tTARGIT AAVs: A sensitive and flexible method to manipulate intersectional neuronal populations

While Cre-dependent viral systems permit the manipulation of many neuron types, some cell populations cannot be targeted by a single DNA recombinase. Although the combined use of Flp and Cre recombinases can overcome this limitation, insufficient recombinase activity can reduce the efficacy of existing Cre+Flp-dependent viral systems. We developed a sensitive dual recombinase-activated viral approach: tTA-driven Recombinase-Guided Intersectional Targeting (tTARGIT) AAVs. tTARGIT AAVs utilize a Flp-dependent tetracycline transactivator (tTA) “Driver” AAV and a tetracycline response element (TRE)-driven, Cre-dependent “Payload” AAV to express the transgene of interest. We employed this system in Slc17a6FlpO;LeprCre mice to manipulate LepRb neurons of the ventromedial hypothalamus (VMH; LepRbVMH neurons) while omitting neighboring LepRb populations. We defined the circuitry of LepRbVMH neurons and roles for these cells in the control of food intake and energy expenditure. Thus, the tTARGIT system mediates robust recombinase-sensitive transgene expression, permitting the precise manipulation of previously intractable neural populations.


Introduction
The molecular heterogeneity of the nervous system requires a rich toolkit for precise study of distinct cell populations. Together with Cre recombinase-expressing mice, the available suite of Cre-dependent viral vectors permits the manipulation of genetically-identified neural types. However, this approach neither permits the study of subpopulations of cells expressing a particular Cre allele (Hodge et al., 2019;Mickelsen et al., 2019) nor the study of Cre-expressing cells within a defined CNS site while excluding the Cre-expressing cells in closely-opposed brain regions.
Restricting transgene expression to cells that express two marker genes can overcome this challenge (Luan and White, 2007). Early attempts at this approach utilized gene-specific, Cre-sensitive transgene-expressing alleles in combination with Cre alleles driven by distinct genes (Chen et al., 2011). Another solution involves expressing two Cre fragments across different transgenes with distinct promoters, reconstituting active Cre only in cells that express both pieces (Hirrlinger et al., 2009).
These approaches generally involve substantial investments of time and resources, however, as they require generating and interbreeding multiple new gene-and experiment-specific alleles.
Transgene expression can also be directed to cell types defined by two marker genes through the use of recombinase-dependent alleles (often inserted into the Rosa26 Locus) containing a ubiquitous promoter and tandem STOP cassettes that are each excised by distinct recombinases (Daigle et al., 2018;Sciolino et al., 2016). 4 Neuroscience research often requires a higher degree of spatial specificity than afforded by the intersectional genetic models outlined above. Stereotaxic injections of recombinase-sensitive viral vectors can restrict transgene expression to a narrow anatomical region. Adeno-associated viruses (AAVs) generally represent the preferred viral system for Cre-dependent transgene expression, given their minimal toxicity and the speed and ease of their generation. Developing AAVs sensitive to multiple recombinases has been challenging because of the limited AAV genome size, which precludes the use of multiple recombinase-sensitive STOP cassettes (Wu et al., 2010).
The INTRSECT system overcomes this limitation by utilizing a single AAV vector that flanks the transgene coding sequence with lox and FRT sites in such a way that combinatorial expression of Cre and Flp permits expression of a functional pre-mRNA that can then be spliced to produce a mature coding sequence (Fenno et al., 2020(Fenno et al., , 2014. The multiple inversion and splicing steps involved in this system can limit transgene expression, however, perhaps due to the relatively poor recombinase activity of Flp (and even optimized FlpO). Furthermore, generating INTRSECT viruses that express new transgenes requires a relatively complex and labor-intensive design and optimization process (Fenno et al., 2020(Fenno et al., , 2017. Seeking a dual recombinase-activated AAV system to overcome these limitations and that could be modified quickly and easily, we generated tetracycline transactivator (tTA)-driven Recombinase-Guided Intersectional Targeting (tTARGIT) AAVs composed of a Flp-dependent tetracycline transactivator (tTA) "Driver" AAV and a tetracycline 5 response element (TRE)-driven Cre-dependent "Payload" AAV to express the transgene of interest.
We applied tTARGIT AAVs to the study of ventromedial hypothalamic LepRb (VMH; LepRb VMH ) neurons, which modulate metabolic adaptations to obesogenic diets (Bingham et al., 2008;Dhillon et al., 2006) but have proven difficult to study directly due to the density and proximity of neighboring LepRb populations. Together with Lepr Cre and Slc16a7 FlpO , tTARGIT AAVs allowed us to overcome these challenges and reveal a specific role for LepRb VMH neurons in suppressing food intake and increasing energy expenditure to promote weight loss.

Results
The density of LepRb neurons in the adjacent arcuate nucleus (ARC), dorsomedial hypothalamus (DMH), and lateral hypothalamic area (LHA) complicated our initial attempts to study LepRb VMH neurons using Lepr Cre mice and Cre-dependent vectors; viruses targeted to the VMH spread to other nearby Lepr Cre neurons, confounding the interpretation of our results (data not shown). Because Slc17a6, encoding the vesicular glutamate transporter 2 (vGLUT2) protein, expression is largely restricted to LepRb VMH neurons and absent from most surrounding LepRb cells (Vong et al., 2011), we generated a Slc17a6 FlpO strain and crossed it with Lepr Cre and a novel Flp-and Cre-dependent reporter (Rosa26 RCFL-eGFP-L10a ). We then tested the potential ability of this combination of Cre and Flp alleles to specify LepRb VMH neurons in the mediobasal hypothalamus. Although these reporter mice identified Cre-and Flp-coexpressing LepRb Slc17a6 neurons elsewhere in the brain, within the mediobasal hypothalamus this approach largely limited eGFP expression to VMH cells (Supplemental Figure 1).
We thus sought to use Flp-and Cre-dependent AAVs to target LepRb VMH neurons and omit manipulation of non-VMH LepRb Slc17a6 cells. We injected the INTRSECT AAV system (Fenno et al., 2014) into the VMH of Slc17a6 FlpO ;Lepr Cre mice in an attempt to express channelrhodopsin (ChR2) in LepRb VMH cells. However, this approach resulted in detectable ChR2 expression in one or fewer cells per section (Supplemental Figure 2). We surmised that while INTRSECT works well in systems with robust Flp and Cre activities, the poor recombinase activity of Flp and the more 7 moderate Flp and Cre expression mediated by Slc17a6 FlpO and Lepr Cre , respectively, might limit INTRSECT-mediated transgene expression in LepRb VMH cells.
We therefore set out to develop a more sensitive AAV system to drive Cre+Flpdependent transgene expression, using as our framework a previously-described inducible gene expression system based on recombinase-dependent expression of the tetracycline transactivator (tTA) in combination with a tetracycline response element (TRE)-driven transgene-expressing allele (Chan et al., 2017;He et al., 2016). We packaged this system into two viral vectors, hereafter "tTA-driven Recombinase-Guided Intersectional Targeting" (tTARGIT) AAVs.
Our tTARGIT system utilizes "Driver" and "Payload" AAVs. The Driver (AAV-hSyn-Flex(FRT)-tTA) utilizes a Flex(FRT) cassette (Fenno et al., 2014) to Flpdependently invert tTA, permitting its expression by a human synapsin I (hSYN1) promoter. This virus also contains two tetracycline operators (TetO) to drive a positive feedback loop and increase tTA expression (Chan et al., 2017). The Payload AAV mediates tTA/TRE-dependent transgene expression following its Cre-mediated inversion into the sense orientation. Hence, only cells that contain both recombinases express the transgene (Figure 1a). Tetracycline inhibits tTA-dependent gene expression (T. Das et al., 2016), so this system mediates constitutive payload expression in target cells in the absence of tetracycline.
To test the recombinase-dependence of this system, we combined the Flpdependent Driver AAV and a Payload AAV that permits the tTA/TRE-mediated Credependent expression of a ChR2-TdTomato fusion protein (ChR2-TdT; AAV-TRE-8 Flex(Lox)-ChR2-TdT). We co-injected these viruses into the VMH of wild-type mice, mice that expressed either Slc17a6 FlpO or Lepr Cre only, or Slc17a6 FlpO ;Lepr Cre mice (Figure 1b,c). We detected no TdT (DSRed-immunoreactivity (-IR)) in the VMH of wildtype or Slc17a6 FlpO animals and minimal expression in Lepr Cre mice. In contrast, the Driver+Payload combination mediated robust DSRed-IR in the VMH of Slc17a6 FlpO ;Lepr Cre mice ( Figure 1c); furthermore, VMH photostimulation in these mice promoted robust colocalization of DSRed-and FOS-IR, consistent with the ability of this system to activate transduced Flp+Cre-expressing cells (Supplemental Figure 3). Importantly, DSRed-IR was restricted to the VMH in these mice.
We also tested the requirement for both the Driver and Payload viruses in this system by injecting each virus alone, or both viruses together, into the VMH of into the antisense orientation. We tested this system with a novel Cre-inactivated Payload virus expressing a hM3Dq designer receptor exclusively activated by designer drugs (DREADD)-mCherry transgene. We coinjected this Cre-OFF Payload and the Flpdependent Driver AAV into the VMH of Slc17a6 FlpO ;Lepr Cre animals on the Credependent Rosa26 LSL-GFP-L10a background (Supplemental Figure 4b). As expected, this modified tTARGIT system drove hM3Dq-mCherry expression almost exclusively in cells that did not express the Cre-dependent GFP (Supplemental Figure 4c). We surmise that the few GFP-IR neurons with detectable mCherry might result from the low Cre expression mediated by Lepr Cre (Patterson et al., 2011) and predict that the Flp-On, Cre-Off tTARGIT system should demonstrate complete Payload inactivation when used in conjunction with a more robustly-expressing Cre allele.
To define the projection targets of LepRb VMH neurons, we developed a payload virus (AAV-TRE-Flex(Lox)-GFP-2A-SynmRuby) that encodes GFP plus a cotranslationally-expressed synaptophysin-mRuby transgene ( Figure 2a). Coinjection of this tTARGIT Payload AAV and the Driver virus into the VMH of Slc17a6 FlpO ;Lepr Cre mice promoted robust VMH-restricted GFP-IR ( Figure 2b). mRuby detection (DSRed-IR) for this virus was much lower than for GFP, however (data not shown); thus, we used GFP-IR to detect projections from LepRb VMH cells. Assessing the entire CNS for the presence of GFP-IR revealed terminals in the periaqueductal gray (PAG), the arcuate, the periventricular thalamic nucleus (PVT), the periventricular hypothalamic nucleus (PVN), the bed nucleus of the stria terminalis (BNST) and the preoptic area (POA) (Figure 2c-d). These are consistent with the known projections of the VMH (Canteras et al., 1994;Meek et al., 2016;Zhang et al., 2020).
VMH-specific (Nr5a1 Cre -mediated) Lepr ablation promotes obesity associated with decreased energy expenditure in high fat diet-fed animals, suggesting a specific role for LepRb in LepRb VMH cells in the control of energy balance via the dietary modulation of energy utilization (Bingham et al., 2008;Dhillon et al., 2006). To define the function of LepRb VMH cells, rather than the function of LepRb in these cells, we developed a Payload virus containing an inverted hM3Dq-mCherry transgene. We coinjected the Driver AAV and this AAV-TRE-Flex(Lox)-hM3Dq-mCherry Payload virus into the VMH of Slc17a6 FlpO ;Lepr Cre animals (LepRb VMH-Dq mice; Figure 3a). This approach promoted robust VMH-restricted expression of functional hM3Dq-mCherry as administration of the DREADD activator (Pei et al., 2008)  To determine the potential modulation of energy expenditure, activity, and food intake by LepRb VMH neuron activation, we placed LepRb VMH-Dq animals in metabolic cages and administered either vehicle or CNO twice daily (Figure 3e-j). Compared to vehicle administration, activating LepRb VMH neurons significantly increased 24-hour oxygen consumption (VO2) and energy expenditure, both primarily due to effects during the light phase, despite decreasing ambulatory activity over 24 hours (primarily due to effects during the dark phase) (Figure 3g-h). Additionally, the hM3Dq-mediated activation of LepRb VMH neurons also suppressed 24-hour food intake, primarily due to decreased light-phase feeding, revealing a previously unsuspected role for these cells 11 in the suppression of feeding. CNO also decreased the respiratory exchange ratio (RER) during the light phase, consistent with the increased metabolism of fat stores due to the combination of increased energy expenditure and decreased food intake.
To understand whether effects on brown adipose tissue (BAT) might contribute to the increased energy expenditure during LepRb VMH neuron activation, we placed temperature probes in the interscapular space of LepRb VMH-Dq animals to monitor BAT thermogenesis. Compared to controls, CNO significantly increased intrascapular temperatures in LepRb VMH-Dq animals, suggesting LepRb VMH neurons promote energy expenditure at least in part by augmenting BAT thermogenesis (Figure 3k-l).
As activating LepRb VMH neurons increased energy expenditure and decreased food intake, we surmised that these neurons should promote weight loss. We thus administered CNO in drinking water to LepRb VMH-Dq mice for three days. During this time, the body weight of LepRb VMH-Dq mice decreased by approximately 10% (Figure   4a), returning to baseline following the cessation of CNO exposure. While CNO treatment decreased food consumption (largely during the second day of treatment) and water intake (Figure 4b-d). The magnitude and timing of these ingestive effects dictates that neither could account for the decreased body weight mediated by CNO, consistent with the notion that increased energy expenditure mediates the major effect of LepRb VMH cells on body weight.
Hence, the use of our dual recombinase-dependent tTARGIT AAV system permitted us to determine that LepRb VMH neuron activation increased energy 12 expenditure and decreased food intake during the inactive phase, suggesting the diurnal control of energy balance by LepRb VMH neurons.

Discussion
The use of sequence-specific DNA recombinases (Cre, Flp and others) in conjunction with recombinase-dependent genetic alleles and viral vectors has revolutionized our ability to manipulate specific circuits and understand the central nervous system. The lack of robust viral systems to manipulate cell populations defined by the expression of multiple genes has impeded the study of more refined neural populations, however-including those identified by single cell RNA-sequencing (Campbell et al., 2017). Our tTARGIT AAV system addresses many shortcomings of previous intersectional tools, including limitations to transgene expression and the difficulty of incorporating novel transgenes into the AAV plasmids.
To facilitate the study of intersectional neural populations, we developed a suite of Cre-dependent tTARGIT Payload plasmids (Table 1). While we have not yet tested the function of all of these, our experience with the transgenes that we have tested predicts similarly robust expression of the various Payload AAV transgenes. The limitations of these Payload vectors are likely to mirror those of standard Cre-dependent viruses, including the requirement for stoichiometric transduction of/recombination in the cell type of interest to observe the effects of interfering with neuronal function. We have additionally generated Cre-inactivated Payload vectors to specifically mark Cre-negative Flp-expressing cells within the injection field; our preliminary findings suggest that these will work most effectively when used in conjunction with a robustly-expressing Cre allele.
14 While the tTARGIT AAV system as we have built it is designed to constitutively express transgenes in Cre-and Flp-expressing cells without the use of tetracycline-like compounds (usually doxycycline), it should be possible to decrease transgene expression from the tTARGIT AAVs by doxycycline treatment. Indeed, we built a Flpdependent rtTA Driver virus that is predicted to require doxycycline treatment to mediate strong transgene expression. The rtTA-based system can drive low-level transcription independent of doxycycline (Zhu et al., 2001), however, and we have not yet tested this system.
The use of tTARGIT AAVs permitted us to target robust transgene expression to LepRb VMH neurons specifically, in isolation from LepRb neurons in adjacent hypothalamic nuclei. While deleting Lepr from the VMH of chow-fed mice or restoring VMH Lepr expression on an otherwise LepRb-deficient background minimally (if at all) alters energy balance, knockout mice fail to increase energy expenditure on high-fat chow and become more obese than controls (Bingham et al., 2008;Dhillon et al., 2006;Gonçalves et al., 2014;Senn et al., 2019, p. 1). While these studies thus suggest a role for leptin action on LepRb VMH cells in the control of energy expenditure, the manipulation of VMH Lepr expression does not otherwise permit the broader study of LepRb VMH cells. In contrast, our use of the tTARGIT system, together with Slc17a6 FlpO ;Lepr Cre mice, identified the projections of LepRb VMH cells, demonstrated their ability to acutely suppress food intake as well as promoting energy expenditure (identifying BAT thermogenesis as a target for these cells), and revealed the diurnal nature of LepRb VMH neuron-mediated control of energy balance. Presumably, the 15 finding that LepRb VMH neuron activation alters food intake and energy expenditure specifically during the light cycle suggests that these neurons may decrease in activity during the inactive/light phase, permitting us to observe the effects of artificial neuron activation during this time.
In summary, we have developed a suite of dyad AAV vectors for the study of

Stereotaxic Surgery
Mice were anesthetized with isoflurane (2%) and mounted in a stereotaxic frame (Kopf). Using standard surgical techniques, 150nL of virus was injected bilaterally via a glass micropipette attached to a microinjector (picospritzer II) targeting the VMH (AP -1.3 mm; ML ±0.25 mm, DV -5.55 mm, relative to bregma).
For DREADD studies, hit sites were verified by mCherry detection (DSRed-IR) following euthanasia. Any data from mice in which mCherry was not detected within the VMH or was detected in other hypothalamic nuclei were discarded. Data from mice with either unilateral or bilateral viral hits were included.

Immunostaining
For control experiments presented in Figure 1, mice were euthanized three weeks following viral delivery. Hit sites were verified using a marker virus (AAV-CMV-Cas9-HA (18)).
Upon the completion of DREADD studies, mice were injected with CNO (1mg/kg), sacrificed two hours post-injection and then perfused with 10% formalin.

Indirect Calorimetry Studies
Mice were singly housed one week prior to indirect calorimetry studies. Mice Data is presented as the average of the two saline days compared to the average of the two CNO days.

Effect of CNO on energy balance
Mice were singly housed for one week prior to study. Mice were given ad libitum access to standard drinking water for 48 hours. For the subsequent 72 hours, standard water was replaced by water containing CNO (2.5 mg/100mL) and 1% glucose (to make the CNO palatable). CNO-laced water was changed daily. For the final 48 hours, mice were returned to standard drinking water. Body weight, food mass and water levels were recorded daily.

Intrascapular temperature measurements
The UM-MMPC placed temperature transponders (IPTT-300 model with corresponding DAS-7007R reader, Bio Medic Data Systems) in the intrascapular subcutaneous tissue directly under the conjunction part of the butterfly-shaped BAT under isoflurane anesthesia. Mice were allowed to recover for 14 d before testing. One day prior to testing, ambient temperatures were increased from 22°C to 30°C. On the day of testing, mice were randomized to either CNO (1 mg/kg) or saline injections and temperatures were recorded at -10, 10,20, 30, 45 60, and 120 min relative to injection time. Following one week, the experiment was repeated and treatment conditions (vehicle or CNO) reversed.
After 3 weeks recovery from surgery, mice were then subjected to optical stimulation using 473 nm wavelength laser using 20 mW/mm 2 irradiance. Light pulses were delivered by 1 s of 20 Hz photo stimulation and 3 s resting with multiple repetitions for one hour.

Statistical Analysis
All Data is displayed as mean +/-SEM. Replicate number is included in each figure legend. Statistical analysis was performed in either Graphpad Prism 8 or R using either t-tests or ANOVAs with Dunnet's post-hoc test or linear mixed model. P<0.05 was considered significant.    shows area under the curve (AUC) for each treatment condition in (k). For metabolic cage studies, statistical significance was determined using either a paired t-test (full-day data), or a linear mixed model for effects by time of day. For interscapular temperature 33 measurements, significance was determined by paired t-test. * P <0.05, ** P <0.01, *** P < 0.001 LepRb VMH-Dq mice receiving two days normal drinking water (days 0-2), followed by three days exposure to CNO-laced drinking water (days 2-5), followed by an additional