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clustering signal in the CNS has focused on three differ-
ent pathways. First, a secreted pentraxin (i.e, a family of
proteins sharing a discoid arrangement of five nonco-
valently linked subunits), Narp, is concentrated at exci-
tatory synapses in cultured spinal and hippocampal
neurons, and ultrastructural studies from hippocampal
neurons in vivo show that Narp is present on both the
presynaptic surface and the dendritic shaft. To deter-
mine whether endogenous Narp participates in gluta-
mate receptor clustering, the C terminus region that
supports axon transport was disrupted such that
axonal transport and secretion at the synapse could be
prevented. When cultured spinal neurons were trans-
fected with this mutant Narp, there was a dramatic
reduction in the clustering of AMPA-type glutamate
receptors at axon-dendrite contacts (O'Brien et al.,
1999; 2002).
A second system that is able to cluster glutamate
receptors is the EphrinB-EphB signaling pathway
which plays such an important role in axon pathfind-
ing and target selection (Chapters 5 and 6). When cul-
tured cortical neurons are exposed to ephrinB1, they
rapidly display clusters of the ephrin receptor, EphB2,
followed by the appearance of NMDA-type glutamate
receptor clusters (Figure 8.21A) (Dalva et al., 2000).
Furthermore, when hippocampal neurons are cultured
from mice that lack 3 EphB receptors, there is a dra-
matic loss of normal spine morphology and glutamate
receptor clustering (Figure 8.21B). However, this sig-
naling pathway does not appear to disrupt inhibitory
GABAergic synapse formation, presumably because
these contacts are not made on dendritic spines
(Henkemeyer et al., 2003).
A third system that participates in receptor clus-
tering is the neurotrophin-Trk signaling pathway,
which also plays a fundamental role in target selection
and neuron survival (Chapters 6 and 7). Once again,
the assay system is a dissociated culture of embryonic
hippocampal neurons which express the BDNF/NT-4
receptor, TrkB, diffusely along their dendrites and
soma. When these neurons are exposed to BDNF,
the number of NMDA receptor clusters doubles,
and they are far more likely to be located adjacent
to a presynaptic terminal (Elmariah et al., 2004).
Conversely, decreasing endogenous BDNF in the cul-
tures produces a loss of NMDA receptor clusters.
Unlike the ephrinB-EphB system, the TrkB pathway
fails to increase AMPA receptor clustering but does
support GABA A receptor cluster formation. To sum-
marize, several candidate signals may be released by
excitatory and inhibitory nerve terminals and may
recruit the aggregation of postsynaptic receptors,
similar to the role of Agrin at the nerve-muscle
junction.
Several studies suggest that the neurotransmitter
itself can promote receptor accumulation. For
example, the largest glutamate receptor clusters occur
opposite the presynaptic terminals that release the
most glutamate at the fly neuromuscular junction
(Marrus and DiAntonio, 2004). Furthermore, when
spontaneous glutamate release is blocked during
development, glutamate receptor clusters do not form
(Saitoe et al., 2001). An effect of activity has also been
observed for glycine receptors and glutamate recep-
tors in tissue culture preparations (Kirsch and Betz,
1998; Shi et al., 1999; Liao et al., 2001; Lu et al., 2001).
It is not yet clear whether activity-dependent receptor
clustering is a developmental mechanism, or is used
largely to adjust synaptic strength throughout the
animal's life (Aoki et al., 2003).
It must be emphasized that an equal number of
studies report little influence of synaptic transmission
on receptor clustering, and this area of research remains
unsettled. In C. elegans , GABA is not required to obtain
synaptically clustered GABA A receptors (Gally and
Bessereau, 2003). A careful in vitro study showed that
glutamate receptors are evenly distributed along the
dendrite at a low density (ª3/mm 2 ) prior to innervation
but form high-density (ª10,000/mm 2 ) aggregates at the
site of presynaptic contacts (Figure 8.22). These gluta-
mate receptor clusters appear even when synaptic or
electrical activity is blocked (Cottrell et al., 2000).
INTERNAL MEMBRANE PROTEINS
AND RECEPTOR AGGREGATION
IN THE CNS
Whatever the clustering signal may be at each central
synapse, there must be a molecular mechanism to hold
the receptors together, similar to the way that Rapsyn
restricts AChR mobility. In fact, a tremendous array of
proteins are located at the cytoplasmic surface and bind
to both membrane receptors and cytoskeletal elements.
Perhaps the strongest case can be made for a protein
called Gephyrin that was discovered during the purifi-
cation of glycine receptor subunits (Kirsch et al.,
1993a,b). Gephyrin displays a high affinity for polymer-
ized tubulin, and a C-terminal domain can bind with
high affinity to the cytoplasmic loops of 2 glycine recep-
tor b subunits (Schrader et al., 2004). Spinal neurons
grown in dissociated culture normally display glycine
receptor clusters. However, when the cells are grown in
the presence of a gepherin antisense nucleotide, which
presumably prevents the translation of gepherin
mRNA, then clusters do not form at the membrane
(Figure 8.23). Similarly, glycine receptors do not aggre-
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