Viral Vectors to Study Synaptic Function - Viral Vector Approaches in Neurobiology and Brain Diseases - page 124

neurotropism. Thus, by expressing GFP along with the protein
of interest (either as a fusion protein or via the use of an IRES),
it is straightforward to identify the cells that have been infected
in living tissue using simple epifl uorescent light microscopy and
perform any number of cell-restricted assays. Here, I show how
we were able to compare the synaptic properties of infected ver-
sus uninfected neighbor neurons in acutely dissected hippocam-
pal slices from infected animals. By performing dual whole-cell
patch clamp recordings of infected and neighbor uninfected neu-
rons (Fig. 4a ), we could compare current amplitudes of AMPARs
and NMDARs (Fig. 4b ). The GFP-infected neurons showed
healthy and stable AMPAR- and NMDAR-mediated excitatory
postsynaptic currents (EPSCs) in CA1 pyramidal neurons com-
parable to those recorded from uninfected neurons in the same
slice preparation and also comparable to neurons from unin-
fected animals [ 14 ]. This demonstrated that sindbis infection per
se did not perturb excitatory synaptic transmission. Figure 4b
displays an example of how the procedure described in this chap-
ter was used to investigate protein function in the regulation of
synaptic transmission. We investigated the role of the transcrip-
tion factor cAMP Response Element Binding Protein (CREB) in
modulating AMPAR and NMDAR EPSCs by expressing a con-
stitutively active form of CREB (CREBCA) along with GFP
using the methods described in Sect. 3 [ 14 ]. We showed that
activating CREB led to a specifi c increase in NMDAR EPSC
amplitude, while leaving AMPAR EPSC amplitude unaffected
(Fig. 4b ). Using this approach, we could perform several other
assays to evaluate how CREB perturbs synaptic function, such as
AMPAR miniature EPSC analysis and diffi cult electrophysiologi-
cal assays such as those that require minimal stimulation tech-
niques [ 14 ].
This technique is also useful to address the role of proteins in
synaptic plasticity. We could maintain whole-cell recordings of
infected neurons for times suffi cient to examine LTP and LTD
(Fig. 5 ). Sindbis virus and GFP expression per se did not affect
these plasticities, while expression of CREBCA specifi cally
enhanced LTP magnitude (Fig. 5 ) [ 14 ]. We have found that a
similar type of analysis could be performed using the less diffi cult
extracellular fi eld potential recording technique provided that the
areas of the slice recorded contained a high proportion of infected
cells [ 26 ], thus confi rming the overall good heath of the slices.
Furthermore, this technique has been successfully applied to
other regions of the brain such as the rat, somatosensory “barrel”
cortex [ 27 ].