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The Journal of Neuroscience, March 1, 2000, 20(5):1883-1892
Disruption of the Olfactoretinal Centrifugal Pathway May
Relate to the Visual System Defect in night blindness b
Mutant Zebrafish
Lei
Li and
John E.
Dowling
Department of Molecular and Cellular Biology, Harvard University,
Cambridge, Massachusetts 02138
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ABSTRACT |
We describe here a dominant mutation, night blindness
b (nbb), which causes an age-related visual
system defect in zebrafish. At 4-5 months of age, dark-adapted
nbb+/
mutants show abnormal visual threshold fluctuations when measured behaviorally. Light sensitizes the animals; thus early dark adaptation of
nbb+/
fish is normal. After 2 hr of dark adaptation, however, visual thresholds of
nbb+/
mutants are raised on average 2-3 log units, and rod system function is not detectable. Electroretinograms recorded from
nbb+/
mutants are normal, but ganglion cell thresholds are raised in prolonged darkness, suggesting an inner retinal defect. The visual defect of
nbb+/
mutants may be likely caused by an abnormal olfactoretinal centrifugal innervation; in
nbb+/
mutants, the olfactoretinal centrifugal projection to the retina is
disrupted, and the number of retinal dopaminergic interplexiform cells
is reduced. A similar visual defect as shown by
nbb+/
mutants is observed in zebrafish in which the olfactory epithelium and
olfactory bulb have been excised. Homozygous nbb fish
display an early onset neural degeneration throughout the CNS and die by 7-8 d of age.
Key words:
dark adaptation; dopamine; escape response; mutation; neuronal degeneration; olfactory bulb; olfactoretinal centrifugal
pathway; terminal nerve neuron; visual sensitivity; zebrafish
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INTRODUCTION |
Zebrafish are ideal for genetic
studies of the vertebrate eye (Brockerhoff et al., 1995
; Baier et al.,
1996; Malicki et al., 1996; Li and Dowling, 1997; Neuhauss et al.,
1999). Zebrafish are highly visual animals whose retinas contain one
type of rod and four types of cone (Branchek and Bremiller, 1984;
Robinson et al., 1993). A number of recessive mutations that affect
zebrafish retinal development or function have been identified recently (Brockerhoff et al., 1995, 1997; Karlstrom et al., 1996; Malicki et
al., 1996; Trowe et al., 1996; Fadool et al., 1997; Neuhauss et al.,
1999). However, virtually all of these mutants demonstrate defects
other than the eye defects. These mutants tend to develop abnormally
and die between 5 and 8 d of age. Thus, it has not been generally
possible to study eye mutations in adult zebrafish.
We have recently developed a behavioral test, based on the
visually mediated escape response of fish to a threatening object, that
permits a quantitative analysis of zebrafish visual sensitivity and the
isolation of visual system mutations in adults (Li and Dowling, 1997).
When challenged by a threatening object, zebrafish display a robust
escape response; as soon as the threatening object comes into view, the
fish turn instantly and swim rapidly away. By varying the light
intensity illuminating the threatening object and its surround,
absolute rod and cone system thresholds as well as the time course of
dark adaptation after bright light adaptation can be measured. Using
our behavioral assay, we have screened for dominant mutations that
cause altered visual sensitivity levels in F1 generation zebrafish that
were derived from N-ethyl-N-nitrosurea (ENU)
mutagenized founders.
We describe here a dominant mutation, designated night blindness
b (nbb), that causes an unusual visual system defect in
adult zebrafish. When fully dark-adapted, the visual threshold of
nbb+/
mutants fluctuates by 2-3 log units from day to day, unlike wild-type fish in which the visual threshold is maintained at a constant level
when measured at the same time on different days. By recording the
electroretinogram (ERG) and ganglion cell discharge, we show that the
defect underlying the
nbb+/
visual deficit appears not to relate to outer retinal function but
rather to abnormalities in the inner retina. In
nbb+/
mutants, the olfactoretinal centrifugal pathway is disturbed, and the
number of retinal dopaminergic interplexiform cells (DA-IPCs) is
reduced. Removing the olfactory epithelium (OE) and olfactory bulb (OB)
in wild-type zebrafish results in a visual system defect similar to
that of
nbb+/
mutant fish. Although heterozygous mutant fish are viable and show
age-related visual system defects, homozygous nbb fish
display an early onset neural degeneration throughout the CNS and die by 7-8 d of age.
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MATERIALS AND METHODS |
Animals and maintenance. Zebrafish (Danio
rerio) were maintained on a 14-10 hr light/dark cycle (light, 8 A.M.-10 P.M.; 1.0-2.5 µW/cm2) as
described (Westerfield, 1995). For some of the circadian experiments,
animals were phase-shifted to a new light/dark cycle (light, 10 P.M.-noon.). Animals were kept in the shifted light/dark cycle for 2 weeks before threshold measurements were made.
The behavioral assay. The apparatus used for behavioral
analysis of adult zebrafish visual sensitivity has been described (Li
and Dowling, 1997). It consists of a stationary transparent container
surrounded by a rotating drum. A black segment (5 × 5 cm) is
marked on the drum that serves as a threatening object. The drum is
illuminated from above with a white light source (maximum intensity,
4.60 × 102
µW/cm2) and turned at 10 rpm by a motor.
Neutral-density filters (in half-log unit steps) are used to change the
light intensity on the drum.
With illumination at or above the visual threshold level, zebrafish
display a robust escape response to the black segment rotating outside
the container (Li and Dowling, 1997). Usually, a judgment as to whether
a fish can see the black segment at a given light intensity can be made
in <10 sec. For mutant screening, the light illuminating the drum was
set at log I = 
5.0, ~1 log unit above the absolute
threshold level of wild-type zebrafish. The F1 fish (derived from ENU
mutagenized founders) were dark-adapted for a minimum of 20-30 min
before a threshold measurement was made. Individuals that failed to
show the escape response under the test level of illumination were
isolated and rescreened on subsequent days.
To measure the course of dark adaptation, the fish were first
light-adapted (3.25 × 103
µW/cm2) for 15-20 min. During
subsequent dark adaptation, the threshold light that was required to
evoke an escape response when the fish was challenged by the rotating
black segment was recorded. The first threshold measurements were made
at 2 min after the start of dark adaptation, then repeated at 2 min
intervals, and completed at 26 min. Log light threshold is plotted as a
function of time of dark adaptation, known as a dark adaptation curve
(Li and Dowling, 1997).
ERG and ganglion cell recordings. Zebrafish were
anesthetized using 4% 3-amino benzoic methylester and immobilized with
10% gallamine triethiodide. Zebrafish were placed on their side on a
sponge with one eye facing toward the light source (maximum intensity,
6.75 × 103
µW/cm2). A slow stream of fish water was
directed into the mouth of the fish to keep the fish well oxygenated.
ERGs were recorded using a glass pipette (filled with a balanced salt
solution) placed on the center of the cornea. Fish were illuminated
with 10 msec flashes (full field). The electrical signals were
amplified and recorded conventionally.
Ganglion cell action potentials were recorded from the optic nerve.
Zebrafish were anesthetized, immobilized, and handled as described
above. The connective tissue surrounding the eye was cut away using a
pair of microsurgery scissors. The eye was pulled out of the orbit
slightly (by 1-2 mm) and held with two glass rods to expose the optic
nerve. A tungsten microelectrode (Frederick Haer and Company) was
inserted into the optic nerve where the ganglion cell activity was
recorded. The eyes were illuminated with 500 msec diffuse light (full
field; the same light source as used for ERG recordings). The
electrical signals were amplified and recorded conventionally.
Histology and immunocytochemistry. Methods used for
histology and immunocytochemistry have been described (Schmitt and
Dowling, 1996). For histology, specimens were first fixed in 1%
paraformaldehyde/2.5% glutaraldehyde and post-fixed in 1% osmium
tetroxide in 0.06 M PBS. Specimens were embedded in
Epon/Araldite (Polysciences, Warrington, PA), cut at 1.0 µm in
thickness using a microtome, stained with 1% methylene blue, and
viewed under a light microscope.
For immunocytochemistry, specimens were fixed in 4% paraformaldehyde
in PBS. Specimens were incubated with primary antibodies [FMRFamide (provided by T. O'Donohue), 1:1,000; 5E11
(provided by J. Fadool), 1:10; tyrosine hydroxylase (Chemicon,
CA), 1:200] and then incubated with secondary antibodies [FITC- or
rhodamine-conjugated secondary antibodies (Boehringer Mannheim, IN)
1:50]. Specimens were mounted on slides and viewed by fluorescence microscopy.
OE and OB excision. Zebrafish were anesthetized using 4%
3-amino benzoic methylester. The connective tissue between the nostril and OE were cut using a microblade. The OE was then stripped out using
a pair of small forceps. The OE excision was later confirmed under a
dissecting microscope. In most cases, not only the OE but also the
olfactory nerve as well as the anterior part of OB were stripped out
using this procedure. Thus we refer to OE/OB excision. The fish were
allowed to recover for 2-3 d before the behavioral visual threshold
measurements were made. No obvious differences in swimming behavior
were observed between control and operated fish.
Acridine orange staining. Zebrafish embryos (2.5 d old) were
dechorionated and stained with 5 µg/ml of acridine orange (Sigma, St.
Louis, MO) in fish water. Embryos were washed with fish water and
viewed by fluorescence microscopy (Furutani-Seiki et al., 1996; Li and
Dowling, 1997).
Confocal microscopy and image analysis. Confocal images were
taken using a Zeiss 410 confocal laser-scanning microscope equipped with a Krypton-argon laser. FITC and rhodamine were excited with the
488 nm and 568 nm emission lines, respectively. Double-labeled specimens were analyzed with sequential scans. Z-series sections were
collected in 1.0 µm steps (n = 12).
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RESULTS |
Isolation of the nbb+/ mutant
We reported previously that zebrafish visual sensitivity is
regulated by an endogenous circadian clock (Li and Dowling, 1998). Over
a 24 hr period, zebrafish are least sensitive to visual stimuli before
dawn and most sensitive before dusk. Although zebrafish visual
sensitivity fluctuates between day and night, it remains constant when
measured at the same time on different days. When evaluating the visual
sensitivity of mutagenized F1 fish, we routinely made threshold
measurements in the late afternoon-early evening hours (4-8 P.M.)
when the fish are most sensitive to light.
The heterozygous night blindness b
(nbbda15) mutant was discovered
because its visual sensitivity fluctuated substantially from day to day
when measured in the late afternoon hours. This is shown in Figure
1A for the F1
generation (9.5 months old) ENU mutagenized zebrafish that we
originally isolated. On day 1, the visual threshold of the
nbb+/
fish was ~2 log units above that of control fish; on the second day
it was even higher, ~2.5 log units above the control level. However,
on day 3, thresholds of the
nbb+/
and control fish were indistinguishable. Two days later (day 5), the
visual threshold of the
nbb+/
fish was 3 log units above the control fish, but then on day 7, it once
again fell to near normal levels. During this time of day, the visual
thresholds of wild-type fish remained at a fairly constant level,
between log I = 6.0 and log I = 6.5.
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Figure 1.
Behavioral visual thresholds of wild-type and
nbb+/
fish. A, Behavioral visual thresholds of a wild-type
fish (9.5 months old,  ) and the original
nbb+/
mutant (9.5 months old,  ) determined at the same time (~6 P.M.) on
5 different days. Each circle indicates a behavioral
visual threshold measurement. Note the threshold fluctuation in the
nbb+/
mutant when measured on different days. The horizontal dashed
line drawn at log I = 5.5 indicates the
highest threshold level of wild-type fish when measured at 6 P.M.
B, Dark adaptation curves of wild-type (,
n = 12) and
nbb+/
fish (, n = 12) measured during late
afternoon-early evening hours. For the first 26 min, visual thresholds
were similar between wild-type and mutant fish. Threshold elevations
were observed in
nbb+/
fish when measured at 1, 2, 4, and 6 hr of dark adaptation. Data
represent the means ± SD. C, Visual thresholds of
wild-type (, n = 6) and
nbb+/
fish (, n = 10) measured with white (left
panel), red (middle panel), or
green (right panel) light. Each
circle indicates a behavioral visual threshold
measurement. Note the similarity in visual thresholds of
nbb+/
mutants when measured using white, red, and green lights.
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The gross morphology and swimming activity of the
nbb+/
fish were indistinguishable from those of wild-type fish. The
nbb+/
fish was crossed with a wild-type zebrafish to generate an F2 generation. In the resulting F2 generation, the
nbb+/
mutants were identified using two criteria: the fluctuation of behavioral visual sensitivity and homozygous lethality. That is, nbb+/
fish when bred together yield 25% offspring that die by 7-8 d of age
(see below).
The visual defect of nbb+/ fish begins in early
adulthood and progresses with age
We measured visual thresholds of F2 generation
nbb+/
fish at different ages to determine when the visual threshold
fluctuations begin and how they relate to age. The threshold
measurements were made in animals that had been dark-adapted for 2 hr
(substantial threshold fluctuations are observed only in prolonged
dark-adapted nbb+/
mutants; see below). At 2 months of age, all of the tested fish (n = 18), which were randomly selected from an outcross
between the original
nbb+/
and a wild-type fish, were normal in terms of visual sensitivity. Defective visual behavior was first detected at 3.5 months; 1 of the 18 test fish showed visual threshold fluctuations similar to that observed
in the original
nbb+/
mutant. The behavioral visual threshold of this individual was log
I = 4.5 on day 1, but it then dropped to log
I = 6.0 when measured at the same time on the
following day. On day 3, its threshold was again raised to log
I = 5.0. As the F2 generation fish grew older, more
individuals showed fluctuations in visual sensitivity. At 4.5 months of
age, for example, 4 of 18 F2 fish showed visual threshold fluctuations,
and by 6.5 months, 10 of 18 showed fluctuating visual thresholds
similar to that seen in the original
nbb+/ mutant.
The extent of visual threshold fluctuation in
nbb+/
fish progressed with age. At 6.5 months, for example, the visual
thresholds of the 10 tested
nbb+/
fish varied by up to 3 log units, and their average visual threshold was ~1.0 log unit higher than the threshold level of wild-type fish
(log I = 5.1 ± 1.3 vs log I = 6.1 ± 0.3). At 11.5 months, the threshold fluctuation of these
nbb+/
fish had increased to 5 log units, and their average visual threshold was ~2 log units above that of wild-type fish (log I = 4.0 ± 1.9 vs log I = 6.0 ± 0.4).
The subsequent studies described below were performed using wild-type
and identified
nbb+/
fish that were 10-14 months old or otherwise specified.
Light sensitizes nbb+/ fish
To explore factors that might affect the visual sensitivity of
nbb+/
fish, we examined the effects of light adaptation and the time course
of subsequent dark adaptation using identified
nbb+/
mutants. The results are shown in Figure 1B. For the
first 20-30 min of dark adaptation after bright light adaptation, the
nbb+/
mutants dark-adapted in a fashion comparable to that of wild-type fish.
During the first 6-8 min of dark adaptation, for example, visual
thresholds of
nbb+/
fish (n = 12) were identical to those of wild-type fish
(n = 12), suggesting that during early dark adaptation
the cone system function in
nbb+/
fish was normal. During subsequent dark adaptation, visual thresholds of both wild-type and
nbb+/
fish continued to decrease until they reached a plateau at 20-22 min.
The final threshold of
nbb+/
fish measured at 26 min of dark adaptation was only slightly higher
(0.4 log unit) than that of wild-type fish. Thus, between 20 and 26 min
of dark adaptation, rod system function in
nbb+/
fish appeared essentially normal.
With additional time in the dark, however, visual thresholds of
nbb+/
fish began to fluctuate, and the average threshold began to rise. After
1 hr of dark adaptation, the visual thresholds of
nbb+/
fish were on average 0.8 log units higher than those of wild-type fish
(Fig. 1B). At 2 hr, they were 2.0 log units above
that of wild-type fish. The average visual thresholds of
nbb+/
fish remained high at 4 and 6 hr of dark adaptation, and the visual
threshold fluctuations among individual mutant fish continued. Visual
thresholds of wild-type fish, on the other hand, remained at a
relatively constant level during this 6 hr dark adaptation period (Fig.
1B).
Rod function of nbb+/ fish is lost during
prolonged darkness
During prolonged darkness, the behavioral visual thresholds of
nbb+/
mutants rose on average by 2-3 log units and reached levels comparable to cone system thresholds in wild-type zebrafish (Fig.
1B). To determine whether the threshold elevations
were caused primarily by a loss of rod system function, we probed the
spectral sensitivity of the cone and rod systems in 2 hr dark-adapted
nbb+/
mutants using phototopically matched red (625 nm) or green (500 nm)
light. Individual
nbb+/
mutants (n = 10) that showed high visual thresholds
(log I > 3.5) under white light illumination (Fig.
1C, left panel) were tested for cone and rod
function using red or green illumination. The results are shown in
Figure 1C (middle and right panels)
With red and green illumination, the average visual thresholds of 2 hr
dark-adapted wild-type zebrafish (n = 6) were log
I = 3.4 (red) and log I = 5.3 (green). This shows that fully dark-adapted wild-type fish are ~2 log units more sensitive to green than to red
light. In other words, at 2 hr of dark adaptation, the behavioral visual sensitivity of wild-type fish is governed by rods (Li and Dowling, 1998). The
nbb+/
mutants, on the other hand, did not show rod-dominated visual behavior
when tested under prolonged dark-adapted conditions. After 2 hr of dark
adaptation, the average thresholds of
nbb+/
mutants were virtually identical when tested using red or green illumination (log I = 2.4). This suggests that the
visual threshold elevations shown by prolonged dark-adapted
nbb+/
mutants are caused primarily by a loss of rod system function. That the
red light thresholds of a number of
nbb+/
mutants were higher than the red light thresholds of wild-type fish
suggests that the cone system is also somewhat affected in nbb+/
mutants when kept in prolonged darkness.
Visual threshold elevation in nbb+/ fish
relates to time in the dark
We next examined visual thresholds of individual
nbb+/
fish as a function of time in the dark. The results are shown in Figure 2A. At 20 min of dark
adaptation, all tested mutants (n = 14) showed normal
visual thresholds similar to those of wild-type fish. After 1 hr in the
dark, threshold elevations were observed in some
nbb+/
mutants; 5 of 14 tested fish showed a rise of visual threshold of
~1.0 log unit. The threshold elevations became more evident when
animals were kept for longer times in the dark. At 2, 4, and 6 hr of
dark adaptation, for example, >70% of the tested mutant fish showed
visual thresholds equal to or above log I = 5.0. Moreover, a number of the tested fish (35-50%) showed no behavioral responses when tested with light 3 log units above the absolute threshold level of wild-type fish (above log I = 3.0).
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Figure 2.
Behavioral visual thresholds of wild-type and
nbb+/
fish as a function of time in dark. A, Visual thresholds
of
nbb+/
fish (, n = 14) during dark adaptation after
bright light adaptation (indicated by the first open bar
shown on the top of the figure). Each
circle indicates a behavioral visual threshold
measurement. Note that at 2, 4, and 6 hr, some of the
nbb+/
fish showed no response to visual stimuli at 3 log units above the
absolute threshold level of wild-type fish (shown above the
horizontal dashed line drawn at log
I = 3.0). The elevated visual thresholds were
decreased by exposure to light (the second open bar
shown on the top of the figure). The black
bars shown on the top of the figure indicate
darkness. Filled circles indicate the average visual
threshold of wild-type fish kept in the same illuminating conditions.
B, Visual thresholds of individual
nbb+/
mutant fish (numbered 1-4)
measured at various times in the dark. Each circle
indicates a behavioral visual threshold measurement. Note the threshold
fluctuations in fish 1, 2, and 4 when measured at different times in
the dark. C, Behavioral visual thresholds of wild-type
(, n = 6) and
nbb+/
fish (, n = 12) measured at subjective dawn and
subjective dusk, Animals were kept in constant dark during the
experiment. Each circle indicates a behavioral visual
threshold measurement. The thresholds measured at subjective dawn were
similar between wild-type and
nbb+/
fish. Significant threshold elevations were observed in
nbb+/
mutants when measured at subjective dusk.
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The elevated visual thresholds of
nbb+/
mutants could be decreased by light adaptation. After 20 min of light
exposure following this 6 hr period of dark adaptation, for example,
all of the tested nbb+/
mutants responded positively to visual stimuli. Moreover, 11 of 14 tested mutants showed essentially normal thresholds similar to those of
wild-type fish (equal to or below log I = 5.5) (Fig. 2A).
We also examined whether a single mutant fish showed consistent visual
thresholds when kept in darkness. Figure 2B shows
that this is not the case. Visual thresholds of individual
nbb+/
fish varied substantially when measured over a 6 hr dark adaptation period. An individual that had a lower visual threshold at 2 hr of dark
adaptation could have a higher visual threshold at 4 and 6 hr of dark
adaptation (fish 1), and the opposite was also observed (fish 2). Fish
that had a high visual threshold at 2 hr of dark adaptation could have
a lower threshold at 4 hr of dark adaptation but a higher one again at
6 hr of dark adaptation (fish 4). Only one of four tested mutants had a
consistent threshold for the entire 6 hr period (fish 3).
The circadian clock that regulates visual sensitivity in
nbb+/ mutants may be abnormal
To conclude our behavioral studies, we asked whether the visual
threshold fluctuation and elevation shown by
nbb+/
mutants relate to the circadian regulation of visual sensitivity. To
this end, we measured visual thresholds of
nbb+/
fish as a function of time of day. Animals were kept in the dark from
lights off the day before the test. The threshold measurements were
made at subjective dawn and subjective dusk.
The results are shown in Figure 2C. In wild-type fish
(n = 6), the average threshold measured at subjective
dawn was ~2 log units higher than the average threshold measured at
subjective dusk (log I = 3.8 vs log I = 6.1). In
nbb+/
mutants (n = 12), however, the average thresholds
measured at these two time points were similar (log I = 3.5 at subjective dawn vs log I = 3.7 at subjective
dusk). At subjective dawn, visual thresholds of wild-type and mutant
fish were similar, and both varied substantially (Fig. 2C).
At subjective dusk, on the other hand, no significant threshold
variations were observed in wild-type fish, but visual thresholds of
nbb+/
mutant fish varied substantially, by as much as 4.5 log units (from log
I = 2.0 to log I = 6.5).
Outer retinal function of nbb+/ fish is
normal
To determine whether the defective visual behavior of
nbb+/
fish is caused by a dysfunction of the outer retina, we recorded ERGs
from mutant fish. The vertebrate ERG consists of two prominent waves: a
corneal negative a-wave, which arises from the photoreceptor cells,
followed by a corneal positive b-wave, which originates from
second-order retinal cells (Dowling, 1987); thus, the ERG monitors
activity primarily from the outer retina. In these experiments, fish
were first light-adapted and then kept in the dark for either 20 min or
2 hr before an ERG was recorded. No obvious differences in ERG
responses were observed when recorded at 20 min or 2 hr of dark
adaptation between wild-type (n = 12) and
nbb+/
fish (n = 8). Over a range of 6 log units of light
stimuli, the ERG waveform as well as the a- and b-wave amplitudes were
similar between wild-type and
nbb+/
fish (Fig. 3A). Furthermore,
the light intensity that was required to elicit a threshold ERG (10-20
µV b-wave) was similar between the wild-type and
nbb+/
fish (Table 1). These data suggest that
the outer retina of nbb+/
mutants functions properly.
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Figure 3.
ERG and ganglion cell recordings.
A, Representative ERGs recorded from 2 hr dark-adapted
wild-type and
nbb+/
fish at dusk. Over a range of 6 log units of stimuli, the ERGs were
similar between wild-type and
nbb+/
animals. a, a-wave; b, b-wave. The light
responses were averaged 6-10 times to increase the response to noise
ratio. Calibration bars (bottom) signify 200 msec
horizontally and 100 µV vertically. B, Representative
histograms of ganglion cell discharges from wild-type and mutant
zebrafish elicited with a full-field flash (intensity, log
I = 3.0). Note both on and
off responses to the 0.5 sec flash. C,
Threshold light levels that were required to fire action potentials of
retinal ganglion cells in wild-type (, n = 6)
and
nbb+/
fish (, n = 11) measured at 20 min and 2 hr of
dark adaptation. Each circle indicates a visual
threshold measurement. Note the threshold elevation in
nbb+/
mutants at 2 hr of dark adaptation. The horizontal dashed
line drawn at log I = 5.75 indicates the
highest threshold level of wild-type fish.
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Table 1.
Threshold levels of ERG and retinal ganglion cell spikes of
wild-type and nbb+/ fish at different times
of dark adaptation
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Inner retinal function of nbb+/ mutants is
abnormal
We next examined the light thresholds of retinal ganglion cells in
nbb+/
mutants to determine whether the
nbb+/
visual defect relates to a dysfunction of the inner retina. Animals were first light-adapted and then dark-adapted for either 20 min or 2 hr before a threshold measurement was made. Ganglion cell discharges
were recorded from the optic nerve using a tungsten microelectrode
(Fig. 3B). In most cases, we recorded ganglion cell activity
simultaneously from four to six ganglion cells. Our criterion for a
threshold response was the observation on the oscilloscope screen of an
increase in action potential discharge. In other words, we measured the
thresholds of the most sensitive ganglion cells in each group of
recorded cells.
Figure 3C shows the results. After 20 min of dark
adaptation, 7 of 11 tested mutant fish showed normal ganglion cell
thresholds, whereas 4 mutant fish showed thresholds above log
I = 5.75, our criterion level for normality. The
average ganglion cell threshold recorded from
nbb+/
mutants was ~0.6 log units above the threshold level recorded from
wild-type fish (n = 6) (Table 1), comparable to the
behavioral visual threshold elevation measured at 20 min of dark adaptation.
When
nbb+/
mutants were kept in darkness for 2 hr, more threshold elevations were
observed in the ganglion cell discharge. For example, only 3 nbb+/
mutants showed normal thresholds (below log I = 5.75)
after 2 hr of dark adaptation, whereas 8 of 11 mutant fish showed
elevated ganglion cell thresholds as compared with those of wild-type
fish (Fig. 3C). The extent of threshold elevation in 2 hr
dark-adapted nbb+/
mutants was as much as 3 log units. The ganglion cell threshold was on
average 1.3 log units higher in
nbb+/
than in wild-type zebrafish (Table 1).
Retinal DA-IPCs are decreased in number in aged
nbb+/ mutants
The
nbb+/
retina is relatively normal in terms of its gross morphology. It shows
normal-appearing nuclear layers as well as plexiform layers. No obvious
defects were seen in the photoreceptor or ganglion cell layers.
However, one possible abnormality in nbb+/
retinas was observed in the lower portion of the inner nuclear layer
(INL) where amacrine and DA-IPCs reside; swollen cells and empty spaces
were sometimes seen (Fig.
4B), although swollen
cells were occasionally seen in wild-type retinas (Fig.
4A). Our impression was that this disruption of
structure was more common in
nbb+/
than in wild-type retinas, although considerable variation in this
regard was noted in the retinas of both types of fish.
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Figure 4.
Histological sections of the retina from wild-type
(A) and
nbb+/
fish (B) showing the inner nuclear layer. Note
that in the lower portion of the inner nuclear layer more swollen cells
and/or empty spaces (arrows) were observed in
nbb+/
than in wild-type retinas. Scale bar, 50 µm.
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To test whether there might be an abnormality in the amacrine cells
and/or DA-IPCs in
nbb+/
mutants, we visualized these cells in retinal whole-mount preparations using antibodies against these two cell types. No obvious differences were observed in the appearance and number of amacrine cells between nbb+/
and wild-type fish [stained with 5E11, an antibody that selectively stains zebrafish amacrine cells (Fadool et al., 1999)]. However, the
number of DA-IPCs in
nbb+/
retinas was reduced somewhat particularly in aged
nbb+/
mutants (stained with an antibody against tyrosine hydroxylase). In
young adults (4.5 months), the number of DA-IPCs was similar between
nbb+/
(n = 8) and wild-type (n = 12) fish
(Table 2). However, as
nbb+/
fish aged, the number of DA-IPCs was reduced. At 11.5 months, DA-IPCs
were decreased by ~20% in
nbb+/
fish (n = 8) as compared with the number found in
4.5-month-old nbb+/
or 11.5-month-old wild-type animals (n = 12) (Table
2).
Centrifugal fiber innervation of the retina is disrupted in
nbb+/ fish
The DA-IPCs in teleost fish retinas receive a rich
innervation from centrifugal fibers (CFs) that originate from the
terminal nerve neurons (TNs) in the olfactory bulb (Zucker and Dowling, 1987). TNs in fish contain two types of neuropeptides: gonadotropin hormone-releasing hormone (GnRH) and molluscan cardioexcitatory tetrapeptide (FMRFamide) (Stell et al., 1984; Walker and Stell, 1986;
Zucker and Dowling, 1987). Using an antibody against FMRFamide, we
identified TNs in zebrafish (Fig.
5A). Similar to other teleost species, TNs (30-40 in number) are found in the anterior/ventral part
of the olfactory bulb in zebrafish (Fig. 5B). Most of the TN
axons terminate in the forebrain and midbrain, but some project into
the optic nerve (Fig. 5C) and enter the retina where they extend along the border of the INL and inner plexiform layer (IPL), in
close apposition to DA-IPCs (Fig. 5D). TN axons that enter the optic nerve and retina are also called CFs.
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Figure 5.
The olfactoretinal centrifugal pathway in
zebrafish. A, A schematic drawing of the forebrain and
midbrain of zebrafish (dorsal view). TNs (red circles)
are found in the anterior/ventral part of the olfactory bulb. Most TN
axons are found in TE and TC.
B, C, and D highlight the
olfactory bulb, the optic nerve, and the retina, respectively, as
indicated in A. OE, Olfactory epithelium;
ON, olfactory nerve; OB, olfactory bulb;
OP, optic nerve; TE, telencephalon;
TC, tectum; RE, retina. B,
A whole-mount olfactory bulb (outlined by the dashed
line; anterior is up) stained with an antibody against
FMRFamide. Both TN cell bodies and axons were stained
(arrows). C, FMRFamide immunostaining of
a retinal section showing CFs in the optic nerve (vertical
arrows) and in the retina (horizontal arrows).
Photoreceptor cells (top left) were nonspecifically
labeled. D, A double-labeled retinal section showing the
CFs (red) and DA-IPCs (green,
arrows). CFs and DA-IPCs were identified using
antibodies against FMRFamide and tyrosine hydroxylase, respectively.
Photoreceptor cells (top) were nonspecifically labeled.
PH, Photoreceptor cell layer; OP, outer
plexiform layer; IN, inner nuclear layer;
IP, inner plexiform layer. Scale bars: B,
100 µm; C, D, 30 µm.
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TNs were identified in
nbb+/
fish. The number and appearance of the TNs in the olfactory bulbs as
well as the distribution of TN axons in the forebrain and midbrain
appeared normal. However, the pattern of CF innervation of the retina
was altered in
nbb+/
mutants. In wild-type retinas, the CFs (five to eight in number at or
near the optic disk) first extend out radially and then branch.
Secondary and tertiary branches of the CFs are often observed, and
prominent varicosities are seen along the processes. The CF branches
tend to run concentrically around the retina, and this is especially
evident in peripheral retinal regions (Fig.
6A,C). In
nbb+/
mutants, the number of CFs that entered the retina was similar to that
of wild-type fish. However, the branching of CFs in the retina occurred
less frequently, and the distribution of CFs was less organized (Fig.
6B,D). Furthermore, the CF
innervation of the retina was reduced in
nbb+/
mutants; the number of FMRFamide-positive varicosities counted from
nasal/ventral regions of
nbb+/
retinas was reduced by ~50% as compared with that counted from the
same regions of wild-type retinas (1471 ± 485/mm2 vs 3086 ± 457/mm2; p < 0.001;
n = 12 for both the wild-type and
nbb+/
fish).
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Figure 6.
Flat-mounted wild-type (A,
C) and
nbb+/
(B, D) retinas. A,
B, Schematic drawing of representative CF distribution
in wild-type (A) and
nbb+/
(B) retinas. Dorsal is up; nasal
is to the left. The black circles marked
in the middle of the retina indicate the optic disk. Note the
disruption and reduction of CFs in the
nbb+/
retina. C, D, Confocal images taken from
nasal/ventral regions (inset boxes in A
and B) of double-labeled wild-type
(C) and
nbb+/
(D) retinas. CFs are shown in red,
and DA-IPCs are shown in green. Note the disruption and
reduction of CFs and the reduction of DA-IPCs in the
nbb+/
retina. Scale bar, 100 µm.
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Visual thresholds are increased in the absence of
olfactory input
To determine whether olfactory centrifugal input plays a role in
the visual system and could relate to the visual defect seen in
nbb+/
mutants, we measured visual sensitivity of zebrafish in which the OE
and OB were removed. After surgery (see Materials and Methods), fish
were allowed to recover for 2-3 d. No obvious differences in behavior
such as feeding or swimming were observed between OE/OB-excised and
control animals.
We first examined dark adaptation. For the first 20-30 min of dark
adaptation after bright light adaptation, the visual thresholds were
similar between OE/OB-excised (4.5 months old, n = 6)
and control animals (4.5 months old, n = 10) (Fig.
7A). However, when kept for
longer times in the dark, OE/OB-excised fish displayed threshold
elevations similar to those seen in the
nbb+/
mutants. After 1 hr of dark adaptation, for example, two of six OE/OB-excised animals showed high visual thresholds; one responded at
log I = 3.0, and the other showed no responses to
visual stimuli at levels 3 log units above the absolute sensitivity
level of the control fish (Fig. 7B). At 2 and 4 hr of dark
adaptation, virtually all of the OE/OB-excised fish showed higher
visual thresholds than control fish. Moreover, one-half to two-thirds
of the OE/OB-excised fish showed no behavioral responses to visual
stimuli at levels 3 log units above the absolute sensitivity level of
control fish (above log I = 3) (Fig. 7B).
Visual threshold fluctuations were also observed in some of the
OE/OB-excised animals; two of six tested fish showed substantial visual
threshold fluctuations (>2 log units) when kept in prolonged
darkness.
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Figure 7.
Behavioral visual thresholds of control (4.5 months old, , n = 10) and OE/OB-excised animals
(4.5 months old, , n = 6) measured during late
afternoon-early evening hours. A, Dark adaptation
curves of the control and experimental animals in 26 min. Note the
similarity in visual threshold between the control and experimental
animals. Data represent the means ± SD. B, Visual
thresholds of experimental animals () during prolonged darkness.
Each circle indicates a behavioral visual threshold
measurement. Note the threshold elevation after 1, 2, and 4 hr of dark
adaptation. Some of the experimental animals showed no response to
visual stimuli 3 log units above the absolute threshold level of
wild-type fish (shown above the horizontal dashed line
at log I = 3.0). Filled circles
indicate the average visual thresholds of wild-type fish kept under the
same illuminating conditions.
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Homozygous nbb mutants are embryonic lethal
To investigate further the nbb mutation, we bred
nbb to homozygosity by crossing individual
nbb+/
mutant fish. Homozygous nbb mutants showed early
degeneration in the brain and retinas, beginning at ~2.5 d
post-fertilization (dpf). At 2.5 dpf, the gross morphology, including
the size of brain and eye, was similar between wild-type and mutant
fish (Fig. 8A,B).
However, cell death in the forebrain and retinas was observed when
nbb/
embryos were stained with acridine orange (Fig.
8B).
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Figure 8.
Photographs of whole-mount zebrafish embryos
(A, B) and histological sections of the
brain and retinas (C-F). A,
B, Photographs of 2.5-d-old wild-type
(A) and
nbb/
(B) embryos stained with acridine orange. Note
the stained cells in the forebrain and eyes of
nbb/
embryos (bright staining in B,
arrow). C-F, Histological sections of
the brain and retinas of wild-type (C, E)
and
nbb/
(D, F) fish. At 3 d of age
(C, D), apoptotic cells were detected in
the brain and retinas in
nbb/
embryos (D, arrow). By 7 d
(E, F), the sizes of the brain
and retinas in
nbb/
embryos were further reduced. Many cells had died (F,
arrow). Scale bars: A, B,
250 µm; C-F, 100 µm.
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Brain and retinal cells in
nbb/
fish continued to die over time so that by 3 dpf,
nbb/
embryos could be readily identified grossly by their small eyes. Figure
8C-F show histological sections across the brain
and eyes of wild-type and
nbb/
embryos at 3 and 7 d of age. At 3 d, the retinas are well
formed in wild-type fish (Fig. 8C) but not so in
nbb/
mutants (Fig. 8D). Dying cells were observed in the
eye and brain in
nbb/
embryos (Fig. 8D). By 7 d, cell degeneration in
nbb/
embryos was widespread; the eye was further reduced in size, and many
cells had died (Fig. 8F). Cell death was also
observed in the developing forebrain/olfactory bulb as well as in the
spinal cord in
nbb/
mutants. The
nbb/
embryos died by 7-8 dpf.
| |
DISCUSSION |
In this paper, we describe a dominant mutation (nbb)
that causes an unusual visual system defect in adult zebrafish. Under fully dark-adapted conditions, visual thresholds of
nbb+/
mutants measured behaviorally fluctuate abnormally. In addition, nbb+/
mutants show gradual elevations in average visual threshold depending on time in the dark. After 2 hr of dark adaptation, for example, visual
thresholds of
nbb+/
mutants are 2-3 log units above the average thresholds measured in
fully dark-adapted wild-type fish. However, the elevated visual thresholds can be decreased by exposure to light. After 20 min of
bright light adaptation, visual thresholds of
nbb+/
fish fall to levels similar to those of wild-type fish.
The threshold elevation shown by
nbb+/
mutants appears to be caused primarily by a loss of rod system
function. By examining the light sensitivity of
nbb+/
fish using phototopically matched color illumination, we show that in
prolonged darkness, the residual visual sensitivity of nbb+/
mutants is mediated mainly by the cones. Several lines of evidence suggest that the visual defect of
nbb+/
mutants does not relate to photoreceptor cell function but rather is
caused by abnormalities proximal to the outer retina. For example, during the first 20-30 min of dark adaptation after bright light adaptation, visual thresholds measured in
nbb+/
fish were similar to thresholds measured in wild-type zebrafish. This
suggests that at least during early dark adaptation, both the cone and
rod photoreceptor cells in
nbb+/
mutants are essentially normal. Furthermore, at all times during dark
adaptation, the ERGs recorded from
nbb+/
mutants showed normal sensitivity levels as well as a- and b-wave amplitudes, suggesting that not only the photoreceptor cells but also
other outer retinal neurons in
nbb+/
fish are functioning properly. On the other hand, light levels required
to fire action potentials in retinal ganglion cells were elevated in
nbb+/
mutants. This suggests that the defect underlying
nbb+/
visual deficit occurs in the inner retina.
Cellular abnormalities were detected in the inner retina in aged
nbb+/
mutants using histological and immunocytochemical methods. In nbb+/
mutants, there is some evidence of cell loss in the lower portion of
the INL where amacrine cells and DA-IPCs normally reside. At 11.5 months of age, the number of DA-IPCs in
nbb+/
retinas was reduced by ~20% in
nbb+/
mutant fish as compared with the number counted from 11.5-month-old wild-type fish or 4.5-month-old
nbb+/ mutants.
The role of centrifugal fiber input to the retina
The observation of reduced DA-IPCs in aged
nbb+/
mutant retinas led to an investigation of the centrifugal fiber
innervation of DA-IPCs in zebrafish. It has long been known that in
many vertebrates, the neural retina receives efferent inputs from other
parts of the brain (Arey, 1916; Brooke et al., 1965; Dowling and
Cowan, 1966; Demski and Northcutt, 1983; Springer, 1983; Fujita et al., 1985). The efferent input originates from the ventral thalamus in
reptiles (Halpern et al., 1976), from the isthmooptic nucleus in bird
(Cowan, 1970), and from the pretectal area in mammal (Itaya, 1980). In
fish, the efferent input originates from the TNs that are located in
the olfactory bulb (Munz et al., 1982; Stell et al., 1984;
Walker and Stell, 1986). Most of the TN axons in teleost fish terminate
in the forebrain and midbrain, but some enter the optic nerve and
retina where they terminate along the border between the inner
plexiform layer and the inner nuclear layer (Stell et al., 1984). Using
electron microscopy, Zucker and Dowling (1987) demonstrated that in the
retina the CFs make conventional synapses onto DA-IPCs.
Although the anatomical connections between the olfactory bulb and
retina have been described, how CFs influence visual function remains
largely unknown. Some in vitro studies performed in isolated fish retinas have investigated the role of CFs in the regulation of
retinal cell activity. In white perch, for example, exogenously applied
GnRH depolarized the retinal horizontal cells and altered their
receptive field size (Umino and Dowling, 1991). It is believed that
these effects are mediated by the DA-IPCs on which the CFs synapse; in
isolated fish retinas in which the DA-IPCs have been destroyed,
exogenous application of GnRH produced no effects on retinal horizontal
cells (Umino and Dowling, 1991). In isolated goldfish retinas,
exogenous application of FMRFamide caused an increase of spontaneous
activity of dark-adapted retinal ganglion cells, but it is not known
whether these effects are mediated directly by the peptide or via the
DA-IPCs (Stell et al., 1984; Walker and Stell, 1986).
Here we report that CF innervation of the retina appears to play
an important role in the regulation of visual sensitivity in zebrafish.
The
nbb+/
mutants showed substantial visual threshold fluctuations and elevations
particularly under prolonged dark-adapted conditions. A visual defect
similar to that shown by
nbb+/
mutants was observed in zebrafish in which the OEs/OBs have been excised. The visual defects shown by
nbb+/
mutant fish may also be related to a depression of DA-IPC activity. We
have found that in the absence of DA-IPCs, zebrafish are less sensitive
to light by up to 3 log units as compared with wild-type fish
[accompanying paper (Li and Dowling, 2000)]. The sensitivity loss in
DA-IPC-depleted animals is attributable primarily to a loss of rod
system function, as is the case for
nbb+/
mutants. At the present time, it is not clear how the olfactory centrifugal input might influence visual sensitivity. One possibility is that olfactory signals trigger excitation of the DA-IPCs. In response, dopamine levels in the retina are raised, enhancing rod
signal transmission through the inner plexiform layer.
The circadian control of nbb+/ visual
sensitivity may be disrupted
The mechanisms that underlie the circadian clock regulation
of visual sensitivity may relate to the defect shown by
nbb+/
fish. In wild-type fish, visual sensitivity is suppressed by up to 2 log units by the circadian clock during early morning hours (Li and
Dowling, 1998). The suppression of visual sensitivity is released
during the day. In
nbb+/
mutants, suppression of visual sensitivity appears to continue during
day and night. As a result, visual thresholds of
nbb+/
fish are on average ~2 log units above the threshold level of wild-type fish as measured before dusk. Of particular note is the fact
that during circadian suppression of visual sensitivity (at dawn),
substantial variation of visual thresholds among wild-type zebrafish is
observed. Little fluctuation in visual system sensitivity is observed
at dusk, when the fish are most sensitive to light. In
nbb+/
fish, substantial visual threshold fluctuations are observed both at
dawn and at dusk. The loss of circadian control of visual sensitivity
in
nbb+/
mutants could be related to the disruption of DA-IPCs in the retina.
For example, in zebrafish in which the DA-IPCs have been ablated, the
circadian control of visual sensitivity is largely abolished
[accompanying paper (Li and Dowling, 2000)].
The nbb gene
Last, what can we say about the role and locus of expression
of the nbb gene? Our data suggest that the
nbb gene is essential for early CNS development and is
required for maintenance of CNS function during adulthood. With half of
the nbb gene product available (heterozygotes), animals are
viable but show age-related defects primarily in the olfactory and/or
visual system. However, when nbb gene function is completely
eliminated (homozygotes), animals show many defects both in the brain
and elsewhere during embryonic development. Homozygous nbb
mutants die by 7-8 d of age. This suggests that the expression of
nbb is not limited to either the olfactory or visual system
but that it must function in many cells and tissues.
| |
FOOTNOTES |
Received Oct. 5, 1999; revised Dec. 16, 1999; accepted Dec. 17, 1999.
This work was supported by National Institutes of Health Grants EY
00811 and EY 00824. We thank A. Adolph for advice on ERG and ganglion
cell recordings, S. Brockerhoff for providing ENU mutagenized
zebrafish, E. Schmitt for protocols of histology and immunocytochemistry, J. Fadool for providing 5E11 antibodies, and T. O'Donohue for providing FMRFamide antibodies. We also thank W. McCarthy, S. Harris, and S. Sciascia for maintenance of zebrafish, and
D. Smith for advice on confocal imaging.
Correspondence should be addressed to Lei Li, Department of Physiology,
University of Kentucky College of Medicine, 800 Rose Street, Lexington,
KY 40536. E-mail: leili{at}pop.uky.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
