Selective Gene Expression by Postnatal Electroporation
during Olfactory Interneuron Neurogenesis
Alexander T. Chesler1, Claire E. Le Pichon1, Jessica H. Brann1, Ricardo C. Araneda2, Dong-Jing Zou1, Stuart Firestein1*
1Department of Biological Sciences, Columbia University, New York, New York, United States of America, 2Department of Biology, University of
Maryland, College Park, Maryland, United States of America
Neurogenesis persists in the olfactory system throughout life. The mechanisms of how new neurons are generated, how they
integrate into circuits, and their role in coding remain mysteries. Here we report a technique that will greatly facilitate research
into these questions. We found that electroporation can be used to robustly and selectively label progenitors in the Subventicular
Zone. The approach was performed postnatally, without surgery, and with near 100% success rates. Labeling was found in all
classes of interneurons in the olfactory bulb, persisted to adulthood and had no adverse effects. The broad utility of
electroporation was demonstrated by encoding a calcium sensor and markers of intracellular organelles. The approach was found
to be effective in wildtype and transgenic mice as well as rats. Given its versatility, robustness, and both time and cost
effectiveness, this method offers a powerful new way to use genetic manipulation to understand adult neurogenesis.
Citation: Chesler AT, Le Pichon CE, Brann JH, Araneda RC, Zou D-J, et al (2008) Selective Gene Expression by Postnatal Electroporation during
Olfactory Interneuron Neurogenesis. PLoS ONE 3(1): e1517. doi:10.1371/journal.pone.0001517
In the central nervous system, postnatal neurogenesis persists into
adulthood and supplies a restricted subset of interneurons in two
tissues: the olfactory bulb and the hippocampus . The most
robust of these appears to be in the olfactory bulb, where
inhibitory interneurons are generated at the astounding rate of 30-
50,000 per day in the mouse . These cells are born from a
population of progenitor cells located in the Subventricular Zone
(SVZ) and then migrate to the bulb along a path known as the
rostral migratory stream (RMS). Within the olfactory bulb,
migrating neuroblasts give rise to a diversity of interneurons that
mediate local signal processing. Among them are periglomerular
cells, a class of interneurons that modulate the transmission of
sensory stimuli at the first synapse in olfactory processing, and
granule cells which modulate the output through numerous
synapses with the projection neurons, mitral cells.
Many investigators have been drawn to study postnatal
neurogenesis in the olfactory bulb and the hippocampus to
address a variety of questions surrounding this important and
unusual process. What determines the fate of precursor cells in the
SVZ and RMS? How do the postnatally generated cells integrate
into existing circuits? What determines whether they will survive?
What controls rates of proliferation? How is this balanced with
rates of cell death? Are there age related factors that are
important? Investigating these and other fundamental questions
require the ability to track cells from birth to maturity, to monitor
activity, and to alter gene expression in individual cells.
Traditionally, transgenic or gene targeted mice have proved a
powerful strategy for the manipulation of gene expression, but suffer
from being time consuming and expensive. Viruses have also proven
useful, but are difficult to generate in high titers, have limitations in
terms of insert and promoter size, and have associated biohazards
that require special handling. More recently, electroporation of
genes in rodents has been gaining as an alternative to these
techniques [3–5]. Electroporation has several advantages over other
approaches: the plasmid construction is simple, versatile, rapid, and
inexpensive, and it is applicable to numerous tissues and species .
Given the wealth of candidate genes generated from genomics and
proteomics, ectopic expression or knockdown of genes by electro-
poration represents a powerful way to elucidate the roles of the
numerous candidate molecules in vivo.
Thus far the use of electroporation in the brain has been largely
limited to embryonic tissue. Injection of plasmids is typically
performed in utero, where the delivery of the electrical pulse enables
the constructs to enter cells . However, this is a surgical
technique with significant risks of pup mortality . More
recently, electroporation has been applied to the postnatal retina
and cerebellum [4,7]. In our efforts to understand postnatal
neurogenesis we developed a highly efficient and non-invasive
procedure that utilizes electroporation for manipulating gene
expression in the subventricular zone (SVZ). Here we introduce a
method of electroporation in early postnatal animals that requires
neither surgery nor stereotaxic apparatus, but which results in
widespread expression of foreign genes in specific cell populations,
with nearly 100% success rates. This methodology makes genetic
manipulation in rodents a bench top procedure, bringing it within
the technical and financial reach of most laboratories.
We investigated the use of in vivo electroporation as an alternative
to viral and transgenic methods for the study of postnatal
neurogenesis. With the aid of a dissecting microscope we found
the illumination of a fiber optic light sufficient for the visualization
of the olfactory bulbs and central sulcus of the brain. Under such
conditions we reproducibly injected lateral ventricles of mice ages
P0-P4 without the requirement of surgery or a stereotactic device
(figure 1a and 1c). A sharp electrode with a 20–50 mm beveled tip
Academic Editor: David Raible, University of Washington, United States of
Received November 12, 2007; Accepted December 31, 2007; Published January
Copyright: ? 2008 Chesler et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Funding: NIH, NIDCD
Competing Interests: The authors have declared that no competing interests
* To whom correspondence should be addressed. E-mail: email@example.com
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Figure 1. Long-term ectopic expression of GFP in neurogenic regions of the postnatal brain by electroporation. A. Animals immobilized by
cooling are injected with plasmid using a dissecting microscope and picospritzer. The injection is made rostral to the olfactory bulb followed by a
series of 5 voltage pulses delivered with tweezer-type electrodes. The pulses are given while sweeping the positive pole upwards from the eyes by 45
PLoS ONE | www.plosone.org2January 2008 | Issue 1 | e1517
was used to pierce through the skin and developing skull into the
brain and inject 1–2 mL of endotoxin-free plasmid (1–5 mg/mL)
into the ventricles. Similar to published methods both in utero and
in postnatal retinas, we determined that a 5 second square pulse
protocol of five 50 ms pulses of 150 V gave optimal results with
minimal side effects . The first pulse was aligned with the center
of the eyes and subsequent pulses were given while sweeping the
positive pole 45 degrees upwards (figure 1a). Electroporation of a
plasmid encoding EGFP under the CAG promoter resulted in
high levels of fluorescence within 24 hours (figure 1c–d). The
preponderance of fluorescence from animals injected was found in
the SVZ compared with the OB (compare figure 1d and 1e).
The pulse protocol resulted in robust ectopic expression with no
detectable adverse effects on pup survival. A litter of 8 pups could be
anesthetized on ice, injected, and returned to their mother in less
than 10 minutes with nearly all (.95%) surviving to adulthood and
expressing the plasmid of interest. The robustness and ease of our
approach offers a major improvement over in utero electroporation in
that the latter requires surgery on expensive timed-pregnant animals
that can lead to significant damage and loss of embryos. Our
approach was also remarkably specific, exclusively labeling the
postnatal neurogenic region of the mouse brain.
Neuroprogenitors in the SVZ give rise to neuroblasts that migrate
along the rostral migratory stream (RMS). 5 days post-electropora-
tion we found strong GFP expression in the RMS (figure 1f). Cells
that had reached the bulb radially migrated throughout the bulb
(figure 1g). GFP positive cells lining the ventricles were also noted,
indicating the persistence of labeled progenitor cells (figure 1h).
Migrating neuroblasts terminally divide to generate the interneurons
in the olfactory bulb. 2 weeks post-electroporation we continued to
observe labeling in the SVZ and in the RMS (figure 1i–j) but now
also found interneurons in all layers of the olfactory bulb (figures 1i
and 1k). Postnatal electroporation rivaled viral techniques, in that it
was sufficient for widespread labeling of both granule and
periglomerular cells (figure 1l–m) as well as for resolution of single
cells (figure 1n). Numerous GFP-positive cells could be found several
months post-electroporation and, although less numerous, persisted
in mice after over a year (figure 1o–q).
We found labeling in every specific cell population we analyzed.
A subset of GFP-positive neuroprogenitors of the SVZ and RMS
were co-labeled by a brief intraperitoneal pulse of BrdU (figure 2a).
Furthermore, we observed GFP expression among the GFAP-
positive progenitors (figure 2b) and among the doublecortin-
positive migrating neuroblasts in the RMS (figure 2c). SVZ
progenitors are a heterogeneous population that gives rise to a
diversity of interneurons in the olfactory bulb [8–9]. Labeled
neurons in the bulb included both granule and periglomerular
cells. We found overlap between GFP and each cell-type specific
marker analyzed (parvalbumin, calbindin, calretinin, and tyrosine
hydroxlyase). Notably, co-staining for all markers tested could be
found from sections prepared from a single animal (figure 2d–g)
showing that electroporation labels diverse progenitor populations
within the SVZ.
Cells ectopically expressing GFP divide, migrate, and morpho-
logically mature. The ease, rapidity, cost effectiveness, and
robustness of the technique suggested postnatal electroporation
to be an ideal approach to study many aspects of neurogenesis, cell
migration, circuit integration, and neurophysiology. Importantly,
in addition to appearing morphologically healthy, we found that
fewer than 0.5% of GFP+ cells were also labeled by activated
caspase 3 antibodies or TUNEL, showing that electroporation
does not accelerate cell mortality. Additionally we found neurons
labeled via electroporation also had the expected electrical
properties. We characterized the membrane properties of
periglomerular (figure 3a) and granule cells (figure 3b) expressing
fluorescent markers (mRFP or GFP) by performing patch clamp
recording in acute bulb slices. Labeled cells were electrophysio-
logically indistinguishable from neurons derived from non-
electroporated animals. Mature granule and periglomerular cells
were recognized by their morphology and their position in
different layers of the olfactory bulb. In current-clamp recordings
granule cells had a resting membrane potential of 6562 mV and
input resistance of 1.5760.22 GV (n=6). Depolarizing stimuli (2–
10 pA) elicited action potentials, which increased in frequency with
larger currents. Mature periglomerular cells cells, like granule cells
had high input resistance (1.1260.58 GV) and hyperpolarized
membrane potentials (6762 mV), but unlike granule cells,
increasing depolarizing stimuli produced a single spike followed
by a plateau potential (n=3). In agreement with these observations,
in voltage-clamp both periglomerular cells and granule cells
exhibited both voltage-dependent inward and outward currents
(not shown). In addition, we recorded from a subpopulation of cells
at various stages of development (as assessed by their dendritic
morphology); most of these cells exhibited small voltage-dependent
inward currents and depolarizing stimuli failed to induce all-or
none action potentials in current-clamp (not shown). Similar
characteristics for granule and periglomerular cells have been
described both in control , as well as in retrovirally GFP-
labeled cells [11-12]. Our results also demonstrate the feasibility of
using recording from postnatally born neurons genetically
manipulated by electroporation to study their functional properties.
In addition to monitoring cells electrically, we also developed
tools to monitor activity optically. Genetically encoded sensors
offer a non-invasive tool to study neuronal function in vivo, are less
technically demanding and time consuming than the use of single-
cell electrophysiology, and allow unequivocal identification of
degrees. B. Sagittal view of a P1 mouse showing localized fluorescence after injection of diO into the lateral ventricles. Scale=1 mm. C–E. Sagittal
views of a P2 mouse 24 hours post-electroporation of the olfactory bulb and ventricle in a P2 mouse 24 hours post-electroporation. C. The
preponderance of GFP is restricted to the ventricular wall; 60 mm sections, scale=1 mm. D–E. Enlarged views of the boxed regions in c highlight the
high levels of expression in along the SVZ relative to the olfactory bulb. Relatively few GFP positive cells can be found in the olfactory bulb (for
example see inset in E). scale=500 mm. F–G. Sagittal views of a P6 mouse 5 days post-electroporation. F. Whole-mount view of an unfixed brain
reveals high levels of GFP expression through the RMS. Scale=1 mm G. A higher magnification view of a different brain showing cells had begun to
migrate throughout the olfactory bulb. Scale=500 mm H. Labeled radial glia persisted in the SVZ; dashed line denotes ventricular wall; 60 mm
sections, scale=50 mm. I–N. Sagittal views of a P15 mouse 2 weeks post-electroporation; 60 mm sections I. In addition to the RMS, GFP-positive cells
can be found throughout the olfactory bulb. scale=1 mm. J–K. Enlarged views of the boxed regions in i showing the RMS (J), and olfactory bulb (K).
Inset in K highlights a periglomerular cell. scale=500 mm. L–N. Z-projections of 60 mm sections TOTO3 staining reveals nuclei (blue). L. Widespread
distribution of GFP positive cells in all layers of the bulb. Scale=200 mm M. Labeling of numerous granule cells. scale=50 mm N. Resolution of a
single morphologically mature granule cell. scale=50 mm. O–Q. Sagittal views of a P430 mouse over 1 year post electroporation; 60 mm sections. O.
labeled PG cell, TOTO 3 (blue) stains all nuclei and inset show a magnified view. Scale=100 mm P–Q.Labeled granule cell, low magnification
counterstained with TOTO3 (P; scale=100 mm) and high magnification of the same cell (Q; scale=25 mm)
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Figure 2. Widespread labeling of specific cell populations by electroporation. 14 mm sections from a P15 mouse electroporated 2 weeks
previously with GFP (green) were stained with a panel of markers (red) labeling several specific cell populations in RMS (A–C) and olfactory bulb (D–
G). 10-fold magnified views are presented for highlighted double-labeled cells. A summary of the locations for each section and the markers used are
in the box on top. scale=50=mm
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Figure 3. Electrical and optical recordings from interneurons labeled by electroporation. Electrical excitability of neurons in current-clamp recordings
in slices from 3–4 weeks-old mice electroporated with EGFP (A) and mRFP (B). A. Example of PG cell recorded in the glomerular layer (bright field, left)
exhibited spontaneous synaptic activity and responded to a depolarizing current (16 pA) with a single action potential followed by an
afterhyperpolarization. The cell input resistance is 1 GV as measured with a negative current stimulus (24 pA). B. Example of a granule cell recorded
in the internal plexiform layer (bright field, left). This cell responded to a depolarizing stimulus (15 pA) with a train of spikes. The cell input resistance is
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morphology. Furthermore, unlike transgenic approaches, electro-
poration of a sensor would be ideal to resolve the detailed
morphology of single cells both in slices and in vivo. Toward this
end, we created a plasmid (pCAG-GCamp2) allowing expression
of the GFP based fluorescent calcium reporter . GCamp2 has
been reported to be a sensitive reporter of calcium transients on
par with dye-based sensors, and its transgenic expression in mice
has been shown to lack any discernable deleterious effects [13–16].
Examination of mice electroporated with pCAG-GCamp2
revealed a distribution of fluorescent cells similar to GFP controls.
GCamp2-expressing cells migrated to destinations throughout the
olfactory bulb where they were found to be morphologically
mature. All cell types labeled were able to respond with an
increase in fluorescence to high potassium (Figure 3c, d) as well as
the physiological ligand, Glutamate (Figure 3e, g), but did not
respond to Ringer application (Figure 3f; N=19 slices from 4
animals). In addition, we were able to monitor intrinsic calcium
waves found in immature cells in the RMS of the OB (see
Supplemental Movie S1). Calcium imaging was also highly
reliable; cells were responsive to repeated applications of stimuli
(data not shown) and to a variety of stimuli.
We also used electroporation to simultaneously express multiple
genes. We demonstrated co-expression by coincident marking of
various intracellular compartments (figure 4a–4c). First we co-
expressed CRE and GFP using a single plasmid containing an
internal ribosome entry site (pCAG-CRE-IRES-GFP). Antibody
staining of electroporated animals produced GFP-positive cells with
CRE labeling in their nuclei (figure 4a). Alternatively, we found co-
electroporation to be equally effective. Co-electroporation of two
plasmids, one encoding GFP and the other either dsRed-Golgi or
dsRed-ER allowed visualization of these organelles without using
c). We found near perfect overlap when a 1:1 ratio of plasmids was
used. While GFP expression labeled the entirety of the cell, both
dsRed-Golgi and dsRed-ER localized to perinuclear rings in the cell
body (figure 4b and data not shown). Expression of dsRed-Golgi also
labeled the dendrites with highly concentrated signal at branch
points (figure 4b inset). Punctate dsRED-ER fluorescence was also
observed throughout the dendritic arbors of interneurons (figure 4c).
Consistent with reported roles for local protein synthesis in spine
morphogenesis and synapse formation, the puncta of dsRed-ER
fluorescence localized to the base of dendritic spines (figure 4c inset).
Given the importance of proteinsynthesis and trafficking in dynamic
neural circuits, in vivo imaging of these organelles should offer insight
into the regulation of these processes [17–18].
One significant advantage over in utero electroporation is that
performing the procedure postnatally obviates the requirement of
surgery on timed pregnant mice. In utero electroporation is
challenging in transgenic or knockin mouse strains that have
smaller litters, are more difficult to breed in large numbers, and
are more sensitive to surgical manipulations. These problems are
overcome by performing electroporation postnatally where it was
asefficient inknockinmiceas wild typestrains (figure 4d–e). Wetook
advantage of an existing strain with fluorescently labeled olfactory
sensory neuron axons, synaptic partners of periglomerular cells .
unique morphology of periglomerular cell subtypes including their
arbors within GFP-positive glomeruli (figure 4d–e). Finally, beyond
its utility in various mouse strains, electroporation is also efficient in
equally effective in rats, thus broadening its application to species
lacking good genetic tools (figure 4f).
Numerous methods to alter gene expression in cells of living animals
have been developed in the last decade or so and many are now in
current use. Some still require significant technical sophistication,
while others are more amenable to common lab practices. In line
with these latter procedures we have here described a technique that
produces reliable results with a minimum of technical sophistication
in molecular biology or genomics. This should make the ability to
alter gene expression available to laboratories normally focused on
physiology or developmental studies.
Among the critical attributes of a gene alteration technique are
reliability and specificity. Control of gene expression is most useful
when it can be specified in particular cells or cell types. With
transgenesis this is usually accomplished by using a cell specific
promoter, if one is available. In the case of electroporation some
specificity can be gained by targeting the plasmids to certain brain
regions, by ‘‘aiming’’ the electrical pulse, or by using the procedure
at specific developmental time points. Here we have made use of
all three of these strategies, resulting in gene expression in a limited
set of interneuronal cells in the olfactory bulb.
In particular our interest here has been to follow the population of
proliferating cells in the SVZ and the integration of their daughter
neurons into existing circuits in the olfactory bulb. Not only does this
technique allow us to follow individual cells over extended periods of
time, but we have also used functional markers that permit
physiological recordings in brain slices. We demonstrate that
GCamp2 is functionally expressed in periglomerular and granule
cells of the olfactory bulb, thus providing an opportunity to observe
the response behavior of a large population of cells simultaneously.
The role of these cells in sensory processing has remained
controversial for some time and it might be expected that observing
population responses will advance that understanding. Finally the
expression of fluorescent markers results in the cell’s entire
morphology being visible – even in living tissue. Although beyond
the scope of this communication, it should be possible to alter
phenotypic characters and function by introducing new genes or by
knockdown of genes with shRNA through electroporation. Addi-
tionally,we havedemonstrated that the procedurecan be performed
in transgenic mice, allowing for the ability to knockout of floxed
genes by electroporation of CRE, or the rescue of targeted
disruptions in specific subpopulations. Further control over expres-
sion can be attained through the use of plasmids with temporal
control elements and cell specific promoters .
1.8 GV as measured with a negative current stimulus (25 pA). The calibration in A and B is 200 ms; 10 and 20 mV respectively. C–G. Calcium imaging of
single cells in slice from 3–6 week old mice electroporated with pCAG-GCamp2. C. PG cell before, during, and after response to KCl (100 mM) presented
during frames 10–25 (36–96 seconds; total of 1 min). Scale bar, 100 mm. (frames 5, 35, and 60 or 16, 136, 236 sec). Scale bar, 5%DF/F, 1 min. D. Granule
cell(attheborderoftheIPL/Mitralcelllayer)respondstoKCl(100 mM)presentedduringframes10–20(36–76 seconds;totalof40 sec).Scalebar=3%DF/
F, 1 min. E. A different granule cell (at the border of the IPL/Mitral cell layer) responds to glutamate (100 mM) presented during frames 10–20 (36–
76 seconds; total of 40 sec). Scale bar=2%DF/F, 1 min. F. Granule cell (same as in e) does not respond to Ringer presented during frames 10–20 (36–
76 seconds; total of 40 sec). Scale bar=2% DF/F, 1 min. G. Migrating cell in RMS during and after response to KCl (100 mM) presented during frames 10–
20 (36–76 seconds; total of 40 sec). Cell showed no response to application of Ringer alone (not shown). Scale bar=5%DF/F, 1 min
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The use of electroporation of the brain has become increasingly
common, but has been generallylimited to inutero delivery or labeling
of single cells postnatally . With the demonstration that electro-
poration can be used in early postnatal animals with high efficiency
an important barrier to its widespread use has been removed. No
surgery is required and there is no need to procure costly timed
pregnant animals. Additionally, electroporation is applicable in a
further developments are likely to proceed at a rapid pace. We
anticipate this will be the case with postnatal electroporation.
MATERIALS AND METHODS
Newborn pups were anesthetized by hypothermia, placed under a
dissecting scope and held by hand. Injection pipettes were beveled
(300) on a grinding stone (Narashige EG-44) to have 20–50 mm
openings Pipettes were guided into the brain rostral to the
olfactory bulb using a micromanipular. 1–2 mL of endotoxin-free
plasmid (1–5 mg/mL) were injected per pup. Immediately after
injection pups were electroporated with tweezer type electrodes
(BTX model 520) using a BTX ECM830. 5 pulses of 150V were
given of 50 ms duration with a 950msec interval. Positive
Electrode was swept 450 upwards from the first pulse aligned
with the center of the eyes. After electroporation, pups were placed
on a heat pad until they recovered full mobility and subsequently
returned to the nest. SVE129 (Taconic) and OMP-GFP mice (kind
gift of Peter Mombaerts, Rockefeller University) and Sprague
Dawley rats (Taconic Farms) were housed according to Columbia
University institutional animal care guidelines.
pCGLH (for expressing GFP) and pCRLH (for expressing mRFP)
were kindly provided by Kenneth Kwan and Nenad Sestan (Yale
University). To make pCAG-Gt, GFP was excised from pCGLH
using EcoRI and replaced with the Gateway Reading Frame
Cassette B (Invitrogen). GCamp2 was kindly provided by Junichi
Nakai (RIKEN Brain Institute). A BglII-GCamp2-NotI fragment
was subcloned into pENTR1a (Invitrogen) to create pENTR1a-
GCamp2, and was subsequnetly recombined with pCAG-Gt to
create pCAG-GCamp2. pCAG-Gt-IRES-EGFP was created by
subcloning IRES-EGFPfor pIRES-EGFP2 (Clontech) behind the
reading frame cassette. nlsCRE was kindly supplied by Andreas
Walz (Rockefeller University), subcloned into pENTR1a and
recombined into pCAG-Gt-IRES-EGFP.
dsRED-ER (Clonetech) were subcloned into pENTR1a and
recombined into pCAG-Gt.
Tissue preparation and staining
Animals were anesthetized with ketamine/xylazine and transcar-
dially perfused with 4% paraformaldehyde in PBS. Brain placed at
4uC, post-fixed for 4 hours and then incubated in 30% sucrose
overnight. Tissue was mounted using TissueTek (Electron
Microscopy Sciences, Hatfield, PA, USA) and section using a Leica
CM1850 cryostat (Bannockburn, IL, USA). 60 mm sections were
hydrated for 5 mins in PBS, incubated in TOTO-3 (1:10,000;
Molecular Probes) for 1 hour in 0.1% Triton-X in PBS (PBS-Tx),
and washed 2 times in PBS. 14 mm sections were processed for
immunohistochemistry by hydrating in PBS for 5 mins, blocking in
10% Normal Donkey Serum in PBS-Tx for 1 hour then incubating
in primary antibody overnight (Mouse anti-BrdU (1:50; Amersham),
rabbit anti-GFAP (1:1000; DAKO), goat anti-doublecortin (1:500;
Santa Cruz), mouse anti-parvalbumin (1:1000, Sigma), mouse anti-
calbindin (1:1000; Sigma), mouse anti-calretinin (1:1000, Chemi-
con), goat anti-tyrosine hydroxylase (1:500; Santa Cruz)). After 4
washes in PBS-Tx, slides were incubated in secondary (1:750 Alexa
488 and 594; Molecular Probes) for 2 hours. After washes, slides
were mounted using Vectasheild, imaged on a confocal, and images
analyzed using ImageJ.
Experiments were performed in olfactory bulb slices obtained from
3 to 4 week old mice. Animals were anesthetized with isofluorane
and decapitated. Brain slices were prepared in a modified artificial
cerebral spinal fluid (sucrose ACSF) of the following composition
(in mM): 222 sucrose, 27 NaHCO3, 1.25 NaH2PO4, 3 KCl, 1
CaCl2and 3 MgSO3. The whole brain was quickly removed and
placed in oxygenated ice-cold sucrose ACSF. A block of tissue,
containing part of the frontal lobes and the olfactory bulbs, was
glued with cyanoacrylate to a microslicer stage and bathed in
chilled sucrose ACSF. Sagittal sections (250–300 mm) of the
olfactory bulb were sliced at using a vibrating microslicer (Leica
VT1000). The slices were then transferred to an incubation
chamber containing normal ACSF (see below) and left to
recuperate first at 37uC for 30 min and then at room temperature
for another hour. In all experiments, unless otherwise indicated,
the extracellular solution is ACSF of the following composition (in
mM): 125 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 3 KCl, 2 CaCl2
and 1 MgSO4, 3 myo-inositol, 0.3 ascorbic acid, 2 Na-pyruvate
and 15 glucose, continuously oxygenated (95% O2and 5% CO2)
to give a pH 7.4 and of osmolarity of ,305 mOsm.
Slices were placed in a submerged recording chamber mounted on
the stage of a Olympus BX51, upright microscope, fitted with
infrared differential interference contrast optics (IR-DIC). Slices
were observed with a 40X water immersion objective and labeled
granule cells were recognized under fluorescent light (LG222
Sutter, CA). All experiments were performed at room tempera-
ture. Images of labeled cells were acquired using a CCD HQ2
camera (Photometrix). Standard patch pipettes (3–7 MW resis-
tance) were pulled on a horizontal puller. Cells were recorded in
Figure 4. Electroporation is effective for the expression of multiple constructs, in transgenic animals, and in rats. A. 20 mm section of an olfactory
bulb of a P6 mouse 5 days post-electroporation with pCAG-CRE-ires-EGFP; anti-CRE staining labels the nucleus (red) and GFP (green) labels the
cytoplasm with a 10X magnified view of the cell body. Scale=25 mm. B–C. Z projections of 60 mm sections from olfactory bulbs co-electroporated
with GFP and either dsRED-Golgi (B; 5 days post-electroporation) or dsRED-ER (C; 14 days post electroporation) showing labeled organelles. 10X
magnified views of highlighted regions show perinuclear golgi staining in the cell body and punctate ER staining at the base of dendritic spines.
Scale=25 mm. D–E. Electroporation of OMP-GFP mice with RFP plasmid. Z-projections of 60 mm sections showing the morphology of single
periglomerular cells (red) and the glomeruli they innervate (green). 2 types of PG cells are shown, one that innervates 3 glomeruli (E; scale=25 mm)
and another other with its processes are confined to a single glomerulus (F; scale=50 mm). F. Highly efficient electroporation of rats. Z-projection of
60 mm section from a P27 rat olfactory bulb electroporated at P1 showing widespread ectopic expression in all layers of the olfactory bulb. TOTO3
(blue) labels all nuclei. Scale=50 mm.
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the current-clamp mode the internal solution had the following Download full-text
composition (in mM); 135 K-gluconate, 10 NaCl, 10 KCl, 10
Hepes-Na, 2.5 ATP and 0.3 GTP adjusted to pH 7.3 with KOH.
The osmolarity of the internal solution was adjusted to 290–
305 mOsm, Single cell voltage (current-clamp) was recorded using
an dual EPC10 patch-clamp amplifier (Heka). Data was acquired
using the Patchmaster software (Heka) and analysed using macros
written for the Igor Pro software (Wavemetrics, Wosego, OR).
Calcium imaging in slices
Experiments were performed in olfactory bulb (OB) slices obtained
from 4 to 5 week-old mice. Animals were anesthetized with
ketamine/xylazine (0.05–0.15 ml 18 mg/ml, and 2 mg/ml, re-
spectively). OB slices (200 um thick) were prepared as described
above (see Slice Preparation). Imaging was carried out at room
temperature with a 20X water immersion objective in a
submerged recording chamber mounted on the stage of an
upright Zeiss Axioskop (Thornwood, NY, USA) equipped with a
CCD camera (C2741-08, Hamamatsu Photonics, Hamamatsu,
Japan) connected to a frame grabber (LG-3, Scion, Frederick,
MD, USA) on a Dell P4 2.4 GHz computer with 1.5 GB RAM
with Windows XP Pro. Scion Image software was used for data
acquisition and analysis (Scion). Customized macros were written
for shutter control (Uniblitz, Vincent Associates, Rochester, NY,
USA) and time-lapse imaging. Images were taken every 4 s (396 s
total). The recording chamber was continuously perfused with
oxygenated Ringer solution and stimuli were applied through a
manifold connected to the perfusion system. Stimuli were applied
for 40 s. Data is shown as the fractional change in fluorescent light
intensity: F/F0 or (F–F0)/F0, where F is the fluorescent light
intensity at each point and F0 is the value of emitted fluorescent
light before the stimulus application (baseline).
Found at: doi:10.1371/journal.pone.0001517.s001 (4.15 MB
We thank Andreas Walz, Kenneth Kwan, Nenad Stestan, and Junichi
Nakai for generously supplying us with reagents. Florencia Marcucci,
Michael Miller, Bolek Zapiec, Cen Zhang and the members of the Firestein
Lab provided helpful comments and technical support.
Conceived and designed the experiments: SF AC DZ. Performed the
experiments: AC CL JB RA. Analyzed the data: SF AC CL JB RA.
Contributed reagents/materials/analysis tools: AC. Wrote the paper: SF
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PLoS ONE | www.plosone.org9 January 2008 | Issue 1 | e1517