1Dipartimento di Biologia, Sezione di Fisiologia Generale, Università di Ferrara, 44100 Ferrara, Italy; and 2Department of Neurobiology, University of Alabama, School of Medicine, Birmingham, Alabama 35294-0021
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ABSTRACT |
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Dmitriev, Andrey, Angela Pignatelli, and Marco Piccolino. Resistance of Retinal Extracellular Space to Ca2+ Level Decrease: Implications for the Synaptic Effects of Divalent Cations. J. Neurophysiol. 82: 283-289, 1999. Ion-sensitive microelectrodes were used to measure the variations of [Ca2+]o induced by application of low Ca2+ media in the superfused eyecup preparation of the Pseudemys turtle. The aim of the experiments was to evaluate the possibility, suggested by previous studies, that in the deep, sclerad, layers of the retina [Ca2+]o may remain high enough to sustain chemical synaptic transmission even after prolonged application of low-Ca2+ saline. It was found that, at depths of 100-200 µm from the vitreal surface, [Ca2+ ]o did not fall below 1 mM even after application for periods of 30-60 min of nominally Ca2+-free media, and it was >0.3 mM after 30-min application of media containing EGTA and with a Ca2+ concentration of 1 nM. Previous studies in isolated salamander photoreceptors have shown that a reduction of [Ca2+ ]o to 0.3-1.0 mM may result in a paradoxical increase of Ca2+ influx into synaptic terminals due to the reduced screening of negative charge on the external face of the plasma membrane. On the basis of these results, the persistence or enhancement of synaptic transmission from photoreceptors to horizontal cells observed in various retinas treated with low-Ca2+ media may be accounted for within the classical Ca2+-dependent theory of synaptic transmission without invoking a Ca2+-independent mechanism.
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INTRODUCTION |
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Among the preparations of nervous tissue suitable for in vitro functional investigations, the vertebrate retina offers the special advantage of being a natural slice of CNS that can be studied easily with minimum experimental alteration of its structure and physiological conditions. This is particularly true for the superfused "eyecup" preparation in which the retinal tissue is not detached from the pigmented epithelium, thus preserving its structure, its viability, and its functions, especially the capability to regenerate photopigment.
To a large extent its suitability for in vitro studies explains why the
retina has been a favorite model of electrophysiological investigation
since the epoch of Svaetichin (1953). However, if morphological and functional integrity of the nervous structure warrants the physiological validity of the results that can be obtained
from the study of isolated retina preparations, from a certain point of
view it may represent an obstacle to experimental investigation. This
is particularly true in experimental studies requiring the manipulation
of the extracellular fluid composition via a change of the perfusing
medium used to maintain the viability of the tissue. In that respect
the in vitro retina stands in contrast to dissociated cell preparations
where the surrounding liquid equilibrates rapidly with the superfusion
medium. Retina may also differ from more conventional slice
preparations where the accessibility of the extracellular space can be
favored by limited tissue thickness and by the possible alterations of
the extracellular space resulting from experimental procedures.
There is abundant experimental evidence indicating that a slow and
incomplete equilibration occurs between the extracellular space and the
superfusing medium in an eyecup preparation. For instance although
glutamate is effective at micromolar concentrations for stimulating
dissociated horizontal cells of the turtle retina, its concentration
must be raised to tens of millimolar to affect the same cells in the
eyecup (Ariel at al. 1984; Cervetto and MacNichol
1972
; Golard et al. 1992
; Ishida and
Neyton 1985
). Intraretinal diffusion barriers and powerful
uptake mechanisms may prevent the penetration of glutamate down to the
more deep (sclerad) layers of retinal tissue (Miyachi
1988
; Normann et al. 1986
). Diffusion problems
and uptake mechanisms also may limit the study in the intact retina of
other neurotransmitters, as for instance GABA (see Yazulla
1991
).
Difficulty in diffusion must be taken particularly into account when
attempting to change the ionic composition of the medium. This is
especially the case for divalent cations because of the existence on
the external surfaces of neuronal membranes of an excess of negative
charges that tends to sequester the ions and limit their mobility in
the extracellular compartment (Harsanyi and Mangel 1993;
McLaughlin 1989
; Morris and Krnjevic
1981
; Piccolino and Pignatelli 1996
;
Piccolino et al. 1999
). There are several indications
that divalent cation diffusion in both retina and in more conventional
slice preparations may be slow and equilibrium may not be achieved in
the conditions of the experiment (Hegstad et al.1989
;
Morris and Krnjevic 1981
; Piccolino et al.
1996
).
Diffusion problems acquire a particular relevance for the
interpretation of experimental results in situations in which the extracellular Ca2+ level is reduced to reduce
Ca2+ influx through the voltage-dependent
Ca2+ channels of the plasma membrane. This is
commonly done, for instance, to block chemical synaptic transmission
(Cervetto and Piccolino 1974; Kaneko and
Shimazaki 1976
; Katz and Miledi 1968
). However, a reduction of external Ca2+ may bring about
opposite effects on Ca2+ influx (Piccolino
and Pignatelli 1996
; Piccolino et al. 1999
). On
the one hand, a [Ca2+]o
decrease tends to reduce Ca2+ influx because of
the reduced driving force and availability of
Ca2+ ions at the channel mouth. On the other
hand, because of the reduced screening of the negative charges on the
external surface of plasma membrane, [Ca2+
]o decrease may result in an increased opening
of Ca2+ channels and thus an increased
Ca2+ influx (Baldridge et al.
1998
; Piccolino et al. 1998
). Whether Ca2+ influx will ultimately decrease or increase
depends on the prevalence of the one or the other of these two
contrasting effects.
In recent experiments on the eyecup preparation of the turtle retina,
it was found that decreased
[Ca2+]o could result in
an increase of transmitter release from photoreceptors via a
surface-charge-mediated augmentation of Ca2+
influx in low-Ca2+ media (Piccolino et al.
1996; see also Baldridge et al. 1998
). On the
basis of this, we suggested that the persistence, or enhancement, of
synaptic transmission during the application of
low-Ca2+ media, as observed in previous studies
(Hankins et al. 1985
; Harsanyi and Mangel
1993
; Piccolino et al. 1996
; Rowe
1987
; Schwartz 1986
; Umino and Watanabe
1987
), does not necessarily imply that transmitter release from
photoreceptor terminals is Ca2+ independent.
There was, however, a difficulty with our interpretation. An increase of Ca2+ current and of intracellular Ca2+ level in isolated photoreceptors was observed only with relatively modest reductions of extracellular Ca2+ (0.3-1.0 mM), whereas synaptic transmission enhancement in the superfused eyecup was seen even when Ca2+ was omitted from the perfusing medium (resulting in Ca2+ concentrations in the micromolar range) or was reduced to extremely low levels (in the nanomolar range) by using Ca2+ buffers. To account for this apparent inconsistency, we assumed that in our preparation [Ca2+]o would remain at levels capable of supporting classical synaptic transmission (hundredths of micromolar), even after prolonged application of low-Ca2+ media, due to incomplete equilibration of divalent cation concentrations between perfusing medium and extracellular compartment near the photoreceptor synapse.
The aim of the present work is to provide an experimental test of our hypothesis. We have measured Ca2+ concentrations in the extracellular space of the retina by using ion-sensitive electrodes in conditions similar to those used in the study of the effects of low-Ca2+ media on synaptic transmission in the retina. Our results show that, in an eyecup, extracellular Ca2+ does not faithfully follow the changes in the superfusing medium but remains at elevated levels even after prolonged perfusion with low-Ca2+ media. The hypothesis that low-Ca2+ media can enhance synaptic transmission from photoreceptor to horizontal cells by promoting Ca2+ entry into photoreceptor synaptic terminals therefore is supported.
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METHODS |
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Preparation
Eyecups were prepared from the retina of turtles,
Pseudemys scripta elegans, obtained from William A. Lemberger (Oshkosh, WI) and kept in outdoor ponds. The animals were
killed by decapitation and pithed after being anesthetized with
ketamine (200 mg/kg). Details of the experimental methods used to
remove the eye and preparing the eyecup are given elsewhere
(Piccolino et al. 1984). The preparation was superfused
with a saline of the following composition (in mM/l): 110 NaCl, 2.6 KCl, 22 NaHCO3, 2 MgCl2, 2 CaCl2, and 10 D-glucose. This
solution was bubbled continuously with a mixture of 95%
O2-5% CO, to a final pH of 7.4. In some experiments, to assess the viability of the preparation, we recorded intracellularly the light responses of retinal neurons (usually horizontal cells) by using fine-tip glass micropipettes prepared with a
Brown-Flaming puller (Model P-77, Sutter Instrument, Novato, CA) filled
with 3 M potassium acetate and 0.2 M KCl (resistance 150-500 M
).
Ca-sensitive microelectrodes
The extracellular concentration of Ca2+ was measured with double-barrelled ion-sensitive microelectrodes based on the Calcium Ionophore I-Cocktail A by Fluka (Switzerland, No. 21048). The microelectrodes were pulled with the P-77 Brown-Flaming puller using double-barrelled borosilicate glass "theta" capillaries (WPI, Sarasota, FL, No. TST150-6). After pulling, the barrel to be used as the reference electrode was backfilled with distilled water. Then several micropipettes were exposed for 4-16 h at room temperature to an atmosphere of silane obtained by dropping a few milliliters of a solution of dimethyldichlorosilane in carbon tetrachloride (10% in volume, both from Sigma) in a tightly closed jar. After silanization the active barrel was backfilled with 0.5 µL of the ion exchange solution. The tip of the microelectrode then was broken gently on a piece of soft paper, thus allowing the ion exchanger to move spontaneously to the very tip. Finally the distilled water in the reference barrel was replaced, by backfilling, with a solution containing (in mM) 140.0 NaCl, 5.0 KCl, 2.6 CaCl2, and 5.0 Tris (pH 7.5). The same solution also was used for filling up the ion selective barrel away from the tip. Typically the microelectrodes used in the experiments had tips with diameters of 10-15 µm.
Solutions and calibration of Ca-selective microelectrodes
The relation between the potential measured by the ion-sensitive
electrodes and the ionic concentration in the medium is described by
the Nicolsky equation (Amman 1986; Amman et al.
1983
), which takes into account the contribution to the
measured potential of the ion investigated (Ca2+
in our case) and the interference by the other ions present in the
solution
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RESULTS |
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Effects of low-Ca2+ medium on [Ca2+]o in the retina
We perfused the retina with a nominally Ca2+-free solution and measured the extracellular Ca2+ concentration within the retina tissue. Figure 1 illustrates the results. With the electrode in the perfusion fluid, above the vitreal side of the retina, the perfusion solution was changed to a Ca2+-free Ringer. The concentration of Ca2+ started decreasing after ~1 min after the application of the test solution, and within ~3 min Ca2+ reached a level a little higher than 2 µM, which obviously corresponded to that of the test medium (Fig. 1A). On reapplying the normal solution, Ca2+ level rapidly reached the control value. Thereafter the electrode was advanced into the retinal tissue to a point 130 µm below the vitreal surface so that its tip resided at the level at which horizontal cells normally are penetrated with intracellular microelectrodes. After the application of test solution (Fig. 1B), Ca2+ began to decrease with a slow time course that reached, after 30 min, a value corresponding to 1.4 mM. In nine experiments in which extracellular Ca2+ was measured at retinal depths of 100-200 µm, the average level of [Ca2+]o reached after 30-min application of Ca2+-free Ringer was 1.41 mM ± 0.33 (mean ± SD). In some experiments, low-Ca2+ application was continued for longer periods (from 40 to 60 min), and even in these circumstances, [Ca2+]o never fell <1 mM.
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Extracellular [Ca2+]o as a function of retinal depth
The values of extracellular Ca2+
concentration changes induced by the application of
low-Ca2+ media depended on retinal depth and were
substantially lower when the electrode was positioned less deep
in the retinal tissue. A typical experiment is illustrated in Fig.
2 where the final level of
[Ca2+]o brought about by
the application of low-Ca2+ medium ranged
from 1.70 at 200 µm depth to 0.23 at 60 µm. This suggested that a
substantial Ca2+ gradient exists vertically
across the retinal tissue in the presence of
low-Ca2+ media, a view verified by experiments in
which electrode position was changed during prolonged application of
low-Ca2+ media. One of these experiments is
illustrated in Fig. 3 where a medium
lacking Ca2+ was applied for ~30 min while the
electrode was deep in the retinal tissue (~100 µm below the vitreal
surface) leading to a final concentration of ~1.05 mM at that
electrode position. Afterward the electrode tip was raised by moving
the microelectrode vertically to different levels in step fashion (the
size of each step is indicated by the numbers near the downward
pointing arrows). The measured concentration decreased rather abruptly
after each movement and became similar to that of the perfusing medium
only after the electrode had been raised by 2.5 mm (i.e., when it was
well outside the retina). Because of the mechanical deformation of the
retinal tissue, the depths reached by the electrode after the different
maneuvers could not be determined precisely. It is clear, however, that
a large vertical gradient of
[Ca2+]o exists in the
retina during the application of low-Ca2+ media
and that Ca2+ levels remain substantial higher in
most of the retinal depths compared with that in the superfusing
medium. Moreover, it is also possible that compared with the bulk flow
of perfusing saline the concentration of Ca2+
remained substantially elevated also outside the retina in the portion
of vitreous that could not be removed by dissection from the retinal
surface and in the adjacent liquid layers. The presence of extra
Ca2+ at this level would be consistent with the
view that retina constantly loses some Ca2+ when
superfused with low-Ca2+ solution. The important
point, however, is that all retinas tested were able to keep a high
level of [Ca2+]o (>1 mM)
in their distal layers in the presence of
low-Ca2+ media for periods of 30 min and, in some
experiments, for periods 1 h.
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In the previous work in which a surface-charge-mediated increase of
Ca2+ influx was produced by decreasing
[Ca2+]o, the
low-Ca2+ media generally were applied in the
presence of divalent cations having a strong influence on surface
charges (Baldridge et al. 1998; Piccolino et al.
1996
). To simulate such conditions, in some experiments the
retina was perfused for 15 min with 0.4 mM Zn2+
(n = 6) before applying a nominally
Ca2+-free solution together with 0.4 mM
Zn2+. The final Ca2+ levels
measured in both the perfusing fluid and inside the retinal tissue did
not significantly differ from those observed in the absence of the
exogenous antagonists. This is illustrated in Fig. 4, which compares the levels reached by
[Ca2+]o at different
retinal depths after 30 min application of a nominally Ca2+-free medium containing 0.4 mM
Zn2+ (
) with the levels reached after
application of a Ca2+-free medium lacking
Zn2+. There appears to be no significant
difference between the two sets of data. Moreover, in spite of the
dispersion of data that blurs the relation between depths and
concentration, it seems that the Ca2+ gradient is
steeper in the proximal part of the retina.
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Effects of low-Ca2+ medium containing EGTA
In some experiments, we measured
[Ca2+]o at various levels
of retina during the application of media in which
Ca2+ had been buffered to very low levels (1 nM)
by adding 5 mM EGTA and appropriate concentrations of
CaCl2. In the experiment illustrated in Fig.
5, the electrode initially was positioned
at a depth of 100 µm with respect to the vitreal surface. After the
application of low-Ca2+ media containing EGTA,
the level of Ca2+ inside the retina dropped
to lower levels than seen in the absence of the buffer. For instance,
after the initial 30-min application of the test solution,
[Ca2+]o at 100 µm depth
dropped to 0.51 mM, a level never reached with low-Ca2+ media lacking EGTA. Nevertheless,
Ca2+ still remained much higher than in the
superfusion medium and well above the level at which
Ca2+ activates transmitter release inside
synaptic terminals (1-100 µM) (see Adler et al. 1991;
Heidelberger et al. 1994
; Matthews 1996
;
Rieke and Schwartz 1996
). In similar experiments in
which extracellular Ca2+ was measured at retinal
depths of 110-200 µm, during the application of
low-Ca2+ solutions containing 5 mM EGTA, the
level of [Ca2+]o reached
after 30 min never fell <0.30 mM (0.48 ± 0.18: mean ± SD,
n = 5). Moreover as in the case of perfusion with
nominally Ca2+-free media lacking EGTA, large
vertical gradients of Ca2+ existed in the retina
during the application of media containing Ca2+
buffers. This appeared clearly when the electrode was pulled up in step
fashion out of the tissue (the size of the steps is indicated in Fig. 5
near the
).
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DISCUSSION |
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The results of the present experiments can be summarized by saying that the Ca2+ concentration in the more sclerad layers of the retina eyecup preparation remains substantially elevated after prolonged superfusion of the vitreal surface with low-Ca2+ media. In particular at the depths where horizontal cells are commonly encountered with intracellular electrodes, [Ca2+]o never decreased to <1 mM after 30- to 60-min application of nominally free Ca2+ media, and it remained at values >0.3 mM even after applications of media containing EGTA that buffered Ca2+ concentration in the nanomolar range.
In general a large concentration difference in the retina preparation
between superfusing medium and extracellular space may depend on the
integrity of the thin and tortuous extracellular space of the retina,
with intimate contact between neurons and glial cells, on the existence
of diffusion barriers from both the vitreal and scleral side and on the
presence of powerful mechanisms of transport on the cell membrane of
both neurons and glia. These elements combine to maintain the constancy
of the extracellular microenvironment and to reduce the possible
influences of changes in the composition of the superfusing medium
(Dreher et al. 1988; Karwoski et al.
1989
; Linsenmeier and Steinberg 1983
;
Stone et al. 1995
; Tout et al. 1993
). It
is indeed well known that retina is able to maintain extracellular ion
concentrations at levels that differ from those of the superfusion
solutions. For instance, it was shown in various in vitro preparations
from different animals that the extracellular concentration of
K+ in subretinal space (i.e., between
photoreceptors and retinal pigment epithelium) is about two times
higher than in the superfusion medium (Bykov et al.
1984
; Dick and Miller 1985
; Dick et al.
1985
; Dmitriev and Bykov 1990
; Miller and
Steinberg 1982
; Oakley and Green 1976
).
Moreover, measurements in intact cat demonstrated that there are
concentration gradients in vitreous and across the retina for
K+ (Steinberg et al. 1980
) and for
Ca2+ (Gallemore et al. 1994
).
Recently it was found that retinal extracellular pH was substantially
different from the pH of the superfusion solution in isolated fish
retina (by 0.23 pH units) (see Dmitriev and Mangel 1997
)
and rabbit eyecup (by 0.62 pH units) (see Dmitriev and Mangel
1998
).
As already mentioned, a limited diffusion of Ca2+
may depend also on the presence of an excess of
Ca2+ on the external surface of neuronal membrane
due to electrostatic interactions with the negative surface charges
(McLaughlin 1989; Morris and Krnjevic
1981
; Piccolino et al. 1999
). This excess of
Ca2+ may act as a functional reserve capable of
effectively buffering the concentration of the ions in the bulk
extracellular medium and thus seriously limiting any attempt to change
it via manipulation of the perfusing medium (Piccolino and
Pignatelli 1996
; Piccolino et al. 1999
).
Moreover, the difficulty in modifying
[Ca2+]o in the deep
retinal layers may depend on the characteristics of
Ca2+ metabolism in the photoreceptor layer and,
in particular, on the intense exchange of Ca2+
between extracellular and intracellular compartment because ~15% of
dark current is carried by Ca2+ (McNaughton
1995
; Yau and Baylor 1989
). This roughly equals
107 Ca2+ ions per second, a
rate such that all extracellular Ca2+ would be
exchanged in ~1 min. Because the total intracellular Ca2+ in photoreceptors is >10 times higher than
the content of extracellular space (taking into account the volume
occupied by cells), it is easy to envision why
Ca2+ losses from the extracellular space could be
compensated for easily in the distal retina.
Even after prolonged application of low-Ca2+
media in the retina eyecup preparation, the deep retinal layers still
retain a sufficient
[Ca2+]o to provide a
large driving force for Ca2+ entry in nerve cells
via the Ca2+ channels of the plasma membrane.
This is still the case for EGTA-containing media because
[Ca2+]o did not fall
<0.30 mM in the distal retina. In general Ca2+
concentration in the presynaptic terminals of chemical synapses must
rise at concentrations of ~20-100 µM to activate transmitter release (Adler et al. 1991; Heidelberger et al.
1994
; Matthews 1996
), and in photoreceptor
synapses the release is likely to occur even at concentrations of <1
µM (Krizaj and Copenhagen 1998
; Rieke and
Schwartz 1996
). As a matter of fact, experiments carried out on
isolated photoreceptors of the salamander retina show that reducing
[Ca2+]o to values
in the range of 0.3-1.0 mM actually may result in an increase of
Ca2+ current at the physiological membrane
potential of photoreceptors in darkness (
35,
30 mV). This effect is
due to a shift to the left (i.e., toward less depolarized
potentials) of the Ca2+ current activation curve
(Baldridge et al. 1998
; Piccolino et al.
1996
), which in turn results from a decrease of the
neutralizing action of Ca2+ ions on the negative
charge at the external surface of the plasma membrane. The shift more
than compensates for the reduced driving force due to decreased
[Ca2+]o and thus may lead
to an increase of [Ca2+]i
in photoreceptor terminals compared with the control conditions.
On the basis of our results it is therefore easy to understand why the
application of low-Ca2+ media to the retina may
lead to an enhancement, rather than to a depression, of synaptic
transmission process from photoreceptor endings (Harsanyi and
Mangel 1993; Rowe 1987
; Piccolino et al. 1996
). In particular, it can be understood why this applies
also to experiments involving the application of superfusing media with
Ca2+ concentrations so low that they would not
support any transmitter release when applied to isolated cell preparations.
Combined with the results of other studies of ionic manipulation
effects on synaptic transmission from photoreceptors to horizontal cells, the present study appears to invalidate the hypothesis that
synaptic transmission at this level is Ca2+
independent (Schwartz 1986). This hypothesis was based
on the observation that synaptic transmission from photoreceptors to second-order neurons of the salamander and toad retina largely persisted in the presence of a superfusing medium nominally lacking Ca2+ and containing Co2+
ions. In interpreting this observation, it was assumed that no significant Ca2+ influx could occur into
presynaptic terminals in a situation in which
Ca2+ channels probably were blocked due to the
presence of Co2+ and, moreover, the
Ca2+ gradient was minimized as a consequence of
the reduced concentration of external Ca2+. This
interpretation largely depended on the assumption that the
concentration of divalent cations in the extracellular space of the
retina followed closely that of the perfusing medium. Later experiments
indicated that this was likely not the case (Piccolino et al.
1996
). First it appeared that in the eyecup preparation experiments the synaptic blocking effect of Co2+,
and of other antagonist divalent cations such as
Zn2+ or Ni2+, could be
accounted for largely by a shift to the right of the channel activation
curve brought about by concentrations of the divalent cations in the
extracellular space of the retina that are much lower than those in the
bathing medium. Because of this shift (which in turn resulted from an
increased screening of the negative surface charges on the plasma
membrane), there was a drastic decrease of the
Ca2+ current at the physiological membrane
potential of photoreceptors, i.e., at the dark potential. In these
conditions, lowering
[Ca2+]o would bring about
an increase of Ca2+ current via a shift to the
left of the activation curve due to the reduction of surface charge
screening. On these grounds, the relative synaptic blocking
inefficacy of low-Ca2+ media containing
Co2+ (and other divalent antagonists) could be
accounted for easily. Moreover, it was also possible to explain why the
synaptic blocking efficacy of Co2+,
Zn2+, and Ni2+ was stronger when a normal
Ca2+ concentration was present in the perfusing medium than
when Ca2+ was omitted from the bathing solution
(Cervetto and Piccolino 1974
; Piccolino et al.
1996
; Schwartz 1986
).
In the context of the interpretation of the effects of
low-Ca2+ media on synaptic transmission in the
retina, the main new information provided by the present results is
that perfusing the retina eyecup preparation with nominally zero
Ca2+ solutions, or with an
EGTA-Ca2+ Ringer in which
[Ca2+]o is estimated to
be ~1 nM, causes a relatively small decrease of
[Ca2+]o in the deep
layers of the retina. That lowered level of
[Ca2+]o is well within the range of the
[Ca2+]o values capable of promoting
Ca2+ influx into photoreceptors at their
physiologically relevant potential (Krizaj and Copenhagen
1998; Rieke and Schwartz 1996
). A strong support
to the notion that synaptic transmission from photoreceptors to
horizontal cells is indeed Ca2+ dependent comes
from a recent work carried out in the Xenopus retina showing
the existence of a fine tuning among the physiologically relevant
presynaptic potential, Ca2+-current activation
curve, and glutamate release (Witkovsky et al. 1997
).
It is worth mentioning here that, as in other regions of the nervous
system, the concentration of Ca2+ in the deep
layers of the retina may vary in physiological conditions, such as
during light-dark adaptation (Bykov et al. 1985;
Gallemore et al. 1994
; Gold and Korenbrot
1980
; Livsey et al. 1990
; Yoshikami et
al. 1980
). It is thus possible that changes of
[Ca2+]o may serve to
modulate the transmission process at the photoreceptor synapse in
physiological conditions.
In a more general context, the presence of a significant difference
between the levels of Ca2+ in the deep extracellular layers
of an in vitro nervous preparation and the perfusing medium, even after
a prolonged application of low-Ca2+ media, invites caution
in interpreting as Ca2+ independent any process that
persists after prolonged application of low-Ca2+ solution.
This is particularly important for thick tissue preparations (wedges or
thick slices), especially when the morphological and functional
integrity of extracellular space is preserved, and when diffusion
barriers are likely to exist between the perfused surface and the layer
of nervous tissue under study (Nicholson 1980;
Nicholson and Hounsgaard 1983
; Tout et al.
1993
). Of course, the interpretation of the results of
low-Ca2+ experiments is more straightforward in the case of
preparations in which diffusion is less restricted by morphological
barriers (e.g., the isolated retina where free diffusion occurs from
the photoreceptor side) and in particular for isolated cell
preparations where the membrane surface is readily accessible to the
perfusing medium. On these grounds, it is likely that a true
Ca2+-independent process operates in the release
of synaptic transmitter from retinal horizontal cells (Schwartz
1987
).
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ACKNOWLEDGMENTS |
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The authors thank O. Belluzzi, K. T. Keyser, and S. C. Mangel for critically reading the manuscript.
This work was supported by Ministero dell' Università e della Ricerca Scientifica e Technologica and the National Research Council of Italy.
Present address of A. Pignatelli: Division of Biological Sciences, Section of Neurobiology, Physiology, and Behavior, University of California Davis, One Shields Ave., Davis, CA 95616-8519.
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FOOTNOTES |
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Address for reprint requests: M. Piccolino, Dipartimento di Biologia, Sezione di Fisiologia Generale, Università di Ferrara, Via Borsari 46, Ferrara, Italy.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 December 1998; accepted in final form 1 April 1999.
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REFERENCES |
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