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The Journal of Neuroscience, April 1, 1999, 19(7):2568-2579
Immunohistological Studies of Metabotropic Glutamate Receptor
Subtype 6-Deficient Mice Show No Abnormality of Retinal Cell
Organization and Ganglion Cell Maturation
Yoshiaki
Tagawa1,
Hajime
Sawai2,
Yoshiki
Ueda1,
Masaki
Tauchi2, and
Shigetada
Nakanishi1
1 Department of Biological Sciences, Kyoto University
Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan, and
2 Department of Welfare System and Health Science, Okayama
Prefectural University, Kuboki, Souja, Okayama 719-1197, Japan
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ABSTRACT |
Immature retinal ganglion cells (RGCs) initially show a
multistratified dendritic pattern, and, during the postnatal period, these dendrites gradually monostratify into ON and OFF sublaminae. The
selective agonist of group III metabotropic glutamate receptors (mGluR), L-2-amino-4-phosphonobutyrate
(L-AP-4), hyperpolarizes ON bipolar cells and reduces
glutamate release. On the basis of L-AP-4-evoked inhibitory
effects on ON-OFF segregation of developing RGCs, it has been
hypothesized that glutamate-mediated synaptic activity is crucial for
formation of the ON-OFF network. Gene-targeted ablation of mGluR6
specifically expressed in ON bipolar cells blocks normal ON responses
but has been predicted to enhance glutamate release from ON bipolar
cells. The mGluR6 knock-out mouse therefore provides a unique
opportunity to investigate whether glutamate release and ON responses
are important factors in the development of ON-OFF segregation. The
combination of several different morphological analyses indicates that
ON bipolar cells, as well as several distinct amacrine cells, in mGluR6
knock-out mice are normally distributed and correctly extend their
terminals to defined retinal laminae. Importantly, both  and  RGCs in adult mGluR6 knock-out mice are found monostratified into cell
type-specific layers. Furthermore, no difference between wild-type and
mGluR6 knock-out mice is observed in the maturation and dendritic
stratification of developing RGCs. Hence, despite a deficit in normal
ON responses, mGluR6 deficiency causes no abnormality in the retinal
cellular organization nor in the stratifications of both ON bipolar
cells and developing and mature RGCs. Based on these findings, we
discuss several possible mechanisms that may underlie ON-OFF
segregation of RGCs.
Key words:
metabotropic glutamate receptor subtype 6; knock-out and
transgenic mice; Lucifer yellow injection; immunohistology; ON
response; dendritic stratification; retinal cells
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INTRODUCTION |
Visual information is segregated
into parallel ON and OFF pathways at retinal bipolar cells (Miller and
Slaughter, 1986 ; Dowling, 1987; Schiller, 1992; Nakanishi, 1995). This
segregation results from two different types of glutamate receptors:
L-2-amino-4-phosphonobutyrate (L-AP-4)-sensitive metabotropic glutamate receptor (mGluR)
for ON bipolar cells and AMPA-kainate receptors for OFF bipolar cells (Nawy and Jahr, 1990, 1991; Shiells and Falk, 1990; Yamashita and
Wässle, 1991; de la Villa et al., 1995; Nakanishi, 1995; Sasaki
and Kaneko, 1996). Light hyperpolarizes photoreceptors, resulting in a
reduction of glutamate release. The reduction of glutamate release
renders AMPA-kainate receptors inactive and hyperpolarizes OFF bipolar
cells. In ON bipolar cells, mGluR also shows reduced activity such that
the downstream signaling elements, G-protein and cGMP
phosphodiesterase, remain in an inactive state. As a result, cGMP
reaches high levels, thereby activating cGMP-gated ion channels and
depolarizing ON bipolar cells. Thus, opposite responses to light
exposure are evoked in ON and OFF bipolar cells.
In the mature retina, the dendrites of ON and OFF retinal ganglion
cells (RGCs) stratify at different levels in the inner plexiform layer
(IPL) (Famiglietti and Kolb, 1976; Nelson et al., 1978; Peichl and
Wässle, 1981). ON RGCs form synaptic contacts with ON bipolar
cells at the inner part of the IPL (sublamina b), whereas OFF RGCs
synapse onto OFF bipolar cells at the outer part of the IPL (sublamina
a) (Wässle and Boycott, 1991). In early retinal development,
immature RGCs possess multistratified dendrites, and these dendrites
gradually monostratify into distinct sublamina during retinal
development (Maslim and Stone, 1986, 1988; Dann et al., 1988; Ramoa et
al., 1988). It has been reported that intraocular injection of
L-AP-4, which hyperpolarizes ON bipolar cells, effectively
suppresses the stratification process of RGCs in developing retina
(Bodnarenko and Chalupa, 1993; Bodnarenko et al., 1995; Bisti et al.,
1998). On the basis of these and other studies, it has been
hypothesized that RGC dendritic remodeling results from
activity-dependent glutamate release from ON bipolar cells.
Considerable evidence has indicated that mGluR6 is essential for
evoking ON responses in both rod and cone systems. mGluR6 is confined
at the postsynaptic site of ON bipolar cells and selectively responds
to L-AP-4 (Nakajima et al., 1993; Nomura et al., 1994). Importantly, ablation of mGluR6 by gene targeting abolishes ON responses recorded from optic nerve terminals, as well as the b-wave of electroretinogram that reflects ON bipolar cell
activity (Masu et al., 1995). Furthermore, loss of mGluR6 function
results in marked impairments in both detecting weak contrasts and
responding rapidly to light intensity (Iwakabe et al., 1997). The
mGluR6 knock-out mouse therefore provides a useful model to test
whether glutamate release from ON bipolar cells is involved in the
formation of the ON-OFF network in the IPL and whether the ON response
might be an important factor in ON-OFF segregation.
In this investigation, we addressed whether mGluR6 deficiency causes
any alteration in the retinal organization of bipolar, amacrine, and
ganglion cells. We also examined whether impairment of ON responses
affects development and stratification of immature RGCs during early
retinal development. Here, we report that, despite a marked deficit of
ON responses, mGluR6 knock-out mice show no abnormality in the cellular
organization of various retinal cells nor in the stratifications of ON
bipolar cells and developing and mature RGCs.
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MATERIALS AND METHODS |
Animals. Homozygous mGluR6 knock-out mice that
expressed the  -galactosidase (lacZ) transgene in ON
bipolar cells under control of the mGluR6 promoter were generated by
mating mGluR6 knock-out mice (Masu et al., 1995) with lacZ
transgenic mice (Ueda et al., 1997). Homozygous disruption of the
mGluR6 gene and integration of the lacZ gene were confirmed
by Southern blot hybridization as described previously (Masu et al.,
1995; Ueda et al., 1997).
X-gal and immunofluorescence stainings. X-gal staining was
performed in PBS supplemented with 1 mg/ml X-gal, 35 mM K3Fe(CN)6, 35 mM K4Fe(CN)6, 2 mM MgCl2, and 0.02% Nonidet P-40
as described previously (Ueda et al., 1997). Immunofluorescence
staining was performed according to the procedures described previously
(Nomura et al., 1994). The primary antibodies (Abs) used were as
follows: mouse monoclonal Abs (mAbs) against protein kinase C (PKC)
-isoform (MC5; 1:100 dilution; Amersham, Buckinghamshire,
UK), choline acetyltransferase (ChAT) (1:250; Chemicon, Temecula, CA)
and calbindin-D28K (CL-300; 1:200; Sigma, St. Louis, MO); and rabbit
polyclonal antibodies against lacZ (1:100; Cappel, Durham,
NC), calretinin (1:2000; Chemicon), and tyrosine hydroxylase (TH)
(1:100; Chemicon). Antibodies used for different mGluR subtypes were as
described previously (Shigemoto et al., 1997). Secondary antibodies
used were as follows: Texas Red (TR)-X-conjugated goat lgGs against
rabbit lgG (Rab-TRX) and mouse lgG (1:200; Molecular Probes, Eugene,
OR); fluorescein isothiocyanate (FITC)-conjugated goat lgG against
rabbit lgG (1:200; Cappel); and FITC-conjugated horse lgG against mouse
lgG (1:200; Vector Laboratories, Burlingame, CA). Biotinylated goat
lgGs against rabbit lgG and mouse lgG (1:200; Vector Laboratories) were
used in some experiments and reacted with FITC-conjugated avidin D (1:1000; Vector Laboratories).
Intracellular injection of Lucifer yellow. Intracellular
injection of Lucifer yellow CH (LY) (Sigma) into RGCs was performed as
described previously (Tauchi and Masland, 1984). Young (postnatal day
12) and adult (7-8 weeks) mice were killed under deep
anesthesia with diethylether, and eyes were removed. Incision was made
along an edge of the sclera, and the retina with the lens was removed and immersed in an oxygenated Ames' medium (A1420; Sigma). The lens
and vitreous body were removed, and the isolated retina was spread on a
filter paper (No. 50; Whatman, Kent, UK), photoreceptor cell-side
upward. After rinsing in Ames' medium, the retina was mounted ganglion
cell-side upward onto a black filter paper (HABP; Millipore, Bedford,
MA), placed in a superfusion chamber on the stage of an Olympus
Opticals (Tokyo, Japan) BH2 microscope, and perfused with Ames' medium
at a flow rate of 0.6 ml/min at 30°C. Two drops of Acridine Orange
(0.001% in Ames' medium; Sigma) were added into the chamber to
visualize somata in the ganglion cell layer (GCL). A glass micropipette
(GC120F-10; Clark Electromedical Instruments, Reading, UK) was inserted
into RGC somata under blue-violet excitation. These cells were
completely filled with 5% LY in 0.1 M LiCl by passing 2-4
nA negative currents (150 msec at 4 Hz) for 2-5 min. Usually, 15-20
RGCs per retina were successfully filled with LY.
Tissue processing and data analysis. LY-injected retinae
were immersed overnight in a fixative containing 4% paraformaldehyde in 0.1 M sodium phosphate buffer. After several washes with
the above buffer, the retinae were cryoprotected in 25% sucrose
overnight and then subjected to freeze ( 80°C)-thaw (37°C) four
times to improve antibody penetration. The retinae were preincubated
with solution A (10% normal goat serum, 0.5% bovine serum albumin, and 0.5% Triton X-100 in PBS) for 4 hr and then incubated with TH-Ab
in solution A for 1-2 d. After a 4 hr washing by gently shaking, the
retinae were incubated with Rab-TRX for 1 day, washed for 4 hr, then
mounted on a slide glass, coverslipped, and sealed with nail enamel.
All procedures were performed at 4°C unless otherwise stated.
Flat-mounted retinae were analyzed with the Olympus Fluoview
confocal microscopy system. Serial images (0.7-1 µm in
z-sections) were taken from the GCL to the border
between the IPL and the inner nuclear layer (INL). The plane of the GCL
was identified by visualizing the somata of RGCs and vessels located in
the GCL. The plane of the IPL-INL border was identified on the basis
of dendritic trees of TH-Ab-immunostained dopaminergic cells. Serial images were processed, and images of the dendritic spread and of the
dendritic stratification were reconstructed on
x-y and x-z planes, respectively.
Stratification levels of RGC dendritic trees were quantified as
follows: for each cell, serial images (z-sections) were
transferred to NIH Image software. The pixels above background
levels were counted for each image and then represented as a percentage
of the z-section that contained the highest pixel count. For
each image, this digital processing was well correlated with direct measurement of dendritic lengths.
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RESULTS |
Stratification of bipolar cell axon terminals
Figure 1A shows a
scheme that illustrates the localization of somata and stratifications
of both dendrites and axon terminals of retinal cells analyzed in this
investigation. The characteristic distribution of these cells in the
retinal cellular organization is discussed in each section described
below. We first examined the localization and stratification of bipolar
cells by crossing mGluR6 knock-out mice with transgenic mice expressing
lacZ under control of the mGluR6 promoter
(mGluR6 //lacZ+).
In a previous study (Ueda et al., 1997), we showed that the mGluR6
promoter is capable of recapitulating a specific expression of
lacZ in rod and cone ON bipolar cells. In addition, when the lacZ protein was analyzed by X-gal staining or
lacZ immunostaining, these transgenic mice displayed a clear
stratification of lacZ-positive axon terminals of both rod
and cone ON bipolar cells at the sublamina b. As shown in Figure 1,
B and C, we confirmed that punctate and intense
mGluR6 immunoreactivity was observed in retinal sections of
mGluR6+/+/lacZ+ mice
but was completely lost in those of
mGluR6//lacZ+
mice.
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Figure 1.
Schematic drawing of retinal cells and
immunostaining of mGluR6. A, The distribution and
dendritic and axonal stratifications of retinal cells. Cone ON bipolar
cells make synaptic contacts with ON RGCs in sublamina b, whereas cone
OFF bipolar cells form synaptic connections with OFF RGCs in sublamina
a. Rod bipolar cells extend their axons to the innermost part of
sublamina b, where they make synaptic contacts with AII amacrine cells
(AII). AII amacrine cells form gap junctions onto
cone ON bipolar cells and also project inhibitory outputs onto cone OFF
bipolar cells and OFF RGCs. a, Sublamina a;
b, sublamina b; Ch, cholinergic amacrine
cell; DA, dopaminergic amacrine cell. B,
C, Immunostaining of mGluR6 in transverse retinal
sections of
mGluR6+/+/lacZ+
and
mGluR6//lacZ+
mice. Punctate and intense mGluR6 immunoreactivity observed in the OPL
of mGluR6+/+/lacZ+
mice was absent in
mGluR6//lacZ+
mice. Scale bar, 20 µm.
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We examined and compared the pattern of X-gal staining and
lacZ immunoreactivity in transverse retinal sections of
mGluR6//lacZ+ and
mGluR6+/+/lacZ+ mice.
In both genotypes, X-gal-positive somata were located at the outer
portion of the INL (Fig.
2A,B).
These cells extended X-gal-positive axon terminals to sublamina b of
the IPL. Furthermore, these axon terminals were separated into two
levels at sublamina b. Intense X-gal reaction products were observed in
the inner part of sublamina b, whereas diffuse X-gal products were
detected in the outer part of sublamina b. The former and the latter
stainings corresponded to axon terminals of rod bipolar cells and cone
ON bipolar cells, respectively. This pattern of X-gal staining was indistinguishable between
mGluR6+/+/lacZ+ and
mGluR6//lacZ+
mice. The segregation of axon terminals of rod and cone ON bipolar cells was more explicitly observed in both genotypes by double immunostaining of lacZ and PKC (Fig.
2C,D). PKC is a specific marker for rod bipolar
cells (Negishi et al., 1988), thereby distinguishing between rod and
cone ON bipolar cells. The axon terminals of
lacZ-positive/PKC-positive rod bipolar cells resided in the
innermost part of the IPL close to the GCL, whereas those of
lacZ-positive/PKC-negative cone ON bipolar cells were
located at the outer half of sublamina b. Axonal stratifications of
both rod and cone ON bipolar cells were unchanged by ablation of mGluR6
expression.
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Figure 2.
X-gal staining and double immunostaining of
bipolar cells in transverse retinal sections of
mGluR6+/+/lacZ+
and
mGluR6//lacZ+
mice. A, B, X-gal staining shows the
localization of dendrites, somata, and axon terminals of rod and cone
ON bipolar cells. C, D, The segregation
of axon terminals of rod and cone ON bipolar cells is visible by double
immunostaining with lacZ-Ab
(green) and PKC-mAb (red);
lacZ-positive/PKC-positive immunostaining at the inner
half of sublamina b (yellow) and
lacZ-positive/PKC-negative immunostaining at the outer
half of sublamina b (green) represent axon
terminals of rod bipolar cells and cone ON bipolar cells, respectively.
Scale bars, 20 µm.
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Because appropriate markers for mouse cone OFF bipolar cells were not
available (Ueda et al., 1997), the stratification pattern of these
cells remains to be determined. However, in this study, wild-type and
knock-out mice showed a comparable width of sublamina a, where cone OFF
bipolar cells form synaptic contacts with RGCs (Fig.
2A,B). This finding suggests that
cone OFF bipolar cells send their axons to the appropriate place in
mGluR6 knock-out mice. Because the lacZ transgene expression
provided a good marker for ON bipolar cells, we used and compared
mGluR6+/+/lacZ+ and
mGluR6//lacZ+ mice
as wild-type and mGluR6 knock-out mice in all subsequent experiments.
Immunohistological characterization of amacrine cells
Many amacrine cells stratify distinctly at the IPL. To examine the
effect of mGluR6 deficiency on stratification patterns of amacrine
cells, we analyzed several distinct amacrine cells, which were well
characterized by immunostaining of specific markers. Cholinergic
amacrine cells represent a well known subpopulation of amacrine cells
that form synaptic contacts with RGCs at the IPL. When these cells were
characterized with ChAT-mAb (Voigt, 1986; Mitrofanis et al., 1988),
ChAT-positive somata were separately located in GCL and INL, and their
dendrites were segregated into two strata in the IPL: one in the middle
of sublamina a and the other in the middle of sublamina b (Fig.
3A,B).
This pattern of immunopositive somata and two dendritic stratifications
was unchanged between wild-type and mGluR6 knock-out mice.
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Figure 3.
Immunostaining of several amacrine cell markers in
transverse retinal sections of wild-type (+/+) and mGluR6 knock-out
(/) mice. A, B, ChAT
immunoreactivity. Blood vessels are marked by asterisks.
C, D, Calretinin immunoreactivity.
Calretinin-positive/ChAT-positive bands and somata as revealed by
double immunostaining with calretinin antibody and ChAT-mAb are marked
by asterisks. E, F,
Horizontal cells in the OPL show intense calbindin-D28K
immunoreactivity (asterisks). Scale bar, 20 µm.
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Immunostaining patterns of two other classes of amacrine cell
containing different calcium-binding proteins (Pasteels et al., 1990)
were also indistinguishable between the two genotypes.
Calretinin-immunoreactive somata were observed in both the INL and GCL,
and three immunopositive bands were seen in the IPL (Fig.
3C,D). Double immunostaining with ChAT-mAb and
calretinin antibody showed that the upper and lower bands, as well as
some somata, were immunoreactive with both antibodies (Fig.
3C,D), indicating that a subpopulation of calretinin-positive amacrine cells are cholinergic amacrine cells. Horizontal cells were strongly immunostained with calbindin-D28K antibody (Fig. 3E,F).
Moreover, weak immunoreactivity in some somata of the INL and two weak
bands in the IPL were observed in both genotypes with this antibody.
Additionally, dopaminergic amacrine cells showed a common innervation
pattern between the two genotypes as described below. The
immunohistological characterization thus indicates that there is no
difference in the cellular organization nor in the stratification
pattern of several representative amacrine cells in mGluR6 knock-out mice.
Expression patterns of mGluR subtypes
Of eight different mGluR subtypes known presently, all but mGluR3
have been shown to be expressed and distinctly localized in different
cell types of the rat retina (Shigemoto et al., 1992; Nakajima et al.,
1993; Ohishi et al., 1993; Akazawa et al., 1994; Nomura et al., 1994;
Duvoisin et al., 1995; Hartveit et al., 1995; Brandstätter et
al., 1996; Koulen et al., 1996, 1997). It has been reported that
ablation of a specific gene alters expression patterns of closely
related genes (Hummler et al., 1994; Blendy et al., 1996). We therefore
addressed this possibility by immunostaining with antibodies directed
against different mGluR subtypes. Immunostaining of mGluR1 showed a
strong immunoreactivity at the inner part of the IPL and a weak
immunoreactive band at the outer part of the IPL (Fig.
4A,B).
mGluR5a immunoreactivity was weaker and was seen at the outer plexiform
layer (OPL) and IPL (Fig. 4C,D). Two strong bands
and one diffuse but weak band of mGluR2 immunoreactivity were observed
at the IPL (Fig. 4E,F). In
agreement with previous data (Koulen et al., 1996), the two bands at
the IPL were immunostained with both ChAT-mAb and mGluR2-Ab, indicating
that mGluR2 is localized in the processes of cholinergic amacrine
cells. It has been reported that mGluR4 is localized at the
postsynaptic targets of cone and rod bipolar cells in the rat retina
(Koulen et al., 1996). Consistent with this observation, mGluR4a was
seen at the IPL (Fig. 4G,H). However,
whether a weak signal detected at the OPL was a specific immunoreaction
to mGluR4a remained unclear. mGluR8 immunoreactivity was clearly seen
at the IPL and separated into two bands (Fig. 4I,J). The important
conclusion of this analysis is that there is no obvious difference in
the expression patterns of mGluR1, mGluR2, mGluR4a, mGluR5a, and
mGluR8 between the two genotypes.
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Figure 4.
Immunostaining of different mGluR subtypes in
transverse retinal sections of wild-type (+/+) and mGluR6 knock-out
(/) mice. Immunoreactivities of mGluR1 (A,
B), mGluR5a (C, D), mGluR2
(E, F), mGluR4a (G,
H), mGluR8 (I,
J), and mGluR7a (K,
L). Note that mGluR7a immunoreactivity can be seen at
the OPL of mGluR6 knock-out (L) but not in
wild-type (K) mice. For other subtypes, no
difference in the immunostaining patterns is observed between the two
genotypes. Asterisks in G-J show
immunostaining of blood vessels. Scale bars, 20 µm.
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In both wild-type and mGluR6 knock-out mice, mGluR7a immunostaining
revealed two major immunoreactive bands at the outer part of the IPL
and two diffuse but weak bands at the inner part of the IPL (Fig.
4K,L). Interestingly, distinct
immunoreactive patches of mGluR7a were detected at the OPL of mGluR6
knock-out mice (Fig. 4L). Because this mGluR7a
expression at the OPL was specific for mGluR6 knock-out mice, we
pursued the cellular origin of this mGluR7a expression. Double
immunostaining with the mGluR7a and PKC antibodies showed no overlap of
the two immunoreactivities (data not shown), suggesting that rod
bipolar cells are not responsible for ectopic expression of mGluR7a at
the OPL. Because lacZ is expressed in cone ON bipolar cells
(Ueda et al., 1997), we double-immunostained dissociated
mGluR6//lacZ+
bipolar cells with the mGluR7a and lacZ antibodies and
attempted to visualize a possible colocalization of mGluR7a
immunoreactivity at dendritic tips of cone ON bipolar cells. However,
we found that cone bipolar cells were fragile during cell dissociation and failed to detect intact dendritic tips of dissociated cone bipolar
cells. The origin of this expression awaits further investigation.
Dendritic morphology and stratification of mature RGCs
We then addressed whether mGluR6 deficiency in ON bipolar
cells causes any change in dendritic morphology and stratification levels of RGCs. RGCs consist of a heterogeneous cell population that
can be subdivided into several morphological and functional classes
(Boycott and Wässle, 1974). We injected LY intracellularly into
RGCs and analyzed the dendritic morphology and stratification levels of
different types of RGCs. Among many different cell types observed, we
focused on RGCs with medium- to large-sized somata (>20 µm in
diameter) because they showed a relatively simple morphology with
monostratified dendrites at the IPL. Using the classification of RGCs
reported in the rat retina as a reference (Peichl, 1989; Tauchi et al.,
1992), we found stained cells that corresponded to and cells on
the basis of dendritic branching patterns and dendritic stratification levels.
ON and OFF bipolar cells distinctly terminate their axons at sublamina
b and sublamina a, respectively. Inner and outer RGCs stratify their
dendrites at sublamina b and sublamina a, respectively, and are thought
to correspond to ON and OFF RGCs (Famiglietti and Kolb, 1976; Nelson et
al., 1978; Peichl and Wässle, 1981). It is therefore critical to
precisely assign stratification levels of individual LY-injected RGCs.
When retinal sections were immunostained with TH-Ab (Mitrofanis et al.,
1988; Wulle and Schnitzer, 1989), the dendritic innervation of
dopaminergic amacrine cells was seen to run along the border of the
IPL-INL (Fig.
5A,B).
Furthermore, whole-mount retinal preparations showed a dopaminergic
innervation, with a meshwork sheet of dendrites over the IPL-INL
border, as well as some sparsely located somata in the inner part of
the INL (Fig. 5F,I). Because
this immunostaining pattern of dopaminergic amacrine cells was
indistinguishable between the two genotypes, we used the TH-Ab-positive
innervation of dopaminergic cells as a reference to determine dendritic
stratification levels of individual LY-injected RGCs. Care was also
taken to compare LY-injected RGCs at the similar eccentricity to avoid
eccentricity-dependent variations of morphology and dendritic field of
RGCs. Examples of this analysis are illustrated in Figure
5D-I.
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Figure 5.
Dendritic morphology and stratification levels of
inner-, outer-, and outer- RGCs in adult wild-type (+/+) and
mGluR6 knock-out (/) mice. A, B,
Dopaminergic amacrine cells immunostained with TH-Ab
(red) were observed along the INL-IPL border in
transverse sections of the two genotypes. This immunostaining was used
as a reference to assign stratification levels of three types of RGCs
(D-I). C, Stratification
levels of three RGCs are schematically illustrated;
i, inner- RGC; o,
outer- RGC; o, outer- RGC;
DA, dopaminergic amacrine cell innervation.
D-I, In each panel, the top shows a
confocal image of dendritic branches of three cell types on the main
dendritic x-y plane, and the
bottom displays a reconstructed image on the
x-z plane. Note that the
x-z plane was processed to cut the GCL
at the middle of RGC somata, thus they appear to protrude toward the
IPL in D and G. green,
LY-injected RGCs; red, immunostained dopaminergic
amacrine cells. Somata of dopaminergic amacrine cells are indicated by
arrowheads in F and I.
J-O, Stratification levels and the distribution of
dendritic branches of three cell types. For each cell, dendritic
branches on each z-section taken from the GCL to the
IPL-INL border (left to right in the
abscissa) were represented as a percentage of the
z-section containing the highest pixel count. The data
were obtained from three types of RGCs shown in D-I.
Eccentricity: D, 1.6 mm; E, 1.7 mm;
F, 1.8 mm; G, 1.3 mm; H,
1.9 mm; I, 1.6 mm. Scale bars: B, 20 µm; I, 50 µm.
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On the basis of this approach, we could identify dendritic morphologies
and stratification levels of and cells. Cells were
subdivided into inner- and outer- cells. Dendrites of inner- RGCs branched at the innermost part of the IPL far from dopaminergic innervation (Fig.
5C,D,G), whereas those
of outer- RGCs ramified in the middle of the IPL near, but not
overlapping with, dopaminergic innervation (Fig.
5C,E,H). Cells have also been classified as inner and outer cells (Peichl,
1989). Outer- RGCs were easily identified; the main dendritic plane
was located in the outermost section of the IPL, which partly
overlapped with dopaminergic innervation (Fig.
5C,F,I). However,
it was difficult to assign inner- cells because of their dendritic
localization at sublamina b close to sublamina a. In addition, as
reported previously (Tauchi et al., 1992), inner- cells, if they
exist, represented a minor population of the cells (Table
1).
Figure 5 also illustrates typical dendritic branching patterns of
inner-, outer-, and outer- cells. Inner- and outer- cells showed smooth, relatively non-overlapping dendrites (Fig. 5D,E,G,H),
whereas cells displayed thinner and more curving branches (Fig.
5F,I). As reported
previously (Huxlin and Goodchild, 1997), inner- cells could be
further subdivided into two types, one showing a dense dendritic field
(Fig. 5D,G) and the other displaying a relatively sparse dendritic field (data not shown). We
classified these two cell types as inner- cells in our
investigation. This morphological analysis indicates that not only the
dendritic branching patterns but also the dendritic fields of the three cell types are indistinguishable between mGluR6/
and mGluR6+/+ genotypes.
To quantify the stratification levels of the three cell types, we
measured the length of dendrites in each serial image section and
plotted it against the thickness of the IPL (Fig.
5J-O). This analysis indicates that all three
RGCs have well defined, monostratified dendrites and show no difference
in their dendritic stratification levels nor in the width of dendritic
distribution between the two genotypes.
Table 1 summarizes the number of the four cell types of wild-type and
mGluR6 knock-out mice according to the cell classification described
above. The data indicates no difference in relative number of the four
cell types between the two genotypes. Thus, the results demonstrate
that, despite absence of mGluR6 expression, both and RGCs
maturate normally in adult retina and are able to extend monostratified
dendrites to cell type-specific layers.
Development of immature RGCs
It is possible that mGluR6 deficiency may affect the maturation
process of RGCs at early stages of retinal development. Therefore, we
examined RGCs at postnatal day 12, just before eye-opening, because it
has been reported that at a similar postnatal period (12-15 d), cat
retinal RGCs exhibit conspicuous immature morphological features but
have already differentiated into cell types closely resembling those of
adult cells (Ramoa et al., 1987, 1988; Dann et al., 1988). To
investigate morphological immaturity and dendritic stratification
levels of developing RGCs, we focused on RGCs with large- to
medium-sized somata and adopted the above mentioned approach, which
combined LY injection of RGCs and TH-Ab immunostaining of dopaminergic
amacrine cells. Developing RGCs identified here exhibited several
immature features (Fig. 6). They showed
extensive dendritic branches and much larger numbers of somatic and
dendritic spines than in adults. Despite these immature properties,
RGCs were well identified using dopaminergic innervation as a
reference. Most of the RGCs, like adult cells, showed distinct
dendritic monostratification in either the inner or outer part of the
IPL. Therefore, immature RGCs were classified as inner--like,
outer--like, and -like RGCs. Remarkably, however, some of the
inner--like RGCs, whose main dendritic branches were located at the
inner part (sublamina b) of the IPL, clearly extended their peripheral branches to the outer part (sublamina a) of the IPL (Fig. 6). Similar
immature multistratification of inner--like RGCs into two sublaminae
were observed in both wild-type and mGluR6 knock-out mice (Fig. 6).
View larger version (21K):
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|
Figure 6.
Confocal images of inner--like RGCs of
wild-type (+/+) and mGluR6 knock-out (/) mice at postnatal day 12. The left images show dendritic branches at the inner
part of the IPL (the main dendritic plane), and the
right images indicate the presence and absence of
peripheral dendritic branches at the outer part of the IPL. These
inner--like RGCs show immature morphology. Cells shown in
A and C monostratify at the inner part of
the IPL, whereas those shown in B and D
also extend their peripheral dendrites to the outer part of the IPL.
Eccentricity: A, 1.9 mm; B, 1.3 mm;
C, 1.2 mm; D, 1.5 mm. Scale bar, 50 µm.
|
|
On the basis of cell classification described above, we calculated the
number of different cell types in wild-type and mGluR6 knock-out mice
at postnatal day 12 (Table 1). In addition to inner--like cells,
multistratified outer--like and outer--like cells were also
observed, but this incidence was very rare in both cell types (Table
1). An important observation of this analysis is that the incidence of
immature dendritic stratifications of different cells is comparable
between the two genotypes. The results strongly suggest that there is
no significant difference in maturation of both and cells in
mGluR6 knock-out mice.
| |
DISCUSSION |
ON-OFF segregation is of fundamental importance in discriminating
light and dark signals (Dowling, 1987) and in promoting the detection
of both weak contrasts and rapid changes in light intensity (Schiller,
1992). Considerable evidence has indicated the role of mGluR6 in
synaptic transmission of ON bipolar cells in both rod and cone systems.
mGluR6 is confined at postsynaptic dendritic tips of both rod and cone
ON bipolar cells (Nomura et al., 1994; Vardi and Morigiwa, 1997).
Furthermore, mGluR6 shows an agonist profile consistent with the
property of the L-AP-4-sensitive mGluR reported in ON
bipolar cells (Nakajima et al., 1993). Ablation of mGluR6 expression by
gene targeting abolishes not only ON responses recorded at the superior
colliculus but also the b-wave of electroretinogram that reflects ON
bipolar cell activity (Masu et al., 1995). In the retina of mGluR6
knock-out mice, the phosphorylated cAMP-responsive element binding
protein and c-fos are not induced in response to light stimulation
(Yoshida et al., 1998). Furthermore, consistent with the role of
ON-OFF segregation in visual detection, these knock-out mice are
markedly impaired in both detecting weak visual contrasts and
responding rapidly to light exposure (Iwakabe et al., 1997).
Nevertheless, mGluR6 knock-out mice can respond to visual inputs and
exhibit light-stimulated induction of c-fos in suprachiasmatic nucleus
(Iwakabe et al., 1997). Sugihara et al. (1997) recently observed that,
under certain illumination conditions, mGluR6 knock-out mice generate a
unique slow ON response that clearly differs from the ON response of
wild-type mice. Although the mechanism underlying the appearance of
such a slowly evoking ON response remains to be determined (but see
Iwakabe et al., 1997; Sugihara et al., 1997; also below), mGluR6
knock-out mice provide a unique opportunity to investigate the role of
glutamate-mediated activity of ON bipolar cells during ON-OFF network
formation. To address this question, we combined several different
approaches: the localization and axonal stratification of ON bipolar
cells with the aid of lacZ-expressing transgenic mice;
immunohistological characterization of the cellular organization of
several amacrine cells and expression patterns of other mGluR subtypes;
and intracellular LY injection to analyze morphology and dendritic
stratifications of mature and developing RGCs. The combination of these
different approaches has allowed us to precisely assign the
stratifications of ON bipolar and of distinct amacrine cells, in
addition to developing and mature RGCs.
Immature cat RGCs initially possess multistratified dendrites, and,
during the time of synaptic formation between bipolar cells and RGCs,
these dendrites gradually monostratify into ON and OFF sublaminae
(Maslim and Stone, 1986, 1988; Dann et al., 1988). On the basis of
L-AP-4-evoked inhibitory effects on dendritic segregation
of developing RGCs, it has been proposed that glutamate-mediated synaptic activation plays a key role in the synaptic formation between
bipolar cells and RGCs (Bodnarenko and Chalupa, 1993; Bodnarenko et
al., 1995; Bisti et al., 1998). In this investigation, we demonstrate
that, despite the dramatic effects of mGluR6 deficiency on ON
responses, this deficiency causes no alteration in the cellular organization of bipolar cells, amacrine cells, and RGCs nor in the
stratifications of both ON bipolar cells and developing and mature
RGCs. In our investigation, we focused on and cells because the
dendritic ON-OFF segregation of these cells could be easily identified
after intracellular LY injection. In comparison, Chalupa and his
associates (Bodnarenko and Chalupa, 1993; Bodnarenko et al., 1995;
Bisti et al., 1998) investigated the inhibitory effect of
L-AP-4 on immature -RGCs, using the lipophilic tracer DiI implanted into optic nerve fibers. In our hands, LY injection into
cells with smaller-sized somata identified a certain number of bushy
and well branched small RGCs, which resembled cat cells. Although
more quantitative experiments are necessary, we observed that these
cells distinctly and comparably monostratify into either sublamina a or
sublamina b in the adult retinae of wild-type and mGluR6 knock-out
mice. One limitation of our study is the lack of direct evidence
indicating that immature -like and -like RGCs differentiate into
corresponding mature cell types during retinal development. However,
the morphological characteristics of mouse -like RGCs completely
agree with those of immature cells characterized in the cat retina
(Ramoa et al., 1987, 1988; Dann et al., 1988). Therefore, similar to
their cat counterparts, it is reasonable to conclude that some immature
-like RGCs multistratify at two laminae. Importantly, such
multistratification of immature -like RGCs is comparable between
wild-type and mGluR6 knock-out mice.
Both intraocular L-AP-4 injection and mGluR6 deficiency
block induction of ON responses. However, the effects of
L-AP-4 injection and mGluR6 deficiency are clearly
different in dendritic ON-OFF segregation of RGCs during retinal
development. This observation, together with the results of Chalupa and
coworkers (Bodnarenko and Chalupa, 1993; Bodnarenko et al., 1995; Bisti
et al., 1998), sheds much insight into possible mechanisms that may
underlie the formation of the ON-OFF network in the IPL. The first
possibility is that glutamate release is indispensable for ON-OFF
segregation in the IPL. L-AP-4 hyperpolarizes ON bipolar
cells and reduces glutamate release. In contrast, it is expected,
although not investigated, that mGluR6 ablation results in a high level
of cGMP within ON bipolar cells, leading to an enhancement of glutamate
release from these cells. Therefore, if the mechanism for ON-OFF
segregation in the IPL depends on glutamate release, the effects of
L-AP-4 injection on ON-OFF segregation should be different
from those of mGluR6 ablation. Second, L-AP-4 acts not only
on mGluR6 but also on other subtypes of group III mGluRs. Therefore, an
alternative possibility is that multiple actions of L-AP-4
in glutamatergic transmission are required for
L-AP-4-mediated retardation of dendritic segregation of
developing RGCs. L-AP-4-mediated glutamatergic neurotransmission may also modulate other neurotransmitters, and such
neurotransmitters may participate in the remodeling of synaptic connections of developing RGCs. It has recently been reported that
GABAergic transmission differentially modulates ON and OFF RGCs during
a period when the axon terminals of RGCs are segregated into ON and OFF
pathways (Fischer et al., 1998). Because a temporally regulated
neuronal activity is thought to be critical for developmental remodeling of synaptic connections, GABAergic transmission may also be
involved in refining the neural network. Third, a unique slow ON signal
remains in mGluR6 knock-out mice (Sugihara et al., 1997). This activity
may be sufficient for the remodeling of immature RGCs. In wild-type
retina, mGluR7 has been shown to be present at presynaptic ribbon
structures of cone ON and OFF bipolar cells but not at postsynaptic
sites of any bipolar cells (Brandstätter et al., 1996).
Interestingly, the present investigation has revealed that mGluR7
immunoreactivity is distributed at the OPL-INL border in mGluR6
knock-out mice. Both mGluR6 and mGluR7 belong to group III mGluRs and
respond to L-AP-4 (Okamoto et al., 1994). This ectopic
mGluR7 may explain the unusual ON response that was observed in mGluR6
knock-out mice under particular illumination conditions (Sugihara et
al., 1997). Because mGluR7 knock-out mice grow normally (Masugi et al.,
1999), it would be interesting to address whether double knock-out mice
of mGluR6 and mGluR7 lose the unusual ON response and, if so, whether
the deficiency of both mGluR6 and mGluR7 may affect developmental
dendritic segregation of ON and OFF RGCs.
| |
FOOTNOTES |
Received Oct. 13, 1998; revised Jan. 12, 1999; accepted Jan. 14, 1999.
This work was supported in part by research grants from the Ministry of
Education, Science, and Culture of Japan, the Sankyo Foundation, the
Yamanouchi Foundation, and the Biomolecular Engineering Research
Institute. We thank Yutaka Fukuda for helpful advice and Kumlesh K. Dev
for careful reading of this manuscript.
Correspondence should be addressed to Shigetada Nakanishi, Department
of Biological Sciences, Kyoto University Faculty of Medicine, Yoshida,
Sakyo-ku, Kyoto 606-8501, Japan.
| |
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January 15, 2003;
23(2):
518 - 529.
[Abstract]
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R. G. Gregg, S. Mukhopadhyay, S. I. Candille, S. L. Ball, M. T. Pardue, M. A. McCall, and N. S. Peachey
Identification of the Gene and the Mutation Responsible for the Mouse nob Phenotype
Invest. Ophthalmol. Vis. Sci.,
January 1, 2003;
44(1):
378 - 384.
[Abstract]
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[PDF]
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J. H. Singer, R. R. Mirotznik, and M. B. Feller
Potentiation of L-Type Calcium Channels Reveals Nonsynaptic Mechanisms that Correlate Spontaneous Activity in the Developing Mammalian Retina
J. Neurosci.,
November 1, 2001;
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8514 - 8522.
[Abstract]
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G.-Y. Wang, L. C. Liets, and L. M. Chalupa
Unique Functional Properties of On and Off Pathways in the Developing Mammalian Retina
J. Neurosci.,
June 15, 2001;
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[Abstract]
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A. Bansal, J. H. Singer, B. J. Hwang, W. Xu, A. Beaudet, and M. B. Feller
Mice Lacking Specific Nicotinic Acetylcholine Receptor Subunits Exhibit Dramatically Altered Spontaneous Activity Patterns and Reveal a Limited Role for Retinal Waves in Forming ON and OFF Circuits in the Inner Retina
J. Neurosci.,
October 15, 2000;
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[Abstract]
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A. J. Olson, A. Picones, and J. I. Korenbrot
Developmental Switch in Excitability, Ca2+ and K+ Currents of Retinal Ganglion Cells and Their Dendritic Structure
J Neurophysiol,
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[Abstract]
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E. Strettoi and V. Pignatelli
Modifications of retinal neurons in a mouse model of retinitis pigmentosa
PNAS,
September 19, 2000;
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190291097.
[Abstract]
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E. Strettoi and V. Pignatelli
Modifications of retinal neurons in a mouse model of retinitis pigmentosa
PNAS,
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[Abstract]
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TOP
ABSTRACT
INTRODUCTION
