Retrograde labeling illuminates distinct topographical organization of D1 and D2 receptor-positive neurons in the prefrontal cortex of mice

The cortex plays an important role in regulating motivation and cognition, and does so by regulating multiple subcortical brain circuits. Glutamatergic pyramidal neurons in the prefrontal cortex (PFC) are topographically organized in different subregions such as the prelimbic, infralimbic and orbitofrontal, and project to topographically-organized subcortical target regions. Dopamine D1 and D2 receptors are expressed on glutamatergic pyramidal neurons in the PFC. However, it is unclear whether D1 and D2 receptor-expressing pyramidal neurons in the PFC are also topographically organized. We used a retrograde adeno-associated virus (AAVRG)-based approach to illuminate the topographical organization of D1 and D2 receptor-expressing neurons, projecting to distinct striatal and midbrain subregions. Our experiments reveal that AAVRG injection in the nucleus accumbens (NAcc) or dorsal striatum (dSTR) of D1Cre mice labeled distinct neuronal subpopulations in medial orbitofrontal or prelimbic PFC, respectively. However, AAVRG injection in NAcc or dSTR of D2Cre mice labeled medial orbitofrontal, but not medial prelimbic PFC, respectively. Additionally, D2R+ but not D1R+ PFC neurons were labeled upon injection of AAVRG in substantia nigra pars compacta (SNpc). Thus, our data are the first to highlight a unique dopamine receptor-specific topographical pattern in the PFC, which could have profound implications for corticostriatal signaling in the basal ganglia. SIGNIFICANCE STATEMENT Corticostriatal connections play an important role in regulating goal-directed and habitual behavior, and neuromodulators such as cortical dopamine play an important role in behavioral flexibility. Dopamine receptor expressing D1R+ and D2R+ projection neurons in the cortex mediate the effects of cortical dopamine, but whether these neurons are anatomically organized in a manner that would explain how these neurons mediate these complex effects, is not clear. Our results show a distinct topographical organization of D1R+ and D2R+ PFC pyramidal neurons that project to distinct striatal and midbrain subregions. These results suggest that effects of cortical dopamine are mediated by anatomically localized distinct receptor- and target-defined subcircuits.

The striatum is the main input center of the basal ganglia, receiving input from cortical, thalamic, limbic and dopaminergic nuclei (Ikemoto and Bonci, 2014). Glutamatergic signals from cortical areas, and dopamine signals from midbrain dopaminergic nuclei act upon spiny projection neurons (SPNs) in the striatum. The SPNs integrate both glutamate and dopamine signals and coordinate various aspects of learning and behavior (Bamford et al., 2018;Horvitz, 2009;Shiflett and Balleine, 2011).
Afferent cortical glutamatergic inputs into the striatum originate within various medial or lateral subregions of the PFC such as the prelimbic, infralimbic, orbitofrontal, and motor cortex.
Although, these studies elegantly outline the role of sub-regions in the PFC or striatum, few studies have explored the neuronal and molecular diversity of PFC pyramidal neurons involved in regulating motivation and cognition.
Dopamine and its receptors in the PFC also regulate motivated behavior and cognitive flexibility (Barker et al., 2013;Goldman-Rakic, 1998;Hitchcott et al., 2007;Natsheh and Shiflett, 2018;Ott and Nieder, 2019). Dopamine activates D1 and D2 class of receptors in the PFC that signal through stimulatory Gαs or inhibitory Gαi proteins, respectively, and through β -arrestins as well, which modulate the activity of both pyramidal neurons and interneurons (Beaulieu et al., 2007;Cousineau et al., 2020;Ferguson and Gao, 2018;Santana et al., 2009;Tomasella et al., 2018;Tseng andO'Donnell, 2004, 2007;Urs et al., 2016). Moreover, pharmacological targeting of D1Rs or D2Rs, or genetic deletion of D2Rs in the PFC can regulate dopamine-dependent behaviors such as locomotion, cognition, and goal-directed behavior (Barker et al., 2013;Del Arco et al., 2007;Hitchcott et al., 2007;Khlghatyan and Beaulieu, 2020;Natsheh and Shiflett, 2018;Tomasella et al., 2018;Urs et al., 2016). Although PFC dopamine receptors play an important role in motivation and cognition, the topographical distribution of D1R+ or D2R+ neurons in specific subregions of the PFC is not known. Here, we use a retrograde AAV (AAVRG)-based approach to identify distinct topographically-organized subpopulations of D1R+ and D2R+ neurons in the PFC based on their target projection areas. Given the role of various subregions of the PFC in motivated behavior and cognition, and the heterogeneity of these regions, the effects of dopamine and its receptors within these subregions will expand our understanding of the molecular and neuronal mechanisms regulating motivated behavior and cognition.
Mice were allowed to recover for 2 weeks to allow for viral expression of GFP or TdT before imaging and counting of cells.
Immunostaining, Imaging and Quantification. 40 μ m thick vibratome cut sections of formalin-fixed mouse brains were processed for imaging. Sections from rostral, rostrocaudal and caudal PFC, and striatal and midbrain sections were imaged using a Nikon AZ100 Zoom microscope, using the same exposure across genotypes for an injection pair. Captured images were used for quantifying number of fluorescent cells for each channel (GFP and TdT) in different subregions of rostral, rostrocaudal and caudal PFC using ImageJ (NIH), and threshold was kept the same between genotypes and injection pairs. At least three sections from rostral, rostrocaudal and caudal PFC were analyzed for each mouse, with an n=5 mice per injection pair. For glutamatergic or GABAergic marker identification, we performed antigen retrieval in citrate buffer at 80 degrees on virally injected PFC sections, and colabeled with antibodies to GFP (Frontier Institute, Japan, Cat# AB_2571575), RFP (Rockland, Cat# 600-401-379), CamKIIa (Enzo Life Sciences, Cat# ADI-KAM-CA002-D), parvalbumin (PV) (Frontier Institute, Japan, Cat # AB_2571613), and GAD 65/67 (Frontier Institute, Japan, Cat# AB_2571698).
Imaging for PV and GAD colabeling were done using the Nikon AZ100 zoom microscope.
Imaging for CamKIIa labeling was done using a Nikon spinning disk confocal (CSU-X1, Yokogawa) with either 10x or 60x objective on an inverted microscope (Nikon Ti2-E), with a back-thinned sCMOS camera (Prime 95B, Photometrics).
Statistical analyses. Data were analyzed by a standard two-way ANOVA test for comparison between genotypes, and injection pairs. Individual genotypes, or injection pairs were compared using a post hoc Tukey's test. Data are presented as mean±SEM. p<0.05 is considered as significant.

RESULTS
For this study we focused primarily on dopamine-related subcortical target regions such as the striatum and midbrain dopamine nuclei i.e. substantia nigra pars compacta (SNpc). The striatum itself can be topographically divided along the dorsoventral or mediolateral axes, into the dorsal and ventral striatum, or dorsomedial (DMS) and dorsolateral (DLS), respectively.
In both D1Cre and D2Cre mice, distinct projection fibers were observed in midbrain regions i.e substantia nigra pars compacta (SNpc) and reticulata (SNpr). In the D1Cre mice, GFP+ projection fibers (from dSTR) were observed in the SNpr, whereas, TdT+ projection fibers were observed in the SNpc (Figure 1, aii). In contrast, for the D2Cre mice only TdT+ projection fibers were observed in the SNpc, and no labeling in the SNpr (Figure 1, bii).
PFC projection neurons are primarily glutamatergic, but some studies have shown that a small percent of these projection neurons can be GABAergic (Lee et al., 2014;Melzer et al., 2017), and contribute to physiological outcomes. To confirm whether these retrogradely labeled neurons are glutamatergic or GABAergic, we performed colocalization studies with known glutamatergic neuron marker CamKIIa, and known GABAergic neuron markers GAD 65/67 and parvalbumin (PV). As seen in Fig. S1, retrogradely labeled neurons in D1 or D2Cre mice predominantly colocalize with CamKIIa but not with GAD or PV, thus confirming a glutamatergic identity of these corticostriatal projection neurons.     Along the dorsoventral axis, PFC D1R+ neurons are topographically organized such that D1R+ neurons in prelimbic regions primarily project to the dSTR, whereas D1R+ neurons in medial OFC and infralimbic regions primarily project to the NAcc core. In contrast, PFC D2R+ neurons have a distinct pattern of organization, such that very few prelimbic D2R+ neurons project to dSTR, but medial OFC and infralimbic D2R+ neurons project to NAcc core, similar to D1R+ neurons. Along the mediolateral axis, D1R+ neurons in the medial prelimbic region primarily project to dorsomedial striatum, whereas D1R+ neurons in M1/M2 motor and agranular insular cortex primarily project to dorsolateral striatum. In contrast very few D2R+ neurons project to either DMS or DLS. However, medial PFC D2R+ but not D1R+ neurons project to midbrain dopamine nuclei. Thus, our data provide, for the first time a detailed insight into the anatomical organization of D1R+ and D2R+ neurons in the PFC.
In this study we use AAVRGs to identify afferent PFC inputs into various striatal and midbrain regions. One potential caveat with using AAVRGs is that we cannot control for variability of infection at the injection site, even if we inject the same volume of AAV. However, one advantage of using AAVRGs is that target neurons can be specifically labeled in a Credependent manner, and therefore label cell bodies of specific populations of neurons using transgenic Cre mice. Our data are consistent with previous findings that D1R+ neurons are     K  h  l  g  h  a  t  y  a  n  ,  J  .  ,  a  n  d  B  e  a  u  l  i  e  u  ,  J  .  M  .  (  2  0  2  0  )  .  C  R  I  S  P  R  -C  a  s  9  -M  e  d  i  a  t  e  d  I  n  t  e  r  s  e  c  t  i  o  n  a  l  K  n  o  c  k  o  u  t  o  f  G  l  y  c  o  g  e  n   S  y  n  t  h  a  s  e  K  i  n  a  s  e  3  b  e  t  a  i  n  D  2  R  e  c  e  p  t  o  r  -E  x  p  r  e  s  s  i  n  g  M  e  d  i  a  l  P  r  e  f  r  o  n  t  a  l  C  o  r  t  e  x  N  e  u  r  o  n  s  R  e  v  e  a  l  s   C  o  n  t  r  i  b  u  t  i  o  n  s  t  o  E  m  o  t  i  o  n  a  l  R  e  g  u  l  a  t  i  o  n  .  C  R  I  S  P  R  J  3  ,  1  9  8  -2  1  0  .   K  i  l  l  c  r  o  s  s  ,  S  .  ,  a  n  d  C  o  u  t  u  r  e  a  u  ,  E  .  (  2  0  0  3  )  .  C  o  o  r  d  i  n  a  t  i  o  n  o  f  a  c  t  i  o  n  s  a  n  d  h  a  b  i  t  s  i  n  t  h  e  m  e  d  i  a  l  p  r  e  f  r  o  n  t  a  l  c  o  r  t  e  x   o  f  r  a  t  s  .  C  e  r  e  b  C  o  r  t  e  x  1  3 ,  S  u  l  z  e  r  ,  D  .  (  2  0  1  1  )  .  H  o  w  a  d  d  i  c  t  i  v  e  d  r  u  g  s  d  i  s  r  u  p  t  p  r  e  s  y  n  a  p  t  i  c  d  o  p  a  m  i  n  e  n  e  u  r  o  t  r  a  n  s  m  i  s  s  i  o  n  .  N  e  u  r  o  n  6  9  ,   6  2  8  -6  4  9  .   T  o  m  a  s  e  l  l  a  ,  E  .  ,  B  e  c  h  e  l  l  i  ,  L  .  ,  O  g  a  n  d  o  ,  M  .  B  .  ,  M  i  n  i  n  n  i  ,  C  .  ,  D  i  G  u  i  l  m  i  ,  M  .  N  .  ,  D  e  F  i  n  o  ,  F  .  ,  Z  a  n  u  t  t  o  ,  S  .  ,  E  l  g  o  y  h  e  n  ,   A  .  B  .  ,  M  a  r  i  n  -B  u  r  g  i  n  ,  A  .  ,  a  n  d  G  e  l  m  a  n  ,  D  .  M  .  (  2  0  1  8  )  .  D  e  l  e  t  i  o  n  o  f  d  o  p  a  m  i  n  e  D  2  r  e  c  e  p  t  o  r  s  f  r  o  m  p  a  r  v  a  l  b  u  m  i