Transgene Expression in Target-Defined Neuron Populations Mediated by Retrograde Infection with Adeno-Associated Viral Vectors

Retrograde infection of OB projection neurons by rAAVs

We first tested the ability of rAAVs to drive transgene expression via retrograde axonal infection in the mouse olfactory pathway. The olfactory bulb (OB) was used as a test bed for characterizing retrograde infection because its principal neurons, mitral/tufted cells (MTCs), constitute an anatomically and functionally heterogeneous group with subpopulations projecting to multiple forebrain targets and having distinct dendritic and laminar organization (Macrides and Schneider, 1982; Nagayama et al., 2010; Igarashi et al., 2012). To selectively target MTCs, we used a transgenic mouse line (PCdh21-Cre) that, within the forebrain, expresses Cre primarily in MTCs (Nagai et al., 2005; Mitsui et al., 2011; Wachowiak et al., 2013). Injection of rAAV containing Cre-dependent expression constructs (e.g., 2/1.FLEX.GCaMP3; 2/1.FLEX.ChR2-tdTomato) into the OB leads to expression that appears restricted to MTCs, with the majority of somata in the mitral cell layer and deep glomerular layer (Fig. 1A) and fewer somata in the external plexiform layer. This expression pattern matches that described for mRNA expression of PCdh21 in the OB (Nakajima et al., 2001), indicating that the rAAV2/1 serotype has sufficient tropism to enable efficient transduction of the Cre-dependent transgene in OB MTCs. The PCdh21+ MTC population likely includes superficial and external tufted cells with somata in the glomerular layer, whose axonal projections either remain intrinsic to the OB or project only to the most anterior portions of olfactory cortex (Schoenfeld et al., 1985; Shipley et al., 2004; Ghosh et al., 2011; Igarashi et al., 2012).

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Figure 1.

Cre-dependent and target-specific transgene expression in OB output neurons via retrograde rAAV infection. A, Expression of GCaMP3 in MTCs after injection of Cre-dependent virus (2/1.FLEX.GCaMP3) into the OB of a PCdh21-Cre mouse. Right image shows higher-magnification view of the dorsal OB. Expression is strongest in the dorsal half of the OB and apparent in somata in the mitral cell layer (MC) and the superficial external plexiform layer (EPL)/deep glomerular layer (GL). Dendritic processes are also apparent throughout the EPL. B, GCaMP3 expression after injection of 2/1.FLEX.GCaMP3 into aPC of a PCdh21-Cre mouse. Left, Low-magnification, epifluorescence image showing heavy expression in MTCs throughout the OB. Middle, Confocal stack showing dense expression in cells predominately in the MC layer (compare with A). Right, Axon terminals of GCaMP3-expressing neurons in piriform cortex. Note the lack of cell bodies expressing GCaMP3 in piriform. C, D, GCaMP3 expression after injection of 2/1.FLEX.GCaMP3 into (C) posterior piriform cortex (pPC) and (D) posterolateral cortical amygdala (PLCo). Injection in both areas leads to preferential expression in mitral and deep tufted cells, with dendritic branching restricted to the deep half of the EPL. E, Expression of GCaMP3 after 2/1.FLEX.GCaMP3 injection into medial amygdala (MeA). Expression is strongest in mitral cells of the accessory OB (AOB).

To test for retrograde infection of OB MTCs with Cre-dependent rAAV2/1 constructs, we injected virus into anterior piriform cortex (aPC), a major target of MTC axons, in PCdh21-Cre mice. We injected a relatively large volume (400–1000 nL) of virus to cover a large extent of aPC. Injection of AAV2/1.FLEX.GCaMP3 into aPC led to widespread expression of GCaMP3 in MTCs throughout the OB (Fig. 1B, left; n = 25 animals). Somata at the injection site in aPC, as well as centrifugal neurons projecting from aPC to the OB (Haberly and Price, 1978; Shipley and Adamek, 1984; Boyd et al., 2012) do not express the transgene as they do not contain Cre (Fig. 1B, right). Transgene expression after retrograde rAAV infection occurred in MTCs throughout the entire extent of the OB, in contrast to localized expression seen with direct OB injection (Fig. 1A, left). Retrograde infection also led to preferential expression in neurons in the mitral cell layer compared with direct OB infection. In retrogradely infected mice, 85% of labeled somata (412 cells, 10 mice) were located in the mitral cell layer, with the remainder in the external plexiform layer. In mice injected directly into the OB, only 61% of counted somata (885 cells, six mice) were located in the mitral cell layer, with the remaining in the external plexiform layer or glomerular layer (Fig. 1A). This difference likely reflects the exclusion of PCdh21+ cell types that do not project to aPC. Retrograde infection was robust, typically leading to expression in a high fraction of all PCdh21+ mitral cells: in PCdh21-Cre mice expressing GCaMP after retrograde infection into aPC, 81% of neurons in the mitral cell layer that expressed the MTC-specific marker Tbx21 (Nagai et al., 2005; Mitsui et al., 2011) were also positive for GCaMP (351 of 433 cells, three mice). Retrograde infection was also reproducible: in >50 injections into aPC in PCdh21-Cre mice, we have achieved widespread expression in MTCs in ∼80% of cases, with most failures attributable to errors in stereotaxic targeting. Retrograde infection was also effective using injection volumes as small as 100 nL (two mice, four injections), though fewer MTCs were labeled.

To establish the usefulness of retrograde infection for isolating subsets of genetically defined neurons based on their axonal projections, we injected virus into different olfactory cortical areas known to be targeted by distinct populations of OB MTCs. These experiments also helped to confirm that transgene expression was in fact due to retrograde infection rather than bulk spread of injected virus from forebrain areas to the OB. One subpopulation of MTCs includes neurons with somata in the mitral cell layer and lateral dendrites that remain restricted to the deep half of the external plexiform layer and which project to posterior piriform cortex and cortical amygdala (Haberly and Price, 1977; Scott et al., 1980; Schneider and Scott, 1983). Another subpopulation of PCdh21+ MTCs includes mitral cells of the accessory OB, which project to medial amygdala but not to piriform cortex (Scalia and Winans, 1975). Consistent with these anatomically defined divisions, we found that Cre-dependent rAAV injection (2/1.FLEX.GCaMP3) into posterior piriform (seven mice, 12 injections) or cortical amygdala (two mice, four injections) led to expression almost exclusively in mitral cells, with 85 and 90% of GCaMP3-expressing neurons (n = 124 and 142 cells) having somata in the mitral cell layer for posterior piriform and cortical amygdala injections, respectively. Furthermore the lateral dendrites of GCaMP3-expressing neurons were restricted to the deep external plexiform layer of the main OB (Fig. 1C,D). Injection into medial amygdala, in contrast, led to expression solely in mitral cells of the accessory OB (Fig. 1E; two mice, four injections). These results argue that the observed expression is not due to bulk spread of virus from the injection site, and that retrograde viral infection can be used to isolate neuronal subpopulations defined on the basis of their axonal projection patterns. We also note that the distance from cortical or medial amygdala to the OB is ∼5.3–6.1 mm, indicating that retrograde infection is effective over a considerable distance.

Robust retrograde infection by rAAVs

Because retrograde infection by rAAVs has been reported as weak or ineffective in some cases and effective in others (Chamberlin et al., 1998; Burger et al., 2004; Wachowiak et al., 2013; Zhang et al., 2013), we evaluated the robustness of this phenomenon by testing for expression of different Cre-dependent transgene constructs and serotypes. We observed similar success of retrograde infection for identical Cre-dependent constructs encoding multiple transgenes and using multiple promoters (Table 1), and across multiple production lots obtained from the same source over 24 months. Both 2/1 and 2/5 serotypes resulted in similarly strong retrograde expression levels (Table 1).

We next tested the ability of constitutive (Cre-independent) constructs to drive transgene expression after retrograde infection using the same injection protocol in PCdh21-Cre mice. Injection of the constitutive GCaMP5G construct (2/1.GCaMP5G) led to expression in neurons throughout aPC and in the terminals of corticofugal axons in the granule cell and glomerular layers of the OB (Fig. 2A), indicating effective viral transduction around the injection site followed by anterograde labeling of axonal projections from piriform. However, this virus failed to drive expression in MTCs of the OB (11 mice, 22 injections; eight cells total could be identified in the MCL in 14 sections in five of the mice). Likewise, injection of 2/1.EGFP, the construct used in a large-scale effort to map anterograde projection patterns across the mouse brain (Allen Brain Institute, http://help.brain-map.org/display/mouseconnectivity/Documentation), also showed robust expression in aPC neurons and their corticofugal projections to the OB but no expression in MTCs (two mice, four injections; no cells could be identified in the MCL in eight sections; Fig. 2B). These data are consistent with previous results showing an absence of retrograde labeling when using rAAVs with constitutive constructs (Chamberlin et al., 1998; Skorupa et al., 1999; Heuer et al., 2002; Passini et al., 2002). These results also suggest that retrograde infection is not facilitated by the presence of Cre recombinase in infected neurons.

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Figure 2.

Differential ability of constitutive rAAV vectors to drive retrograde expression in OB projection neurons. A, GCaMP5G expression in centrifugal axons in the OB after injection of the constitutive rAAV 2/1.GCaMP5G into aPC of a PCdh21-Cre mouse. Left, Middle, Low-magnification and high-magnification images showing strong labeling of axonal processes in the granule cell layer (GCL) throughout the OB but not cell bodies expressing GCaMP5G. Right, Image of aPC, near injection site, showing numerous GCaMP5G-expressing cell bodies. B, Similar labeling of centrifugal axons in the OB after injection of rAAV2/1.EGFP into aPC. Here, axon terminals are apparent in the GCL as well as around the periphery of glomeruli in the glomerular layer (GL), but no cell bodies are apparent in the OB. C, Robust retrograde expression mediated by the constitutive rAAV 2/1.mCherry. 2/1.mCherry was coinjected with 2/1.FLEX.GCaMP5G into aPC of a PCdh21-Cre mouse. Left image shows GCaMP5G expression in the OB (green). Middle image shows mCherry expression (red). Right image shows overlay. GCaMP5G expression is apparent primarily in mitral cells, while mCherry expression is apparent in mitral cells and in axons in the granule cell layer, consistent with both retrograde infection of mitral cells and direct infection of neurons projecting from aPC to the OB.

Surprisingly, however, a third Cre-independent construct, 2/1.mCherry, was highly effective at driving transgene expression via retrograde infection. To characterize this, we coinjected 2/1.mCherry virus into aPC of PCdh21-Cre mice along with the Cre-dependent 2/1.FLEX.GCaMP3 vector described above (two mice, four injections). In these experiments, coexpression of mCherry and GCaMP3 was prevalent in OB MTCs (Fig. 2C), with clear coexpression in 92% (292 of 317 GCaMP-expressing cells) of visible cells in the mitral cell layer. While mCherry was visible in the axon terminals of corticofugal projections from aPC to the OB, we observed no mCherry expression in neurons of the granule cell layer, as might be expected if the virus spread by bulk movement to the OB. These experiments demonstrate that at least some Cre-independent rAAV vectors are capable of driving robust transgene expression through retrograde infection via axonal projections, consistent with a recent report for 2/1.hChR2 (Zhang et al., 2013). Notably, these results also demonstrate that simultaneous infection by two different rAAVs and retrograde expression of distinct transgenes is possible in the same neuron.

Retrograde transgene expression in target-defined cortical pyramidal neuron subsets

We next assessed the ability of rAAVs to retrogradely infect other projection neuron types. Layer 5 pyramidal neurons are a major source of output from neocortex and project to diverse targets. There is evidence that layer 5 subpopulations projecting to different targets have distinct functional and morphological properties (Hattox and Nelson, 2007; Larsen et al., 2007). To assess the ability of retrograde rAAV infection to define target-specific cortical neuron populations, we investigated projections from mouse primary somatosensory cortex (S1), in which different subpopulations of layer 5 pyramidal neurons have been previously identified using retrograde tracing with a recombinant rabies virus (Larsen et al., 2007). We used the constitutive rAAV 2/1.mCherry construct, injected into three different targets of S1 layer 5 pyramidal neurons: superior colliculus, thalamus, and contralateral S1.

2/1.mCherry injection into these three targets (six mice; one injection in each) led to mCherry expression patterns that largely recapitulated those achieved with recombinant rabies virus (Larsen et al., 2007). Injection into superior colliculus led to strong expression in layer 5 pyramidal neurons within a spatially restricted region of S1 (Fig. 3A,D). Injection into thalamus also drove strong mCherry expression in layer 5 pyramidal neurons, although across a broader area of S1 (Fig. 3B,E). Injection into S1 led to mCherry expression in layer 5 pyramidal neurons in contralateral S1 (Fig. 3C,F). S1 injection also drove strong expression in layer 5 neurons around the injection site, whose axons could be seen coursing through the corpus callosum and terminating throughout contralateral S1 (Fig. 3C,F). This last experiment demonstrates the ability of rAAVs to infect neurons directly through their somata, in contrast to recombinant rabies variants, which selectively infect neurons via their axon terminals (Kelly and Strick, 2000). The number of retrogradely infected pyramidal neurons apparent after superior colliculus or thalamus injection appeared similar to or greater than those reported using recombinant rabies virus (Larsen et al., 2007), indicating a robust ability of this construct to drive transgene expression after axonal infection. Finally, we demonstrated that the retrograde infection capabilities of these rAAVs enables the differential labeling of pyramidal neuron subpopulations projecting to distinct targets in the same animal. We used Emx1-Cre mice, which express Cre in glutamatergic neurons of neocortex (Gorski et al., 2002) and injected AAV2/1 Cre-dependent vectors into contralateral S1 and superior colliculus in the same mouse (n = 2 mice). As predicted from the single-injection experiments and from prior retrograde tracing studies (Larsen et al., 2007), these injections resulted in labeling of nonoverlapping populations of pyramidal neurons (Fig. 3G).

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Figure 3.

Retrograde transgene expression in cortical pyramidal neurons projecting to different targets. A–F, Low-magnification epifluorescence images (top) and higher-magnification confocal stacks (bottom) of mCherry fluorescence in S1 after injection of rAAV2/1.mCherry into superior colliculus (A, D, SC), thalamus (B, E), and contralateral S1 (C, F, cS1). For all images, the pial surface is toward the top. mCherry-expressing layer 5 pyramidal neurons are apparent in each preparation. In the contralateral-injected animal (C, F), the brightest fluorescence is from anterograde labeling of contralateral-projecting neurons infected at the injection site, but mCherry-expressing cell bodies are apparent in layer 5 (F). G, Injection of two different Cre-dependent viruses into SC and cS1 in the same Emx1-Cre mouse labels distinct populations of pyramidal neurons. 2/1.FLEX.tdTomato (red) was injected into cS1; 2/1.FLEX.GCaMP3 into SC. Low-magnification image (top) shows GCaMP3-expressing somata and anterograde mCherry labeling in contralaterally projecting axon terminals. Retrogradely expressing somata from cS1 are less clear at this magnification and in this section, but are apparent in higher-magnification confocal stacks (bottom). Retrogradely labeled pyramidal neuron populations from SC and cS1 are nonoverlapping.

Retrograde expression in target-defined subsets of centrifugal projection neurons

Retrograde viral infection is also useful for isolating subsets of neurons that mediate centrifugal feedback or neuromodulation in a particular brain region. We thus tested the ability of rAAVs to isolate target-defined populations of centrifugal neurons. First we tested two systems that modulate olfactory processing in the OB: descending projections from the anterior olfactory nucleus (AON; Shipley and Adamek, 1984; Markopoulos et al., 2012) and GABAergic projections from the basal forebrain (Ma and Luo, 2012; Zaborszky et al., 2012). A large population of descending AON projection neurons target the OB, with neurons throughout AON projecting to the ipsilateral and, to a lesser extent, contralateral OB (Shipley and Adamek, 1984). AON projections also target other forebrain areas, such as piriform cortex (Brunjes et al., 2005; Hagiwara et al., 2012). Because of evidence that AON projection neurons may contain the peptide transmitter cholecystokinin (CCK; Burgunder and Young, 1990; Schiffmann and Vanderhaeghen, 1991), we used CCK-Cre mice to attempt to retrogradely label neurons projecting from AON to the OB by injecting Cre-dependent rAAV (2/1.FLEX.GCaMP3) into the OB on one side (two mice, two injections). In addition to expression in CCK-positive tufted cells of the OB due to direct infection (Seroogy et al., 1985), virus injection led to strong expression in large numbers of neurons with somata throughout the ipsilateral AON (Fig. 4A), as well as sparser expression in neurons of the contralateral AON (Fig. 4A, inset). Likewise, we isolated GABAergic projections from the horizontal limb of the diagonal band of Broca (HDB), the source of GABAergic basal forebrain projections to the OB (Shipley and Adamek, 1984; Zaborszky et al., 2012) by injecting 2/1.FLEX.GCaMP3 into the OBs of mice expressing Cre in GAD65-positive neurons (GAD2-Cre mice; three mice, six injections). In addition to the expected expression in GABAergic interneurons of the OB (Wachowiak et al., 2013), GCaMP3 expression also occurred in neurons throughout HDB (Fig. 4B). Retrograde expression was more sparse than was evident after direct virus injection into HDB (Fig. 4B; 32% of neurons labeled by direct HDB injection; mean of 75 vs 235 neurons per counted section; four injections, two mice) consistent with only a subpopulation of these neurons projecting to the OB.

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Figure 4.

Retrograde transgene expression in diverse populations of centrifugal projection neurons. A, Retrograde expression of GCaMP3 in CCK-positive neurons projecting from AON to the OB. Left image shows confocal stack of GCaMP3 fluorescence in the ipsilateral AON after injection of 2/1.FLEX.GCaMP3 into the OB of a CCK-Cre mouse, with dense expression in neurons throughout AON. Right images show confocal stack of GCaMP3 fluorescence in the contralateral AON of the same mouse, showing weaker but still clear expression in numerous AON neurons. Right, Zoom of outlined region showing details of GCaMP3-expressing neurons. B, Retrograde expression of GCaMP3 in GABAergic neurons projecting from the basal forebrain to the OB. Left, Image of GCaMP3 fluorescence in the nucleus of the diagonal band of Broca (DB) after injecting 2/1.FLEX.GCaMP3 into DB of a GAD2-Cre mouse. Right, Image of GCaMP fluorescence in the HDB after injection of rAAV2/1.Flex.GCaMP3 into the OB of a GAD2-Cre mouse, showing appreciable numbers of GCaMP3-expressing neurons and their processes. Section from atlas (Paxinos and Franklin, 2001) illustrates targeted injection sites and source of labeled projections. C, Retrograde expression of ChR2-EYFP in serotonergic neurons projecting to basolateral amygdala. Image shows EYFP fluorescence in the dorsal raphe after injection of 2/9.DIO.hChR2(H134R)-EYFP into the basolateral amygdala (BLA) of a SERT-Cre mouse. Numerous ChR2-EYFP-expressing somata are apparent clustered along the midline.

We also tested for retrograde infection of serotonergic projection neurons, which project to multiple targets throughout the brain from the midbrain raphe nuclei. Cre-dependent rAAV (2/9.DIO.ChR2-EYFP) was injected into the basolateral amygdala (three mice, six injections) of Slc6a4 (SERT)-Cre mice, which express Cre selectively in serotonergic neurons (Zhuang et al., 2005). Basolateral amygdala receives relatively strong innervation from serotonergic raphe neurons (Vertes, 1991). Consistent with this, virus injection led to ChR2-eYFP expression in substantial numbers of neurons throughout the raphe nuclei (n = 671 cells; 17 sections, two mice), with the majority of expressing somata in the dorsal raphe (Fig. 4C). Together these results indicate that retrograde infection using rAAVs can also be used to isolate otherwise indistinguishable subpopulations of centrifugal or neuromodulatory projection neurons based on the target of their axonal projections.

Retrograde transgene expression in neurons projecting to and from midbrain nuclei

We next targeted projections to and from specific nuclei of the midbrain, a phylogenetically more primitive brain structure that receives input from and sends projections to diverse brain regions. Isolating subsets of midbrain projection neurons based on projection target or source is thus a useful application of retrograde viral tracing. First we used the constitutive AAV 2/1.mCherry to test for retrograde infection of neurons projecting to the periaqueductal gray (PAG). The PAG represents an interface between the forebrain and the lower brainstem and receives input from diverse brain areas (Benarroch, 2012). AAV 2/1.mCherry injection into the PAG resulted in, besides expression in PAG neurons themselves, mCherry expression in substantial numbers of neurons with somata in frontal association cortex, cingulate cortex, hypothalamus, and the amygdala (Fig. 5A). Neurons with somata along the ventricles between the midbrain and frontal cortex showed no infection, indicating specific retrograde tracing and not an unspecific diffusion along the ventricle.

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Figure 5.

Retrograde transgene expression of projections to and from midbrain nuclei. A, mCherry fluorescence after injection of 2/1.mCherry into PAG. Left, Middle, Low-magnification epifluorescence (top) and higher-magnification confocal stacks (bottom) from frontal association (FrA) and cingulate cortices (Cg). Right, Confocal stacks from amygdala (top) and hypothalamus (bottom). All images are taken from the same mouse, indicating retrograde infection of somata in widespread regions that project to PAG. B, Retrograde expression of GCaMP3 in dopaminergic neurons projecting to the striatum. Image shows GCaMP3 fluorescence in the substantia nigra pars compact (SNC) after injection of 2/1.FLEX.GCaMP3 into the striatum (CPu) of a TH-Cre mouse. Numerous GCaMP3-expressing somata are apparent in SNC. C, Retrograde expression of GCaMP3 and tdTomato in separate neuron subsets of the VTA after injection of 2/1.FLEX.GCaMP3 into the nucleus AcbSh and 2/1.FLEX.tdTomato into PrL/IL cortex of the same TH-Cre mouse.

We next tested for retrograde infection of dopaminergic midbrain projection neurons, which originate in several midbrain nuclei and project to multiple forebrain targets. First, injection of 2/1.FLEX.GCaMP3 into the striatum (caudate putamen) of TH-Cre mice (four mice, four injections) labeled neurons in substantia nigra pars compacta (Fig. 5B) that were pyramidal in shape with rostrocaudally oriented dendrites, matching earlier descriptions of substantia nigra neurons projecting to striatum (Fallon et al., 1978; Faull and Mehler, 1978). Notably, these results differ from recent studies finding that constitutive vectors using the native AAV2 serotype failed to drive retrograde expression in nigrostriatal projection neurons (Ciesielska et al., 2011; Salegio et al., 2013) but appear similar to the levels of retrograde expression mediated by other retrogradely infecting AAV serotypes (Ciesielska et al., 2011; Masamizu et al., 2011; Salegio et al., 2013). Second, we labeled neurons projecting from the ventral tegmental area (VTA) to distinct forebrain targets (Margolis et al., 2006) by injecting 2/1.FLEX.tdTomato or 2/1.FLEX.GCaMP3 into either prelimbic/infralimbic cortex (PrL/IL; three mice, three injections) or nucleus accumbens shell (AcbSh; four mice, four injections) of TH-Cre mice. In each case, injection led to expression in sparsely distributed neurons in VTA. Injection of virus into PrL/IL and AcbSh in the same TH-Cre mouse resulted in labeling of distinct (though still sparse) subsets of VTA neurons (Fig. 5C); these results are consistent with previous retrograde labeling studies using histochemical markers (Margolis et al., 2006).

Table 2 summarizes all virus injection sites used to evaluate retrograde infection capabilities of rAAV constructs. Together these results indicate that retrograde infection by a variety of rAAVs can be used to isolate diverse subpopulations of neurons based on the target of their axonal projections.

Optogenetic identification of target-defined projection neurons in vivo using retrograde expression of ChR2

We have recently demonstrated that expression of GCaMPs via retrograde infection yields sufficient expression levels to support the selective imaging of spontaneous and odorant-evoked activity from aPC-projecting MTCs in vivo (Wachowiak et al., 2013). Here, we tested whether this approach yields sufficient expression levels to support the identification and physiological characterization of cell types based on projection target. This process is equivalent to the ChR2-assisted circuit mapping approach described recently (Petreanu et al., 2007) but in the retrograde direction. ChR2(H134R) was expressed in MTCs projecting to aPC via retrograde rAAV infection and “blind” whole-cell or extracellular recordings were obtained from neurons in the OB. For in vivo extracellular or whole-cell recordings, neurons in the dorsal OB were targeted and brief (5–10 ms) light pulses were delivered to the dorsal OB to test for ChR2 expression in the recorded cells. For in vitro whole-cell recordings, mitral cells were visually targeted in OB slices using differential interference contrast optics and light pulses (3–5 ms) delivered to the entire slice. In all three cases, ChR2-expressing neurons were distinguished by reliable (95–100%) light-evoked spikes (for extracellular recordings) or depolarizing potentials and spike bursts (for whole-cell recordings) occurring with latencies of <10 ms and low jitter (SD of spike time, 0.2–1.9 ms; Fig. 6), consistent with other characterizations of responses of ChR2-expressing neurons to light flashes (Arenkiel et al., 2007; Zhang et al., 2013). In the OB slice recordings, five of seven recorded mitral cells (in two mice) showed light-evoked responses meeting these criteria. In the in vivo whole-cell recordings, five of eight cells (in five mice) responded reliably to light stimulation; one of these five was recovered and confirmed to be a mitral cell. Two of the three cells showing no or longer-latency responses to light stimulation were recovered and identified as a superficial tufted cell and a granule cell (data not shown). These results demonstrate that retrograde viral infection with optogenetic transgenes can enable the isolation of genetically specified cell types projecting to specific target locations for in vivo or in vitro analysis.

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Figure 6.

Optogenetic identification of target-defined OB projection neurons after retrograde expression of ChR2. A, In vivo extracellular recording from an MTC in the mouse OB after injection of 2.1.DIO.hChR2(H134R)-EYFP into aPC of a PCdh21-Cre mouse. Top, Three trials of 50 light pulses (10 ms duration, 10 Hz, 200 μm optical fiber, ∼1 mW total power at the fiber tip) elicits a single spike following each pulse with near 100% reliability. Bottom, Pulse-triggered spike histogram showing spike latencies of 8–10 ms with little jitter. B, In vitro whole-cell recording from a mitral cell in an OB slice after retrograde ChR2 expression in MTCs (PCdh21-Cre mouse, as in A). Top trace shows responses to a train of light pulses (5 ms duration, 5 Hz, 1 mm optical fiber, ∼24 mW total power), with each pulse eliciting a spike. Lower trace shows overlay of the response to each pulse in the train, showing short latency to depolarization onset and low jitter in spike time. C, In vivo whole-cell recording from an MTC after retrograde ChR2 expression, as in A and B. Left trace shows continuous recording of response to train of 10 pulses (11 mW total power) delivered as in B. In this cell, the light pulse does not always evoke a spike; right traces show overlay of responses to pulses that fail to evoke a spike. Failed responses consist of a depolarization with short latency (2–3 ms) and no detectable jitter in onset time. Higher intensities (≤25 mW total power) or longer durations elicited spikes to each pulse (data not shown).