Monosynaptic Retrograde Tracing From Prelimbic Neuron Subpopulations Projecting to Either Nucleus Accumbens Core or Rostromedial Tegmental Nucleus

The prelimbic (PL) region of the medial prefrontal cortex (mPFC) has been implicated in both driving and suppressing motivated behaviors, including cocaine-seeking in rats. These seemingly opposing functions may be mediated by different efferent targets of PL projections, such as the nucleus accumbens (NAc) core and rostromedial tegmental nucleus (RMTg), which have contrasting roles in reward-seeking behaviors. We sought to characterize the anatomical connectivity differences between PL neurons projecting to NAc core and RMTg. We used conventional retrograde tracers to reveal distinct subpopulations of PL neurons projecting to NAc core vs. RMTg in rats, with very little overlap. To examine potential differences in input specificity for these two PL subpopulations, we then used Cre-dependent rabies virus (EnvA-RV-EGFP) as a monosynaptic retrograde tracer and targeted specific PL neurons via injections of retrograde CAV2-Cre in either NAc core or RMTg. We observed a similar catalog of cortical, thalamic, and limbic afferents for both NAc- and RMTg-projecting populations, with the primary source of afferent information arising from neighboring prefrontal neurons in ipsilateral PL and infralimbic cortex (IL). However, when the two subpopulations were directly compared, we found that RMTg-projecting PL neurons received a greater proportion of input from ipsilateral PL and IL, whereas NAc-projecting PL neurons received a greater proportion of input from most other cortical areas, mediodorsal thalamic nucleus, and several other subcortical areas. NAc-projecting PL neurons also received a greater proportion of contralateral cortical input. Our findings reveal that PL subpopulations differ not only in their efferent target but also in the input specificity from afferent structures. These differences in connectivity are likely to be critical to functional differences of PL subpopulations.


INTRODUCTION
The prelimbic (PL) region of the medial prefrontal cortex (mPFC) plays roles in both driving and suppressing motivated behaviors, including drug-seeking and conditioned fear (Moorman et al., 2015;Gourley and Taylor, 2016). Opposing behavioral functions for PL may be mediated by distinct efferent projections. This is supported by previous work demonstrating bidirectional behavioral effects after optogenetic stimulation of distinct PL projection pathways, including PL projections to lateral habenula vs. dorsal raphe in a forced swim task (Warden et al., 2012), nucleus accumbens (NAc) vs. basolateral amygdala (BLA) in an active avoidance task (Diehl et al., 2020), and NAc vs. paraventricular nucleus of the thalamus during cue-induced reward-seeking (Otis et al., 2017). Additionally, whereas PL projections to NAc core have been shown to drive cue-induced reinstatement of cocaine-seeking (McFarland et al., 2003;Stefanik et al., 2013Stefanik et al., , 2016McGlinchey et al., 2016;James et al., 2018), we recently showed that PL projections to the rostromedial tegmental nucleus (RMTg) play a suppressive role in cue-induced reinstatement (Cruz et al., 2020).
Here, we sought to characterize anatomical connectivity differences between PL neurons projecting to NAc core and RMTg, given that these two efferent targets often have opposing influences on reward-seeking behavior. The NAc core is critically involved in driving motivated behavior and action initiation, particularly when guided by incentive stimuli (Du Hoffmann and Nicola, 2014;Floresco, 2015;Hamid et al., 2016;Syed et al., 2016;Mohebi et al., 2019;Sicre et al., 2019). Conversely, the RMTg, or tail of the ventral tegmental area (tVTA), is involved in behavioral inhibition and aversive valence encoding, via its inhibitory influence on dopamine neurons in the ventral tegmental area (Jhou et al., 2009a(Jhou et al., ,b, 2013Kaufling et al., 2009Kaufling et al., , 2010Balcita-Pedicino et al., 2011;Barrot et al., 2012;Bourdy et al., 2014;Vento et al., 2017;Li et al., 2019;Smith et al., 2019). Previous work has demonstrated that mPFC projections to a variety of brain structures typically arise from different subpopulations of mPFC neurons (Akintunde and Buxton, 1992;Pinto and Sesack, 2000;Gabbott et al., 2005). Therefore, we hypothesized that distinct subpopulations of PL neurons project to NAc core vs. RMTg, and we sought to investigate connectivity differences for these subpopulations, as differences in input/output specificity are likely to be critical to their functional differences.
Using conventional retrograde tracers, we show that PL neurons projecting to NAc core and RMTg are markedly separate, forming two largely distinct sublayers of pyramidal cells in rats (Figure 1). Therefore, we investigated these two PL projection pathways in terms of afferent connectivity, considering that input specificity might influence differential recruitment of these populations during behavior. We used Cre-dependent rabies (EnvA-RV-EGFP) as a monosynaptic retrograde tracer to determine direct afferents to the two different PL projection neuron subpopulations (Wickersham et al., 2007a(Wickersham et al., ,b, 2010Callaway, 2008;Wall et al., 2010Wall et al., , 2013Watabe-Uchida et al., 2012). Cre was expressed specifically in RMTg-or NAc-projecting neurons using retrograde CAV2-Cre microinjected into the efferent target (RMTg or NAc), followed by microinjection of AAV helper viruses and EnvA-RV-EGFP into PL. Our results revealed that PL neurons projecting to either NAc core or RMTg receive input from similar afferents, but differ in the proportions of input arising from these afferents.

Animals
Male Sprague Dawley rats (initial weight 250-300 g; Charles River, Raleigh, NC, USA) were single-housed under a 12-h reverse light/dark cycle (ZT0 = 19:00) and had access to food and water ad libitum. Animals were housed in a temperature-and humidity-controlled animal facility with AAALAC accreditation. All experiments were approved by the Institutional Animal Care and Use Committee at Texas A&M University and conducted according to specifications of the National Institutes of Health as outlined in the Guide for the Care and Use of Laboratory Animals.

Viruses
The transduction and monosynaptic spread of glycoprotein (G)deleted rabies virus (RV) with EnvA pseudotyping (EnvA-RV-EGFP) is limited to neurons expressing TVA receptor and rabies glycoprotein (RG) in a Cre-dependent manner (Wickersham et al., 2007a,b;Watabe-Uchida et al., 2012). We targeted specific PL neurons by injecting either NAc core or RMTg with retrograde Cre-expressing virus (CAV2-Cre) and injecting PL with Cre-dependent helper AAV viruses (TVA and RG) and EnvA-RV-EGFP.
EnvA-RV-EGFP (titer of 4.3 × 10e8) was provided by Ed Callaway via the Gene Transfer, Targeting, and Therapeutics Facility at the Salk Institute (Wickersham et al., 2007a). AAV-TVA (AAV1-EF1a-FLEX-TVAmCherry, a titer of 4 × 10e12) and AAV-RG (AAV1-CA-FLEX-RG, a titer of 4 × 10e12) were obtained through the viral vector core at the University of North Carolina (developed by Watabe-Uchida et al., 2012). CAV2-Cre (CAV2-CMV-Cre recombinase) was provided by Eric J. Kremer at the Institut de Genetique Moleculaire de Montpellier (Kremer et al., 2000). We diluted CAV2-Cre 1:10 in 10% glycerol in PBS for a final titer of 10.3 × 10e11 ppml, based on preliminary experiments indicating cell toxicity at higher titers. All viral procedures were approved by the Institutional Biosafety Committee at Texas A&M University and conducted according to specifications of the National Institutes of Health.

Stereotaxic Surgery
Rats were anesthetized with vaporized isoflurane (induced at 5%, maintained at 1-2%), given the analgesic ketoprofen (2 mg/kg, s.c.), and placed in a stereotaxic frame (Kopf, Tujunga, CA, USA). A skin incision was made over the skull and holes were drilled in the skull over the target sites. Intracranial injections were made using a pulled glass micropipette and Nanoject 2010 injector (World Precision Instruments, Sarasota, FL, USA). After the injection, the pipette was slowly raised from the brain and the skin was stapled closed.

Tissue Processing
Rats were deeply anesthetized with isoflurane and then transcardially perfused with 0.9% NaCl followed by 10% neutralbuffered formalin via a peristaltic pump (Cole Parmer, Vernon Hills, IL, USA). Brains were collected, post-fixed in 10% formalin overnight at 4 • C, placed in 20% sucrose in phosphate-buffered saline (PBS) with 0.02% sodium-azide at 4 • C for ≥2 days, frozen in dry ice, cut into 40-µm thick coronal sections on a cryostat, and collected into PBS-azide for storage before immunohistochemistry (IHC). For IHC, all incubations and rinses took place at room temperature on a shaker. Sections were rinsed in PBS three times between steps.

Data Analysis
Fluorescent and brightfield images were acquired using an Olympus BX51 microscope. For CTB and FG tracing, neurons were counted in PL on two sections per rat. Four rats were excluded from CTB/FG analysis due to small/missing RMTg injections. One rat was removed from CTB/FG analysis after being identified as a significant outlier (Grubb's method) due to a higher percentage of double labeling. The remaining data were analyzed with a paired t-test. For rabies tracing, all EGFP-labeled neurons were counted on all sections stained with DAB (every 12th section) and categorized into brain regions according to boundaries identified by Paxinos and Watson (2007). RMTg boundaries were defined (Figures 1B, 2C) according to Smith et al. (2019). A neuron was only counted if a soma was clearly visible. Fluorescent sections were used to count all doublelabeled neurons (EGFP + mCherry) on every 12th section. Photo location is shown in the inset (magenta box). Scale bar = 100 µm. (B) Location and spread for retrograde tracer injections in NAc core (FG, red) and RMTg (CTB, blue). Each rat is represented by one translucent outline, and outlines are overlapped. Anterior-posterior (AP) levels represent mm from bregma. Scale bar = 1 mm. (C) Percentage of PL neurons that project to both NAc core and RMTg (i.e., double-labeled for both CTB and FG), as a proportion of the total number of PL neurons projecting to NAc core (all FG-labeled cells) or RMTg (all CTB-labeled cells). Averages (± SEM) are shown, as well as individual data points for each rat (n = 11, *p < 0.05). Expression of EGFP alone indicated input cells, and expression of both mCherry and EGFP indicated starter cells. Inputs were normalized within each animal and calculated as a percentage of total inputs per rat. Brain regions were only included in analysis and figures if normalized inputs were ≥0.2% in ≥2 rats. Data were analyzed using two-way ANOVAs (with repeated measures when appropriate) and Sidak post hoc analyses.

Conventional Retrograde Tracing in PL Targets
Sparse double labeling was seen in PL ( Figure 1A) when conventional retrograde tracers (FG and CTB) were injected into ipsilateral NAc core and RMTg (Figure 1B; n = 11). We observed NAc-projecting PL neurons in layers II/III and upper layer V, whereas RMTg-projecting neurons were located in deeper layer V (Figure 1A). Double-labeled neurons (projecting to both NAc core and RMTg) represented only 0.5% ± 0.1 of PL neurons projecting to NAc core and 1.1% ± 0.3 of PL neurons projecting to RMTg (Figure 1C; t (10) = 3.004, p = 0.013). These data indicate that PL neurons projecting to NAc core vs. RMTg are predominantly separate subpopulations.

Monosynaptic Retrograde Tracing in PL Subpopulations
To determine whether these PL subpopulations differ in terms of input specificity, we conducted monosynaptic retrograde tracing in each projection subpopulation via retrograde Cre delivery (Figure 2A). CAV2-Cre was injected into either NAc core (n = 4) or RMTg (n = 4; Figures 2B,C), and Cre-dependent helper AAVs driving expression of TVA-mCherry and RG were injected into PL. Three weeks later, EnvA-RV-EGFP was injected into PL. Rabies only infects and monosynaptically spreads from neurons expressing both TVA and RG.
We mapped and counted the number of starter cells (mCherry + EGFP) using fluorescent labeling (representative images in Figures 2D-G). We counted the number of input cells (EGFP) across different brain regions using immunohistochemistry with DAB labeling (representative images in Figure 4). Starter cells projecting to either NAc core or RMTg were primarily in PL, but also in neighboring infralimbic cortex (IL; Figure 3A). Although the average number of starter cells differed for NAc vs. RMTg (t (6) = 5.50, p = 0.0015), the number of transsynaptically labeled cells was correlated with the number of starter cells (Figure 3B; r = 0.91, p = 0.0019).
We also saw prominent inputs from the dorsal endopiriform nucleus, claustrum (Figures 4E,G), BLA (Figures 4F,H), and CA1 in the ventral hippocampus (Figures 4I,J). For all of these areas, NAc-projecting PL neurons received a greater percentage of input ( Figure 3C). In contrast, the two subpopulations of PL neurons were similar in terms of afferent input arising from the lateral septum, diagonal band, and navicular nucleus, ventral pallidum, globus pallidus, and ventral subiculum ( Figure 3C).

DISCUSSION
We found that PL neurons projecting to NAc vs. RMTg are distinct subpopulations (Figure 1). Pathway-specific monosynaptic retrograde tracing for PL neurons projecting to either NAc vs. RMTg (Figure 2) revealed differences in afferent information to the two PL subpopulations (Figures 3, 4). While RMTg-projecting PL neurons received a greater proportion of inputs from nearby sources in ipsilateral PL and IL, NAc-projecting PL neurons received a greater proportion of inputs from other cortical areas, thalamic nuclei, and subcortical areas. Also, NAc-projecting PL neurons received a greater proportion of contralateral cortical input. These data are the first to demonstrate anatomical connectivity differences between PL neurons projecting to NAc vs. RMTg, which may contribute to opposing functions of these pathways.

PL Projection Subpopulations
We found very little overlap in PL neuron subpopulations projecting to NAc core vs. RMTg, with NAc-projecting PL neurons located in layers II/III and upper layer V, and RMTg-projecting neurons located in deeper layer V (Figure 1A). This corresponds with previous studies using conventional retrograde tracers (e.g., CTB) injected into various mPFC targets, which showed that distinct mPFC neuron subpopulations project to different subcortical targets, including NAc, BLA, MD, lateral hypothalamus, and numerous brainstem areas (Akintunde and Buxton, 1992;Pinto and Sesack, 2000;Gabbott et al., 2005). mPFC neurons in layers II/III, V, and VI have been shown to project to the striatum, while mPFC neurons in layer V project to multiple brainstem targets, including the parabrachial nucleus, periaqueductal gray, ventral tegmental area, dorsal raphe, the nucleus of the solitary tract, and ventrolateral medulla (Pinto and Sesack, 2000;Ding et al., 2001;Gabbott et al., 2005).

Afferent Inputs to PL Subpopulations
Previous studies have mapped afferents to rat PL using conventional retrograde tracers (Condé et al., 1995;Hoover and Vertes, 2007). However, unlike conventional tracers, RV-EGFP allows the separation of starter cells from input cells, so that short-range projections within PL and IL can be assessed. Although previous studies have used RV-EGFP in mouse mPFC, they targeted all projection neurons (Ährlund-Richter et al., 2019), interneuron populations (Ährlund-Richter et al., 2019;Sun et al., 2019), or PL layer V (DeNardo et al., 2015. Here, we used RV-EGFP in rat mPFC and targeted PL neurons projecting to NAc core vs. RMTg to provide more detailed complexity about input specificity to PL subpopulations. We observed prominent cortical inputs from mPFC (PL, IL, cingulate cortex, secondary motor cortex, dorsal peduncular cortex), OFC (with the highest percentage in medial), and agranular insular cortex, and limited labeling in the anterior olfactory nucleus, dorsal tenia tecta, piriform cortex, and ento/ecto/perirhinal cortex (Figures 3C, 4A,B). This parallels previous studies with conventional tracers (Condé et al., 1995;Hoover and Vertes, 2007). We found that for both NAc-and RMTg-projecting populations, the primary source of afferent information was neighboring prefrontal neurons in ipsilateral PL and IL, as shown previously with RV-EGFP targeting layer V neurons in PL (DeNardo et al., 2015). However, we also identified a major distinction between afferent inputs to NAc-and RMTg-projecting neurons. RMTg-projecting PL neurons received a higher percentage of input from ipsilateral PL and IL, as well as medial OFC, but a similar or lower percentage of input from all other cortical areas. In contrast, NAc-projecting PL neurons received a higher percentage of input from ipsilateral dorsal tenia tecta, lateral OFC, and agranular insular cortex. Additionally, NAc-projecting PL neurons received a greater percentage of contralateral input, including from PL, IL, cingulate cortex area 1, secondary motor cortex, OFC, and dorsal peduncular cortex. These findings indicate that RMTg-projecting neurons may be under greater local control, while NAc-projecting neurons have greater integration of information across hemispheres.
We found that NAc-projecting PL neurons received a higher proportion of inputs from all thalamic regions (Figures 3C,  4C,D). We observed the greatest thalamic labeling in MD and moderate labeling in midline thalamic nucleus, as per previous work (Condé et al., 1995;Hoover and Vertes, 2007). We also observed moderate labeling in intralaminar, ventral, anterior, and reticular nuclei, and some labeling in the lateral nucleus (but only in NAc-projecting PL neurons).
Notably, unlike previous studies using conventional retrograde tracers in PL (Condé et al., 1995;Hoover and Vertes, 2007), the current study using RV-EGFP as a tracer did not detect inputs from the monoaminergic nuclei of the brainstem, including ventral tegmental area, dorsal raphe, median raphe, and locus coeruleus, indicating that RV-EGFP does not spread transsynaptically to all retrograde afferent neurons. This is confirmed by previous studies showing a lack of brainstem inputs when using RV-EGFP in PL (DeNardo et al., 2015), and a paucity of dopamine inputs when using RV-EGFP in dorsal striatum as a monosynaptic tracer but not as a traditional retrograde tracer (Wall et al., 2013). These results indicate that the transsynaptic spread of RV-EGFP has reduced efficiency at monoaminergic synapses, possibly due to differences in synapse type, location, or distance, as compared to glutamatergic and GABAergic synapses, and this represents a significant limitation of transsynaptic tracing with RV-EGFP. Alternatively, these data may indicate that these PL subpopulations do not receive direct input from monoamine neurons. However, previous work has shown direct synaptic contact between NAc-projecting mPFC neurons and axon terminals containing tyrosine hydroxylase (Carr et al., 1999). Further studies are needed to determine whether RMTg-projecting PL neurons receive direct synaptic input from monoamine neurons.

Technical Considerations
In this study, we used a two-helper-virus system, and this may have resulted in overestimating starter cells due to the assumption that each cell was successfully infected with both viruses. We did not demonstrate Cre dependence of the helper viruses in the current study (e.g., by including control animals lacking injection of CAV2-Cre), but previous work has shown limited expression of helper viruses in the absence of Cre (Watabe-Uchida et al., 2012;Wall et al., 2013;Schwarz et al., 2015). Additionally, it is important to note that our injections of CAV2-Cre likely were not restricted solely to RMTg, given the small size of RMTg as compared to NAc core and given that many injections show some spread outside the intended target ( Figure 1B). It is important to note as well that these studies were performed in male rats; future studies are necessary to determine whether sex differences exist for these PL subpopulations. Finally, although RV-EGFP tracing reveals the presence of a connection between two neurons, it does not reveal information about the strength or function of the connection. One neuron may form many synapses onto many neurons and may provide a strong influence despite limited labeling with RV-EGFP. Therefore, additional studies are necessary to explore potential functional differences in afferent input to these PL subpopulations.

Conclusions
The current work shows that PL subpopulations differ not only in their efferent target but also in the proportion of inputs they receive from a variety of afferent structures. When directly compared, RMTg-projecting PL neurons receive a greater proportion of input from nearby cortical input (ipsilateral PL and IL), whereas NAc-projecting PL neurons receive a greater proportion of input from other mPFC and cortical areas, thalamic nuclei, amygdala, and ventral hippocampus, as well as contralateral mPFC. These differences in afferent information may be critical to potential functional differences of these PL subpopulations and may be related to the contrasting roles played by NAc core and RMTg during reward-related behavior, including cocaine seeking. More generally, our data indicate that projection neuron subpopulation may be an important organizational feature of mPFC that has been mostly overlooked, and that differences in afferents, efferents, and function may be critical to the diverse functionality of mPFC across a range of behavioral responses.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee at Texas A&M University.

AUTHOR CONTRIBUTIONS
RS designed the research. AC, TK, and RS performed the research. AC and RS analyzed the data and wrote the article. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by National Institutes of Health grant R21 DA037744 (RS).