Strategy for Genetically Accessing Neuronal Populations Based on Immediate Early Gene Expression
TRAP utilizes two genetic components: 1) a transgene that takes advantage of IEG regulatory elements to express a drug-dependent recombinase, such as the tamoxifen-dependent Cre recombinase CreERT2
(Feil et al., 1997
), in an activity-dependent manner; and 2) a transgene or virus that expresses an effector protein in a recombination-dependent manner (). For the first component, we generated knock-in mice in which CreERT2
is expressed from the endogenous Fos
loci ( and Figure S1
). These knock-ins retain all sequences 5′ to the translational start site but replace the endogenous 3′UTRs, which contribute to mRNA destabilization and to Arc
mRNA dendritic trafficking (see Supplemental Experimental Procedures
), with an exogenous SV40 polyadenylation signal to promote high-level expression; the introns and coding regions are also displaced ( and S1
). Although these alleles are predicted to be null for Arc
, we have not observed any gross behavioral or anatomical abnormalities in the resulting heterozygous ArcCreER/+
mice (see Discussion). For the second component, we used AI14, a knock-in allele of the Rosa26 (R26)
locus that allows high-level, ubiquitous expression of the red fluorescent protein tdTomato following excision of a loxP-flanked transcriptional stop signal (Madisen et al., 2010
Strategy of Targeted Recombination in Active Populations (TRAP)
In the absence of tamoxifen (TM), CreERT2 is retained in the cytoplasm of active cells, and no recombination can occur (, top). TM administration causes active CreERT2-expressing cells to undergo Cre-mediated recombination (to be “TRAPed”), resulting in permanent expression of the effector gene (e.g., tdTomato; , bottom). Non-active cells do not express CreERT2 and do not undergo recombination even in the presence of TM. Due to the transient nature of IEG transcription, CreERT2 is present for only a limited time following neuronal activation, and the lifetime of TM is limited by metabolism and excretion; as a result, only neurons that are active within a limited time window around drug administration can be TRAPed.
Background Recombination Is Very Low in FosTRAP Mice and Is Limited to Specific Cell Types in ArcTRAP Mice
Since many CreERT2
lines have drug-independent recombination as a result of leaky CreERT2
activity (e.g. Madisen et al., 2010
), we first examined recombination in FosTRAP (FosCreER/+R26AI14/+
) and ArcTRAP (ArcCreER/+R26AI14/+
) mice that were not treated with TM. Under these conditions, we observed very few labeled cells (from zero to a few cells per 60 μM sagittal section) in both young adult (, top; , left column) and aged (6-7-month-old; Figure S2B
, top; Figure S2C
, right column) FosTRAP mice. Thus, despite CreERT2
expression in response to neuronal activity throughout the life of the animal, cytoplasmic retention of the CreERT2
protein in the absence of TM prevented CreERT2
-induced recombination (, top). Labeling in untreated ArcTRAP mice is significant but is restricted to a few specific cell types, including layer 6 neurons in neocortex and granule cells in the dentate gyrus (, bottom; , left column). The TM-independent recombination in ArcTRAP mice is likely caused by Arc
’s relatively high level of expression (Lyford et al., 1995
). Consistent with this assumption, the frequency of labeled cells in untreated ArcTRAP mice increased with the animal’s age (Figure S2B
, bottom; Figure S2D
, right column). The remaining experiments in this paper were performed in mice that were 6-8 weeks of age.
Background and Homecage Recombination in FosTRAP and ArcTRAP Mice
Fos and Arc Loci Drive CreER Activity in Partially Overlapping Neuronal Populations in the Homecage
Treatment of both FosTRAP and ArcTRAP mice with TM (150 mg/kg i.p.) in the homecage induced labeling in restricted regions throughout the brain when mice were examined one week post-injection (; , right columns). Because tdTomato fills cell bodies and processes, the identities of recombined cells could readily be determined by morphology. In FosTRAP mice, we observed recombination in cells lining the brain and ventricle surfaces, in blood vessels, and in putative oligodendrocytes in white matter. Within the grey matter, recombination occurred almost exclusively in cells with neuronal morphologies; recombination in grey matter glial cells was rarely observed. In ArcTRAP mice, TM treatment induced labeling most dramatically in forebrain regions and was exclusively neuronal. Compared to uninjected controls, injection with vehicle produced no increase in the numbers of labeled cells in either line, indicating that the stimulus of injection alone was insufficient to trigger recombination in the absence of TM (Figure S2A
; Figure S2C and D
, left columns).
Following homecage TM treatment, ArcTRAP and FosTRAP mice had similar patterns of recombination in many brain areas (, right columns), including in neocortex, where labeled cells were relatively sparse in layer 5; in the hippocampus, where labeled cells were enriched in the dentate gyrus and in CA1; in the piriform cortex; and in the olfactory bulb, where granule cells were heavily TRAPed. Even for those cell types that had high background recombination in untreated ArcTRAP mice, TM treatment increased labeling (e.g. compare left and right columns in for the hippocampus and for neocortical layer 6). In most brain regions, the recombination frequency was higher in ArcTRAP mice than in FosTRAP mice, but FosTRAP was more efficient in some areas, such as the cerebellum. In the thalamus of ArcTRAP mice, no recombination in intrinsic thalamic neurons was detected despite densely labeled corticothalamic axons; in contrast, FosTRAP mice show efficient recombination in some thalamic nuclei. On the other hand, medium spiny neurons of the striatum were efficiently labeled with ArcTRAP but not with FosTRAP.
The high frequency of recombination under homecage conditions in both FosTRAP and ArcTRAP mice contrasts with the low levels of Fos
expression under similar conditions (Lyford et al., 1995
; Morgan et al., 1987
). Since CreERT2
-mediated recombination is irreversible, TRAPed cells accumulate as long as TM is present; in addition, perdurance of CreERT2
mRNA or protein may allow TRAPing of cells activated prior to TM injection. The final TRAPed population is thus a result of activity integrated over a time window determined by CreERT2
stability and by TM metabolism and excretion. In contrast, endogenous Arc and Fos are rapidly degraded after induction and thus report activity over a more limited time period prior to sacrifice.
The above experiments demonstrate that, with the exception of a small subset of cell types in the ArcTRAP mice, recombination in TRAP mice is TM-dependent. They also show that Arc and Fos loci differ to some extent in their cell type specificities. Finally, although ArcTRAP has higher background recombination than FosTRAP, it also has higher TM-induced recombination (compare the bottom panels of ). The two lines may thus be preferred for certain types of experiments depending on the relative importance of specificity versus efficiency and on the cell types of interest.
Recombination in the Primary Somatosensory Cortex is Dependent on Sensory Input
To determine whether neurons that are activated by specific sensory stimuli can be TRAPed, we performed sensory deprivation experiments in the whisker-barrel system of TRAP mice. Somatosensory information from the facial vibrissae are relayed via brainstem and thalamic nuclei to contralateral primary somatosensory cortex (S1), where thalamic afferents representing individual whiskers innervate discrete somatotopically organized “barrels” in layer 4 (Petersen, 2007
). Stimulation of a single whisker induces IEG expression selectively in the corresponding barrel (Staiger et al., 2000
). We describe below results on FosTRAP mice (); however, qualitatively similar results were obtained with ArcTRAP (Figure S3
FosTRAP in Barrel Cortex of Whisker-Plucked Mice
After manipulating sensory input to the barrel cortex by plucking specific whiskers, we injected mice with TM and returned them to the homecage with tubes and nesting material to stimulate whisker exploration (). When all whiskers were left intact, labeled processes and cells were distributed uniformly across all barrels (, left), which were visible both in coronal sections (, bottom) and in sections tangential to layer 4 (, top). In contrast, when all large whiskers except C2 were plucked, a dense collection of cells and processes was apparent in the C2 barrel, with only scattered labeled cells present in other barrels (, right). This restriction of labeled cells to the C2 barrel extended up to layer 2/3 but not down to layer 6, where a large number of cells outside the C2 barrel were labeled (, right). TRAPing of cells in barrel cortex is thus dependent on specific sensory input.
Layer 4 barrel neurons can be activated by deflections of adjacent whiskers (Armstrong-James et al., 1992
). To test the contributions of these non-principal inputs to TRAPing, we repeated the above experiment in mice that had only the C2 whisker removed. We found that under these conditions the corresponding C2 barrel was devoid of labeled cells and processes and that this effect was strongest in layer 4 (, middle). This observation suggests that Fos
expression in layer 4 is evoked mainly by thalamocortical input, either directly by thalamocortical synapses or indirectly by intracortical connections within a barrel.
Different Forms of Tamoxifen Allow Activity to Be TRAPed Over Different Time Windows
We performed additional characterization of TRAP in the visual system, where IEG expression can be robustly induced by light (Kaczmarek and Chaudhuri, 1997
), focusing on FosTRAP because of its low TM-independent background. Light stimulation increased the numbers of TRAPed cells in the dorsal lateral geniculate nucleus (dLGN) and primary visual cortex (V1) by 4.2-fold and 8.3-fold, respectively, relative to mice maintained in the dark ( and S4A-C
). The TRAPed cells were distributed across all layers of V1 but were most dense in layer 4, and more than 96% expressed the neuronal marker NeuN; the remaining ~4% of cells included putative endothelial cells and glia (Figure S4E
). Fewer than ~3% of V1 cells were GABAergic (Figure S4E
). Most TRAPed cells in V1 are thus excitatory neurons.
Time Window for Effective TRAPing Relative to Drug Injection in Primary Visual Cortex
To determine the time window around a TM injection during which active cells are efficiently TRAPed, we examined V1 in FosTRAP mice that had been stimulated with 1 h of diffuse bright light at various times relative to the injection (). TRAPing was maximal when light stimulation occurred 23-24 h after injection. No TRAPing above the level of the dark control occurred when light was given 6-7 h before the injection or 35-36 h after injection (). Labeling in a control region, S1, was similar across all time points (). Thus, under these conditions, TRAP appears to be sensitive to neuronal activation that occurs less than 6 h prior to injection and up to 24-36 h after injection.
A long time window may be desirable in cases where it is beneficial to TRAP cells based on integration of activity over a long period of time. However, applications that utilize stimuli and experiences of short duration could benefit from a shorter time window. After injection, TM is metabolized to its principal active form, 4-hydroxytamoxifen (4-OHT; Robinson et al., 1991
). Directly injecting 4-OHT shortened the TRAPing time window to <12 h (): optimal TRAPing in V1 was observed when light was administered in the hour immediately before injection of 4-OHT, and minimal TRAPing was observed when light was delivered 6-7 h before or 5-6 h after the injection.
To determine the dependence of TRAP on stimulus duration, we delivered light pulses of varying durations beginning 1 h before a 4-OHT injection. Relative to mice left in the dark, mice exposed to light pulses of 5 min, 15 min, and 60 min in duration had 2.6-fold, 4.9-fold, and 8.3-fold more TRAPed cells in V1 (Figure S5A-C
). Thus, even short (5 min) stimuli are sufficient for TRAPing, although longer duration stimuli increase the total numbers of TRAPed cells; these results are consistent with prior findings that induction of Fos protein in V1 is dependent on stimulus duration (Amir and Robinson, 1996
The time course of effector expression after TRAPing determines the earliest time point at which subsequent experimental manipulations are possible. Although this parameter is likely to be dependent on effector and cell type, we found that it took at least 72 h following light stimulation and 4-OHT injection for TRAPed V1 cells to express sufficiently high levels of tdTomato to be reliably identified (Figure S5D-F
TRAP Provides Selective Genetic Access to Cochlear Nucleus Neurons Tuned to Specific Sound Frequencies
Next, we took advantage of the tonotopic organization of the auditory system to evaluate whether TRAP can provide genetic access to cell populations that are activated by particular features of sensory stimuli. We focused on the cochlear nucleus (CN), all three subdivisions of which receive input from spiral ganglion neurons (SGNs) that carry auditory information from the cochlea. SGNs that innervate the apex or the base of the cochlea are tuned to low- and high-frequency sounds, and terminate their axons in the ventral or dorsal regions of each CN subdivision, respectively. SGN axons are thus arrayed in a high to low frequency tonotopic map along the dorsoventral axis of the CN (Young and Oertel, 2004
). Similar tonotopy is observed in CN neuronal responses themselves, determined both electrophysiologically (Luo et al., 2009
) and by Fos
induction (Friauf, 1992
; Saint Marie et al., 1999
We injected FosTRAP mice with 4-OHT during a 4 kHz or 16 kHz continuous pure tone stimulus to TRAP CN neurons tuned to those frequencies; to increase the total number of TRAPed cells, we took advantage of TRAP’s ability to integrate IEG expression over time by using a 4 h pure tone stimulus during the TRAPing period. 4-5 days later, we delivered a second 4 kHz or 16 kHz stimulus for 1 h and then sacrificed the mice 1 h later and processed the tissue for Fos immunostaining (). Thus, TRAPed cells represent neurons activated by the first stimulus and Fos protein immunopositive (Fos+) cells represent neurons activated by the second stimulus.
TRAPing Cells that Respond to Specific Frequencies of Auditory Stimuli
Consistent with prior results, we found that 4 kHz stimulation during the second epoch induced Fos expression in clusters of cells in all three CN subdivisions that were located more ventrally than the clusters that were Fos+ after 16 kHz stimulation. Similar results were observed for TRAPed cells. When the tone frequency was the same for the two stimulus epochs, the TRAPed and Fos+ populations overlapped, with the 4 kHz cluster localized more ventrally than the 16 kHz cluster (, first and third columns). Within mice receiving stimuli of two different frequencies, the cells TRAPed by the 16 kHz stimulus were dorsal to Fos+ cells induced by the 4 kHz stimulus (, second column), while the reverse was true when the 4 kHz stimulus was TRAPed and the 16 kHz representation was revealed by Fos immunostaining (, last column). These qualitative impressions were confirmed by quantification of the numbers of TRAPed and Fos+ cells in bins spanning the dorsoventral axis of the central dorsal cochlear nucleus (DCN; ). In general, the populations of TRAPed cells were less sharply confined along the dorsoventral axis than the population of Fos+ cells; this may reflect the longer stimulus used for TRAPing (4 h, versus 1 h for Fos immunostaining) or some general noise in the TRAP approach. Regardless, this analysis supports the observations from individual sections that both TRAP and Fos immunostaining reveal similar tonotopic maps along the dorsoventral axis of the DCN.
We also quantified the overlap between TRAPed and Fos+ cells for the different treatment groups across the entire extent of the DCN. As expected, the overlap between the two populations was greater when the stimuli during the two epochs were the same (4kHz-4kHz and 16kHz-16kHz groups) than when the stimuli during the two epochs were different (16kHz-4kHz and 4kHz-16kHz groups; ). The partial overlap in the 16kHz-4kHz and 4kHz-16kHz groups is not unexpected given the complexity of the tuning curves for some types of CN neurons (Luo et al., 2009
; Young and Oertel, 2004
). The fact that ~70% of Fos+ cells were also TRAPed in the 16kHz-16kHz and 4kHz-4kHz groups (, left) suggests that TRAP can provide genetic access to the majority of cells that express Fos in response to a particular stimulus. Our finding that only ~30-40% of TRAPed cells were Fos+ in these groups (, right) could be due to some noise intrinsic to the TRAP approach or to greater sensitivity of TRAP relative to Fos immunostaining; alternatively, it could be due to TRAPing of cells that expressed Fos in response to the long-duration stimulus used during the TRAPing period but that did not express Fos in response to the shorter stimulus delivered prior to sacrifice.
Neurons Activated by Complex Experiences Can Be Effectively TRAPed
While the experiments in the somatosensory, visual, and auditory systems suggest that TRAP can have high signal-to-noise ratio in the context of sensory deprivation and controlled stimulation, we wanted to evaluate whether it would also be possible to TRAP neurons activated by complex experiences. Toward this end, we allowed FosTRAP mice to explore a novel environment for 1 h, injected them with either 4-OHT or vehicle, and then allowed them to continue exploring the novel environment for another 1 h. An additional group of mice received 4-OHT injections in the homecage. Mice were sacrificed one week after treatment. Virtually no cells were TRAPed in any brain region in mice given an injection of vehicle during novel environment exploration ( and S6A
), confirming that CreER activity is tightly regulated by tamoxifen. Compared to 4-OHT-injected homecage controls, mice injected with 4-OHT in a novel environment had more TRAPed cells throughout the brain. For instance, novel environment exploration increased the numbers of TRAPed cells in piriform and barrel cortices by 1.9-fold and 3.5-fold, respectively (Figure S6
), consistent with prior studies using in situ
hybridization or immunohistochemistry to detect IEGs (Hess et al., 1995
; Staiger et al., 2000
). Interestingly, the TRAPing of oligodendrocytes in the white matter was not affected by novel environment exposure (Figure S6
), suggesting that the differences in neuronal TRAPing were not due to variability in 4-OHT dosing or metabolism.
TRAPing Cells Activated by Exploration of a Novel Environment
We also found that exploration of the novel environment increased the numbers of TRAPed dentate gyrus (DG) granule cells and CA1 pyramidal cells by 2.4-fold and 2.9-fold, respectively, compared to homecage controls (). This result is consistent with previous work using in situ
hybridization to detect IEGs (Guzowski et al., 1999
; Hess et al., 1995
). TRAPed cells in CA3 were very sparse in all conditions. In the DG, more TRAPed cells were located in the upper (suprapyramidal) blade than in the lower (infrapyramidal) blade (). The increased TRAPing of DG granule cells with novel environment exploration was also greater in the upper blade than in the lower blade (), consistent with prior reports of an upper blade-selective increase in Arc
expression in rats exploring a novel environment (Chawla et al., 2005
). Although the significance of this apparent functional difference between upper and lower blades is unclear, our data, taken together with prior results, suggest that it is consistent for different IEGs and across rats and mice. Moreover, TRAP can capture patterns of DG activity consistent with those obtained using classical methods, and TRAP has sufficient signal-to-noise ratio in the absence of sensory deprivation to detect neuronal activity associated with complex experiences.