Functional Requirement for Class I MHC in CNS Development and Plasticity

Abstract

Class I major histocompatibility complex (class I MHC) molecules, known to be important for immune responses to antigen, are
expressed also by neurons that undergo activity-dependent, long-term structural and synaptic modifications. Here, we show
that in mice genetically deficient for cell surface class I MHC or for a class I MHC receptor component, CD3ζ, refinement
of connections between retina and central targets during development is incomplete. In the hippocampus of adult mutants,N-methyl-d-aspartate receptor–dependent long-term potentiation (LTP) is enhanced, and long-term depression (LTD) is absent. Specific
class I MHC messenger RNAs are expressed by distinct mosaics of neurons, reflecting a potential for diverse neuronal functions.
These results demonstrate an important role for these molecules in the activity-dependent remodeling and plasticity of connections
in the developing and mature mammalian central nervous system (CNS).

Full text

Functional Requirement for
Class I MHC in CNS
Development and Plasticity
Gene S. Huh,* Lisa M. Boulanger, Hongping Du, Patricio A. Riquelme,
Tilmann M. Brotz,† Carla J. Shatz*
Class I major histocompatibility complex (class I MHC) molecules, known to be
important for immune responses to antigen, are expressed also by neurons that
undergo activity-dependent, long-term structural and synaptic modifications.
Here, we show that in mice genetically deficient for cell surface class I MHC or
for a class I MHC receptor component, CD3␨, refinement of connections be-
tween retina and central targets during development is incomplete. In the
hippocampus of adult mutants, N-methyl-
D-aspartate receptor–dependent
long-term potentiation (LTP) is enhanced, and long-term depression (LTD) is
absent. Specific class I MHC messenger RNAs are expressed by distinct mosaics
of neurons, reflecting a potential for diverse neuronal functions. These results
demonstrate an important role for these molecules in the activity-dependent
remodeling and plasticity of connections in the developing and mature mam-
malian central nervous system (CNS).
The development of precise connections in
the CNS is critically dependent on neural
activity, which drives the elimination of in-
appropriate connections and the stabilization
of appropriate ones. In the visual system of
higher mammals, the refinement of initially
imprecise axonal connections requires spon-
taneously generated activity early in develop-
ment and visually driven activity later (1–4).
Fine-tuning of neural connectivity is thought to
result from changes in synaptic strength, driven
by patterned impulse activity (1, 2, 5, 6).
To identify molecules critical for activity-
dependent structural remodeling, we previ-
ously conducted an unbiased screen for
mRNAs selectively regulated by blocking
spontaneously generated activity in the devel-
oping cat visual system. This manipulation
prevents the remodeling of retinal axons from
each eye into layers within the lateral genic-
ulate nucleus (LGN) (7–9). Although many
known neural genes were not detectably reg-
ulated by activity blockade, this screen re-
vealed to our surprise that members of the
class I MHC protein family are expressed by
neurons and are regulated by spontaneous
and evoked neural activity (10). Neuronal
class I MHC expression corresponds to well-
characterized times and regions of activity-
dependent development and plasticity of
CNS connections, including retina, LGN, and
hippocampus. Furthermore, the mRNA for
CD3␨ [a class I MHC receptor subunit in the
immune system (11)] is also expressed by
neurons (10), consistent with its interaction
with class I MHC during activity-dependent
remodeling and plasticity. Although class I
MHC is primarily known for its function in
cell-mediated immune recognition, the above
findings from our differential screen suggest
that class I MHC molecules may play roles in
structural and synaptic remodeling in the de-
veloping and mature CNS.
To explore these possibilities by genetic
means, we first confirmed by in situ hybrid-
ization that class I MHC and CD3␨ were
expressed in the developing mouse CNS. Be-
cause numerous class I MHC genes exist in
the mouse genome (12), we used a pan-
specific cDNA probe expected to detect
many class I MHC molecules (13). This
probe detected elevated amounts of mRNAs
in the dorsal LGN (dLGN) during the first
two postnatal weeks, exactly when ganglion
cell axons sort into eye-specific layers in the
mouse (14); mRNA levels declined at later
ages (Fig. 1A, compare postnatal days P6 and
P40). Expression was also evident in the gan-
glion cell layer of the retina (Fig. 1A, P6 eye),
in neocortex (in layer 4 at early ages and in
deeper layers later; Fig. 1A), and in granule
and pyramidal cell layers of the hippocam-
pus (Fig. 1A, P40, and Fig. 2). CD3␨
mRNA, like that of class I MHC, was
expressed in the mouse dLGN during the
first two postnatal weeks (Fig. 1B); expres-
sion appeared higher medially. CD3␨
mRNA was also detected in small amounts
in P40 hippocampus (15). Therefore, as in
cat (10), class I MHC and CD3␨ transcripts
are present in the developing murine CNS at
locations and times consistent with a role for
these molecules in activity-dependent struc-
tural remodeling and synaptic plasticity.
Strikingly, different class I MHC genes
are expressed in unique subsets of neurons
throughout the mature CNS, as revealed by
using probes (13) that react more specifically
with each of two class Ia (H–2D, H–2K)or
two class Ib MHC genes (Qa-1, T22). For
example, within the somatosensory cortex,
H–2D probe signal was distributed through
many layers but was strongest in layer 4;
Qa-1 signal was specific to layer 6, and T22
signal was evident in both layers 5 and 6 (Fig.
2). H–2D and T22 signals were both strong in
the pyramidal layers of the hippocampus and
in the habenula; in contrast, that of Qa-1 was
weak in those locations (Fig. 2). Transcripts
detected by the T22 probe were particularly
abundant in the thalamic reticular nucleus,
globus pallidus, and substantia nigra [Fig. 2
and (15)]. H–2K signal paralleled that of
H–2D but was much lower throughout the
brain (16). The distinct expression patterns
detected by these probes extended prior in-
ferences from RNase protection experiments
in cat (10) and demonstrated conclusively
that several class I MHC mRNA subtypes are
differentially expressed by distinct subsets of
neurons in the CNS. These findings suggest a
potential for functional diversity among class
Ia and Ib genes within the CNS. Such heter-
ogeneity of function occurs among these
genes within the immune system (17).
To test directly our hypothesis that class I
MHC is required for activity-driven structural
remodeling and synaptic plasticity, mice de-
ficient either for cell surface class I MHC
expression or for CD3␨ were analyzed. Be-
cause numerous class I MHC genes may be
expressed by specific subsets of neurons (Fig.
2), we examined mice lacking two molecules
required for the stable cell-surface expression
of nearly all fully assembled class I MHC
molecules: ␤
2
-microglobulin [␤
2
M, a class I
MHC cosubunit (18)], and TAP1 [a compo-
nent of the transporter that supplies peptides
to class I MHC enroute to the cell surface (19,
20)]. ␤
2
-M is expressed by neurons in LGN,
cortex, and hippocampus (10) and, as in non-
neuronal cells, induction of class I MHC on
the cell surface of neurons requires expres-
sion of ␤
2
M and TAP1 mRNAs (21). In
addition, to examine whether CD3␨-contain-
ing receptors were involved in class I MHC–
mediated signaling in the CNS, we analyzed
mice lacking CD3␨ (22). When raised in a
germ-free facility, all mutant mice are out-
wardly normal and are not obviously differ-
ent from wild-type mice in weight, body
length, appearance, or behavior.
We hypothesized that mice deficient in
class I MHC–mediated signaling might have
abnormal patterns of retinogeniculate projec-
tions because blockade of neural activity si-
multaneously prevents the segregation of ret-
inal ganglion cell axons into eye-specific lay-
Department of Neurobiology, Harvard Medical
School, 220 Longwood Avenue, Boston, MA 02115,
USA.
*To whom correspondence may be addressed. E-mail:
gshuh@alum.mit.edu or carla_shatz@hms.harvard.edu
†Present address: Experimental Immunology Branch,
National Cancer Institute, National Institutes of
Health, Building 10, Room 4B36, Bethesda, MD
20892, USA.
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ers and reduces class I MHC expression in
the LGN (7–10). The normal adult mouse
dLGN has a small layer that receives inputs
from ganglion cells in the ipsilateral eye;
inputs from the contralateral eye occupy the
remainder of the dLGN (Fig. 3A). The refine-
ment of these eye-specific connections in the
mouse occurs between postnatal day 4 (P4)
and P8 (14). We therefore examined the dis-
tribution of retinal inputs at P13, 5 days after
segregation was complete, using the antero-
grade transport of horseradish peroxidase–
conjugated wheat germ agglutinin (WGA-
HRP) injected into one eye (23). Compared
with wild-type animals (Fig. 3, A and F,
␤
2
M
⫹/⫹
), the pattern of the retinogeniculate
projection was significantly altered in all
three mutant genotypes tested. This point is
best appreciated by inspecting the size and
shape of the ipsilateral retinal projection to
the dLGN (Figs. 3, A to E). Although all
mutants still form an ipsilateral patch located
approximately normally in the mediodorsal
dLGN, the area of this patch was significantly
larger in mutant mice and, in extreme cases,
was accompanied by multiple ectopic clusters
of inputs that were never observed in wild-
type mice (Fig. 3, C and E, arrowheads).
These ectopic clusters appeared in medial
areas of the dLGN, where the highest levels
of CD3␨ mRNA are normally present (com-
pare Fig. 3, C and E, with Fig. 1A). In these
extreme cases, ectopic clusters were also ob-
served in the ipsilateral superior colliculus,
another retinorecipient target that expresses
low-to-moderate levels of class I MHC
mRNA in mouse (15).
To assess quantitatively the altered retino-
geniculate projection in mutant mice, com-
puterized image analysis was used to measure
the fraction of dLGN area occupied by the
ipsilateral projection. All image analyses
were carried out by an observer blind to
genotype (24 ). In all mutant genotypes, there
was a significant increase in area occupied by
the ipsilateral projection over that of wild-
type controls [Fig. 3F: ␤
2
M
–/–
, 130.3 ⫾ 7.3%
(n ⫽ 10); ␤
2
M
–/–
TAP1
–/–
, 133.3 ⫾ 5.7%
(n ⫽ 13); CD3␨
–/–
, 122.7 ⫾ 4.2% (n ⫽ 13);
wild-type ␤
2
M
⫹/⫹
, 100.0 ⫾ 9.1% (n ⫽ 12);
P ⬍ 0.05, Student’s two-tailed t-test]. These
observations support the hypothesis that class
I MHC function is required for the develop-
mental refinement of the retinal projections
and the formation of precise eye-specific re-
gions in the LGN.
Although the refinement of retinogenicu-
late axons was abnormal in mutant mice,
many other aspects of LGN development ap-
pear to proceed normally. The histological
appearance, size, shape, and location of
the dLGN and thalamus, as viewed in
Nissl-stained sections, were indistinguishable
among all experimental groups (15). The
bulk of the ipsilateral projection was posi-
tioned, as expected, in the binocular region of
the dLGN. At the ultrastructural level, the
synaptic organization of the LGN in ␤
2
M
–/–
TAP1
–/–
mice appeared qualitatively indistin-
Fig. 1. Class I MHC expression in mouse CNS. (A) Expression of class I MHC transcripts in coronal
sections of the mouse CNS at P6 and P40 and in a cross section of P6 eye (13). Left column,
adjacent Nissl-stained section; middle column, hybridization with antisense riboprobe under
dark-field optics; right column, hybridization with control sense probe. D, dorsal; L, lateral; hc,
hippocampus; ctx, neocortex; gcl, ganglion cell layer. Arrowheads and dashed lines indicate dLGN.
Scale bar for P6 and P40 brains, 0.5 mm; scale bar for P6 eye, 250 ␮m. (B) Expression of CD3␨ in
the dLGN during eye-specific layer formation. Upper panel, adjacent Nissl-stained coronal section
of P6 mouse brain (arrowhead, dLGN). Middle panel, hybridization with CD3␨ antisense probe
(dashed lines, dLGN); hybridization is also present in the ventroposterior nucleus of thalamus (down and
to right of dLGN). Lower panel (cptr), excess of unlabeled competitor probe. Scale bar, 200 ␮m.
Fig. 2. Expression of multiple class I MHC sub-
classes in distinct regions of the mature CNS.
Coronal sections of P40 mouse brain analyzed
by in situ hybridization, using subclass-specific
probes indicated at top of each panel (13). S1,
somatosensory cortex; hb, habenula; hc, hip-
pocampus; rs, retrosplenial cortex; tr, thalamic
reticular nucleus; gp, globus pallidus. Numerals
(4, 6, 5⫹6) indicate neocortical layers. Scale
bar, 1 mm.
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guishable from that of wild type (23). Reti-
nogeniculate axons were well-myelinated,
and glomeruli and R-type synaptic boutons
[hallmarks of retinogeniculate synapses; (25–
27)] were present, indicating that normal ret-
inal synapses do form in the LGN (Fig. 3G).
These observations suggest that many activ-
ity-independent processes (1, 2, 28, 29) are
not perturbed in mice with abnormal class I
MHC function.
Because similar abnormalities in the ipsi-
lateral projection result from blockade of
spontaneous activity at comparable ages in
the cat or ferret visual system (7–9), we tested
whether the mutant retinogeniculate pheno-
types were secondary to abnormal retinal ac-
tivity. Calcium imaging of mutant retinas
revealed spontaneous retinal waves with spa-
tiotemporal properties indistinguishable from
those of normal mice (30). Thus, we ascribe
abnormalities in the mutant retinogeniculate
projection directly to a loss of class I MHC
signaling downstream of neural activity.
Because activity-dependent structural re-
organizations during development are thought
to arise from cellular mechanisms of synaptic
plasticity (1, 2, 6), we next asked whether
synaptic plasticity is altered in mutant mice.
Because little is known about such mecha-
nisms in the developing LGN, we used a
well-characterized model system for studying
long-lasting changes in the strength of syn-
aptic transmission: the Schaffer collateral-
CA1 synapse of the hippocampus (31, 32).
Class I MHC and CD3␨ were both expressed
in the adult hippocampus (Fig. 1) (10, 15).
Furthermore, class I MHC immunoreactivity
can be detected in synaptosome preparations,
suggesting that some class I molecules are
synaptically associated (33). We therefore as-
sessed hippocampal synaptic plasticity in
wild-type and mutant mice. Data collection
was performed by an observer blind to geno-
type (34 ).
In wild-type mice (C57BL/6), tetanic
stimulation (4 ⫻ 100 Hz) resulted in a sus-
tained increase in the slope of the field exci-
tatory postsynaptic potential (f EPSP) (167 ⫾
13% of pretetanus baseline; n ⫽ 15; Fig. 4, A
and C). In contrast, in CD3␨
–/–
mutant ani-
mals, LTP in response to the same tetanus
was significantly enhanced relative to that in
wild-type mice (248 ⫾ 29% of baseline; n ⫽
8; P ⬍ 0.05; Fig. 4, A and C). A similar
enhancement of LTP was observed in ␤
2
M
–/–
TAP1
–/–
mutant mice (227 ⫾ 22% of base-
line; n ⫽ 10; P ⬍ 0.05; Fig. 4C). Basal
synaptic transmission is not significantly dif-
ferent among all experimental groups (35).
Enhanced LTP in gene knockout animals was
not due to changes in inhibition, because
GABA
A
-mediated transmission was blocked
with 100 ␮M picrotoxin in all experiments.
Nor was the enhanced LTP due to induction
of an N-methyl-
D-aspartate (NMDA) recep-
tor-independent form of LTP, because LTP
was completely abolished in all genotypes in
the presence of the NMDA antagonist 2-ami-
no-5-phosphonovalerate [50 ␮M D-APV;
Fig. 4B and (36)].
It is conceivable that enhancement of LTP
seen in these genotypes is due to some non-
specific effect of immune compromise on the
CNS. Thus we also examined LTP in a more
severely immunodeficient strain of mice that
lacks recombination activating gene-1
(RAG1). RAG1 is required for production of
B and T cells and is also transcribed by
neurons in the CNS (37, 38). LTP in RAG1
–/–
mice was indistinguishable from that of wild
type [153 ⫾ 13% of baseline (n ⫽ 10),
compared with 167 ⫾ 13% in wild type; P ⫽
0.48; Fig. 4C], indicating that the LTP abnor-
malities seen in ␤
2
M
–/–
TAP1
–/–
or CD3␨
–/–
mice are specific to their genotypes rather
than to immune status.
Synaptic plasticity in the hippocampus is
dependent on stimulation frequency, with
high frequencies producing LTP and low fre-
quencies producing LTD (31, 39–41). We
therefore examined the effect of other stimu-
lation frequencies on synaptic plasticity in
animals deficient for class I MHC signaling.
In adult wild-type slices, the delivery of 900
pulses at 0.5 Hz induced significant LTD
(82 ⫾ 6% of baseline; n ⫽ 8; P ⬍ 0.05; Fig.
4D). In adult slices from both mutant geno-
types, however, there was no significant
change in f EPSP slope upon 0.5 Hz stimula-
tion [CD3␨
–/–
, 107 ⫾ 7% of baseline (n ⫽ 5,
P ⫽ 0.29); ␤
2
M
–/–
TAP1
–/–
,99⫾ 5% of
baseline (n ⫽ 8, P ⫽ 0.78); Fig. 4D]. Fur-
thermore, after 900 pulses at 1 Hz, transmis-
sion was significantly enhanced over baseline
in both CD3␨
–/–
(141 ⫾ 14% of baseline, n ⫽
5, P ⬍ 0.05) and ␤
2
M
–/–
TAP1
–/–
slices
(128 ⫾ 9%, n ⫽ 6, P ⬍ 0.05) but was
unchanged in wild-type slices (94 ⫾ 5%, n ⫽
14, P ⫽ 0.41; Fig. 4D). Thus, in mutant mice,
LTD could not be detected, and the frequen-
cy-response curve of hippocampal synaptic
plasticity was consistently shifted across a
broad range of stimulation frequencies.
These results indicate that class I MHC/
CD3␨ signaling is important for mediating
Fig. 3. Abnormal retinogeniculate projections but normal dLGN ultrastructure in mice deficient in
class I MHC signaling. At P12, one eye was injected with WGA-HRP (23); after 1 day, anterograde
axonal transport results in labeling of the entire retinal projection to the LGN. Labeling pattern in
the dLGN is shown in bright-field optics (label is black) or as dark-field composites [label is white;
see (24)]. (A) Representative projection from retina to dLGN contralateral (dashed lines; coronal
section; dorsal is up; lateral is left) or ipsilateral to eye injected with WGA-HRP (asterisks indicate
labeled area from ipsilateral eye: lateral is to right) in a P13 ␤
2
M
⫹/⫹
wild-type mouse and a ␤
2
M
–/–
mutant mouse. (B and C) Representative (B) and extreme (C) examples of the projection from the
ipsilateral eye observed in ␤
2
M
⫺/⫺
TAP1
⫺/⫺
mice. (D and E) Representative (D) and extreme (E)
examples of the projection in CD3␨
–/–
mice. Arrowheads indicate ectopic projections, which appear
extensive under the more sensitive dark-field optics. Scale bar, 200 ␮m. (F) Graph of areas (⫾SEM)
occupied by the ipsilateral retinal projection to the LGN for ␤
2
M
⫹/⫹
(wild-type), ␤
2
M
–/–
, ␤
2
M
–/–
TAP1
–/–
, and CD3␨
⫺/⫺
mice (24), normalized to total dLGN area. The ipsilateral projection area in
␤
2
M
⫹/⫹
animals is set as 100% (horizontal dashed line). Asterisks indicate significant differences
from ␤
2
M
⫹/⫹
mice (P ⬍ 0.05, Student’s two-tailed t test). (G), Electron micrograph of the dLGN
from a ␤
2
M
–/–
TAP1
–/–
mouse (at P24), showing a typical R-type synaptic bouton (R) making
contacts with a dendrite (d). A well-myelinated axon (ax) is also present in this field. Scale bar, 1
␮m.
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activity-dependent synaptic depression, be-
cause, in mutants, there is a shift in the
bidirectional regulation of synaptic strength
[i.e., the frequency response function (39–
41)] that favors potentiation. In the absence
of class I MHC or CD3␨, patterns of neural
activity that normally have no effect on syn-
aptic strength or that lead to synaptic depres-
sion result, instead, in abnormal synaptic
strengthening. Likewise, in the dLGN, en-
hanced LTP and lack of LTD at the develop-
ing retinogeniculate synapse could account
for the structural phenotype observed: a per-
sistence of inappropriate connections that
would be normally be removed via an activ-
ity-dependent process of synaptic weakening
during eye-specific segregation (14, 42–44 ).
Class I MHC and CD3␨ are expressed in the
CNS by specific sets of neurons that undergo
activity-dependent changes (10). Here, we
show that mice lacking these molecules exhibit
abnormalities in connections between these
neurons, suggesting a direct neuronal function
for class I signaling. In addition, both mutants
have strikingly similar phenotypes, implying
that class I MHC signaling in the brain is
transduced via a CD3␨-containing receptor, ei-
ther an unknown CNS-specific or a known
immune receptor. The expression patterns of
class I MHC and CD3␨ in the CNS are consis-
tent with signaling via a number of possible
receptor-ligand configurations. For example,
both class I MHC and CD3␨ are expressed by
neurons in the hippocampus; in addition, class I
MHC mRNA is also expressed by retinal gan-
glion cells when CD3␨ is detected in the dLGN
[Fig. 1A and (10)]. Detailed information con-
cerning the ultrastructural localization of these
molecules will be needed to resolve this issue.
Whatever the case, the evidence to date
supports a model in which class I MHC func-
tions in the CNS by engaging CD3␨-containing
receptors to signal activity-dependent changes
in synaptic strength that ultimately lead to the
establishment of appropriate synapses. Class I
MHC may act directly at the synapse to pro-
mote the elimination of inappropriate connec-
tions, by using signaling mechanisms already
characterized in immune cells (11), possibly via
phosphorylation of CD3␨ by fyn [a kinase pre-
viously implicated in hippocampal plasticity
(45)]. Because different class I MHC mem-
bers are expressed by different subsets of
CNS neurons, additional signaling specificity
may be furnished by the particular repertoire
of MHC molecules present in any given neu-
ron. In the immune system, recognition of
class I MHC by T cell receptors can result in
functional elimination of inappropriate self-
reactive T cell populations (46, 47). Our
results demonstrate that class I MHC is also
required for normal regressive events in the
developing and adult CNS, including activi-
ty-dependent synaptic weakening and struc-
tural refinement.
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␤
2
M
–/–
(5⫻ backcrossed to C57BL/6) and ␤
2
M
–/–
TAP1
–/–
double mutant mice (5⫻ backcrossed to
C57BL/6) were obtained from D. Raulet (University of
California at Berkeley) (18, 19). CD3␨
–/–
mice (22)(8⫻
backcrossed to C57BL/6) and RAG1
⫺/⫺
mice (38) (10⫻
backcrossed to C57BL/6) were obtained from Jackson
Laboratories (Bar Harbor, ME). As part of the blind
study, ␤
2
M
⫹/–
heterozygotes (from ␤
2
M
–/–
⫻ C57BL/6
crosses) were intercrossed; ␤
2
M
⫹/⫹
and ␤
2
M
–/–
pups
were not revealed until after image analysis was com-
plete. P12 mouse pups were anesthetized with isoflu-
rane, and one eye was injected with 1 to 2 ␮l WGA-HRP
(4 to 10% in saline; L7017 from Sigma, St. Louis, MO, or
PL-1026 from Vector Laboratories, Burlingame, CA).
After 22 to 26 hours, 50-␮m brain sections were pre-
pared for histology essentially as described (9); the
nitroprusside solution used to stabilize the reaction
product was ice-cold and included 10 mM sodium ac-
etate (pH 3.3). For electron microscopy of ␤
2
M
–/–
TAP1
–/–
mice, P24 animals were perfused first with
buffer (0.1 M sodium cacodylate pH 7.35, 5 U/ml hep-
arin) and then 1% paraformaldehyde, 2% glutaralde-
hyde, 0.2% acrolein, and 4 mM CaCl
2
in buffer. The
thalamus was fixed overnight at 4°C; dLGN was isolated
from 150-␮m Vibratome sections and processed for
electron microscopy.
24. The following series of steps was carried out on all
slide sets by an observer blind to genotype. Only sets
exhibiting equivalent degrees of anterograde labeling
Fig. 4. Enhanced hippocam-
pal LTP in mice deficient ei-
ther for cell surface class I
MHC expression or for
CD3␨.(A) Field EPSP
(fEPSP) slopes in wild-type
versus CD3␨
–/–
-deficient
mice. Tetanus was applied
at time 0. (Insets) Superim-
posed sample fEPSPs re-
corded 10 min before or
180 min after tetanic stim-
ulation from individual
wild-type (left) and
CD3␨
–/–
(right) slices. Scale
bar, 10 msec/0.25 mV. (B)
NMDA receptor depen-
dence of LTP in CD3␨-defi-
cient mice. Tetanus was ap-
plied at time 0 either in the
absence [filled circles; from
(A)] or presence (hollow cir-
cles) of 50 ␮M D-APV. All points in (A) and (B) are averages of four consecutive fEPSPs (means ⫾
SEM, normalized to 15-min baseline) recorded from CA1. (C) Graphs summarizing degree of
potentiation in wild-type, ␤
2
M
–/–
TAP1
–/–
, CD3␨
–/–
, or RAG1
–/–
mice after 100-Hz tetanus. Data are
shown for mice with histologically normal brains (48). Asterisks indicate significant differences
from wild type (one-way ANOVA, P ⬍ 0.05). (D) Relation (logarithmic plot) between synaptic
enhancement and stimulation frequency. Points at 0.033 Hz (test pulse frequency) indicate
baseline values (horizontal dashed line). Points at 100 Hz are taken from (C). Values in (C) and (D)
are mean fEPSP slopes for each genotype over the 1-hour period following tetanus. See text and
(34) for methods.
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15 DECEMBER 2000 VOL 290 SCIENCE www.sciencemag.org2158

were selected for analysis. Eight-bit TIFF images con-
taining the dLGN were acquired on a Macintosh-
linked charge-coupled device camera (MTI VE1000)
attached to a Nikon Microphot FXA. Using NIH Image
(v1.62b7), images of the dLGN ipsilateral and con-
tralateral to the injected eye were cropped to exclude
ventral LGN, intrageniculate leaf and extrageniculate
optic tract; images of ipsilateral dLGN were also
modified to eliminate the optic tract running above
the dLGN. NIH Image macros were used to eliminate
background blood vessel-derived staining (very
heavily stained blood vessels were removed by hand)
and to calculate areas occupied by retinal projections
(9). For each brain, an internally controlled measure
of the area occupied by the ipsilateral projection was
obtained by dividing the average of the four largest
ipsilateral areas (corresponding to the middle third of
the dLGN) by the average of the four largest total
dLGN areas (assessed by the outer boundaries of the
contralateral projection zones). Sections in Fig. 3, A
to E, were photographed in both bright-field and
dark-field optics. Although dark-field optics are more
sensitive and reveal lightly labeled regions as white,
very heavily labeled regions become saturated and
appear black. Therefore, for accuracy, composites of
bright-field and dark-field images of the same section
were constructed to ensure that heavily labeled re-
gions appeared white, while detailed information
about lightly-labeled regions revealed in dark-field
was preserved.
25. N. Aggelopoulos, J. G. Parnevelas, S. Edmunds, Anat.
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31. R. C. Malenka, R. A. Nicoll, Science 285, 1870 (1999).
32. T. V. Bliss, G. L. Collingridge, Nature 361, 31 (1993).
33. Supplementary data are available on Science Online
at www.sciencemag.org/cgi/content/full/290/5499/
2155/DC1
34. Slices of mouse brain 400 ␮m thick (from 8- to
17-week-old animals, killed with halothane) were
maintained at 25°C in a submerged recording cham-
ber (perfused at 2 to 3 ml/min) with artificial cere-
brospinal fluid (ACSF: 126 mM NaCl, 2.5 mM KCl,
1.25 mM NaH
2
PO
4
, 1.3 mM MgSO
4
, 2.5 mM CaCl
2
,
26 mM NaHCO
3
, and 10 mM glucose). Connections
to the CA3 region of the hippocampus were cut, and
100 ␮M picrotoxin (Sigma) was added to the bath
ACSF. Stainless-steel bipolar electrodes were used to
stimulate Schaffer collateral/commissural fibers;
glass microelectrodes filled with ACSF (2 to 6 M⍀)
were inserted into the stratum radiatum to record
currents from populations of CA1 pyramidal cells.
Test pulses (0.033 Hz) were applied at a stimulation
intensity required to produce an fEPSP that was 30%
(for 100 Hz stimulation) or 50% (for 0.5 and 1 Hz
stimulation) of the maximal response for each re-
cording. High-frequency stimulation (tetanus) con-
sisted of four trains of 100 pulses at 100 Hz (inter-
train interval 15 s), applied at time 0. All values are
reported as means ⫾ SEM, n is the number of slices
(one slice per mouse). Data collection was performed
by an observer blind to genotype. Before the blind
was dropped, recordings were omitted from analysis
if the extracellular resistance changed significantly
(3/94) or if the stimulating electrode had visibly
drifted over the course of the recording (4/94). LTP
was calculated as the average of responses between
0 and 60 min after tetanus, normalized to a 15-min
pretetanus control period. Stimulus intensity was
relatively high because of the use of electrodes with
uninsulated tips to maximize the number of fibers
stimulated. Stimulus artifacts were clearly complete
well before fEPSP onset and so were easily excluded
from analysis. In experiments using D-APV, drug was
added at least 30 min before tetanic stimulation and
was present throughout the entire recording. Statis-
tical significance was assessed by two-tailed one-way
ANOVA or Student’s t-test.
35. Pretetanus test pulse fEPSP slopes for mice with
normal ventricles (millivolts per millisecond) were as
follows: wild type, 0.091 ⫾ 0.007 (n ⫽ 14); CD3␨
–/–
,
0.091 ⫾ 0.009 (n ⫽ 8, P ⫽ 0.95 compared with wild
type); ␤
2
M
–/–
TAP1
–/–
, 0.089 ⫾ 0.010 (n ⫽ 9, P ⫽
0.85). Stimulation intensities required to evoke an
fEPSP at 30% of the maximal response (in microam-
peres) were as follows: wild type, 136 ⫾ 27; CD3␨
–/–
,
134 ⫾ 24 (P ⫽ 0.75 compared with wild type);
␤
2
M
–/–
TAP1
–/–
, 128 ⫾ 17 (P ⫽ 0.59).
36. Posttetanus fEPSP slopes, averaged over 180 min, did
not differ significantly from baseline in the presence
of 50 ␮M D-APV: wild type, 107 ⫾ 14% (n ⫽ 3; P ⫽
0.64); CD3␨
–/–
,97⫾ 9% (n ⫽ 3; P ⫽ 0.93); and
␤
2
M
–/–
TAP1
–/–
, 106 ⫾ 10% (n ⫽ 5; P ⫽ 0.56, Stu-
dent’s t-test).
37. J. J. Chun, D. G. Schatz, M. A. Oettinger, R. Jaenisch, D.
Baltimore, Cell 64, 189 (1991).
38. P. Mombaerts et al., Cell 68, 869 (1992).
39. M. F. Bear, Neuron 15, 1 (1995).
40. K. Deisseroth, H. Bito, H. Schulman, R. W. Tsien, Curr.
Biol. 5, 1334 (1995).
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153, 85 (1990).
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45. S. G. Grant et al., Science 258, 1903 (1992).
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(1998).
47. E. Sebzda et al., Annu. Rev. Immunol. 17, 829 (1999).
48. When brains of otherwise normal-appearing animals
at age P13 were examined, 52% (16/31) of ␤
2
M
–/–
TAP1
–/–
mice and 22% of CD3␨
–/–
mice (10/45) had
enlarged lateral ventricles. This phenotype is unlikely
to be due to immunocompromise because severely
immunodeficient RAG1
–/–
mice, when cohoused in
our facility, do not exhibit this phenotype (0/18). This
phenotype also occurs in 57% (12/21) of ␤
2
M
–/–
TAP1
–/–
and 20% (2/10) of CD3␨
–/–
adult mice. Al-
though ventricular enlargement does not affect the
appearance of the dLGN and thalamus (assessed by
Nissl stains), the size, placement, and appearance of
extrathalamic structures such as the hippocampus
can be altered. In the LTP analysis, animals with
enlarged ventricles were treated separately because
in these animals, LTP measurements could be con-
founded by abnormal hippocampal architecture and
the known reduction of LTP by hydrocephalus [T.
Tsubokawa, Y. Katayama, T. Kawamata, Brain Inj. 2,
19 (1988)]. Consistent with the latter idea, LTP at
100 Hz in ␤
2
M
–/–
TAP1
–/–
mice with dilated ventri-
cles, while still present, is significantly lower than
that of ␤
2
M
–/–
TAP1
–/–
mice with normal-appearing
brains (168 ⫾ 15% relative to 227 ⫾ 22%; P ⬍ 0.05).
CD3␨
–/–
mice with dilated ventricles also displayed
diminished LTP (data not shown).
49. We thank S. Wiese, C. Cowdrey, and H. Aaron for
expert technical assistance; D. Stellwagen for exam-
ination of retinal waves in mutant mice; A. Toroian-
Raymond for assistance with electron microscopy; M.
Bennett and E. Choi for advice and assistance with
synaptosome preparations; D. Raulet for generously
providing ␤
2
M
–/–
and ␤
2
M
–/–
TAP1
–/–
mice. Support-
ed in part by grant NIH MH48108 and an Alcon
Research Institute Award to C.J.S. G.S.H. and L.M.B.
were Howard Hughes Associates. L.M.B. was support-
ed by NRSA 1F32EY07016. H.D. was supported by
NRSA EY06912.
31 July 2000; accepted 14 November 2000
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