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thesis occurs in neuronal dendrites, often near synapses,
and polyribosomal aggregates appear in dendritic spines
during development (Miyashiro et al., 1994; Steward
and Shuman, 2001). However, it has been difficult to
determine whether the synthesis of new glutamate re-
ceptor subunits occurs subsynaptically in the dendrites
of central neurons (Steward, 1994; Craig et al., 1993).
Injection of mRNA directly into isolated dendrites
showed that local synthesis was possible (Kaharmina
et al., 2000).
An imaginative set of techniques was used to prelabel
existing AMPA receptors in cultured hippocampal
neurons, and then counterstain with a second label to
identify the location of newly synthesized subunits. In
concept, this approach is nearly identical to that
employed on AChRs by Role et al. (see Figure 8.24). Hip-
pocampal neurons were first transfected with a gluta-
mate receptor subunit (GluR1 or GluR2) that was
modified to contain a tetracysteine motif on its intracel-
lular C-terminal (Figure 8.25). This permitted the use of
two different dyes that fluoresced (red or green) only
when bound to tetracysteine. When exposed to the red
dye, the majority of GluRs expressed over a 24-hour
period were labeled in the dendrites. The red dye was
then removed, and the neurons were exposed to the
second green dye eight hours later. Newly expressed
GluRs were labeled primarily in the soma, the presumed
site of synthesis, and a few were found in the dendrite,
presumably due to rapid transport. To show that some
receptors are synthesized locally, the dendrite was tran-
sected from the soma after prelabeling with red dye.
Thus, any GluR aggregates that were subsequently
labeled with only the green dye must have been synthe-
sized within the dendrite (Figure 8.25). This turned out
to be the case (Ju et al., 2004). Finally, it was possible to
show that the GluRs synthesized in the dendrite were
inserted into the membrane. Since the mRNA for GABA
receptors is located within the dendrites of cortical
pyramidal neurons (Costa et al., 2002), it is likely that
receptor expression can be regulated by a local cue, as
occurs for AChRs at the neuromuscular junction.
NEURONAL ACTIVITY REGULATES
RECEPTOR EXPRESSION
The increase in receptor synthesis that accompanies
synapse formation suggests that the presynaptic ter-
minal initiates this process. One simple possibility is
that the transmitter itself can regulate expression of
synaptic proteins. At the neuromuscular junction,
HA Thr
Glu
S
S
S
S
S
S
S
S
As
As
As
As
O -
O -
O
O
O
O
N
E
A
A
A
R
CO -
A
A
ReAsH-EDT 2
R
ReAsH
C
C
E
A
E
C
R
C
FlAsH
FlAsH-EDT 2
FIGURE 8.25 Local synthesis of AMPA receptors in the dendrite. (Left) Schematic showing the
membrane topology of GluR1/2 and the location of the intracellular tetracysteine and extracellular
HA/thrombin tags. The tetracysteine binds to FIAsH-EDT 2 or ReAsH-EDT 2 , yielding a green or red fluores-
cent signal, respectively. (Right) An example of a hippocampal neuron in which all GluR1 protein was pre-
labeled with red dye, followed by transection of the dendrite (white arrow) and labeling with green dye.
Note that some GluR aggregates are labeled only with the green dye (inset, white arrowheads), indicating
that they must have been synthesized after the dendrite was detached from the soma. Preexisting aggregates
that are labeled with both red and green dye are orange. (Ju et al., 2004)
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