Neurons in the mammalian brain are a highly diverse population with a complex assortment
of electrophysiological, morphological and molecular properties, which has hindered
efforts to classify them into genetically and functionally meaningful subtypes and
to understand their various roles in the normal or pathological brain. Nowhere is
this issue more acutely felt than in the study of inhibitory cortical interneurons,
whose classification is still a source of contention and confusion (Markram et al.,
2004; Petilla Interneuron Nomenclature et al., 2008; Defelipe et al., 2013). A major
boon to investigators has been the development of mouse lines in which genetically
defined subsets of interneurons express fluorescent proteins, allowing their identification
and targeting during electrophysiological recordings or imaging experiments (Oliva
et al., 2000; Meyer et al., 2002; Chattopadhyaya et al., 2004; Ma et al., 2006). More
recently, investigators funded by the NIH Neuroscience Blueprint project have developed
a toolbox of “driver” lines in which the Cre recombinase gene is inserted immediately
downstream to genes that are known markers of specific interneuron subsets (Taniguchi
et al., 2011). These Cre lines can be bred with mice carrying floxed genes, to generate
cell type-specific knockouts of genes of interest. In addition, by breeding these
lines with mice from a parallel toolbox of Cre reporter lines in which a gene coding
sequence is inserted after a lox-STOP-lox cassette, or by transfecting them with viral
vectors carrying similar constructs, investigators can induce cell-type specific expression
of any gene of interest, from inert fluorescent proteins to calcium probes and light-activated
ion channels or pumps (Madisen et al., 2010, 2012; Zariwala et al., 2012). While these
technologies carry great promise and have already enabled some important findings,
the rush to use them also carries considerable risk, if the relevant expression patterns
are not fully characterized. A case in point is the somatostatin–IRES-Cre (SOM-Cre)
mouse line (Taniguchi et al., 2011), in which Cre expression was targeted to cells
containing the neuropeptide somatostatin (SOM). In the cerebral cortex, SOM-containing
neurons are a well-studied population of dendritic-targeting inhibitory interneurons
(Ma et al., 2006; Silberberg and Markram, 2007; Fanselow et al., 2008; Tan et al.,
2008; Ma et al., 2010; Fino and Yuste, 2011). The SOM-Cre line has already been used
in several high-profile studies, and in most of these the authors tacitly assumed—but
did not validate—that Cre-mediated recombination was restricted to SOM interneurons
(Adesnik et al., 2012; Gentet et al., 2012; Lee et al., 2012; Wilson et al., 2012;
Chiu et al., 2013; Kvitsiani et al., 2013; Xu et al., 2013). We found, however, that
6–10% of neurons expressing a Cre-dependent reporter in any given cortical layer were
fast-spiking/ parvalbumin-expressing (FS/PV) interneurons, a subtype quite distinct
from SOM interneurons in electrophysiological etc., morphological and molecular properties
(Rudy et al., 2011) [Note that there is another SOM-Cre line reported in the literature
(Lovett-Barron et al., 2012), which we did not test].
Our experiments complied with all relevant institutional and federal animal use guidelines
and regulations and were approved by the West Virginia University Institutional Animal
Care and Use Committee, and the methods have been previously published (Hu et al.,
2011). We crossed SOM-Cre males with females of the Ai14 reporter line (Madisen et
al., 2010), to generate double transgenic progeny in which SOM interneurons express
td-Tomato, a red fluorescent protein (RFP). We refer to such double transgenics as
“SOM-RFP mice.” We conducted whole-cell recordings in brain slices prepared from the
somatosensory cortex of SOM-RFP mice, and were surprised to find that 18% (20/112)
of RFP-expressing neurons in cortical layers 3 through 5 exhibited an electrophysiological
“fingerprint” typical of FS interneurons and clearly distinct from that of SOM interneurons
(Beierlein et al., 2003; Hu et al., 2011) (Figure 1A). To verify the subclass identity
objectively, we developed a simple non-linear classifier based on three electrophysiological
parameters: spike width at half height (SWHH), after-hyperpolarization (AHP) and adaptation
ratio (AR), measured as previously described (Ma et al., 2006). Each cell was tested
for three conditions: SWHH < 0.26 ms, AHP ≥ 14.5 mV and AR > 0.56, and was classified
as FS if at least 2 conditions were true and as SOM otherwise. We first tested this
classifier on a “ground truth” dataset of 91 GFP-expressing interneurons from the
X94 line (Ma et al., 2006); all but one were classified correctly as SOM interneurons.
Since in the X94 line GFP-expressing SOM interneurons have quasi fast-spiking firing
properties, separating them correctly from FS interneurons was a stringent test of
the classifier. We then tested the classifier on a sample of 15 GFP-expressing interneurons
from the G42 line (Chattopadhyaya et al., 2004) and 96 RFP-expressing interneurons
from progeny of a PV-Cre line (Hippenmeyer et al., 2005); all 111 interneurons were
classified correctly as FS. Finally, we applied the classifier to our sample of SOM-RFP
interneurons; 21 interneurons were classified as FS, including the 20 initially identified.
Recordings from progeny of SOM-Cre mice bred with a different reporter line [Ai39
(Madisen et al., 2012)] yielded a similar, though smaller percentage of FS interneurons
(2 out of 19).
Figure 1
RFP-expressing fast-spiking interneurons in SOM-RFP mice. (A) Voltage responses (upper
panels) of an RFP-expressing layer 5 barrel cortex neuron to the intracellular current
steps shown in the lower panel. Note the pronounced after-hyperpolarization (arrowhead)
and the non-adapting spike train, typical of FS interneurons. (B) The same neuron
was found post-hoc to contain biocytin and RFP and to be immunopositive for PV. (C)
A projection of a confocal stack from cortical area A1, showing five RFP-expressing
cells immunopositive for PV (circled). The cells labeled #1 and #2 are shown in (D).
(D) Single optical sections from subregions of the same field of view as in (C), in
the vicinity of cell #1 (left panels, arrows) and cell #2 (right panels, arrows),
separated into red and green channels.
FS interneurons are uniquely characterized by their PV expression (Kawaguchi and Kubota,
1993); we fixed a subset of slices in which we recorded RFP-expressing FS interneurons,
stained them with an antibody to PV and with fluorescent streptavidin (to label the
biocytin-filled neurons recorded from), and imaged them on a confocal microscope.
Out of 7 RFP-expressing FS neurons recovered, five were immunopositive for PV (Figure
1B), substantiating their electrophysiological identification. The remaining two neurons
were likely false negatives, due to wash-out of the cytoplasmic PV protein during
the whole-cell recording.
Since electrophysiological sampling can be biased (for example, FS interneurons may
be more likely to be targeted for recordings because they are typically larger than
SOM cells), we sectioned fixed brains from four SOM-RFP mice and dually immunostained
them against PV and SOM. Two brains were from third postnatal week pups (the age range
used in our recording experiments), and two were from ~1 month old animals. We imaged
sections representing five cortical areas (cingulate, M1, S1, A1, and V1) on a confocal
microscope and counted RFP+, PV+, and RFP+/PV+ double-labeled cells in confocal stacks,
verifying that double labeled cells were indeed so in single optical sections (Figures
1C,D). In total, about 18,000 RFP-expressing neurons were examined. Of these, on average
6% were immunopositive for PV. This number is a lower estimate, because weakly double-labeled
cells (cells with fluorescence intensity in either channel weaker than the average
intensity of single-labeled cells in the same optical section) were not counted as
double-labeled. When averaged by layer, cortical area and age group, percentage of
RFP+/PV+ double-labeled cells was highest in layer 4 of S1, reaching 14% in one animal
and averaging 11.5 and 10% in the younger and older animals, respectively. Percentage
of double-labeled cells was somewhat lower in other cortical areas and layers, with
the lowest (2%) found in layers 2/3 of V1 of the younger animals, but in the older
animals we observed 6–10% double labeled cells in all areas and layers except layers
2/3 of V1 (3%). We carefully examined all RFP+/PV+ double-labeled neurons for potential
SOM expression, but only 0.4% of them appeared to be immunopositive for SOM. Finally,
in the two brains with the best SOM immunostaining (one from each age range) we also
examined RFP+/PV− cells for SOM immunolabeling, and found that 8% of RFP-expressing
neurons, on average, were immunonegative for both SOM and PV. To what neuronal subtype
these immunonegative neurons belonged remains to be determined; however, some of them
could have been false-negative for PV, suggesting that the fraction of FS/PV interneurons
in these animals could have been higher than our estimate, closer to the percentage
observed in our electrophysiological recordings.
Our observation of RFP expression in FS interneurons cannot be explained by leaky
expression of the RFP gene (i.e., expression in cells that did not undergo recombination)
or by non-specific expression of the Cre gene (i.e., expression not under the control
of the endogenous SOM promoter), because we would then expect to see widespread RFP
expression in excitatory neurons, which are the majority cortical cell type. Thus,
RFP expression must be under the same genetic control as the endogenous SOM gene;
however, we found no SOM expression in the subset of double-labeled RFP+/PV+ neurons,
consistent with previous studies which observed no overlap between SOM and PV protein
expression in mouse and rat cortical interneurons (Gonchar and Burkhalter, 1997; Xu
et al., 2010). This apparent paradox can be explained in two different ways. First,
both SOM and FS/PV interneurons are born from embryonic progenitors in the medial
ganglionic eminence (Batista-Brito and Fishell, 2009), and it is possible that a subset
of progenitors (or of post-mitotic neuroblasts) destined to become FS/PV interneurons
transiently express SOM. In the SOM-Cre mice these cells will also transiently express
Cre recombinase, undergo Cre-mediated recombination and then express RFP for life,
even after losing their SOM expression and attaining their mature FS phenotype. Alternatively,
it has been reported that a subset of adult mouse cortical interneurons co-express
PV and SOM mRNA (Lee et al., 2010). In the SOM-Cre line these neurons will co-express
PV mRNA and the bicistronic SOM-IRES-Cre transcript. It is possible that the Cre transcript
is translated into protein even though the SOM transcript is not, or that both transcripts
are translated but at very low levels, sufficient for Cre-mediated recombination but
not for detection of SOM protein.
The two alternative mechanisms above imply slightly different risks to investigators:
the first implies that recombination in FS interneurons is expected when breeding
SOM-Cre mice with Cre reporter lines, but not necessarily when transfecting adult
neurons with viral vectors, while the second mechanism would result in off-target
recombination regardless of the mode of delivery of the reporter construct. Either
way, our findings underscore an important caveat for researchers using “subtype specific”
mouse driver lines, including those in which the Cre coding sequence is presumed to
be under the control of the endogenous gene promoter—these lines should be used with
caution and with proper validation. Most studies using the SOM-Cre line appear to
use it without validation; an exception is a recent study that tested recombination
specificity and found that 5% of recombined neurons in the visual cortex were immunopositive
for PV (Pfeffer et al., 2013). We found, by a lower estimate, 6–10% off-target recombination
in FS interneurons in most cortical areas and layers. When using the SOM-Cre line
to express optogenetic constructs for activation or silencing of SOM interneurons,
this degree of contamination by FS interneurons can potentially affect the results
and lead to erroneous conclusions. For example, a recent study Kvitsiani et al. (2013)
observed that about one third of SOM-Cre interneurons tagged by Cre-dependent channelrhodopsin
(ChR2) exhibited fast spike waveforms and high firing rates reminiscent of FS interneurons;
but the possibility that some of these were actually ChR2-expressing FS interneurons
was not considered. We submit that investigators ignoring the potential for off-target
recombination when using the SOM-Cre line, or indeed any other Cre driver line that
has not been fully characterized, are doing so at their own risk.