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The Journal of Neuroscience, July 1, 1998, 18(13):5019-5025
Blockade of Glutamate-Mediated Activity in the Developing Retina
Perturbs the Functional Segregation of ON and OFF Pathways
Silvia
Bisti1, 2,
Claudia
Gargini1, 3, and
Leo
M.
Chalupa4
1 Institute of Neurophysiology, Consiglio Nazionale
delle Ricerche, Pisa 56127, Italy, 2 Dipartimento di
Scienze e Tecnologie Biomediche, University of L'Aquila, L'Aquila
67100, Italy, 3 Dipartimento di Psichiatria, Neurobiologia,
Farmacologia, e Biotechnologie, University of Pisa, Pisa, Italy, and
4 Center for Neuroscience and Section of Neurobiology,
Physiology and Behavior, Division of Biological Sciences, Davis,
California 95616
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ABSTRACT |
The dendrites of ganglion cells initially ramify throughout the
inner plexiform layer of the developing retina before becoming stratified into ON or OFF sublaminae. This ontogenetic event is thought
to depend on glutamate-mediated afferent activity, because treating the
developing retina with the glutamate analog 2-amino-4-phosphonobutyrate (APB), which hyperpolarizes ON cone bipolar cells and rod bipolar cells, thereby preventing their release of glutamate, effectively arrests the dendritic stratification process. To assess the functional consequences of this manipulation, extracellular recordings were made
from single cells in the A laminae of the dorsal lateral geniculate
nucleus and from the optic tract in mature cats that had received
intraocular injections of APB during the first postnatal month. Such
recordings revealed that stimulation of the APB-treated eye evoked both
ON as well as OFF discharges in 37% of the cells tested. (As expected,
when the normal eye was activated, virtually all cells yielded only ON
or OFF responses.) The proportion of ON-OFF cells found here
corresponds closely to the incidence of multistratified dendrites
observed previously in anatomical studies of APB-treated cat retinas.
This suggests that the ganglion cells with multistratified dendrites
receive functional inputs from ON as well as OFF cone bipolar cells.
This interpretation is further supported by the finding that the
proportion of ON-OFF cells was very similar in the geniculate layer
innervated by the treated eye and in the optic tract. The cells
activated by the APB-treated eye were also found not to show response
suppression when flashing stimuli of increasing size were used. This
suggests that exposing the developing retina to APB perturbs the neural
circuitry mediating the antagonistic center-surround organization
found in normal receptive fields. The functional changes evident after
treating the developing retina with APB suggest that it should now be
feasible to assess how the segregation of ON and OFF retinal pathways
relates to organizational features at higher levels of the visual
system, such as orientation selectivity in cortical cells.
Key words:
inner plexiform layer; ON/OFF responses; dorsal lateral
geniculate nucleus; retina; ganglion cells
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INTRODUCTION |
In the vertebrate retina, the
dendrites of most ganglion cells are stratified into two distinct
sublaminae of the inner plexiform layer (IPL). Retinal ganglion cells
with dendrites ramifying proximal to the soma are activated by
increments of light, whereas ganglion cells with distal dendrites
respond to light decrements (Famiglietti and Kolb, 1976 ; Nelson et al.,
1978). This functional distinction reflects the fact that the dendritic
processes in the two sublaminae of the IPL are differentially
innervated by bipolar cells that either depolarize or hyperpolarize on
exposure to light. Such an organization provides the structural basis
for the segregation of ON and OFF retinal pathways (for review, see
Wässle and Boycott, 1991). Moreover, in some species such as the
tree shrew, ferret, and mink, ON and OFF channels remain segregated
within different sublaminae of the dorsal lateral geniculate nucleus
(dlgn) as well as in layer 4 of the visual cortex (Stryker and Zahs,
1983; Norton et al., 1985; Conway and Schiller, 1993).
The formation of segregated ON and OFF retinal pathways involves the
gradual restriction of initially multistratified ganglion cells (Maslim
and Stone, 1986, 1988; Bodnarenko and Chalupa, 1993; Bodnarenko et al.,
1995). This process takes place relatively late in development so that
in the newborn cat retina ~40% of the ganglion cells are still in
their immature, multistratified state (Bodnarenko et al., 1995).
Moreover, the restriction of dendrites into ON and OFF sublaminae of
the IPL occurs during the time when bipolar cells establish synaptic
contacts with ganglion cells (Maslim and Stone, 1986, 1988). Such a
temporal coincidence suggested the possibility that synaptic events
within the IPL could provide a signal for the retraction of initially
multistratified processes. This hypothesis was tested recently by
treating the developing cat retina with the glutamate analog
2-amino-4-phosphonobutyrate (APB) (Bodnarenko and Chalupa, 1993;
Bodnarenko et al., 1995). This drug hyperpolarizes ON cone bipolar
cells as well as rod bipolar cells, thereby preventing their release of
glutamate (Slaughter and Miller, 1981; Muller et al., 1988; Euler et
al., 1996). Daily treatment with APB from postnatal day 2 (P2) to P12
was found to prevent the stratification of ganglion cell dendrites into ON and OFF sublaminae of the IPL. Interestingly, earlier studies had
reported that neither blockade of action potentials by intraocular injection of TTX (Dubin et al., 1986; Wong et al., 1991) nor dark rearing (Leventhal and Hirsch, 1983; Lau et al., 1990) affects the
normal incidence of stratified ganglion cells. Taken together, the
available evidence lends support to the idea that glutamate-mediated afferent activity plays a role in the stratification of ganglion cell
dendrites, resulting in the segregation of ON and OFF retinal pathways.
The functional consequences of arresting the dendritic stratification
of retinal ganglion cells by treating the developing retina with APB
remains to be established. One possibility is that the multistratified
ganglion cells respond to flashing spots of light with either ON or OFF
discharges, as is the case in the normal retina. This would imply that
bipolar cell innervation patterns are not altered appreciably, so that
ON or OFF bipolar cells selectively innervate a given multistratified
ganglion cell. Alternatively, there could be an abnormally high
proportion of ganglion cells responding to flashing stimuli with
ON-OFF discharges. The presence of such neurons would suggest that
multistratified ganglion cells in the APB-treated eye receive
functional synaptic inputs from ON as well as OFF bipolar cells. To
distinguish between these possibilities, intraocular injections of APB
were made in cats during the first postnatal month. Responses to
flashing spots of light were subsequently assessed when these animals
reached maturity by means of extracellular recordings from the optic
tract (OT) and the A laminae of the dorsal lateral geniculate
nucleus.
Some of our findings have been summarized in abstract form (Gargini et
al., 1996).
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MATERIALS AND METHODS |
Experiments were performed on six cats obtained from an in-house
colony. Four of the animals received unilateral intraocular injections
of APB during the first postnatal month, and two were normal controls.
All procedures were in compliance with National Institutes of Health
guidelines and approved by the Animal Care and Use Committee of the
Consiglio Nazionale delle Ricerche.
Intraocular injections. APB was injected into the
right eyes of newborn cats at P2 and continued daily until P32, with a
2 d respite for the weekends. The animals were anesthetized with 4% halothane in oxygen, and intraocular injections were made with a 10 µl syringe, containing 0.092 mg of APB diluted in sterile saline, and
a 28 gauge needle. This dosage is the same as that used in previous
electrophysiological and anatomical studies (Slaughter and Miller,
1981; Bodnarenko et al., 1995). The injections were made into the
temporal portion of the sclera at the level of the ora serrata, and
great care was taken to insert the needle through the initial opening
for all subsequent injections. After the last injection at P32, the
animals were allowed to reach maturity in the colony.
Animal preparation for recordings. When an animal was at
least 4 months of age, it was used for the electrophysiological
experiments. Anesthesia was initially induced by an injection of
ketamine (Ketalar; Parke-Davis, Courbevoie, France; 30 mg/kg, i.m.),
and an endotracheal tube and venous cannula were inserted. During
surgery and throughout the recording session, anesthesia was maintained
by Farmotal (sodium thiopental, Famitalia; 1.5 mg · kg 1 · hr1)
delivered intravenously. Bilateral openings were made in the skull to
allow insertion of microelectrodes into the dlgn and the OT,
contralateral and ipsilateral to the treated eye. After infiltration of
exposed tissue with local anesthetic (Novocain), paralysis was induced
with intravenal infusion of Pavulon (pancuronium bromide, 0.2%;
Organon Teknika Cappel, Durham, NC; 0.1-0.2
ml · kg1 · hr1), and
the animal was artificially ventilated to maintain expired CO2 at 4.0-4.2%. Both EEG and ECG were monitored
continuously to assess the adequacy of the anesthesia and the general
condition of the animal. Body temperature was also monitored and
maintained at 38°C with an electric heating pad. The pupils were
dilated with atropine sulfate (0.5%), and the corneas were protected
by contact lenses with artificial pupils of 3 mm diameter. The
refraction of the eye was determined by means of retinoscopy and
corrected with appropriate spectacle lenses placed in front of the eye. The positions of the papilla and area centralis were determined at the
beginning of the experiment and were checked periodically.
Electrophysiological recordings. Single-unit recordings were
made from cells in laminae A and A1 of the dlgn, using a micropipette filled with NaCl (3 M) and fibers in the OT with tungsten
microelectrodes. When the action potentials of a single cell were
isolated from background activity, the unresponsive eye was covered,
and the position of the receptive field was plotted on a tangent screen using slits or spots of light. Responses of the cell were assessed by
flashing stimuli in a region of the visual field corresponding to the
receptive field of the cell. This region was subdivided into separate
elements (usually between 9 and 25) of equal size in which the
luminance was locally modulated in time either sinusoidally or in a
square-wave manner. The computer program sampled each element in the
stimulus matrix according to a linear or random sequence, allowing for
a recovery time between successive modulations during which the entire
field remained at mean luminance. For each element in the matrix, light
was modulated at a frequency of 1-10 Hz for at least 10 periods. The
responses of the cell triggered by the modulation frequency were
conventionally amplified and displayed on an oscilloscope. For
quantitative analyses, each action potential triggered a standard pulse
from a window discriminator that was sent to a computer for storage and
analysis. The mean luminance of the field was 28 cd/m2, and the amplitude of modulation was always
maintained at 100%. The sinusoidal or square-waves were rectified to
obtain stimuli reversed in contrast. Stimuli above and below background
were generated by the VSG2/3 computer graphics card (Cambridge Research Systems) displayed on the face of a Barco CDCT6551 color monitor at a
frame rate of 120 Hz with 5122 pixel spatial
definition and 14 bits per color per pixel. The acquisition system was
synchronized with the temporal frequency of each stimulus to provide
peristimulus time histograms and corresponding rasters depicting
individual response epochs.
After recordings were completed on one side of the brain, a pressure
injection of horseradish peroxidase was made into the dlgn and OT
before recordings were made from these structures on the other side. At
the end of all recordings, the animal was administered a lethal dose of
barbiturate and perfused transcardially with solutions appropriate for
peroxidase histochemistry. The retinas were removed and processed for
an anatomical investigation (S. Bisti, S. Deplano, C. Gargini, and L. M. Chalupa, unpublished observations).
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RESULTS |
The results are based on recordings from 113 cells: 73 from the
dlgn and 40 from the OT. Thirty-five of the cells responded to
stimulation of the normal eye (23 dlgn and 12 OT), whereas 78 cells
were activated by the APB-treated eye (50 dlgn and 28 OT). Cells were
classified on the basis of their responses to a flashing stimulus
centered on the receptive field with the size of the spot chosen to
evoke clear and reliable responses. When the normal eye was stimulated,
all but one of the neurons discharged to either the onset or the offset
of a flashing stimulus. The atypical cell, encountered in the dlgn
within layer A1, discharged to both stimulus onset as well as offset.
In all other cases, the response properties of the units activated by
the normal eye changed in a predictable manner when the contrast of the
stimulus shifted from above to below background levels. Thus, cells
that yielded ON discharges to bright spots produced OFF responses of approximately equal magnitude when dark spots were used.
By contrast, stimulation of the APB-treated eye produced ON-OFF
discharges in 37% of the neurons tested (Fig.
1), and such cells were encountered at
about the same frequency within the OT (9 of 28) as in the dlgn (20 of
50). In all these cases, ON-OFF discharges were evident when flashing
stimuli were above as well as below background levels. Figure
2 shows two examples of such ON-OFF
cells, one recorded from the optic tract (A) and the
other from the dlgn (B). Also illustrated are the
responses of a third neuron (C) that yielded only ON
discharges to stimulation of the APB-treated eye in a manner
indistinguishable from a normal response pattern.
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Figure 1.
Percentage of ON, OFF, and ON-OFF cells in
response to stimulation of the normal eye (open columns,
n = 35) and the APB-treated eye (shaded
columns, n = 78). The proportions of
ON-OFF cells encountered in recordings from the OT (32.1%) and the
dlgn (40%) were similar, so the data have been combined in the
histograms for purposes of clarity.
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Figure 2.
PSTHs and response rasters for three neurons in
response to stimulation of the APB-treated eye. A,
ON-OFF cell isolated in the OT (10 stimulus presentations of a
20 diameter spot). B, ON-OFF cell
isolated in layer A of the dlgn (30 stimulus presentations of a
10 spot). C, ON cell in layer A of
the dlgn (18 stimulus presentations of a 2.50 spot).
The light stimulus was modulated at 1 Hz by a half-wave rectified
sinusoid above (left panels) or below (right
panels) the mean luminance (28 cd/m2). The
contrast was 100%, and the time bin was 31.3 msec.
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Because our goal was to sample the response patterns of as many cells
as possible, we did not perform the battery of tests required to
distinguish between different classes of cells (e.g., X and Y).
However, there was no indication that the ON-OFF cells differed in
terms of response briskness or receptive field size from the overall
sample; moreover, such units were characterized by sustained as well as
transient discharge patterns. Thus, most likely the ON-OFF cells we
recorded encompassed more than one cell class, but the proportion of
the different cell classes manifesting such discharge patterns remains
to be established.
In 15 units that yielded ON-OFF discharges to stimuli centered on the
receptive field (nine dlgn and six OT), we also assessed responses to
small flashing spots positioned within different regions of the
receptive field. In five of these cells (three dlgn and two OT) the
response pattern was the same (i.e., ON-OFF) irrespective of the
position of the stimulus in the receptive field, although response
magnitude did vary with stimulus position. An example of one such cell
showing relatively uniform spatial responsivity is depicted in Figure
3.
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Figure 3.
Uniform spatial organization within the receptive
field. A, PSTHs recorded at nine different positions
within the receptive field of an ON-OFF cell isolated in dlgn, lamina
A, to 24 stimulations of the APB-treated eye. Note that ON-OFF
discharges were obtained at all stimulus positions, except at the top
right position, which was outside the responsive region. The stimulus
was a 1.40 spot modulated at 2 Hz by a half-wave
rectified sinusoid below the mean luminance, indicated by filled
bars. B, PSTHs to a larger stimulus
(30 spot) centered on the receptive field. In this
case, the stimulus was modulated at 2 Hz by a half-wave rectified
sinusoid above the mean luminance of 28 cd/m2
(open horizontal bar). Contrast was 100%, and the time
bin 15.2 msec.
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In 10 other cells tested in the same manner (six dlgn and four OT),
there were pronounced variations in discharge patterns with stimulus
position. In the example shown in Figure
4, the cell yielded ON-OFF discharges
when the stimulus was in the upper left, middle, and lower right
positions of the receptive field, but only ON responses were evoked at
other stimulus positions. This neuron, and all other cells exhibiting
such a patchy spatial organization, manifested clear ON-OFF discharge
patterns to large flashing stimuli covering the entire region of the
receptive field sampled by the smaller stimuli (Fig.
4B).
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Figure 4.
Patchy spatial organization within the receptive
field. A, PSTHs to a 20 spot at nine
stimulus positions within the receptive field of an OT cell showing a
patchy internal organization when the APB-treated eye was activated.
B, ON-OFF responses in the same cell to a
60 spot covering the entire receptive field. In all
cases, the PSTHs are based on 10 stimulus presentations, and the
stimulus was modulated at 2 Hz by a half-wave rectified sinusoid below
the mean luminance (indicated by the filled bar). Mean
luminance, 28 cd/m2; contrast, 100%; and time bin,
15.2 msec.
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The receptive field properties of the cells driven by the APB-treated
eye also differed from normal in their responses to stimuli of
increasing size. A common feature of these neurons was that they did
not show response suppression. This is illustrated in the series of
peristimulus time histograms (PSTHs) depicted in Figure
5A-C for three ON-OFF cells.
In each case, the magnitude of the response can be seen to increase
with stimulus size. There were differences, however, in the manner that
responses changed with stimulus size. In Figure 5A, an
ON-OFF discharge pattern of increasing magnitude is evident as the
size of the stimulus increased. This pattern was found in the majority
of cells (13 of 16). In the remainder of the neurons (3 of 16),
responsivity also increased when the stimulus was increased in size,
but in these cells the response pattern was found to vary as a function of stimulus size. In the cell shown in Figure 5B, an ON
discharge is apparent with the smallest stimulus, whereas ON-OFF
responses are evident with large stimuli. In Figure 5C, the
two smaller stimuli elicited weak ON-OFF responses, but with the
largest stimulus a robust OFF discharge can be seen. The receptive
field locations of the three cells manifesting changes in response
pattern did not differ from the overall sample of cells. Such
variations in response pattern with stimulus size might relate to the
spatial organization of receptive fields described above (Fig. 4), but this remains to be established.
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Figure 5.
Lack of an inhibitory surround in three ON-OFF
cells driven by the APB-treated eye. In A (dlgn
cell) and B (OT
cell), the same response patterns were evident in the
PSTHs at all stimulus dimensions; in C (dlgn
cell) the pattern changed from ON-OFF to OFF (onset of
dark stimulus) when the largest stimulus was used. The PSTHs are based
on 20 stimulus presentations; all other conventions are as in Figure
4.
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Interestingly, the cells that responded to stimulation of the
APB-treated eye with only ON or OFF discharges also showed no evidence
of a suppressive surround in their receptive fields. This was found in
every cell tested with stimuli of increasing size (n = 16). Examples of the response properties of two such cells, one ON
and the other OFF, are shown in Figure
6, A and B,
respectively. For comparative purposes, Figure 6C depicts
the responses of a cell to stimulation of the control eye demonstrating the normal decrease in responsivity when the dimensions of the flashing
stimulus exceeded the receptive field center.
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Figure 6.
Lack of an inhibitory surround in an ON
(A) and an OFF (B) cell
driven by the APB-treated eye. In C, normal response
suppression in an ON cell activated by the control eye with stimuli of
increasing size. The stimuli were modulated at 1 Hz by a half-wave
rectified sinusoid below (A) or above (B,
C) the mean luminance of 28 cd/m2. The PSTHs
are based on 10 stimulus presentations, the contrast was 100%, and the
time bin was 31.3 msec.
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DISCUSSION |
In the mature cat retina, ganglion cells respond to flashing
stimuli centered on their receptive fields with either ON or OFF
discharge patterns (Kuffler, 1953). This fundamental feature of visual
processing relates to the stratification pattern exhibited by these
neurons: cells with dendrites stratifying distal to the soma respond to
light decrements, whereas those with proximal dendrites are activated
by light increments (Famiglietti and Kolb, 1976; Nelson et al., 1978).
By contrast, early in development the dendrites of retinal ganglion
cells ramify throughout the IPL. In the cat retina, such stratification
of dendrites begins ~2 weeks before birth and continues throughout
the first postnatal month. This process can be perturbed by treating
the developing retina with the glutamate analog APB (Bodnarenko and
Chalupa, 1993; Bodnarenko et al., 1995), which hyperpolarizes ON-cone
and rod bipolar cells (Slaughter and Miller, 1981; Bolz et al., 1984; Muller et al., 1988). Thus, after short-term APB treatment, the dendrites of ~40% of the cells remain multistratified. An almost identical proportion of ON-OFF cells was found in the present study
when the APB-treated eye was activated by flashing stimuli. Taken
together, these observations suggest that nearly all of the
multistratified dendrites in the APB-treated retina are innervated by
ON as well as OFF bipolar cells and that both sets of interneurons form
functional synapses.
The present study also reveals that treating the developing retina with
APB throughout the first postnatal month has relatively long-term
consequences. Our recordings were made several months after such
treatments were discontinued, yet the incidence of ON-OFF discharges
was virtually the same as the proportion of multistratified cells found
in the postnatal cat retina that sustained short-term treatment with
APB (Bodnarenko and Chalupa, 1993; Bodnarenko et al., 1995). Moreover,
in the earlier study, we showed that when short-term treatment of APB
was discontinued, the dendritic stratification process was found to
resume, so that by 3 months of age the normal incidence of stratified
ganglion cells was apparent. Thus, whereas the effects of short-term
APB treatment appear to be entirely reversible, treating the developing
retina for a longer period (the first postnatal month) alters
functional organization into adulthood. Although we have yet to
determine the proportion of multistratified cells in the retinas of the
animals from which recordings were made, preliminary observations
indicate that the incidence of such neurons is abnormally high in these
retinas (Bisti, Deplano, Gargini, and Chalupa, unpublished
observations).
Convergence of bipolar cells in APB-treated retinas
Recordings were made from both the OT and the dlgn, and in both
structures the proportion of ON-OFF cells was found to be essentially
the same. Most likely, this outcome reflects the convergence of ON and
OFF bipolar cells onto multistratified ganglion cell dendrites in the
APB-treated retinas. Recordings limited only to the geniculate would
not have allowed such an inference, because we could not have ruled out
the possible convergence of ON and OFF retinal fibers onto geniculate
neurons within the A layers. Such an abnormal innervation pattern is
thought to occur after treatment of the developing cat retina with TTX
(Dubin et al., 1986). It is also the case that some W cells (which
innervate only the C-laminae of the dlgn) respond to flashing spots of
light with ON-OFF discharges (Stone and Fukuda, 1974). Thus,
recordings limited to the OT would be subject to the concern that the
presence of an abnormally high number of ON-OFF cells could have
reflected a high sample of W fibers from the treated eye. We did not
encounter neurons with such properties in our OT recordings from normal animals. This was probably attributable to the fact that our electrodes were not suitable for isolating the thin-caliber W-cell fibers. Thus,
our finding that the proportion of ON-OFF cells was similar in the OT
and dlgn can only be interpreted as reflecting a convergence of ON and
OFF bipolar cells onto ganglion cells with multistratified dendrites.
There are two means by which APB treatment of the developing retina
could result in a high incidence of ON-OFF ganglion cells. One
possibility is that, during normal development, the dendrites of the
multistratified cells could be transiently innervated by the axon
terminals of ON as well as OFF bipolar cells. If this was the case, APB
treatment might simply arrest such immature retinal circuitry,
resulting in ganglion cells with ON-OFF responses at maturity. The
alternative scenario is that either ON or OFF bipolar cells selectively
innervate the multistratified dendrites of individual ganglion cells,
and APB treatment then induces the ingrowth of bipolar cells of the
opposite response polarity. One way to differentiate between these two
possibilities is to assess the incidence of ON-OFF ganglion cells in
the developing retina when many cells are still in their immature
state. In the former case, one would expect a high incidence of ON-OFF
responding ganglion cells, whereas in the latter, the prevalence of
such neurons in the developing retina should be no greater than at
maturity.
Functional state of developing retinal ganglion cells
Extracellular recordings from ganglion cells in the newborn cat
retina have been made by Tootle (1993), using an eyecup preparation. He
found no evidence for ON-OFF cells, suggesting that early in development the dendrites of multistratified ganglion cells are selectively innervated by either ON or OFF bipolar cells, but not by
both sets of interneurons. This provides support for the instructional
model proposed to explain how glutamate-mediated afferent activity
might act to regulate dendritic stratification (Bodnarenko et al.,
1995, their Fig. 8). The model postulates an asymmetrical distribution
of synaptic inputs favoring either distal or proximal dendrites of
initially multistratified ganglion cells so that during normal
development, activity-mediated afferent inputs would "instruct"
immature neurons which dendritic process to retract and which to
maintain. It was hypothesized that blocking such afferent inputs by APB
causes an arrest of dendrites in their multistratified state. Taken
together, the results of the present study and the findings of Tootle
(1993) suggest that APB treatment induces bipolar cell innervation onto
multistratified dendrites (i.e., the second of the two alternatives
discussed above). The fact that many cells driven by stimulation of the
APB-treated eye showed a "patchy" organization in their receptive
fields could reflect a nonuniform pattern of such ingrowth. It is also
possible that some segment of the multistratified dendrites may have
retracted in the APB-treated retinas. The resolution of this issue
requires a careful morphological assessment of the treated retinas,
and, as mentioned earlier, this work is now in progress.
The results of the present study also suggest that APB treatment
induces alterations in neuronal circuitry of the developing retina
other than the maintenance of multistratified ganglion cells and the
subsequent ingrowth of bipolar cell axonal terminals. This is indicated
by the finding that all of the cells driven by the APB-treated eye
lacked a suppressive surround. Unlike in the normal retina, the
responses of these neurons did not decrease with the size of flashing
stimulus. Because it is commonly assumed that horizontal and amacrine
cells are responsible for generating the receptive field surrounds of
mature ganglion cells (Wässle and Boycott, 1991), this implies
that treating the developing retina with APB might also alter the
synaptic organization of these interneurons.
APB treatment of developing retina affects both ON and
OFF cells
In the mature retina, APB has been shown to selectively block the
ON pathway when the retina is light adapted (for review, see Schiller,
1992). At the same time, our previous anatomical studies revealed that
both ON and OFF retinal ganglion cells are affected about equally when
the developing retina is treated with this glutamate agonist
(Bodnarenko and Chalupa, 1993; Bodnarenko et al., 1995). The present
functional results are entirely in accord with these observations,
because the incidence of ON and OFF cells was found to be reduced by a
nearly equal extent from the normal 50:50 ratio. The lack of
selectivity for the ON pathway observed here and in the previous
anatomical studies could reflect one of two factors. First, our APB
injections undoubtedly blocked the release of glutamate from ON cone
bipolar cells as well as rod bipolar cells. The rod bipolar cells form
part of the circuitry for both ON and OFF retinal pathways because
these interneurons terminate on AII amacrine cells, which connect with
ON cone bipolar cells via gap junctions and with OFF ganglion cells via
conventional synapses (Kolb and Famiglietti, 1974; Famiglietti and
Kolb, 1976; McGuire et al., 1984, 1986). Thus, under scotopic
conditions hyperpolarization of rod bipolar cells by APB in the retina
has been shown to completely eliminate visual responses in both ON and
OFF ganglion cells in the adult cat retina (Wässle et al., 1991).
In our studies, the direct OFF cone bipolar pathway would be expected
to remain functional, but apparently this is not sufficient to induce
stratification in the OFF ganglion cells.
It may also be the case that, unlike in the mature retina, ON as well
as OFF cone bipolar cells are APB-sensitive, and such expression is
transient in the OFF bipolars. This could be assessed by making
patch-clamp recordings from these interneurons during the developmental
period when APB injections were made in the present study. Recently, we
showed by means of such recordings that developing ganglion cells are
not sensitive to APB (Liets and Chalupa, 1996). This finding rules out
the possibility that the effects documented here could be attributable
to a direct influence of this ligand on multistratified ganglion cells,
but whether other retinal neurons manifest a transient expression of
APB-sensitive receptors remains to be established.
Implications for functional changes at higher levels of the
visual system
The functional utility of segregated ON and OFF retinal pathways
is unknown, yet this appears to be a common feature of all vertebrates.
From this perspective, it would be of interest to use behavioral
measures to assess the processing of visual information by the
APB-treated eye. It would seem reasonable to think that such an
approach could reveal some degree of impairment in terms of the ability
to discriminate light from dark stimuli and, quite possibly, other
visual impairments stemming from reorganized connections at higher
levels of the visual system. In particular, it has recently been shown
that treating the developing ferret retina with APB disrupts the
formation of ON and OFF retinogeniculate sublaminae (Yeung et al.,
1997). This may relate to the observations of Wong and Oakley (1996),
who found separate waves of ON and OFF ganglion cell activities in the
developing ferret retina during the developmental period when these
sublaminae are normally formed in the dlgn. It has also been suggested
that the orientation selectivity exhibited by visual cortical cells
reflects the sequential innervation pattern of ON or OFF dlgn cells
onto single cortical cells (Chapman et al., 1991). In view of our
results, one might expect orientation-tuning properties of some
cortical cells to be abnormal after APB treatment of the developing
retina. The observations reported here may prove useful for examining
the contributions of segregated ON and OFF retinal channels to the
organization of the visual system at higher levels.
| |
FOOTNOTES |
Received Feb. 9, 1998; revised April 13, 1998; accepted April 22, 1998.
This work was supported by National Institutes of Health Grant EYO3391,
National Science Foundation Grant IBN12593, the Fogarty International
Center, and NATO. We thank Paolo Martini and Mario Pirchio for computer
software, Daniela Morconi for animal care, and Julio Cappagli for
technical assistance.
Correspondence should be addressed to Leo M. Chalupa, Section of
Neurobiology, Physiology, and Behavior, University of California, Davis, CA 95616.
| |
REFERENCES |
-
Bodnarenko SR,
Chalupa LM
(1993)
Stratification of ON and OFF ganglion cell dendrites depends on glutamate-mediated afferent activity in the developing retina.
Nature
364:144-146[Medline].
-
Bodnarenko SR,
Jeyarasasingam G,
Chalupa LM
(1995)
Development and regulation of dendritic stratification in retinal ganglion cells by glutamate-mediated afferent activity.
J Neurosci
15:7037-7045[Abstract].
-
Bolz J,
Wassle H,
Thier P
(1984)
Pharmacological modulation of on and off ganglion cells in the cat retina.
Neuroscience
12:875-885[ISI][Medline].
-
Chapman B,
Zahs KR,
Stryker MP
(1991)
Relation of cortical cell orientation selectivity to alignment of receptive fields of the geniculocortical afferents that arborize within a single orientation column in ferret visual cortex.
J Neurosci
11:1347-1358[Abstract].
-
Conway JL,
Schiller PH
(1993)
Laminar organization of tree shrew dorsal lateral geniculate nucleus.
J Neurophysiol
50:1330-1342.
-
Dubin MW,
Stark LA,
Archer SM
(1986)
A role for action-potential activity in the development of neuronal connections in the kitten retinogeniculate pathway.
J Neurosci
6:1021-1036[Abstract].
-
Euler T,
Schneider H,
Wässle H
(1996)
Glutamate responses of bipolar cells in the rat retina.
J Neurosci
16:2934-2944[Abstract/Free Full Text].
-
Famiglietti Jr EV,
Kolb H
(1976)
Structural basis for ON- and OFF-center responses in retinal ganglion cells.
Science
194:193-195[Abstract/Free Full Text].
-
Gargini C,
Chalupa LM,
Bisti S
(1966)
Reorganization of receptive field properties after treatment of the developing retina with APB.
Soc Neurosci Abstr
22:1725.
-
Kolb H,
Famiglietti EV
(1974)
Rod and cone pathways in the inner plexiform layer of the cat retina.
Science
186:47-49[Abstract/Free Full Text].
-
Kuffler SW
(1953)
Discharge patterns and functional organization of mammalian retina.
J Neurophysiol
16:37-68[Free Full Text].
-
Lau KC,
So KF,
Tay D
(1990)
Effects of visual or light deprivation on the morphology, and the elimination of the transient features during development, of type I retinal ganglion cells in hamsters.
J Comp Neurol
300:583-592[Medline].
-
Leventhal AG,
Hirsch HV
(1983)
Effects of visual deprivation upon the morphology of retinal ganglion cells projecting to the dorsal lateral geniculate nucleus of the cat.
J Neurosci
3:332-44[Medline].
-
Liets LC,
Chalupa LM
(1996)
The metabotropic glutamate agonist 2-amino-4-phosphonobutyric acid (APB) does not activate currents in postnatal retinal ganglion cells.
NeuroReport
7:2873-2877[Medline].
-
Maslim J,
Stone J
(1988)
Time course of stratification of the dendritic fields of ganglion cells in the retina of the cat.
Dev Brain Res
44:87-93[Medline].
-
Maslim J,
Webster M,
Stone J
(1986)
Stages in the structural differentiation of retinal ganglion cells.
J Comp Neurol
254:382-402[ISI][Medline].
-
McGuire B,
Stevens J,
Sterling P
(1984)
Microcircuitry of bipolar cells in cat retina.
J Neurosci
4:2920-2038[Abstract].
-
McGuire B,
Stevens J,
Sterling P
(1986)
Microcircuitry of beta ganglion cells in the cat retina.
J Neurosci
6:907-918[Abstract].
-
Muller F,
Wässle H,
Voigt T
(1988)
Pharmacological modulation of the rod pathway in the cat retina.
J Neurophysiol
59:1657-1672[Abstract/Free Full Text].
-
Nelson R,
Famiglietti Jr EV,
Kolb H
(1978)
Intracellular staining reveals different levels of stratification for on- and off-center ganglion cells in cat retina.
J Neurophysiol
41:472-483[Abstract/Free Full Text].
-
Norton TT,
Rager G,
Kretz R
(1985)
ON and OFF regions in layer IV of striate cortex.
Brain Res
488:348-352.
-
Schiller PH
(1992)
The ON and OFF channels of the mammalian visual system.
Prog Retina Eye Res
15:173-195.
-
Slaughter MM,
Miller RF
(1981)
2-amino-4-phosphonobutyric acid: a new pharmacological tool for retina research.
Science
211:182-185[Abstract/Free Full Text].
-
Stone J,
Fukuda Y
(1974)
Properties of cat retinal ganglion cells: a comparison of W-cells with X- and Y-cells.
J Neurophysiol
37:722-748[Free Full Text].
-
Stryker MP,
Zahs KR
(1983)
ON and OFF sublaminae in the lateral geniculate nucleus of the ferret.
J Neurosci
3:1943-1951[Abstract].
-
Tootle JS
(1993)
Early postnatal development of visual function in ganglion cells of the cat retina.
J Neurophysiol
69:1645-1660[Abstract/Free Full Text].
-
Wässle H,
Boycott B
(1991)
Functional architecture of the mammalian retina.
Physiol Rev
71:447-480[Free Full Text].
-
Wässle H,
Yamashita M,
Greferath U,
Grunert U,
Muller F
(1991)
The rod bipolar cell of the mammalian retina.
Vis Neurosci
7:99-112[ISI][Medline].
-
Wong RO,
Oakley DM
(1996)
Changing patterns of spontaneous bursting activity of on and off retinal ganglion cells during development.
Neuron
16:1087-1095[ISI][Medline].
-
Wong RO,
Herrmann K,
Shatz CJ
(1991)
Remodeling of retinal ganglion cell dendrites in the absence of action potential activity.
J Neurobiol
22:685-697[ISI][Medline].
-
Yeung G,
Miller L,
Stoddard A,
Bodnarenko SR
(1997)
Role of glutamate-mediated afferent activity in the development of on/off retinal ganglion cell (RGC) morphology in ferrets.
Soc Neurosci Abstr
23:640.
Copyright © 1998 Society for Neuroscience 0270-6474/98/18135019-07$05.00/0
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Top
Abstract
Introduction
