversal potential for the lightinduced c u r r e n t , the response still adapts to the stimulus (Millecchia and
Mauro, 1969b). T h e r e f o r e , a light-induced rise in intracellular Na + cannot
account entirely for light adaptation (Lisman and Brown, 1972b). T h e experiments described in this p a p e r were designed to evaluate quantitatively the role
that intracellular changes in sodium concentration may have in both light and
dark adaptation. We have addressed ourselves to two questions: (a) to what
extent do the effects o f the intracellular iontophoretic injection o f sodium ions
mimic light an q! dark adaptation? and (b) to what extent do intracellular
changes in sodium concentration contribute to light and dark adaptation?
FgXN ANn CrlARLTON IntracellularSodium and PhotoreceptorAdaptation
603
Downloaded from jgp.rupress.org on July 31, 2017
the clamp was working adequately are described in Fein and Charlton, 1977a. Na ÷ was
injected into the photoreceptor by passing c u r r e n t between the two intracellular pipettes.
This was accomplished by connecting the switch in Fig. 1 A in the current clamp
position (the clamp being set for zero m e m b r a n e current). Amplifier A2 in the
electrometer amplifier (Fig. 1 A) was used to inject square pulses of current into the cell.
T h e feedback pathway from amplifier Aa to amplifier A2 (Fig. 1 A) insured that these
current pulses were not affected by differences or changes in pipette and m e m b r a n e
resistance. T h e current clamp circuit insured that whatever current was injected out of
the electrometer pipette did not pass across the cell m e m b r a n e (current clamped for
zero m e m b r a n e current). Fig 1 B, C illustrates how we checked that this circuit was
working correctly. Fig. 1 B, C shows the input voltage of the electrometer (VI) and the
current (i4) measured by the current-to-voltage converter when we injected square
current pulses (il) through the electrometer pipette. Fig, 1 B illustrates the waveforms
we measured when we injected current through the electrometer pipette and the other
pipette was disconnected from the clamp amplifier. The electrometer measured a
voltage drop across the pipette and the cell membrane. The current-to-voltage converter
measured a current flowing across the m e m b r a n e into the bath. When the other pipette
was connected to the clamp amplifier the waveforms of Fig. 1 C were measured. T h e
voltage drop across the m e m b r a n e and the current flowing out across the cell m e m b r a n e
were absent (as illustrated in Fig. 1 C) or greatly reduced. For injection currents as large
as 25 nA we never passed more than 0.5 nA across the cell membrane. The effects
described in this paper are specific effects of injecting Na + into the photoreceptor;
similar effects are not observed when K + is injected into the cell (Lisman and Brown,
1972b; Fein and Charlton, 1977b). T h r o u g h o u t this paper we display the photoresponse
(inward m e m b r a n e current) as an upward deflection of the trace. Injection currents for
Na ÷ ions are given as iNa+, where iNa+ is the total current passing through the NaCl-filled
electrode. Not all the current passing through the electrode would be expected to be
carried by Na ÷ ions, however.
Light intensities I are given as log10 I/I0 where I0 is the intensity of the u n a t t e n u a t e d
beam of white light which was used to stimulate the photoreceptor. T h e intensity of the
u n a t t e n u a t e d beam was found to be equivalent to 1.2 x 1015520 nm photons/cm2-s (Fein
and Charlton, 1977a). For uniform illumination of the photoreceptor (Figs. 2-5 and
Fig. 8) the n u m b e r of equivalent 520-nm photons incident on the photoreceptor for the
u n a t t e n u a t e d beam was found to be 6 x 101°/s. T h e threshold for producing one
quantal event on the average with a 20-ms flash of white light corresponds to a log
intensity of - 6 . 2 5 to - 6 . 3 5 in Figs. 2-5 and Fig. 8 (uniform illumination of the
photoreceptor) and to a log intensity of -4.45 to - 4 . 5 5 (this is a lower bound) in Figs. 6
and 7 (stimulation with 10-g.m diam spots of light).
Fig. 1 D shows the timing sequence for the different events that occurred d u r i n g an
experiment. T h e photoreceptor was stimulated once every 11 s by a 20-ms test flash
(chosen to be below the integration time of the photoreceptor) of variable intensity. The
photoreceptor was voltage clamped to its resting (dark) potential for an interval that
overlapped the time when the response to the test flash occurred. During the interval
between test flashes the photoreceptor was either: (a) in darkness; (b) light adapted by a
5-s adapting flash whose onset preceded the test flash by 9 s; (c) iontophoretically
injected with Na + for a 5-s interval whose onset preceded the test flash by 9 s. The
current clamp was turned on for an interval that overlapped the time when the Na +
injection occurred.
Sometimes d u r i n g the course of an experiment (a series of light adaptations, Na +
injections, and recoveries) the resting potential would drift. This drift was in addition to
the reversible hyperpolarization due to Na + injection. When this drift occurred the
6O4
THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 70 " 1977
A.
Electrometer
Amplifier
+
I00 K
I__
I
I Clamp
I Amplifier
EP o
_.m_
I
V- Clamp
I
I
I
i
I- Clamp
!
14 =LL
,,~OB t
D ~ Q
F - ~ Condenser
~ C ' u r r e n t to
Voltage Converter
Unclamped
B
v,
f
k
.,,k. &i
C
i, ~J
I
i4 --J
"~'~---'A
Current
/
~-
-&iRm(I-e
J
L & j R m e -(t-'#')/RmCm
"L-
i
Clamped
D.
~__ / ' , i R E
.L
Interval during which light
injection may occur
{4---- 5s
!~
_l
9 s
,-lis
20ms
Flash
'tAi
i4
t
or current
|
|
i
I
-t/Rmcm)
,t.
adaptation
_J
Z~i RE
-I-
1.5
s --'*!
20
i{
i!
V- Clamp
ms
Test
Flash
{~'--
i~,- 3 s -..-.w!~
=
!
,I
h
8s
I - Clamp
O<t<T
"E<t
Downloaded from jgp.rupress.org on July 31, 2017
i
FEIN AND CHARLTON
IntraceUular Sodium and Photoreceptor Adaptation
605
photoresponse was measured throughout the experiment with the photoreceptor
clamped to its initial resting potential. The drift in resting potential never amounted to
more than 15 mV and was typically under 5 mV.
RESULTS
FIGURE 1. (Opposite) A, Apparatus used for voltage clamping (V-clamp) and current clamping (/-clamp) Limulus ventral photoreceptors. Amplifiers AI-A5 are conventional commercial operational amplifiers. Amplifier A2 was used to inject constant current pulses (i~) through the micropipette. The feedback path between
amplifier A~ and As insured that current i~ was determined by the voltage at point 2
and the l0 s 1"1resistor. Any voltage drop across the pipette or cell membrane (V~)
was compensated for by the feedback from A~ to A2. The preparation was observed
using the eyepiece (EP) and the objective (OB2). The photoreceptor was stimulated
by light projected onto it by the condenser (OB1). The photostimulator is described
in detail by Fein and Charlton (1975b). B, Input voltage (V~) measured by the
electrometer and the current (i4) measured by the current-to-voltage converter
when square current pulses (il) are passed through the electrometer pipette.
These waveforms are measured when the other pipette is disconnected from the
clamp amplifier. The voltage (V~) measured by the electrometer is made up of two
components: the voltage across the electrode, and the voltage across the cell
membrane. C, V~, i4, and il when the other electrode is connected to the clamp
amplifier (as shown) and the clamp amplifier is connected for current clamping to
zero membrane current (see text for further details). D, Timing diagram for
events that take place during an experiment.
Downloaded from jgp.rupress.org on July 31, 2017
Fig. 2 c o m p a r e s the c h a n g e s in sensitivity a n d in the time course o f the
p h o t o r e s p o n s e p r o d u c e d by light a d a p t a t i o n a n d intracellular Na + injection.
Control responses (solid lines, Fig. 2 A , B) were m e a s u r e d in the d a r k both
b e f o r e a n d a f t e r each light a d a p t a t i o n a n d s o d i u m injection. Control responses
were only m e a s u r e d a f t e r the cell had fully r e c o v e r e d f r o m the desensitizing
effects o f light a d a p t a t i o n or sodium injection. T h e data o f Fig. 2 were obtained
as follows: (a) a set o f control responses were m e a s u r e d for t h r e e intensities o f
the test flash d i f f e r i n g by a factor o f 2 (log intensity - 5 . 5 , - 5 . 2 , - 4 . 9 ) ; (b) then
the p h o t o r e c e p t o r was r e p e a t e d l y stimulated (for 5 s every 11 s, see Materials
a n d Methods) by an a d a p t i n g flash o f log intensity - 3 . 0 , a n d the test flash
intensity was adjusted (log intensity - 3 . 1 ) to give a r e s p o n s e equal in a m p l i t u d e
to the control r e s p o n s e elicited by the d i m m e s t test flash (log intensity - 5 . 5 ) .
T w o additional responses were o b t a i n e d by d o u b l i n g the test flash intensity
twice (log intensity - 2 . 8 , - 2 . 5 ) . T h e s e t h r e e responses (log intensity o f test
flash - 3 . 1 , - 2 . 8 , - 2 . 5 ) are given by the dots in Fig. 2 A and C; (c) the a d a p t i n g
light was t u r n e d off, the p h o t o r e c e p t o r was allowed to recover, a n d a new set
o f control responses were m e a s u r e d ; (d) t h e n the p h o t o r e c e p t o r was r e p e a t e d l y
injected with a 15-nA, 5-s s q u a r e pulse (every 11 s, see Materials a n d Methods)
f r o m the Na+-containing pipette. A f t e r 15 min o f injection the r e s p o n s e to a
test flash o f log intensity - 3 . 1 was equal in a m p l i t u d e to the r e s p o n s e o b t a i n e d
with the same test flash d u r i n g the previous light a d a p t a t i o n . T w o additional
responses were m e a s u r e d by d o u b l i n g the test flash intensity twice (log intensity
- 2 . 8 , - 2 . 5 ) . T h e s e t h r e e responses (log intensity o f test flash - 3 . 1 , - 2 . 8 , - 2 . 5 )
606
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
70 • 1977
" VOLUME
are given by the x symbols in Fig. 2 B, C; (e) the injection c u r r e n t was then
t u r n e d off, the cell was allowed to recover, and a new set o f controls were
measured. T h e controls, shown by the solid lines in Fig. 2 A, B, were obtained
after the light adaptation a n d before sodium injection. T h e time course o f the
sensitivity changes for Na + injection and light and dark adaptation will be
discussed subsequently (see Fig. 5).
-55
-31
A
Y " : . . . ~
- 2 . 8 ; / ~
.._
-
~zt s
-3,1
-5.5
-$)8 ,
-52
I
I
200
C
Light
x.1,1
~
r"\
-2,8
-3.1
•sj~'l'x,
s
ms
-2.5:%
,~';
s.m.x.l.x-i
[7
~-l-~ J.14
- ~
,.I-x..-t ~
"~-~-x .s.s.s
[7
Monitor
FIGURE 2. Comparison of changes in sensitivity and the time course of the
photoresponse produced by light adaptation and intracellular Na + injection. The
number next to each waveform gives the log intensity of the 20-ms test flash. The
light monitor shows the time course of the test flash. A, Comparison of lightadapted responses (dots) with control responses (solid lines). The adapting light
(log I = -3.0) desensitized the cell 2.4 log units and the light-adapted response
occurred sooner than the control. B, Comparison of responses obtained after 15
min of Na ÷ injection (×) with control responses. Injection of 15 nA from the Na +
containing pipette desensitized the photoreceptor by 2.4 log units and the desensitized response occurred sooner than the control. C, Superposition of light-adapted
responses (dots) from A with responses obtained during Na ÷ injection (×) from B.
The threshold for producing on the average one quantal event per 20-ms test
flash corresponds to a log intensity of between -6.25 and -6.35 (see Materials and
Methods, and Fein and Charlton, 1977a). See Results and Materials and Methods
for details of experimental methods used in obtaining these data.
Fig. 2 A shows the changes in sensitivity a n d the time course o f the p h o t o r e sponse associated with light adaptation. T h e a d a p t i n g light desensitized the
p h o t o r e c e p t o r by 2.4 log units and the desensitized response o c c u r r e d s o o n e r
than the control. Fig. 2 B shows the changes in sensitivity and p h o t o r e s p o n s e
time course associated with Na ÷ injection. Injection o f Na ÷ for 15 min resulted
in a 2.4 log unit decrease in sensitivity and the desensitized response o c c u r r e d
sooner than the control. In Fig. 2 C we c o m p a r e the responses for light
adaptation and Na ÷ injection. For the two dimmest flashes (log intensity - 3 . 1
Downloaded from jgp.rupress.org on July 31, 2017
13
FEIN AND CHARLTON Intracellular Sodium and Photoreceptor Adaptation
607
Downloaded from jgp.rupress.org on July 31, 2017
and -2.8) the responses were essentially superimposable. For the brightest
flash (log intensity -2.5) the falling phase of the light-adapted response was
more rapid than that of the response obtained during Na ÷ injection. This
difference was consistently seen in all the photoreceptors we studied. We do
not know what factors are responsible for this difference. Similar results were
obtained when the Na + injection was carried out first and the light adaptation
was equated to the Na + injection. The findings presented in Fig. 2 suggest that
light adaptation and Na + injection desensitize the photoreceptor in a similar
but not identical manner.
In the stimulus paradigm described for Fig. 2 the photoreceptor is stimulated
with a more intense test flash during a light adaptation or a Na ÷ injection than
during a control run. This raises the possibility that the test flash might
significantly alter the adaptational state of the photoreceptor produced by light
adaptation or Na ÷ injection. This possibility was checked by using the procedure
described in Fein and Charlton (1977b). We found that during light adaptation
or Na ÷ injection the test flash did not significantly alter the adaptational state
of the photoreceptor.
In Fig. 3 we quantitatively compare Na + injection and light adaptation at two
different times during the Na ÷ injection. A procedure similar to that described
for Fig. 2 was followed and similar data were obtained. As a measure of the
photoreceptor sensitivity we plot the log of the peak amplitude of the response
to the 20-ms test flash against the log of the test flash intensity (Fig. 3 A). As a
measure of the photoresponse time course we plot the log of the photoresponse
time delay (time from stimulus onset until photoresponse first reaches 10% of
peak amplitude) against the log of the test flash intensity (Fig. 3 B). Our results
remain essentially unchanged for other definitions of time delay between 10%
and 100% of peak amplitude. Fig. 3 A shows that a template curve with slope of
1 fits all the peak amplitude data reasonably well (Lisman and Brown, 1975 a;
however, see Fein and Charlton, 1977a). Both light adaptation and Na ÷
injection appear to desensitize the photoreceptor by causing the peak amplitude
response curve to shift along the log stimulus intensity axis. Fig. 3 B shows that
both light adaptation and Na + injection decrease the response time delay.
Taken together Fig. 3 A and B show that for nearly equal desensitizations
produced by either light adaptation or Na ÷ injection the changes in time delay
are nearly equal. The effects of both Na + injection and light adaptation were
found to be completely reversible. Fig. 3A shows a decrease in the variability of
threshold response amplitude associated with the desensitization produced by
light adaptation and Na ÷ injection. Similarly, Fig. 3 B shows a decrease in the
absolute value of the variability in time delay associated with both light
adaptation and Na ÷ injection. Both these decreases in threshold response
variability were found to be completely reversible. A possible basis for this
decrease in threshold response variability will be considered in the Discussion.
In Fig. 4 we present composite data from six photoreceptors for which we
compared Na ÷ injection and light adaptation. We have also included in Fig. 4
data from a previous study (Fein and Charlton, 1977b) where we compared
Ca ++ injection and light adaptation. In order to combine data from different
photoreceptors injected with Na + we arbitrarily chose a 2-nA photocurrent as
608
THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 70 • 1977
the criterion r e s p o n s e . O u r results r e m a i n essentially u n c h a n g e d f o r o t h e r
values o f the criterion r e s p o n s e (see Fig. 3). I n the Ca ++ injection e x p e r i m e n t s
the p h o t o r e s p o n s e was n o t m e a s u r e d u n d e r voltage c l a m p . I n those e x p e r i m e n t s
log IA= - 4 4
iNo+ = 20 no
,~,
0Q.
8
6
O
O
4
~
z
? /,/>:
O/
b~
l0
Z
O
O
0
13 rain
///
no
// "
ii
o
20
22 rain
/I
/Y
/,'-
///
v< 0.5
hl
[
-61
i
i
i
i
1
i
-5.5
-4.9
-4.3
-3.7
-5.t
-2.5
LOG INTENSITY OF 20 ms T E S T F L A S H
B
b_
0 A
120
--
~ I00
0 v 90
80
---[3
~8~o
--
- -
,,I
I-
60
~
50
~0 ~
40
V-
30 ~
i
-61
i
-5,5
LOG
i
i
-49
-4.3
0
Controls
No
zx Light
Injections
Adaptations
i
-57
INTENSITY OF 2 0 ms
i
-3.1
TEST
i
-25
FLASH
Comparison of light adaptation and Na + injection at two times during
the Na + injection. A, Log-log plot of peak amplitude of photoresponse versus
intensity of test flash. B, Log-log plot of response time delay versus intensity of
test flash. For both A and B, log IA gives the intensity of the adapting light, iNa+
gives the current passing through the NaCI electrode, and the time in minutes
gives the time after the onset of the Na + injection at which the Na + injection data
were obtained. In both A and B, the controls are given by O, the light-adapted
data by A, and the Na + injection data by D. These data were obtained from a
photoreceptor different from that shown in Fig. 2. The same experimental
methods as described in the text for Fig. 2 were used in obtaining these data.
FIGURE 3.
we m e a s u r e d the p h o t o r e s p o n s e by m o n i t o r i n g the t r a n s m e m b r a n e d e p o l a r i z a tion. By c h o o s i n g to c o m p a r e a 10-mV r e s p o n s e with a 2 - n A r e s p o n s e in Fig. 4
we have m a d e the r e a s o n a b l e a s s u m p t i o n that the i n p u t resistances o f the
p h o t o r e c e p t o r s are o n the a v e r a g e 5 Mf~ (Millecchia a n d M a u r o , 1969a). N o t e
in Fig. 4 that b o t h the o r d i n a t e a n d abscissa are absolute ( u n - n o r m a l i z e d )
Downloaded from jgp.rupress.org on July 31, 2017
/~o
Q
log IA= -5.0
iNo +=
609
FEIN AND CHARLTON Intracellular Sodium and Photoreceptor Adaptation
scales. T h e r e s u l t s o f Fig. 4 i n d i c a t e t h a t u n d e r t h e c o n d i t i o n s o f t h e s e
e x p e r i m e n t s l i g h t a d a p t a t i o n , N a + i n j e c t i o n a n d C a ++ i n j e c t i o n p r o d u c e c h a n g e s
in s e n s i t i v i t y a n d t i m e d e l a y t h a t a r e to a first a p p r o x i m a t i o n q u a n t i t a t i v e l y
-6,1
150
140
<[ o_
0
Z
0
-4.13
-3.7
I
l
I
-2.5
I
-I.9
l
F_° c°ntr°js
Injections
Experiments L& Light Adaptation
0
13o
•
O00
o
ooo
n~
ca.
(I
oe
~
o
"4
70
:Coo,ros
ExperimentsLa Injections
o n
Light Adaptation
6O
A
0
1
0
I
"1- n"
•
t ,x,
'~ U_ 4O
W 0
n"
(Z) UJ
a
--
I
No*
o~
80
-3.1
IAI
I1
20
I
-6.1
-5.5
!
I
-4.9
-4.3
l
-3.7
I
-3.1
I
l
-2.5
-I.9
FIGURE 4. Log-log plot of the experimentally d e t e r m i n e d relationship between
photoreceptor sensitivity and response time delay for light adaptation, Na ÷
injection, and Ca ++ injection. Voltage clamp data from this study (unfilled symbols)
are combined with u n c l a m p e d data from a previous study (filled symbols). For
both types of data the response time delay is plotted on the same log scale. For the
voltage clamp data (Na + experiments o f this study) the log of the response time
delay is plotted against the log o f the test flash intensity needed to p r o d u c e a 2 nA
criterion response. For the u n c l a m p e d data, Ca ++ experiments o f a previous study
(Fein and Charlton, 1977b), the log of the response time delay is plotted against
the log of the test flash intensity needed to p r o d u c e a 10-mV criterion response.
T h e straight line was drawn through the data points by eye. T h e data indicate that
a 3 log unit decrease in sensitivity is associated with about a four-fold decrease in
time delay. Composite data from 16 photoreceptors are plotted (6 for Na +
experiments and 10 for Ca +÷ experiments). T h e experimental methods used in the
Na + experiments (unfilled symbols) are the same as described in the text for Fig.
2. T h e experimental methods used in the Ca ++ experiments (filled symbols) are
given in Fein and Charlton (1977b).
Downloaded from jgp.rupress.org on July 31, 2017
w 09
n ~
-4.9
I
o
%
130
~
120
E rio
I00
W
u)
9O
Z
'-tO
-5.5
610
THE JOURNAL
Or
GENERAL PHYSIOLOGY
" VOLUME
70 • 1977
equivalent. Also, t h e r e d o n o t a p p e a r to be a n y systematic d i f f e r e n c e s b e t w e e n
the c l a m p e d a n d the u n c l a m p e d data.
I n Fig. 5 we c o m p a r e the onset of, a n d r e c o v e r y f r o m , desensitization
p r o d u c e d by Na + injection to light a n d d a r k a d a p t a t i o n . T h e m e t h o d o f
injecting N a + a n d light a d a p t i n g the p h o t o r e c e p t o r was the s a m e as d e s c r i b e d
f o r Fig. 2. T h e d a t a in Fig. 5 (also see Fig. 8) clearly show that the onset o f a n d
ON
OFF
-3.5 -']
^ ~~r.~-~,...,,
X
iNo* = 15no
0
iNa. : 2 5 no
-4.o
oJ
~
- 6.0
0
-6.5
°.9
I
i
0
5
I
Io
1
Is
i
20
TIME (min)
i
i
i
i
I
z5
30
35
40
45
FXCURE 5. Comparison'of the time course of light and dark adaptation with the
onset of and recovery from desensitization produced by Na + injection. The log
threshold (intensity of 20-ms test flash needed to produce a 2 nA criterion
response) is plotted as a function of time for light and dark adaptation and Na +
injection. The arrow labeled ON denotes the onset of the adapting light and the
sodium injection. The arrows labeled OFF denote the time at which the adapting
light and Na + injection were turned off. The current passing through the NaCI
electrode is denoted by iNa÷ and I A is the intensity of the adapting light. The same
experimental methods as described in the text for Fig. 2 were used in obtaining
these data.
r e c o v e r y f r o m desensitization f o r N a + injection are m a r k e d l y slower t h a n the
time c o u r s e o f light a n d d a r k a d a p t a t i o n .
We have previously s h o w n that local illumination o f p a r t o f a ventral
p h o t o r e c e p t o r leads to a localized flow o f m e m b r a n e c u r r e n t (Fein a n d Charlt o n , 1975a). F u r t h e r m o r e , it has b e e n s h o w n that the light a d a p t a t i o n p r o d u c e d
by local illumination is localized to the r e g i o n o f illumination (Fein, 1973;
Downloaded from jgp.rupress.org on July 31, 2017
-4.5
FEIN AND CHARLTON IntraceUular Sodium and Photoreceptor Adaptation
611
Spiegler a n d Yeandle, 1974; Fein a n d C h a r l t o n , 1975b). Also, Fein a n d Lisman
(1975) have shown that injection o f calcium ions into ventral p h o t o r e c e p t o r s
locally desensitized the r e c e p t o r . A n d we (Fein and C h a r l t o n , 1977a) have
shown that e n h a n c e m e n t is spatially localized in these r e c e p t o r s . T h e s e findings
led us to investigate w h e t h e r Na + injection would locally desensitize these
receptors.
Fig. 6 shows the data f r o m an e x p e r i m e n t w h e r e we tested for localized
desensitization d u r i n g Na + injection. Fig. 6 D is a schematized version o f the
p h o t o r e c e p t o r showing the two stimulus spots (nominally 10 t t m in diameter)
a n d the location o f the NaCI and KCI microelectrodes. We have previously
described the p h o t o s t i m u l a t o r a n d e x p e r i m e n t a l m e t h o d s used in this type o f
e x p e r i m e n t (Fein a n d C h a r l t o n , 1975b). Fig. 6 A, B shows that b o t h regions 1
a n d 2 o f the p h o t o r e c e p t o r can be light a d a p t e d locally, whereas Fig. 6 C shows
that Na + injection desensitizes region 1 a n d 2 equally. Fig. 6 shows that unlike
Light
Na +
Adaptation
Znjection
B
Control
After
Odopting
stimulus
at position I ~ ' "
] ~
j \
After
After
odoptin9
stimulus
at position 2
NO÷ injection
01 position 2
J~
Recovery
Light
Monitor
I
i
I
--["
i/2
2
tl
I
__t__|
i
2
Test Flash Position
KCI
Electrode
T
I0 na J
J.
Photoreceptor
log I~= -21
~"'
is
log 12:-I.6
Na CI
Electrode
FIGURE 6. A comparison of local adaptation (A, B) and Na + injection (C). D,
Schematized representation of the photoreceptor showing the position of the two
stimulus spots labeled 1 and 2 (nominally 10/zm in diameter), and the position of
the NaCI and KCI electrodes. I1 and 12 give the intensity of the 20-ms test flash
located at positions 1 and 2, respectively. A and B show that both regions 1 and 2
of the photoreceptor can be adapted locally. The adapting spot of light had a log
intensity of -2.1 in A and -1.6 in B. In both A and B the adapting light was on
for 8 s and was turned off 2 s before the first test flash. C shows that a 15 nA Na +
injection for about 9 min (see Fig. 7) equally desensitized both regions of the
photoreceptor.
Downloaded from jgp.rupress.org on July 31, 2017
Local
612
THE JOURNAL OF GENERAL PHYSIOLOGY" VOLUME 7 0 " 1977
local i l l u m i n a t i o n , t h e i n t r a c e l l u l a r i n j e c t i o n o f N a + d o e s n o t p r o d u c e l o c a l i z e d
d e s e n s i t i z a t i o n . I n Fig. 7 we c o m p a r e , f o r t h e two r e g i o n s o f t h e p h o t o r e c e p t o r
s h o w n s c h e m a t i c a l l y in Fig. 6 D, t h e o n s e t o f a n d r e c o v e r y f r o m d e s e n s i t i z a t i o n
p r o d u c e d by N a + i n j e c t i o n . T h e d a t a o f Figs. 6 a n d 7 a r e f r o m t h e s a m e
p h o t o r e c e p t o r . F i g 7 s h o w s t h a t we fail to f i n d l o c a l i z e d d e s e n s i t i z a t i o n d u r i n g
the onset of and recovery from the Na + injection.
W e c o n s i s t e n t l y f i n d t h a t t h e d e s e n s i t i z i n g e f f e c t o f N a + i n j e c t i o n is d e l a y e d
ON
15no N + injection
OFF
25
--~ z0
t'15
O
k'-
O
T
Qt
,O
o
o
x
OXx x
o,,o
X~ 0
' x ,
I0
y
,
o ,,o
~(~ X XX x -
, Cpo'~ xx
OGD
5
00~
x x
4
0
EL
LL
x
3
2
(330
O0
Ox
...J
EL
X
0
a,?~o
XX O(~t.
CK-~)
°
x
0
0
x
0 X)al,
o Response
to stimulus
at position
x Response
to
at
log
stimulus
I
position
2
log I z = - I . 6
I,=-2.1
Oxx
x
OXx
,<
,y.
<t
LIJ
EL
0
O0 x x
m= (:DO
CX x
o
x
o o
Ox
xx
00ll~l~'rJO
0 0
o x
x 0
x 0
x x
o
laJ
a
0
0
x
x
0.5
i
0
i
2
i
4
i
6
i
8
i
[0
i
12
i
14
1
16
i
18
I
20
I
Z2
TIME (rain)
FIGURE 7. Onset o f a n d recovery from desensitization p r o d u c e d by Na + injection
(as measured at two spatially separated regions o f a photoreceptor). These data
are from the same cell as shown in Fig. 6 and the experimental conditions are as
shown in part D o f that figure. T h e arrows indicate when the 15 nA Na + injection
was turned on and off. I1 and I2 are intensities o f the test flashes at positions 1 and
2 on the photoreceptor. Note that there is no localized desensitization at any time
d u r i n g the onset o f or recovery from the desensitization p r o d u c e d by the Na +
injection.
after the onset o f the injection; in the e x p e r i m e n t s h o w n in Fig. 7, this delay
was about 2 min (also see Fig. 8). This time delay could indicate that the Na ÷
concentration must attain s o m e critical value before desensitization occurs.
DISCUSSION
A. Sodium Injection, Light Adaptation, and Dark Adaptation
It h a s p r e v i o u s l y b e e n s h o w n t h a t t h e i n t r a c e l l u l a r i n j e c t i o n o f N a + r e v e r s i b l y
d e c r e a s e d t h e r e s p o n s e to a c o n s t a n t i n t e n s i t y s t i m u l u s f o r Limulus v e n t r a l
p h o t o r e c e p t o r s ( L i s m a n a n d B r o w n , 1972b). T h e r e s u l t s o f o u r s t u d y e x t e n d
I
24
Downloaded from jgp.rupress.org on July 31, 2017
LtJ
03
Z
O
EL
U~
taJ
o x
FEIN AND CHARLTON Intracellular Sodium and PhotoreceptorAdaptation
613
Downloaded from jgp.rupress.org on July 31, 2017
the findings of these investigations. Specifically, we have shown the following:
(a) both light adaptation and the intracellular injection of sodium are associated
with a decrease in the variability of the threshold response amplitude (Fig. 3 A);
(b) both light adaptation and the intracellular injection of Na + are associated
with a decrease in the absolute value of the variability in threshold response
time delay (Fig. 3 B); (c) a template curve with a slope of 1 (Fig. 3 A) fits all the
data (controls, light adaptation, Na + injection) reasonably well in the response
range of 0-10 nA; (d) both light adaptation and sodium injections produce
similar changes in response time delay for desensitizations as great as 3 log
units (Figs. 2-4). This last result suggests that, except for some small differences
in the falling phase of the photoresponse (see Fig. 2 C), the intracellular
injection of sodium quantitatively mimics the changes in sensitivity and time
delay produced by light adaptation.
These findings can be interpreted in terms of the "bumps" (quantal events)
that are believed to make up the photoresponse in Limulus receptors (Fuortes
and Yeandle, 1964; Adolph, 1964; Dodge et al., 1968; Millecchia and Mauro,
1969a; Spiegler and Yeandle, 1974). Dodge et al. (1968) have proposed that: (a)
the photoresponse arises from a superposition of bumps which are triggered by
the absorption of light; (b) the average size of the bumps decreases with
increased illumination and is the major mechanism of light adaptation. The
results presented in Figs. 2-4 can be interpreted in terms of the above-stated
ideas if the following are true. (i) Both Na + injection and light adaptation cause
a reversible decrease in the size of a bump, thereby reversibly decreasing
sensitivity and the variability in response amplitude (Fig. 3 A). The variability
for a constant amplitude response is decreased when the cell is desensitized
because the desensitized response is made up of a greater number of smaller
bumps (Dodge et al., 1968). (ii) Both Na + injection and light adaptation are
associated with a reversible decrease in the time delay and the absolute temporal
dispersion of bump occurrence, thereby reversibly decreasing response delay
and the absolute value of time delay variability (Fig. 3 B). qii) The rate of bump
production is to a first approximation a linear function of light intensity,
therefore a template curve with a slope of 1 fits all the peak amplitude data
reasonably well (Fig. 3A); however, see Fein and Charlton, 1977a. (iv) Both
Na + injection and light adaptation produce similar changes in bump amplitude
and bump time delay for desensitization up to three log units (Figs. 2-4).
We have suggested (on the basis of the previous work of Dodge et al., 1968)
that the variability in response amplitude is decreased when the cell is desensitized because the response to a brighter flash is made up of a greater number
of smaller bumps. If a photoreceptor is tested with a constant intensity test
flash during desensitization the average number of bumps elicited by the test
flash should remain constant provided the light adaptation or Na + injection
does not affect the quantum efficiency of bump production (Dodge et al.,
1968). Then during desensitization by light or Na + injection the response to a
constant intensity stimulus should only reflect changes in the average size of a
bump. If this assertion is true, the absolute variation in response amplitude to
constant intensity stimulus should decrease during desensitization but the
percentage variation in response amplitude should remain essentially un-
614
THE JOURNAL
OF GENERAL
PHYSIOLOGY
" VOLUME
70 " 1977
B. Sodium Injection and Local Adaptation
Local illumination o f ventral p h o t o r e c e p t o r s leads to a local influx o f Na + (Fein
a n d C h a r l t o n , 1975a) a n d to local a d a p t a t i o n (Fein, 1973; Spiegler a n d Yeandle,
1974; Fein a n d C h a r l t o n , 1975b). This m a y suggest that a local rise in intracellular Na + concentration gives rise to local a d a p t a t i o n . T h e data in Figs. 6 a n d 7
Downloaded from jgp.rupress.org on July 31, 2017
c h a n g e d . T h e p e r c e n t a g e variability, in the sense we are using it, is a dimensionless quantity a n d is equivalent to taking the ratio o f the s t a n d a r d deviation to
the m e a n . We have tested the above assertion a n d the results are p r e s e n t e d in
Fig. 8. In Fig. 8 A the p e a k a m p l i t u d e o f the r e s p o n s e to a constant intensity
stimulus is plotted on a linear scale as a function o f time for equal desensitizations p r o d u c e d by light a d a p t a t i o n or Na + injection. In Fig. 8 B the same data
as in Fig. 8 A are p r e s e n t e d by use o f a logarithmic scale to plot the a m p l i t u d e .
It can be seen in Fig. 8 A that d u r i n g desensitization p r o d u c e d either by light
a d a p t a t i o n or Na + injection the absolute variability in r e s p o n s e a m p l i t u d e is
d e c r e a s e d as suggested above. W h e n the same data are plotted on a logarithmic
scale as in Fig. 8 B , it can be seen that the p e r c e n t a g e variation in r e s p o n s e
a m p l i t u d e r e m a i n s essentially u n c h a n g e d t h r o u g h o u t the desensitization, as
suggested above. T h e findings p r e s e n t e d in Fig. 8 are consistent with the idea
that both light a d a p t a t i o n a n d Na + injection desensitize the p h o t o r e c e p t o r by
similarly affecting the b u m p s that are believed to underlie the p h o t o r e s p o n s e .
T h e findings discussed above point out s o m e very striking similarities between
the effects o f light a d a p t a t i o n a n d sodium injection. H o w e v e r , t h e r e are also
some very striking differences. We consistently f o u n d that the time course o f
light a d a p t a t i o n a n d d a r k a d a p t a t i o n was faster than the onset o f a n d recovery
f r o m desensitization p r o d u c e d by Na + injection (see Figs. 5 and 8). This
finding suggests that a rise in intracellular Na + concentration does not m a k e a
large quantitative c o n t r i b u t i o n to a d a p t a t i o n in these r e c e p t o r s . Specifically,
d u r i n g the first 5 rain o f d a r k a d a p t a t i o n in Fig. 5 the threshold (A) d r o p s
nearly 2.3 log units, whereas in the s a m e p e r i o d d u r i n g recovery f r o m the 25nA Na + injection the threshold (O) only d r o p p e d 0.5 log units. T h e r e f o r e , on
the basis o f the 25-nA Na + injection, recovery f r o m intracellular sodium acc u m u l a t i o n could only account for < 2 % o f the recovery o f threshold d u r i n g the
first 5 min o f d a r k a d a p t a t i o n in Fig. 5. I f one a r g u e s similarly, the 15 nA Na +
injection ( x ) suggests that recovery f r o m intraceUular Na + a c c u m u l a t i o n could
account for at most 5% o f the recovery o f t h r e s h o l d d u r i n g the first 5 min o f
d a r k a d a p t a t i o n in Fig. 5. T h u s it would a p p e a r that o v e r 95% o f the recovery
o f threshold that occurs d u r i n g the first few minutes o f d a r k a d a p t a t i o n is not
caused by a decrease in intracellular Na + accumulation.
T h e time course o f light a d a p t a t i o n c a n n o t be simply c o m p a r e d to the
desensitization p r o d u c e d by a series o f constant Na + injections. T h i s is because
the p h o t o r e s p o n s e to each o f a series o f constant a d a p t i n g flashes is not
constant. T h e first o f the series o f a d a p t i n g flashes p r o d u c e s a m u c h larger
r e s p o n s e t h a n s u b s e q u e n t flashes (Spiegler a n d Yeandle, 1974). T h e r e f o r e we
do not draw any quantitative conclusions based on the d i f f e r e n c e between the
time course o f light a d a p t a t i o n a n d the onset o f desensitization p r o d u c e d by
Na + injection.
A
c ~ eJ
ON
20
615
Intracellular Sodium and Photoreceptor Adaptation
FEIN AND CHARLTON
OFF
i
¥
i
n,I
$8
7 _
(2) ~
Ck
16
m z
©
H- ~
O ._a
T
el._ ~
'
~
!.
L2
10
:%
LI_
..
,
m
• .......
:l
'~,.",
I
~
U_
•
4/" :V
6
!.,,,
ii
I=
.....
'.
i
.
Light adapt (10g IA : -5.3)
13_
~
O
~
,<, , o
0
O
4
8
12
16
20
24
28
32
36
40
44
48
52
56
Q_
TIME (minutes)
B
ON
20
v
18
Ld
CO >-
16
z_F
14
I:L
i2
rrZ_
10
o~
O
o~
T
8
d
6
!
I'"":-
11~
b~
,,,'I,
tm u._
OFF
i
4
F
i
.~,!'
i
r
,~ '!i~-
::,:,<': "
."
.
.,e
,..,.
'
r
-~- Light adept(10g IA= -5.3)
, ~% j :
,~ ' i
~
....
12 nA No +injection
0.-
•~
O
oa
LaJ o
n
0
4
B
12
16
20
24
28
32
36
40
44
48
52
TIME (minutes)
FIGURE 8. T h e effects o f light a d a p t a t i o n and Na + injection on p h o t o r e s p o n s e
variability for a constant intensity test flash. A, T h e peak a m p l i t u d e o f the
r e s p o n s e to a constant intensity (log It = - 4 . 2 ) test flash plotted on a linear scale
as a f u n c t i o n o f t i m e for equal desensitizations p r o d u c e d by light a d a p t a t i o n o r
Na + injection. B, T h e s a m e data as in A r e p i o t t e d with a l o g a r i t h m i c scale for the
peak a m p l i t u d e . T h e arrows labeled ON d e n o t e the onset o f the a d a p t i n g light
and the s o d i u m injection. T h e arrows labeled OFF d e n o t e the time at which the
a d a p t i n g light and Na + injection were t u r n e d off.
56
Downloaded from jgp.rupress.org on July 31, 2017
12 nA NQ+ injection
<
616
THE JOURNAL
OF GENERAL
PHYSIOLOGY " VOLUME
70 " 1977
C. Relationship between Sensitivity and Time Delay
Fuortes and H o d g k i n (1964) were the first to point out that for d i f f e r e n t levels
o f light a d a p t a t i o n a quantitative relationship exists between the sensitivity a n d
time to peak o f the p h o t o r e s p o n s e in Limulus lateral eye. T h e results p r e s e n t e d
in Fig. 4 indicate that such a relationship exists for ventral p h o t o r e c e p t o r s as
well. T h e straight line d r a w n t h r o u g h the data points in Fig. 4 indicates that a 3
log unit decrease in sensitivity is associated with a b o u t a f o u r f o l d decrease in
time delay. Brown a n d Lisman (1975) have shown that both light a d a p t a t i o n
and the intracellular injection o f Ca ++ cause a decrease in the latency o f the
p h o t o r e s p o n s e o f Limulus ventral p h o t o r e c e p t o r s . T h e data in Fig. 4 indicate
that the intracellular injection o f both sodium and calcium p r o d u c e s a decrease
in sensitivity a n d a time delay that are quantitatively similar to those p r o d u c e d
by light a d a p t a t i o n . T h i s finding suggests that changes in b o t h intracellular
Na + a n d Ca ++ concentration cause their effects by s o m e h o w acting at a point or
points in the t r a n s d u c t i o n process close, or identical, to those at which light acts.
It might be t h o u g h t that any process that desensitizes the p h o t o r e c e p t o r
causes changes in sensitivity and the time course o f the p h o t o r e s p o n s e that are
similar to light a d a p t a t i o n . H o w e v e r , this is not the case. Lisman and B r o w n
(1975b) have shown that the intracellular injection o f a Ca ++ b u f f e r (EGTA)
desensitizes the p h o t o r e c e p t o r but slows the rate o f rise o f the p h o t o r e s p o n s e .
Downloaded from jgp.rupress.org on July 31, 2017
indicate that this is not the case. T h a t is, the local injection o f Na + does not
locally desensitize the p h o t o r e c e p t o r . We had previously speculated (Fein and
C h a r l t o n , 1975 b) that a local influx o f Na + ions would not sustain an intracellular
Na + g r a d i e n t , . o n the basis o f the m e a s u r e d mobility o f intracellular Na + in
o t h e r tissues. T h e r e f o r e we concluded that a localized c h a n g e in intracellular
Na + concentration could not account for local a d a p t a t i o n (Fein a n d C h a r l t o n ,
1975b). T h e results p r e s e n t e d in Figs. 6 and 7 indicate that this conclusion was
correct. T h e r e f o r e , the results o f Figs. 6 a n d 7 can be i n t e r p r e t e d as follows: (a)
injection o f Na + out o f the intracellular pipette gives rise to increased concentration o f Na + n e a r the tip o f the pipette; (b) diffusion o f Na + will cause the Na +
concentration to equilibrate t h r o u g h o u t the cell body; this will occur with a
half-time o f several seconds (Fein and C h a r l t o n , 1975b); (c) w h e n the intracellular Na + concentration t h r o u g h o u t the cell b o d y reaches some critical value,
the sensitivity o f the p h o t o r e c e p t o r begins to fall simultaneously t h r o u g h o u t
the cell b o d y (Figs. 7 a n d 8); (d) w h e n the Na + injection is t u r n e d off, the
p h o t o r e c e p t o r begins to recover sensitivity as the sodium p u m p (Brown a n d
Lisman, 1972) begins to r e d u c e the intracellular sodium c o n c e n t r a t i o n . S o m e
effect o f local illumination o t h e r t h a n local accumulation o f Na + m u s t account
for local adaptation: possibly a local accumulation o f Ca ++ ions (Fein a n d
Lisman, 1975).
We have previously shown that local light a d a p t a t i o n can induce m o r e than a
20-fold d i f f e r e n c e o f sensitivity o v e r a distance o f 80 t~m (Table I, Fein and
C h a r l t o n , 1975b). On the basis o f the a r g u m e n t s given above we can conclude
that this 20-fold d i f f e r e n c e in sensitivity is not d u e to a local a c c u m u l a t i o n o f
Na +. T h e r e f o r e , no m o r e t h a n 5% o f the light a d a p t a t i o n p r o d u c e d by local
illumination is caused by an increase o f intracellular Na +.
FEIN AND CHAaLTON
Intracellular Sodium and Photoreceptor Adaptation
617
Also, Lantz and Mauro (1977) have shown that anoxia, DNP, and 100% CO2
cause a reversible decrease in p h o t o r e c e p t o r sensitivity that is associated with a
slowing o f the p h o t o r e s p o n s e . T h e r e f o r e , the c o r r e s p o n d e n c e shown in Fig. 4
between the decrease in sensitivity and time delay p r o d u c e d by light adaptation,
Ca ++ injection, and Na + injection appears to be specific and not shared by
every process that may desensitize the p h o t o r e c e p t o r .
D. Mechanism of the Na + Effect
E. Summary and Conclusion
We had two questions in mind while carrying out these e x p e r i m e n t s . First, to
what extent does the intracellular iontophoretic injection o f Na + mimic adaptation? A n d second, to what extent do changes in intracellular Na + concentration
contribute to adaptation?
In answer to the first question, the results o f Figs. 2-4 indicate that Na +
injection quantitatively mimics changes in sensitivity, p h o t o r e s p o n s e time
course, and response variability associated with light adaptation. Also, the
same template curve adequately fits the intensity response relationships measured for light adaptation and Na + injection (Fig. 3 A). On the o t h e r h a n d , the
results o f Figs. 5 and 8 indicate that the time course o f light and d a r k
adaptation is faster than the onset o f and recovery f r o m desensitization
p r o d u c e d by Na + injection. Moreover, Figs. 6 and 7 show that, unlike local
illumination, Na + injection does not p r o d u c e local adaptation. T h u s Na +
injection mimics certain aspects o f adaptation while failing to mimic others.
Previous studies have indicated that an increase in intracellular Na + cannot
account entirely for light adaptation (Millecchia and Mauro, 1969 b; Lisman
and Brown, 1972b; Lisman, 1976). T h e r e f o r e we have answered, in quantitative
terms, o u r second question. T h e results o f Fig. 5 indicate that recovery from
intracellular Na + accumulation can account for at most 5% o f dark adaptation.
Similarly, the results o f Figs. 6 and 7, t o g e t h e r with the results o f Fein and
C h a r h o n (1975 b) (see Discussion, section B), indicate that at most 5% o f the
Downloaded from jgp.rupress.org on July 31, 2017
Lisman and Brown (1972b) have suggested that the desensitizing effect o f
intracellular Na + injection in Limulus ventral p h o t o r e c e p t o r s is not direct. T h e y
have shown that in Ringer's solution containing less than 0.1 mM Ca ++ there is
almost no decrease in p h o t o r e s p o n s e d u r i n g intracellular Na ÷ injection. T h e y
have also shown that the intracellular injection o f Ca ++ also desensitized the
p h o t o r e c e p t o r . On the basis o f these findings they p r o p o s e d that a rise in
intracellular Na + leads to an increase in intracellular Ca ++ and thereby to
desensitization o f the p h o t o r e c e p t o r (Lisman and Brown, 1972b). Also, Waloga
et al. (1976) have shown that intracellular Na ÷ injection leads to a rise in
intracellular Ca ++. T h e results p r e s e n t e d in Figs. 2-4 are consistent with the
above proposal.
Lisman and Brown (1972a) have p r o p o s e d that a rise in intracellular Ca ++ is
a factor leading to light adaptation in Limulus ventral p h o t o r e c e p t o r s . T h e
results p r e s e n t e d in Fig. 4 are consistent with this hypothesis. T h e Ca ++
injection data o f Fig. 4 are m o r e fully discussed elsewhere (Fein and Charlton,
1977b).
618
T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y • V O L U M E
70
" 1977
light a d a p t a t i o n p r o d u c e d by local illumination can be accounted for by a rise
in intracellular Na + concentration. T h e s e conclusions hold only for the r a n g e
o f light intensities c o n s i d e r e d in these e x p e r i m e n t s ( - 3 log units above absolute
threshold).
F. Speculation: the Role of Intracellular Na + in Light and Dark Adaptation
We thank Drs. R. D. DeVoe, J. Lisman, E. F. MacNichoi, Jr., and E. Szuts for their constructive
criticisms during the preparation of this manuscript. We thank Dr. E. F. MacNichol, Jr., for the
use of his equipment.
Downloaded from jgp.rupress.org on July 31, 2017
We have concluded that changes in intracellular Na + concentration m a k e at
most a small quantitative contribution to the total a d a p t a t i o n o b s e r v e d in
Limulus ventral p h o t o r e c e p t o r s o v e r the first 3 log units o f a d a p t a t i o n above
b u m p threshold. H o w e v e r , we do not m e a n to imply that a rise in intracellular
Na + concentration does not m a k e any contribution to a d a p t a t i o n .
Previous studies (Fein a n d DeVoe, 1973) a n d the results p r e s e n t e d in Fig. 5
indicate that t h e r e is an initial fast c o m p o n e n t a n d a later slow c o m p o n e n t o f
d a r k a d a p t a t i o n . We have also f o u n d that with p r o l o n g e d intense a d a p t a t i o n
(log I = 0, d u r a t i o n 10-20 min) the slow c o m p o n e n t o f d a r k a d a p t a t i o n can be
g r e a t e r than 1 log unit ( u n p u b l i s h e d observation). F u r t h e r m o r e , in Fig. 5 t h e r e
is a slow increase in t h r e s h o l d d u r i n g the 20 min o f light a d a p t a t i o n . Also, in
o u r study o f local a d a p t a t i o n (Fein a n d C h a r l t o n , 1975 b) we f o u n d that t h e r e
was a c o m p o n e n t o f a d a p t a t i o n (that increased with a d a p t i n g intensity) which
was not localized a n d which could not be accounted for by light scatter. T h u s
t h e r e a p p e a r to be a slow c o m p o n e n t a n d a nonlocalized c o m p o n e n t o f
a d a p t a t i o n that increase with the intensity o f the a d a p t i n g stimulus. T h e s e
c o m p o n e n t s are not very p r o m i n e n t at the threshold elevations (3 log units) we
have investigated in this study (Figs. 5-7). B r i g h t e r a d a p t i n g lights a n d t h e r e b y
g r e a t e r elevations o f threshold are n e e d e d to b r i n g out these c o m p o n e n t s . We
speculate that p a r t or all o f these c o m p o n e n t s o f a d a p t a t i o n may be associated
with changes in intraceUular Na + concentration.
T h e slow c o m p o n e n t o f light a d a p t a t i o n (Fig. 5) may be d u e to the intracellular accumulation o f Na +, a n d the time course o f the slow c o m p o n e n t o f d a r k
a d a p t a t i o n (Fig. 5) may possibly reflect the rate at which a c c u m u l a t e d Na + is
p u m p e d out o f the cell. Similarly, the nonlocalized c o m p o n e n t o f light a d a p t a tion that is not d u e to light scatter may reflect a rise in intracellular s o d i u m
t h r o u g h o u t the cell. T h e i n v o l v e m e n t o f Na + in these c o m p o n e n t s o f a d a p t a t i o n
is c u r r e n t l y b e i n g investigated.
It is a p p r o p r i a t e to ask what are the possible connections between the
internal concentrations o f s o d i u m a n d calcium, a n d how they m a y be related to
a d a p t a t i o n . As s u m m a r i z e d by Fein a n d C h a r l t o n (1977 b), all the available
evidence is consistent with the suggestion that a rise in intracellular Ca ++ is a
factor controlling a d a p t a t i o n (Lisman a n d Brown, 1972 a). Also, as discussed in
part D, the desensitization o f the p h o t o r e c e p t o r p r o d u c e d by intracellular Na +
injection a p p e a r s to be d u e to a rise in intracellular Ca ++ (Lisman a n d Brown,
1972 b; Waloga et al., 1976). We have suggested above that certain c o m p o n e n t s
o f a d a p t a t i o n m a y be d u e to a rise in intracellular Na +.
FEIN ANn CHARLTON Intracellular Sodium and Photoreceptor Adaptation
619
This work was supported by National Institutes of Health grant EY01362 to Alan Fein and partially
supported by a Sloan Fellowship to Alan Fein and a grant from the Rowland Foundation to the
Laboratory of Sensory Physiology, Marine Biological Laboratory, Woods Hole, Massachusetts. J.
Sherwood Charlton was supported by National Institutes of Health grant 1 F32 EY 05109-01.
Receivedfor publication 10 February 1977.
REFERENCES
ADOLPH, A. R. 1964. Spontaneous slow potential fluctuations in the Limulus photoreceptor. J. Gen. Physiol. 48:297-322.
Downloaded from jgp.rupress.org on July 31, 2017
BADER, C. R., F. BAUMANN, and D. BERTRAND. 1976. Role of intracellular calcium and
sodium in light adaptation in the retina o f the honey bee d r o n e (Apis mellifera, L.). J.
Gen. Physiol. 67:475-491.
BROWN, H. M. 1976. Intracellular Na +, K +, and C1- activities in Balanus photoreceptors.
J. Gen. Physiol. 68:281-296.
BROWN, H. M., S. HAGIWARA, H. KOIKE, and R. M. M•ECH. 1970. M e m b r a n e
properties o f a barnacle p h o t o r e c e p t o r examined by the voltage clamp technique.
J. Physiol. (Lond.). 208:385-413,
BROWN, J. E., and J. E. LISMAN. 1972. An electrogenic sodium p u m p in Limulus ventral
p h o t o r e c e p t o r cells. J. Gen. Physiol. 59:720-733.
BROWN, J. E., and J. E. LISMAN. 1975. Intracellular Ca ++ modulates sensitivity and time
scale in Limulus ventral photoreceptors. Nature (Lond.). 258:252-253.
DODGE, F. A., B. W. KNIGHT, and J. TOYODA. 1968. Voltage noise in Limulus visual cells.
Science (Wash. D.C.). 160:88-90.
FEIN, A. 1973. Local adaptation in the ventral photoreceptor o f Limulus. Biol. Bull.
(Woods Hole). 145:432.
FEIN, A., and J. S. CUAaLTON. 1975 a. Local m e m b r a n e current in Limulus photoreceptors. Nature (Lond. ). 258:250-252.
FERN, A., and J. S. CHARLTON. 1975 b. Local adaptation in the ventral photoreceptors o f
Limulu~. J. Gen. Physiol. 66:823-835.
FERN, A., and J. S. CHARLTON. 1977 a. Enhancement and phototransduction in the
ventral eye of Limulus. J. Gen. Physiol. 69:553-569.
FE1N, A., and J. S. CHARLTON. 1977 b. A quantitative comparison o f the effects o f
intracellular calcium injection and light adaptation on the photoresponse o f Limulus
ventral p h o t o r e c e p t o r s . J . Gen. Physiol. 70:591-600.
FERN, A., and R. D. DEVoE. 1973. Adaptation in the ventral eye of Limulus is functionally
i n d e p e n d e n t o f the photochemical cycle, m e m b r a n e potential, and m e m b r a n e resistance. J. Gen. Physiol. 61:273-289.
FEIN, A., and J. LISMAN. 1975. Localized desensitization o f Limulus photoreceptors
p r o d u c e d by light or intracellular calcium ion injection. Science (Wash. D.C.). 187:10941096.
FUORTES, M. G. F., and A. L. HODGKIN. 1964. Changes in time scale and sensitivity in
the ommatidia o f Limulus. J. Physiol. (Lond.). 172:239-263.
FUORTES, M. G. F., and P. M. O'BRYAN. 1972. G e n e r a t o r potentials in invertebrate
photoreceptors. In H a n d b o o k o f Sensory Physiology. Vol. VII/2. Physiology o f
Photoreceptor Organs. Springer Verlag, BerLin. 279-319.
FUORTES, M. G. F., and S. YEANDLL 1964. Probability o f occurrence o f discrete potential
waves in the eye ofLimulus. J. Gen. Physiol. 47:443-463.
KOIKE, H., H. M. BROWN, and S. HAGtWARA. 1971. Hyperpolarization o f a barnacle
620
T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M E
70
- 1977
Downloaded from jgp.rupress.org on July 31, 2017
photoreceptor m e m b r a n e following illumination. J. Gen, Physiol. 57:723-737.
LANTZ, R. C., and A. MAURO. 1977. Alteration of sensitivity and time scale in invertebrate
photoreceptors exposed to anoxia, dinitrophenol and carbon dioxide. Biophys. J. 17(2,
Pt. 2):15 a. (Abstr.).
LISMAN, J. E. 1976. Effects o f removing extracellular Ca 2+ on excitation and adaptation
in Limulus ventral photoreceptors. Biophys. J. 16:1331-1335.
L1SMAN, J. E., and J. E. BROWN. 1972 a. T h e effects of intracellular Ca 2+ on the light
response and light adaptation on Limulus ventral photoreceptors. In T h e Visual
System: Neurophysiology, Biophysics and their Clinical Applications. G.B. A r d e n ,
editor. Plenum Publishing Corp., New York. 23-33.
LISMAN, J. E., and J. E. BROWN. 1972 b. T h e effects of intracellular iontophoretic
injection of calcium and sodium ions on the light response o f Limulus ventral
photoreceptors. J. Gen. Physiol. 59:701-719.
LXSraAN,J.E., and J. E. BROWN. 1972 a. Light-induced changes of sensitivity in Limulus
ventral photoreceptors. J. Gen. Physiol. 66:473-488.
LISraAN, J. E., and J, E. BROWN. 1975 b. Effects of intracellular injection of calcium
buffers on light adaptation in Limulus ventral photoreceptors. J. Gen. Physiol. 66:489506.
MILLECCHIA, R., and A. MAURO. 1969 a. T h e ventral photoreceptor cells of Limulus. II.
T h e basic p h o t o r e s p o n s e . J . Gen. Physiol. 54:310-330.
MILLECCrIIA, R., and A. MAURO. 1969 b. T h e ventral photoreceptor cells of Limulus. I l l .
A voltage-clamp s t u d y . J . Gen. Physiol. 54:331-351.
SPIEOLER, J. B., and S. YEANDLE. 1974. I n d e p e n d e n c e of location of light absorption
and discrete wave latency distribution in Limulus ventral nerve photoreceptors. J. Gen.
Physiol. 64:494-502.
WALOGA, G., J. E, BROWN, and L. H. PXN'rO. 1975. Detection of changes of Cain from
Limulus ventral photoreceptors using arsenazo III. Biol. Bull. (Woods Hole). 149:449450.