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The Journal of Neuroscience, November 1, 1999, 19(21):9399-9411
Disruption of Laminin  2 Chain Production Causes Alterations in
Morphology and Function in the CNS
Richard T.
Libby1,
Christopher R.
Lavallee1,
Grant W.
Balkema1,
William
J.
Brunken4, 1, and
Dale D.
Hunter2, 3
1 Departments of Biology, Boston College, Chestnut
Hill, Massachusetts 02167, 2 Cutaneous Biology Research
Center, Massachusetts General Hospital and Harvard Medical School,
Charlestown, Massachusetts 02129, and 3 Departments of
Neuroscience, Anatomy and Cell Biology, and Ophthalmology, Tufts
University School of Medicine, Boston, Massachusetts 02111
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ABSTRACT |
From the elegant studies of Ramon y Cajal (1909) to the current
advances in molecular cloning (e.g., Farber and Danciger, 1997),
the retina has served as an ideal model for the entire CNS. We
have taken advantage of the well described anatomy, physiology, and
molecular biology of the retina to begin to examine the role of the
laminins, one component of the extracellular matrix, on the processes
of neuronal differentiation and synapse formation in the CNS. We have
examined the effect of the deletion of one laminin chain, the  2
chain, on retinal development. The gross development of retinas from
laminin 2 chain-deficient animals appears normal, and photoreceptors
are formed. However, these retinas exhibit several pathologies:
laminin 2 chain-deficient mice display abnormal outer segment
elongation, abnormal electroretinograms, and abnormal rod photoreceptor
synapses. Morphologically, the outer segments are reduced by 50% in
length; the outer plexiform layer of mutant animals is disrupted
specifically, because only 7% of observed rod invaginating synapses
appear normal, whereas the inner plexiform layer is undisturbed;
finally, the rate of apoptosis in the mutant photoreceptor layer is
twice that of control mice. Physiologically, the electroretinogram is
altered; the amplitude of the b-wave and the slope of the b-wave
intensity-response function are both decreased, consistent with
synaptic disruption in the outer retina. Together, these results
emphasize the prominence of the extracellular matrix and, in
particular, the laminins in the development and maintenance of synaptic
function and morphogenesis in the CNS.
Key words:
synapse development; laminin; photoreceptor; ERG; apoptosis; extracellular matrix
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INTRODUCTION |
The development and maintenance of
functional connections within the CNS is dependent on a wide variety of
processes. These include, but are not limited to, the coordinated
production of different cell types, the establishment of proper
connections among different cell types, and the subsequent maintenance
of these connections. Many environmental factors have been suggested to
play roles during these three processes (for review, see Chiba and
Keshishian, 1996 ; Pearlman and Sheppard, 1996; Higgins et al., 1997;
Hunter and Brunken, 1997). Because it is well described and continues
to mature postnatally, the retina is an excellent model system in which
to dissect the role of environmental factors in these three processes.
We have begun to analyze the role of one element of the extracellular
matrix, the laminins, in CNS development. The laminins are a complex
family of extracellular glycoproteins, each composed of three
independent gene products: an  , a , and a  chain. Eleven
laminin chain isoforms (five , three , and three ) are known
(Timpl, 1996; Koch et al., 1999), and at least 14 potential heterotrimers have been postulated (Timpl, 1996; Miner et al., 1997;
Koch et al., 1999) with more, no doubt, to be identified.
Mutations in several of the laminin chains are correlated with human
disease. These include mutations in the 3, 3, and 2 chains in
the blistering disorders known as junctional epidermolysis bullosas
(for review, see Ryan et al., 1996) and mutations in the 2 chain in
certain types of muscular dystrophy (for review, see Arahata et al.,
1995). The potential pathologies in the CNS have not been extensively
studied; however, because 2 is expressed in several parts of the CNS
(Morissette and Carbonetto, 1995; Hagg et al., 1997; Raabe et al.,
1997; Tian et al., 1997), and muscular dystrophies frequently are
associated with CNS dysfunction, it is reasonable to postulate that at
least the 2 chain is important in maintenance of CNS function
(Arahata et al., 1995).
2-containing laminins are expressed on the apical surface of the
retina (Hunter et al., 1992; Libby et al., 1996, 1997a,b) and in the
outer plexiform layer (OPL; R. T. Libby, Y. Xu, E. P. Gibbons, M. F. Champliaud, M. Koch, R. E. Burgeson, D. D. Hunter, and W. J. Brunken, unpublished data) from early
in development through adulthood and appear to be the product of the
retinal Müller glial cell (Libby et al., 1997). We have shown
that 2-containing laminins are important in retinal development
(Hunter et al., 1992; Libby et al., 1996, 1997a; Hunter and Brunken,
1997). In the peripheral nervous system, 2-containing laminins have
been shown to be important for the development and maintenance of
synaptic contacts: mice that are deficient in production of the laminin 2 chain (Noakes et al., 1995a) exhibit abnormalities in their neuromuscular junctions. However, the effects of disruption of laminin
2 chain production in the CNS have not, as yet, been examined. Using
histological and physiological techniques, we characterize the retinas
of laminin 2 chain-deficient mice. Our data demonstrate that
photoreceptor morphogenesis and synaptogenesis in the OPL are disrupted
in these animals.
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MATERIALS AND METHODS |
Animals
All procedures involving animals were approved by the Boston
College and Tufts University Animal Care Committees and were in
accordance with the National Institutes of Health Guide for the
Care and Use of Animals. Mice heterozygous for a null mutation in
the laminin 2 chain gene (Noakes et al., 1995a) were a gift from
Joshua Sanes (Washington University, Saint Louis, MO). These mice were
created by a homologous recombination that targeted the second coding
exon of the mouse laminin 2 chain gene. The mutation was made by
inserting a neo cassette into exon 3 (the second coding exon) of the
Lamb2 gene resulting in a gene that is 1.5 kb longer than the wild type
(Noakes et al., 1995a). DNA transfer blot analysis has confirmed the
presence of a disrupted laminin 2 chain gene (Noakes et al., 1995a).
Stop codons were placed in all three frames of the inserted cassette,
and skipping by alternate splicing of exon 3 would result in a frame
shift and a stop in translation. Thus, no full-length protein should be
produced in these animals. Protein transfer blot analysis has confirmed
the absence of laminin 2 chain protein in homozygous nulls (Noakes
et al., 1995a), which was confirmed in the kidney (Noakes et al.,
1995b). However, parts of exons 2 and 3 could be translated, resulting
in the production of the most N-terminal domains (V and VI) of the 2
chain. There are no reagents available to test for the production of
this protein fragment. Nevertheless, if the short N terminus were made,
it is highly unlikely that it would be exported, because only fully
assembled trimers with intact long arms are exported (Yurchenco et al.,
1997). Heterozygous animals, maintained in a 12 hr day/night cycle,
were bred in our colony at Boston College. The day of birth was defined
as postnatal day 0 (P0). Genotypes of offspring of heterozygous matings
were determined as described previously (Noakes et al., 1995a) with minor modifications; to identify the mutated gene we used the following
primer pairs: 5'-ccgggcgcccctgcgctgacagc-3' and
5'-cgaattcgaacacgcagatgcag-3'; these primers yield a 250 bp PCR product.
In all respects, heterozygous animals appear indistinguishable from
homozygous normal mice (data not shown); this observation is consistent
with previous reports that characterized animals carrying the laminin
2 chain-null mutation. Thus, as in those previous reports,
heterozygous (+/ ) and homozygous (+/+) animals, both of which are
phenotypically wild type, were used as controls.
Histology
Tissue preparation. Unfixed tissue was prepared, embedded,
and frozen as described previously (Libby et al., 1996). For
preparation of paraformaldehyde-fixed tissue, eyes were removed, and a
hole was made at the ora serrata using a hypodermic needle. The tissue was then placed in 4% paraformaldehyde (Sigma, St. Louis, MO) in
PBS (137 mM NaCl, 2.68 mM KCl, 10 mM Na2HPO4, 1.76 mM
KH2PO4, pH 7.4) at 0°C. After ~30 min, the
anterior chamber and lens were removed, and the resultant eyecup was
fixed for 2 hr or overnight. The tissue was then placed into an
ascending series of sucrose solutions (5, 10, 15, and 20% in PBS),
incubated overnight at 4°C in a mixture of 20% sucrose and 80% OCT
(Miles, Elkhart, IN), and then changed to a fresh solution of 20%
sucrose and 80% OCT. The tissue was then frozen and cut at 10 µm, as
described previously (Libby et al., 1996). For some histological
examinations, the tissue was fixed in 3% glutaraldehyde (Polysciences,
Warrington, PA) and 2% formaldehyde in PBS and then treated as
described above.
For electron microscopy, eyes were prepared as above but fixed in 3%
glutaraldehyde and 2% formaldehyde in 0.1 M sodium
cacodylate, pH 7.4, for 1 hr. Eyecups were sectioned into quadrants,
washed in 0.1 M sodium cacodylate, pH 7.4, fixed in 2%
glutaraldehyde and 1% osmium tetroxide for 1 hr, and then dehydrated
in a graded series of ethanols, embedded in plastic, and sectioned.
Semithin sections were taken to assure the orientation of the tissue,
and then ultrathin sections were made and viewed and photographed on a
Philips (Eindhoven, The Netherlands) CM-10 transmission electron microscope. Synaptic morphology was scored and counted from working prints. Synaptic ribbons were identified and the number of postsynaptic elements counted; synapses were grouped into four categories: triads,
requiring the presence of three postsynaptic processes; dyads, in which
either one or two distinct postsynaptic processes are present; floating
ribbons, in which a ribbon but no apposed postsynaptic process is
present; and two on one, in which two ribbons are apposed to one
postsynaptic element.
Immunohistochemistry on unfixed and paraformaldehyde-fixed tissue was
performed as described previously (Libby et al., 1996, 1997a).
Antibodies and lectins. A mouse monoclonal antibody that
recognizes rhodopsin (Ret-P1; Fekete and Barnstable, 1983) was a gift
from C. Barnstable (Yale University, New Haven, CT). Anti-laminin antibodies were a polyclonal anti-laminin 4 chain from J. R. Sanes; a monoclonal anti-laminin 2 chain (Hunter et al., 1989); and
a polyclonal anti-laminin 3 chain from R. E. Burgeson (Harvard University, Charlestown, MA). A mouse monoclonal antibody that recognizes synaptic ribbons was raised in our laboratories (B16; Balkema, 1991; Balkema and Rizkalla, 1996). A mouse monoclonal anti-synaptophysin antibody was obtained commercially (Boehringer Mannheim, Indianapolis, IN). Secondary antibodies were obtained from
Sigma and Incstar (Stillwater, MN). Texas Red-labeled peanut agglutinin
was obtained from Boehringer Mannheim.
Assay for apoptotic cell death
For consistency, sections from P15 and P20 animals were chosen
that included the optic nerve or were close to it. Apoptotically dying
cells in these sections were detected using an in situ cell death detection kit (Boehringer Mannheim). This kit detects dying cells
by the terminal deoxynucleotidyl transferase-mediated dUTP nick
end-labeling method (Gavrieli et al., 1992), which labels the ends of
fragmented DNA with labeled UTP (here, the UTP is coupled to
fluorescein), using the enzyme terminal deoxynucleotidyl transferase.
Paraformaldehyde-fixed sections (10 µm; see above) were prepared for
labeling by fixation onto slides with 4% paraformaldehyde in PBS for
20 min at room temperature and then were permeabilized and labeled
according to the manufacturer's instructions. After labeling, sections
were mounted in a glycerol-based solution containing paraphenylenediamine (1 mg/ml) to reduce photobleaching.
Electroretinography
Electroretinograms (ERGs) were performed on animals between
postnatal days 20 and 26. All preparative procedures were performed in
normal room light. Animals were anesthetized with Taractan (chlorporthixene; Roche, Nutley, NJ; ~12.5 mg/kg), followed by Nembutal (~65 mg/kg), and then placed into a stereotactic holder. A
reference electrode, made from silver wire, was placed between the skin
and the skull near bregma and secured with cynoacrylate. After suturing
the eye open, a drop of atropine (0.54 mg/ml) was placed onto the eye
for ~1 min. The animal was then placed into a light-tight Faraday
cage. After aligning the animal with the light source, a cotton wick
electrode coupled to a silver-silver chloride half-cell was placed onto
the animal's cornea in a position that did not attenuate the light
flashes. A test flash was presented to the animal to check for proper
electrode placement. After a 21 min dark adaptation, animals were
presented 10 separate 50 msec flashes that were separated by 2 sec
(PS22 Photopic stimulator; Grass Instruments, Quincy, MA); the
responses were amplified using a Dam 50 differential amplifier (World
Precision Instruments, Sarasota, FL) and recorded and averaged using
MacLab 4S (AD Instruments, Mountain View, CA). For each animal, at
least one set of recordings was made over a 4.2 log range of
intensities, starting with least bright. A 21 min period of dark
adaptation preceded any subsequent recording sessions.
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RESULTS |
Mice that are homozygous for the laminin 2-null mutation (here
referred to as laminin 2 chain-deficient) have been previously reported to have disrupted neuromuscular junctions and to suffer from
proteinuria (Noakes et al., 1995a,b). Laminin 2 chain-deficient mice
stop gaining weight during the second postnatal week and normally live
only into the fourth postnatal week. Here, we have characterized
disruptions in one portion of the CNS of these laminin 2
chain-deficient animals through the fourth postnatal week. The animals
lactate normally but are lethargic and move about with difficulty. No
extensive behavioral tests were performed on these animals.
Histology
During the first 10 or 11 postnatal days, retinas from laminin
2 chain-deficient and control mice were not grossly different (results not shown). By P13, however, the first differences between the
laminin 2 chain-deficient and control mice become evident; in
particular, the thickness of the retinas in laminin 2
chain-deficient mice is reduced (results not shown). This difference in
thickness is easily detectable by P15 (Fig.
1). This reduction is attributable almost
entirely to a decrease in photoreceptor length: although nuclear
and plexiform (synaptic) layers in laminin 2
chain-deficient retinas appear largely normal (as judged by width), the
photoreceptor outer segments, and possibly inner segments, are clearly
shorter (Fig. 1). The period during which this difference becomes
apparent is the period when outer segments are undergoing their maximal lengthening.
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Figure 1.
Comparison of wild-type and laminin 2
chain-deficient retinas at postnatal day 15. Sections of wild-type
(A, C) and laminin 2 chain-deficient littermate
(B, D) retinas from areas adjacent to the optic nerve
head are shown. There are no gross effects of laminin 2 chain
deficiency on the outer nuclear layer (ONL), inner
nuclear layer (INL), IPL, or ganglion cell layer
(GCL). However, the outer segments (OS)
and inner segments of the laminin 2 chain-deficient animal are
considerably smaller than those of the wild-type animal, as judged by
the distance from the external limiting membrane (arrow)
to the retinal pigmented epithelium (RPE). C,
D, Enlargements of A and B for
clarity. Scale bar: A, B, 50 µm; C, D,
12.5 µm.
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In control mice, i.e., phenotypically wild type and genotypically
either +/ or +/+, outer and inner segments reach their maximum length
over the subsequent 2 postnatal weeks (weeks 3 and 4 after birth). In
contrast, in laminin 2 chain-deficient mice, outer and inner
segments fail to increase dramatically over this same period. At P25 in
control mice, photoreceptor outer and inner segment lengths (~32 and
16 µm, respectively) have reached the normal range for adult mice
(LaVail, 1973); in contrast, outer and inner segments in retinas from
laminin 2 chain-deficient mice are ~50% shorter (Fig.
2). Thus, by the fourth postnatal week
(that is, at their maximal survival time), laminin 2 chain-deficient mice have severely retarded development of outer and inner segments or
a disposition to shorter outer and inner segments, presumably as a
consequence of the laminin 2 chain deficiency.
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Figure 2.
Comparison of wild-type and laminin 2
chain-deficient retinas at postnatal day 25. Sections of wild-type
(A, C) and laminin 2 chain-deficient littermate
(B, D) retinas, from similar regions, were examined
histologically (A, B) or were reacted with an antibody
specific for rhodopsin (Ret-P1; C, D). Inner segments
(IS) and outer segments (OS) of the
wild-type mouse have reached their adult length by postnatal day 25 (A); both inner and outer segments of the laminin
2 chain-deficient mouse are shorter than those of its littermate
(B). Wild-type and laminin 2 chain-deficient
retinas express rhodopsin and localize it to their outer segments
(C, D). However, comparison of rhodopsin expression
emphasizes the fact that wild-type OS (C) are
significantly longer than those in laminin 2 chain-deficient retinas
(D). RPE, Retinal pigmented
epithelium; ONL, outer nuclear layer. Scale bars:
A, B, 10 µm; C, D, 15 µm.
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Although the morphology of the photoreceptors is clearly altered in
laminin 2 chain-deficient mice, these mice still express the
photopigment rhodopsin in their outer segments. An antibody to
rhodopsin reveals rhodopsin in laminin 2 chain-deficient mice that
is properly targeted to the outer segments (Fig. 2). Although immunohistochemistry cannot determine whether laminin 2
chain-deficient mice produce control amounts of rhodopsin, we predict
that, judging by the production and location of photopigment, these
photoreceptors would be capable of responding to light.
Ultrastructurally, the outer segments of the 2 chain-deficient
animals are normal (Fig. 3). The
membranous disks are well formed and appear normal (Fig. 3, compare
A, B). We did note that the disks were more
loosely packed in some 2 chain-deficient mice than in others, but we
cannot rule out fixation artifacts as the source of this observation
(data not shown). The outer segments of the 2 chain-deficient
animals arise from basal bodies (Fig. 3C, arrows) in the
inner segment, and the ciliary stalk appears normal. Another
abnormality in the outer retina of the 2-deficient animals was the
presence of aberrant processes in the interphotoreceptor matrix (IPM;
Fig. 3D, boxed region shown in higher power in
E). These processes appear to arise from deep in the retina
and extend up to the retinal pigmented epithelium (Fig. 3D),
where they appear to make tight junctions with the overlying retinal
pigemented epithelial cells (Fig. 3E). Although we have not
as yet identified the source of these fibers, we believe that they
arise from Müller cells, which have been shown to sprout in
response to retinal injury (Lewis et al., 1999). We did not observe any
remarkable differences between control and laminin 2 chain-deficient
mice in the ultrastructure of the retinal pigmented epithelium in the
electron microscope, nor did we see any change in their attachment to
their basement membrane, Bruch's membrane (data not shown).
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Figure 3.
Ultrastructural comparison of wild-type and
laminin 2 chain-deficient outer retinas at postnatal day 21. Sections of wild-type (A) and laminin 2
chain-deficient (B-E) outer retinas were viewed
with transmission electron microscopy. Although photoreceptor inner and
outer segments are shorter in the laminin 2 chain-deficient retinas
(B), they appear structurally normal, including
at the basal bodies (C, arrows). Aberrant processes were occasionally observed in the
laminin 2 chain-deficient retinas (D), which
extended through the outer nuclear layer and to the retinal
pigmented epithelium. The boxed area in D
is shown at higher power in E. Scale bar: A,
B, 4.4 µm; C, 1.1 µm; D, 8.5 µm; E, 2.5 µm.
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In a screen of laminin 2 chain-deficient retinas, we did not detect
any marked changes in the numbers or morphology at the light-microscopic level of several other cell types, including bipolar
cells (using an antibody against protein kinase C; Greferath et
al., 1990), horizontal cells (using an antibody against calbindin; Massey and Mills, 1996), and Müller cells (using an antibody to
vimentin; Dräger et al., 1984) (data not shown). These data, when
coupled with the apparent normal anatomy of the inner retinas from
laminin 2 chain-deficient mice (see above), suggest that mainly
photoreceptor development is altered by the removal of the laminin 2 chain.
Apoptosis
Programmed cell death, i.e., apoptosis, is a normal part of
retinal morphogenesis, occurring during specific periods of retinal development. In the mouse retina, the number of cells undergoing cell
death generally follows a Gaussian distribution, beginning before
birth, peaking at ~P9, and ceasing at ~P25 (Young, 1984). Also,
within this distribution, inner retinal cells (i.e., ganglion cells and
amacrine cells) die earlier than outer cells (i.e., photoreceptors and
bipolar cells). To test whether the laminin 2 chain-deficient mice
were undergoing these normal developmental processes, we assayed for
programmed cell death on two different critical days, P15 and P20.
At P15 in our control wild-type mice, the type (primarily rod
photoreceptors, based on location) and number (16.5 ± 1.5 cells per section, mean ± SEM) of cells undergoing programmed cell
death is similar to that previously reported (Young, 1984). Laminin 2 chain-deficient mice had approximately twice the amount of programmed cell death (35.7 ± 6.1 cells per section).
In normal mouse development, by the 20th postnatal day the number of
cells undergoing cell death declines rapidly and is limited mainly to
photoreceptors. At P20, wild-type retinas contained fewer apoptotic
cells (3.6 ± 1.2 cells per section) than at P15; laminin 2
chain-deficient mice also have fewer apoptotic cells at P20 (9.3 ± 2.2 cells per section) than at P15.
Therefore, laminin 2 chain-deficient mice do have elevated
programmed cell death; however, they are still following the basic developmental trend: a decrease in dying cells with age. Moreover, the
rate of decrease in cell death is parallel to that of the rate in the
littermate controls, suggesting that cell death in the laminin 2
chain-deficient mice is slowing as it should. In addition, the increase
in dying cells in the laminin 2 chain-deficient mice is relatively
small in number when compared with the total number of retinal cells
(many thousands per retinal section), so that there is probably little
or no effect on retinal function merely because of a paucity of cells.
It should also be noted that at both ages examined there is no apparent
clumping of apoptotic cells that would suggest necrosis (Vaux, 1993);
apoptotic cells are generally surrounded by viable cells. This suggests
that the retina of the laminin 2 chain-deficient mouse is not
significantly affected by the other pathologies (e.g., kidney
disturbances) occurring in the animal.
Laminins in the IPM of laminin 2 chain-deficient mice
Our early work on retinal laminins focused on the apical
expression of the laminin 2 chain, i.e., within the
interphotoreceptor matrix (Hunter et al., 1992, Libby et al., 1996,
1997). More recently, we have shown that the laminin 2 chain is
co-expressed with the laminin 3, 4, and 3 chains on the apical
surface of the retina but also in the outer plexiform layer; the inner
plexiform layer appears to contain other laminins, because of the known
laminin chains only 4 is expressed there (Libby et al., 1997b;
Libby, Xu, Gibbons, Champliaud, Koch, Burgeson, Hunter, and Brunken, unpublished data).
We have proposed that the retina contains two novel laminin
heterotrimers, which we have termed laminin 13 (323) and
laminin 14 (423) (Libby, Xu, Gibbons, Champliaud, Koch,
Burgeson, Hunter, and Brunken, unpublished data). Does the lack of the
laminin 2 chain result in a decreased synthesis or deposition of its
potential partners in the retina? For example, disruptions in the gene
encoding the 3 chain of laminin 5 (332) result in a failure
of the other chain partners to be assembled and secreted (Matsui et
al., 1995, 1998).
We have examined the retinas of wild-type and laminin 2
chain-deficient animals for the presence of the other components of
laminins 13 (323) and 14 (423), i.e., the laminin
3, 4, and 3 chains. We examined the expression of RNAs
encoding these three chains (by in situ hybridization). We
have not detected any changes in expression of RNA encoding these
chains (results not shown), suggesting that there are no gross changes
in transcription of these chains as a result of the removal of the
laminin 2 chain.
We have only been able to examine the protein expression of two of the
chain partners of 2, because there is currently no mouse-reactive
anti-3 chain reagent available. As we have shown elsewhere for
normal rat and human retinas (Libby et al., 1997b; Libby, Xu, Gibbons,
Champliaud, Koch, Burgeson, Hunter, and Brunken, unpublished data), the
laminin 4 and 3 chains are present in the retinas of wild-type
mice (Fig.
4A,C).
In all three species, these laminin chains are present in three
distinct locations: (1) the interphotoreceptor matrix, (2) the outer
plexiform layer, and (3) the inner plexiform layer, where only the 4
chain is expressed. Although full-length laminin 2 chain protein is
lacking in laminin 2 chain-deficient mouse, we cannot detect any
gross disturbances in the distribution of its presumed partners, the laminin 4 and 3 chains, in the laminin 2 chain-deficient
retinas in any of these locations (Fig.
4B,D). These data are consistent with the apparent lack of change in expression of RNA encoding these
chains and suggest that the loss of the laminin 2 chain protein does
not result in a gross change in the deposition of the laminin 4 and
3 chains.
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Figure 4.
Other laminin chains are not disrupted by laminin
2 chain deficiency. Unfixed, transverse sections of wild-type and
laminin 2 chain-deficient (2) littermate retinas
were reacted with several antibodies that recognize other laminin
chains. The expression pattern of the laminin 4 (A,
B) and 3 (C, D) chains, potential laminin
trimer partners of the laminin 2 chain in the neuronal retina,
appear to be identical in wild-type (A, C) and laminin
2 chain-deficient (B, D) mice: the laminin 4 and
3 chains are still present in the subretinal space around inner
segments (arrowheads) and in the outer plexiform layer
(arrows). The laminin 4 chain (A, B)
is also present in both mice throughout the inner retina, through the
inner nuclear layer (INL) and the ganglion cell layer
(GCL). ONL, Outer nuclear layer. Scale
bar, 25 µm.
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Laminins are assembled intracellularly, first as dimers, to
which chains are added, allowing export (Matsui et al., 1995; Yurchenco et al., 1997). Notably, in the absence of the laminin 2
chain, the laminin 4 and 3 chains continue to be expressed in the
IPM, wherein they are almost certainly extracellular (Hunter et al.,
1992; Libby et al., 1997b). These data suggest that there is a
compensatory expression of another, perhaps unknown, laminin chain
in trimers containing the 4 and 3 chains. Such compensatory mechanisms have been suggested to be present in the kidney of laminin
2 chain-deficient animals (Noakes et al., 1995b).
In the kidney of laminin 2 chain-deficient mice, the laminin 1
chain has been shown to be ectopically expressed in what would normally
be a laminin 2 chain pattern. Therefore, we examined the
distribution of the laminin 1 chain in the retina of these animals
to ascertain whether it could substitute for the laminin 2 chain in
retinal laminin trimers containing the 4 and 3 chains. Using a
polyclonal antiserum that reacts with all three chains of laminin-1
(1, 1, and 1), we can only detect the laminin chains that are
associated with the retinal vasculature; we found no ectopic laminin
1 chain (or laminin 1 and 1 chains) in the laminin 2
chain-deficient retinas (results not shown). We have no mouse-reactive
anti-laminin 3 chain antibodies, leaving open the possibility that
the laminin 3 chain, which is not normally associated with the adult
neuronal retina (Libby et al., 1997b), may substitute for the laminin
2 chain in the laminin 2 chain-deficient retinas. However,
in situ hybridizations using probes that detect RNA
encoding the laminin 3 chain suggest that there are no alterations in expression of the RNA encoding this chain. It is, therefore, plausible that a novel laminin chain may substitute for the laminin
2 chain in the interphotoreceptor matrix and outer plexiform layer
of laminin 2 chain-deficient mice.
Light responses
An ERG documents the summed electrical activity of the retina,
thereby yielding information about its overall physiology. In
particular, an ERG can describe independently the response of
photoreceptors and outer retinal interneurons to light (Dowling, 1960;
Brown and Wiesel, 1961): photoreceptor responses are present as an
initial downward deflection, known as the a-wave, whereas the
transmission to second-order retinal interneurons, dominated by the
contribution of depolarizing bipolar cells, is present as a subsequent
upward deflection, known as the b-wave (Fig.
5). The amplitude of these waves, their
shape, and the time it takes to reach a peak voltage ("time to
peak" or "implicit time") can be used as diagnostic tools to
determine the physiological state of the retina.
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Figure 5.
Electroretinography of wild-type and laminin 2
chain-deficient retinas. A, Top, The ERGs
of a P21, wild-type mouse (left) and a P20, laminin 2
chain-deficient mouse (right) are shown at maximum light
intensity. The laminin 2 chain-deficient mouse exhibits a normal
a-wave; however, its b-wave is markedly attenuated.
Bottom, Intensity-response series (over 4.2 log
units, after 21 min of dark adaptation) of a P18, wild-type mouse
(left) and a P20, laminin 2 chain-deficient mouse
(right). The a- and b-waves of the wild-type mouse
increase with increasing levels of light. The laminin 2
chain-deficient mouse shows a similar intensity-response profile, but
the b-wave is clearly altered. Calibration: 20 µV
(vertical); 100 msec
(horizontal). B, The average
a-wave amplitudes (triangles) and b-wave amplitudes
(squares) for wild-type mice (open
symbols) and laminin 2 chain-deficient mice
(filled symbols) are shown over a wide range of
light intensities. The a-waves of the wild-type mice and the laminin
2 chain-deficient mice are similar; in contrast, the b-waves are
attenuated in laminin 2 chain-deficient mice over the entire
range.
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We performed ERGs on phenotypically wild-type (both +/+ and +/) and
laminin 2 chain-deficient (/) mice. At maximum stimulus intensity, the ERG of a control, wild-type mouse (Fig. 5A, top left trace) is typical, with a photoreceptor-driven a-wave
overtaken at ~50 msec by the field potential arising from the
second-order interneurons, the b-wave. The b-wave reaches its peak at
~100 msec, and then the ERG falls quickly back to baseline.
On the other hand, the ERGs obtained from the laminin 2
chain-deficient mouse are abnormal (Fig. 5A, top right
record). The a-wave appears largely normal in terms of its
amplitude at maximal light intensity; although the example shown in
Figure 5A has a slightly increased amplitude, this is not
representative of the population as a whole (see below). The mean
a-wave amplitude for 2 chain-deficient mice (n = 7)
was 69.9 ± 7.7 µV (mean ± SEM) compared with 85.3 ± 18.9 µV in control mice (n = 9). The time to peak
(implicit time) was also unaltered (44.5 ± 1.4 msec in the 2
chain-deficient mice compared with 49.6 ± 2.4 msec in the wild-type mice). This suggests that, although the morphology of the
photoreceptor outer segments is clearly affected by the lack of the
laminin 2 chain, the overall ability of the photoreceptors to
respond to light is relatively unaffected (see below).
In contrast to the largely normal a-wave, there is a marked disruption
of the b-wave of ERGs from laminin 2 chain-deficient animals (Fig.
5A, top right record). Although the b-waves have normal
implicit times (98.8 ± 4.2 msec in / mice compared with 100.2 ± 7.7 msec in wild-type mice), their mean amplitudes at maximal light intensity are significantly reduced (to 136 ± 13.4 µV in the / mice from 269 ± 51.8 µV in control mice;
p < 0.05). Moreover, the ERG does not return to
baseline as in the wild-type mouse; rather, it generally remains well
above baseline for several seconds. This standing wave is frequently
persistent and can be seen at the outset of subsequent stimulation (2 sec after the previous stimulation); this is most evident at
particularly high light intensities.
Next we examined the intensity-response functions of the ERG in both
wild-type and mutant animals. Wild-type (+/ and +/+) mice exhibit the
normal response of the retina to increases in light intensity (Fig.
5A, bottom left records, plotted in B, open symbols): (1) at low light levels, the b-wave is the only
electrical activity observable in the ERG; as the light intensity
increases, its amplitude increases and its implicit time decreases; and
(2) a-waves are first detectable at brighter light levels than b-waves; like b-waves, the a-wave amplitude increases and implicit time decreases with increasing light intensity.
The response of laminin 2 chain-deficient retinas to increases in
light intensity is fundamentally similar to that of wild-type mice: the
amplitudes of both a- and b-waves increase with increasing light
intensity (Fig. 5A, bottom right records, plotted in
B, filled symbols). However, at maximal
intensities, although the a-waves of the laminin 2 chain-deficient
retinas are similar to those of wild-type controls, the b-waves are
greatly attenuated. The response-intensity functions for both a- and
b-waves show that, over the whole range of light intensities, the
laminin 2 chain-deficient retinas exhibit near-normal a-waves (Fig.
5B, filled triangles). There is some apparent reduction at
the maximal intensity of light in the a-wave amplitude, but this
reduction is not statistically significant. Such a reduction would be
expected given the reduction in the volume of the photoreceptors outer segment.
On the other hand, the b-waves of the laminin 2 chain-deficient mice
are attenuated at all light intensities tested (Fig. 5B).
The ratio of the b- to a-wave amplitudes is a sensitive measure of the
transfer function between photoreceptors and second-order neurons. The
b- to a-wave ratios (taken at maximal light intensity) are
significantly lower in the laminin 2 chain-deficient mice (1.99 ± 0.13 vs 3.33 ± 0.19; p < 0.01). Together,
these data suggest that the laminin 2 chain-deficient retinas are
capable of detecting light nearly normally, but that information
transfer from photoreceptors to second-order cells (the retinal bipolar
cells) is compromised. Further support for this suggestion comes from
the shape of the b-wave intensity-response curve in laminin 2
chain-deficient retinas (Fig. 5B, filled squares,
heavy line), which is considerably different from that in
control animals; the b-wave in 2 chain-deficient mice is flatter and
approaches a linear function, rather than the exponential shape in the
control (Fig. 5B, open squares). The exponential shape of
the b-wave response-intensity function reflects the transfer
properties of the synapse; the change from an exponential to a linear
function in the mutant mouse suggests that the photoreceptor to bipolar
transmission is not as efficacious in the mutant mice.
Synaptic integrity in laminin 2 chain-deficient retinas
The physiological data suggest that there is reduction in synaptic
transmission in the outer retina, i.e., in the OPL. Therefore, we
examined the OPL in detail for any defects in synaptic formation with
both the light and electron microscopes. Our previous studies on
laminin expression have focused on the apical surface of the retina
(Hunter et al., 1992, Libby et al., 1996); however, more recently, we
showed that the component chains of laminin 13 and 14 are expressed not
only on the apical surface of the retina but also in the OPL of rat and
human retinas (Libby et al., 1997b; Libby, Xu, Gibbons, Champliaud,
Koch, Burgeson, Hunter, and Brunken, unpublished data). Unfortunately,
we have no mouse-reactive anti-laminin 2 chain antibodies; however,
in the rat OPL, laminin 2 chain immunoreactivity co-localizes with
that of B16, a marker of the synaptic ribbon (Fig.
6A-C). Because the and chains of laminin 14 are present in the mouse OPL (see Fig. 4),
we presume that the laminin 2 chain is expressed in the mouse OPL as
it is the rat retina. Both reverse transcription PCR and in
situ hybridizations (data not shown) have established that laminin
2 chain RNA is expressed in mouse retina, and, thus, we have no
reason to think there any species difference among mouse, rat, and
primate. Thus, laminins containing the 2 chain are localized to the
photoreceptor synapse and ideally situated to mediate some of the
processes of synaptic formation and stabilization.
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Figure 6.
The OPL is disrupted in laminin 2
chain-deficient mice. As a mouse-reactive laminin 2 chain antibody
was not available, 2 chain expression is shown in rat OPL
(A-C). B16 is an antibody that recognizes
photoreceptor ribbon synapses (A) and
co-localizes with the laminin 2 chain (B) in
the OPL (images in A and B are merged in
C). Note that the laminin 2 chain is also present in
capillaries (*), whereas the B16 antigen is not. D-G,
Transverse sections of postnatal day 25 wild-type (D,
F) and laminin 2 chain-deficient littermate
(E, G) retinas were reacted with a lectin (peanut
agglutinin; D, E) that recognizes cone outer segments
(OS) and cone photoreceptor terminals
(arrows) or an antibody that recognizes all
photoreceptor synaptic termini (anti-synaptophysin; F,
G) within the OPL, located between the outer nuclear layer
(ONL) and the inner nuclear layer (INL).
Photoreceptor synapses in the laminin 2 chain-deficient retina
appear disorganized. Scale bars, 25 µm.
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Peanut lectin, which specifically labels cone photoreceptors, is
normally expressed in discrete patches in the OPL, reflecting the
regularly, and widely, spaced cone pedicles (Fig. 6D,
arrows point to individual pedicles). In contrast, in the
mutant mice, peanut lectin binding appears to be a nearly continuous
band, as if adjacent cone pedicles have expanded their bases (Fig.
6E). Synaptophysin, which is expressed at presynaptic
sites, is confined to the terminals of wild-type mice (Fig.
6F); in the 2 chain-deficient mouse, it is
expressed along the axon of the photoreceptor as well as in the
terminal, suggesting a disruption in the molecular anatomy of the
terminals (Fig. 6G). These alterations, albeit minor, of the
synapse suggested that ultrastructural investigations of the
photoreceptor anatomy were warranted.
To describe further the synaptic phenotype, we examined retinas from
seven animals (four mutants and three controls) at P21-P25, an age
when the OPL is mature (Olney, 1968a,b; Blanks et al., 1974; McArdle et
al., 1977). In wild-type mice, photoreceptor synapses usually are seen
as a classic "triad," in which the photoreceptor terminal, complete
with its synaptic ribbon and underlying arciform density demarcating
the active zone, are apposed to the dendrites of three postsynaptic
cells: one central, bipolar element and two lateral, horizontal cell
elements (Fig. 7A,
*). This structure predominates in normal retinas (Table
1). However, as a result of the
complexity of the invaginations into the photoreceptor terminal, the
central bipolar dendrite can be missed if the plane of section is not
through the long axis of the terminal; under this circumstance, one
only sees the horizontal processes (Table 1, dyads). Together, triads
and dyads represent ~95% of the synapses in the wild-type retina and
confirm that OPL development is largely complete at this age.
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Figure 7.
Ultrastructural comparison of synapses within the
outer plexiform layer of wild-type and laminin 2 chain-deficient
retinas at postnatal day 21. Sections of wild-type
(A) and laminin 2 chain-deficient
(B-H) retinas were viewed with transmission
electron microscopy. In the normal retina, rod photoreceptors make
synapses, called triads (T), with three
postsynaptic elements (*); the ribbon is a presynaptic specialization
that marks the active site for release; the three postsynaptic elements
invaginate into the base of the photoreceptor such that two horizontal
cell dendrites lie laterally, and one bipolar cell dendrite lies
centrally. The photoreceptor wraps around the bipolar cell
(arrows). In the 2 chain-deficient animal
(B-H), several different types of synapses are
present. Triads (T) like those in the wild type
animal are rare; dyads, with only one or two horizontal cell processes
apposed to the ribbon, are more common (D); dyads
are seen in wild-type retinas as well (not illustrated). Also common in
the 2 chain-deficient retinas are floating synapses
(F), wherein a fully formed ribbon, often with
vesicles associated, is seen without any postsynaptic element apposed.
This type of synapse is seen extremely rarely in the wild-type retina
(see Table 1). Finally, occasionally, two ribbons from the same
photoreceptor will be apposed to a single postsynaptic element
(2); this was also rarely observed in the
wild-type animal. Scale bar: A, D, E, 850 nm; B,
C, F-H, 630 nm.
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In marked contrast, the OPLs of laminin 2 chain-deficient mice
rarely contain fully formed triads (Table 1); instead, a variety
malformations are present (Fig. 7B-H). A few triads
(T in Fig. 7B,D) are present; these represented
<8% of all synapses. There were numerous examples of a ribbon complex
apposed to one or two postsynaptic elements, which we could identify as
horizontal cell dendrites by the presence of synaptic vesicles
(D in Fig. 7B,C,E); these were scored together as
dyads (Table 1) and accounted for nearly 50% of all synaptic
complexes, which is not significantly different from the control
retinas. Together, triads and dyads, relatively mature synapses,
account for only ~55% of the ribbon synapses in the mutant mice. We
observed with low frequency multiple ribbons apposed on one
postsynaptic element (2 in Fig. 7E,H). However,
the most conspicuous malformations we saw were structures we call
floating ribbons (Fig. 7C,D,F-H); these are fully
assembled ribbon complexes with associated synaptic vesicles that are
unapposed to any postsynaptic element; these structures account for
44% of all ribbon structures in the laminin 2 chain-deficient
retinas (Table 1). This profound disruption in the anatomical
arrangement of the photoreceptor output synapse is likely to account
for the disruption in the b-wave in the mutant animals.
To ask whether the defect is a result of malformation or failure for
bipolar cells to develop properly, we examined the inner plexiform
layer (IPL). In the IPL, bipolar cells make output synapses with a
ribbon structure onto amacrine and ganglion cells (Fig. 8A,E, R).
The IPL also contains conventional synapses between amacrine cells and
from amacrine cells onto bipolar and ganglion cells (not illustrated
for control). Examination of the IPL of the 2 chain-deficient mouse
revealed no striking abnormalities; both well formed ribbon synapses
(Fig. 8B,F, R) and conventional synapses
(Fig. 8C,D,G, C) were present. Although not
exhaustive both light (data not shown) and these electron microscopic
studies suggest that the IPL is well developed, which is not
unexpected, because the laminin 2 chain is not expressed in this
region. Therefore, the apparent defect in the 2 chain-deficient
mouse is in the outer retina, consistent with the expression pattern of
this molecule, i.e., in the IPM and in the matrix of the OPL.
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Figure 8.
The ultrastructure of the inner plexiform layer of
wild-type and 2 chain-deficient animals is similar. Bipolar cell
output synapses are ribbon-type synapses (R),
whereas amacrine cells make conventional synapses
(C) onto other amacrine cells. Two examples of
ribbon synapses in wild-type animals (A, E) are shown;
apposing the ribbon are one or two postsynaptic processes of unknown
origin. Synapses in 2 chain-deficient animals (B-D, F,
G) illustrate that the IPL of the mutant animal has both normal
ribbon synapses (B, F) and normal conventional
synapses (C, D, G), one of which contains a dense-core
vesicle (arrowhead). These data suggest that
synaptogenesis has proceeded normally in the IPL. Scale bar:
A, 340 nm; B-G, 250 nm.
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DISCUSSION |
Laminin 2 chain-deficient mice have two basic morphological
abnormalities: (1) shortened inner and outer segments and (2) disrupted
synaptic connections between photoreceptors and second order cells.
These mice also have a basic functional abnormality: ineffective
transmission of light stimuli from photoreceptors to retinal
interneurons, presumably as a result of the disrupted photoreceptor
synapses. We believe that these disruptions result from a block of
normal photoreceptor differentiation in laminin 2 chain-deficient
retinas. Synaptogenesis and outer segment morphogenesis occur in the
first and second postnatal weeks and happen simultaneously (Fig.
9). First, an apical process is
elaborated as the growth cone of the axon makes contact with horizontal
cell processes (P4); later, the outer segment arises, and horizontal
cell dendrites invaginate into the terminal (P7); maturation is
complete when the bipolar cell invades between flanking horizontal
cells and the outer segments reach their adult length (P14). In the
2-deficient mouse, final stages of photoreceptor morphogenesis are
halted.
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Figure 9.
Disruption of laminin 2 chain production leads
to disruption of photoreceptor development. During normal development,
photoreceptors begin to develop from an undifferentiated progenitor
cell between P0 and P4, at which time they begin to form contacts with
processes from interneurons within the outer plexiform layer. The
normal developmental program begins to falter in laminin 2
chain-deficient retinas at this stage, with poor development of outer
segments and synaptic contacts by P7 and little progress past this
point. The net result is that, by P14, the morphogenesis of the
photoreceptor is markedly disrupted. This is likely to contribute to
the inadequate function that is readily apparent in laminin 2
chain-deficient retinas.
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Differences in disruption of the 2 chain in vitro
and in vivo
Using an in vitro system, we have suggested that the
2 chain, directly or indirectly, is at least partly responsible for the differentiation of rod photoreceptors (Hunter et al., 1992; Hunter
and Brunken, 1997). This result is consistent with the in
vivo data reported here, wherein a lack of the 2 chain results in poor differentiation of rods, i.e., their outer segments and synapses. Previously, we showed that the 2 chain modulates a choice
between rod photoreceptor and rod bipolar cell fates in vitro (Hunter and Brunken, 1997), a choice that is not reflected in the data reported here.
Why are the results of our in vitro and in vivo
disruptions of the 2 chain different? Perhaps the most unsatisfying,
but most likely, explanation is that there are redundant developmental pathways in vivo that are absent in vitro. FGF-2
(Hicks and Courtois, 1992), taurine (Altshuler and Cepko, 1992), and
retinoic acid (Kelly et al., 1994) have all been shown to promote rod
photoreceptor development, whereas TGF- (Lillien and Cepko, 1992),
leukemia inhibitory factor (Neophytou et al., 1997), and CNTF
(Ezzeddine et al., 1997; Neophytou et al., 1997) have all been shown to
inhibit photoreceptor development. With so many different factors
potentially influencing the development of photoreceptors, it is not
surprising that the absence of any single factor may not be sufficient
to prevent a progenitor cell from differentiating into a photoreceptor but may be sufficient to cause a disruption in the overall
differentiation program.
Another possibility is that, although we have functionally removed the
2 chain in vitro and in vivo, the retinal
laminins have been disrupted in different ways in the two systems. As
noted above, several of the laminin chains that are thought to be chain partners with the 2 chain are present in the adult retina (Fig. 4);
therefore, in vivo, a non-2 chain may substitute in a
laminin that may subserve some of the functions of 2
chain-containing laminins.
Is the health of the 2 chain-deficient mice contributing to the
retinal phenotypes?
2 chain-deficient mice stop growing during the second week of
life and eventually die, probably as a result of proteinuria, during
the fourth week of life. Because the animal is unhealthy during this
period, it is possible that the alteration in phenotype that we have
attributed to a direct effect of the lack of the 2 chain in the
retina is, instead, secondary to extraretinal pathologies. However, it
is unlikely that slow growth affects photoreceptor outer segment
length: rodents that are malnourished and have growth curves similar to
those of 2 chain-deficient mice do not have altered photoreceptor
outer segment length during their third week of life (Sinning et al.,
1984), the period when the 2 chain-deficient mice show considerably
shorter outer and inner segments. Thus, simple nutritional deficiency
is not sufficient to cause stunted outer and inner segments.
In situations of malnutrition, particularly vitamin A deficiency, ERG
abnormalities are markedly different from those of 2 chain-deficient
animals: the a-wave almost completely disappears before the b-wave
begins to attenuate. In later stages of vitamin A deficiency, the
photoreceptors begin to degenerate (Dowling, 1960). Similarly, in the
photoreceptor degenerative disorders, generally, the a-wave is the
first to be attenuated, followed by the b-wave. The normal a-wave in
the ERG of the 2 chain-deficient mice suggests that the
photoreceptors are healthy and, importantly, not undergoing degeneration.
Disruption of retinal morphology
The OPL is disrupted in retinas of 2 chain-deficient animals,
whereas the IPL remains largely unaffected. There are at least two
potential loci where the loss of the 2 chain could affect development of this layer. First, removal of the 2 chain from the
ventricular surface of the retina could adversely affect not only outer
segment formation but also overall photoreceptor development, such that
all photoreceptor structures, including terminals, might be disrupted.
Second, because 2 chain protein is expressed, along with its chain
partners, in the matrix of the OPL, it is possible that the disruption
of the OPL is a direct result of the loss of the 2 chain at or near
the synapses.
It is novel, but not surprising, to find an effect of the 2 chain
associated with a synaptic layer in the CNS. Laminins containing the
2 chain are found at a peripheral synapse, the neuromuscular junction (e.g., Hunter et al., 1989; Patton et al., 1997). Furthermore, the neuromuscular junction is similarly disorganized in the 2 chain-deficient mouse, suggesting that it is an important factor in
differentiation and stabilization at this peripheral synapse. Thus, by
analogy, the 2 chain could be important in the differentiation and
function of particular central synapses, including the rod photoreceptor synapses within the OPL of the retina. The apparent disruption in the process of the Müller glial cell in the outer retina is also consistent with the finding at the neuromuscular junction in which the glial cell behavior is aberrant (Patton et al.,
1998).
Disruption of retinal physiology
The ERG a-wave, a measure of photoreceptor activity (Dowling,
1960; Brown and Wiesel, 1961), has been related to the rhodopsin content of photoreceptors and, therefore, their size. Thus, the lack of
an altered a-wave in the 2 chain-deficient mice, which have shorter
outer segments, is counterintuitive. However, although rhodopsin
concentration is likely an important determinant of the a-wave, during
development in the rat a near-normal a-wave function can be achieved
with only 50% of the normal content of rhodopsin (Fulton et al.,
1995). Thus, it is possible that there is enough rhodopsin in the
shortened outer segments of the 2 chain-deficient retinas to
generate a nearly normal photoresponse.
The most striking alteration in the ERGs of 2 chain-deficient mice
is in their severely attenuated b-wave, the wave generated by the
activation of the second-order neurons. A likely explanation for the
selective attenuation of the b-wave is the synaptic disturbance in the
OPL itself. Because the 2 chain is associated with the OPL in
mammals, a deficiency in the laminin 2 chain could result in an
improperly formed or functioning OPL, which then could directly explain
the attenuated b-wave. Moreover, the intensity-response relationship of the b-wave is markedly altered in the mutant mice (Fig.
4B); in particular, the function becomes nearly
linear with a marked attenuation at the higher intensities. These
observations suggest that the photoreceptor output synapse is not able
to function effectively at the higher rates of vesicular fusion seen at
high light levels. These alterations in b-wave properties are analogous to the electrophysiological alterations observed at the 2
chain-deficient neuromuscular junction, where both a decrease in
synaptic efficacy and rates of spontaneous release were seen (Noakes et
al., 1995a).
The alterations in ERGs seen in the 2 chain-deficient retina are
remarkably similar to those reported after disruptions in the protein
dystrophin, as seen in humans (in Duchenne and Becker muscular
dystrophy) and in mice (in the mdx and
mdxCv3 mutants): a normal a-wave and
an attenuated b-wave. Dystrophin is present in photoreceptor terminals
in many mammals, including mouse and human, and the attenuated b-wave
in dystrophin mutant mice is thought to be the result of disruptions at
those terminals (Schmitz et al., 1993; Fitzgerald et al., 1994; Pillars
et al., 1995; Ueda et al., 1997); however, disruptions of the synaptic terminals on the scale shown here are not reported in the
mdxCv3 mutant (Blank et al.,
1999).
Dystrophin can interact with laminin via the dystroglycan complex (for
review, see Sunada and Campbell, 1996); at least part of this complex
is present at photoreceptor synapses. Because genetic disruptions in
dystrophin and the laminin 2 chain cause similar attenuations of the
b-wave, and the outer plexiform layer contains at least part of the
complex that may link dystrophin and laminins, we speculate that these
two components may act in concert in the differentiation and
stabilization of photoreceptor synapses. This, again, would be
analogous to the neuromuscular junction, where disruptions in both the
laminin 2 chain and dystrophin result in neuromuscular pathologies.
We further speculate that such interactions may be important at
additional central synapses.
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FOOTNOTES |
Received Feb. 18, 1999; revised Aug. 12, 1999; accepted Aug. 18, 1999.
This work was supported by funds provided by Boston College (W.J.B.,
G.W.B.), Tufts University and the National Eye Institute (D.D.H.), and
the Foundation Fighting Blindness and the E. Matilda Ziegler Foundation
(W.J.B.). Some of this work was supported by the core facilities of the
Cutaneous Biology Research Center (CBRC) under a Massachusetts General
Hospital-Shiseido Co., Ltd. agreement. We thank Josh Sanes for the
generous gift of the founder mice for our 2 knock-out colony, Colin
Barnstable for the gift of the antibody against rhodopsin; Josh Sanes
and Jeff Miner for the gift of the antibody against the laminin 4
chain, and Bob Burgeson and Marie-France Champliaud for the gift of the
antibody against the laminin 3 chain. W.J.B. thanks Bob Burgeson for
encouragement and generosity during his leave of absence at the CBRC.
Correspondence should be addressed to Dr. William J. Brunken, Cutaneous
Biology Research Center, Massachusetts General Hospital, Harvard
Medical School, Building 149, 13th Street, Charlestown, MA 02129. E-mail: bill.brunken{at}cbrc2.mgh.harvard.edu.
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ABSTRACT
INTRODUCTION
