Identification of Novel BDNF-Specific Corticostriatal Circuitries

Abstract Brain-derived neurotrophic factor (BDNF) is released from axon terminals originating in the cerebral cortex onto striatal neurons. Here, we characterized BDNF neurons in the corticostriatal circuitry. First, we used BDNF-Cre and Ribotag transgenic mouse lines to label BDNF-positive neurons in the cortex and detected BDNF expression in all the subregions of the prefrontal cortex (PFC). Next, we used a retrograde viral tracing strategy, in combination with BDNF-Cre knock-in mice, to map the cortical outputs of BDNF neurons in the dorsomedial and dorsolateral striatum (DMS and DLS, respectively). We found that BDNF-expressing neurons located in the medial prefrontal cortex (mPFC) project mainly to the DMS, and those located in the primary and secondary motor cortices (M1 and M2, respectively) and agranular insular cortex (AI) project mainly to the DLS. In contrast, BDNF-expressing orbitofrontal cortical (OFC) neurons differentially target the dorsal striatum (DS) depending on their mediolateral and rostrocaudal location. Specifically, the DMS is mainly innervated by the medial and ventral part of the orbitofrontal cortex (MO and VO, respectively), whereas the DLS receives projections specifically from the lateral part of the OFC (LO). Together, our study uncovers previously unknown BDNF corticostriatal circuitries. These findings could have important implications for the role of BDNF signaling in corticostriatal pathways.


Introduction
Brain-derived neurotrophic factor (BDNF) is a member of the nerve growth factor family of neurotrophic factors, which is highly expressed in the CNS (Hofer et al., 1990;Yan et al., 1997;Kowia nski et al., 2018). The majority of BDNF in neurons is stored in presynaptic dense core vesicles and is released upon neuronal depolarization (Dieni et al., 2012;Song et al., 2017). Once BDNF is released at axon terminals, it binds the receptor tyrosine kinase, tropomyosin-related kinase B (TrkB). Activation of TrkB stimulates extracellular regulated kinase 1/2 (ERK1/2), Protein kinase C (PKC) and/or phosphoinositide 3 kinase (PI3K) signaling cascades resulting in the initiation of transcriptional and translational machineries (Huang and Reichardt, 2003;Leal et al., 2014;Zagrebelsky et al., 2020). In the adult brain, BDNF plays a crucial role in synaptic and structural plasticity (Panja and Bramham, 2014;De Vincenti et al., 2019), as well as in learning and memory (Bekinschtein et al., 2014;Miranda et al., 2019).
BDNF is highly expressed in the cerebral cortex of both rodents and humans (Hofer et al., 1990;Timmusk et al., 1993;Conner et al., 1997). Studies in rodents suggest that BDNF in the cortex contributes to learning and memory paradigms (Miranda et al., 2019). For example, absence of BDNF in the prelimbic cortex (PrL) alters fear expression in mice indicating a role for consolidation and expression of learned fear (Choi et al., 2010). In the orbitofrontal cortical (OFC), BDNF is critical for goal-directed decision-making and in selecting actions based on their consequences (Gourley et al., 2013). In the motor cortex, BDNF contributes to motor learning (Andreska et al., 2020).
The cerebral cortex is also the major source of BDNF in the striatum (Altar et al., 1997;Baquet et al., 2004;Strand et al., 2007). BDNF released from cortical terminals, binds to, and activates its receptor, TrkB, in the striatum (Altar et al., 1997;Baydyuk and Xu, 2014). BDNF/TrkB signaling in the striatum has important cellular and behavioral roles (Besusso et al., 2013;Engeln et al., 2020). For example, TrkB signaling is required to control inhibition of locomotor behavior in enkephalin (ENK) positive medium spiny neurons (MSN; Besusso et al., 2013), and Lobo and colleagues provided data to suggest that BDNF/TrkB signaling in dopamine D1 receptor expressing (D1) MSN plays a role in stereotypy behaviors (Engeln et al., 2020).
Finally, rodent studies have suggested that BDNF in corticostriatal circuitries is linked to addiction. For example, numerous studies investigated the role of BDNF in the prefrontal cortex (PFC) in relation with cocaine use (for review, see Pitts et al., 2016). Specifically, cocaine exposure regulates BDNF signaling in the PFC and in turn BDNF influences the development and maintenance of cocaine-related behaviors Gourley et al., 2013;Zhang et al., 2015;Pitts et al., 2018). In addition, activation of BDNF signaling in the dorsolateral striatum (DLS) keeps alcohol intake in moderation (Jeanblanc et al.,. 2009(Jeanblanc et al.,. , 2013, whereas malfunctioning of BDNF/TrkB signaling in the corticostriatal regions promotes compulsive heavy use of alcohol and other alcohol-mediated behaviors (Logrip et al., 2009;Darcq et al., 2015Darcq et al., , 2016Moffat et al., 2023).
As detailed above, BDNF is crucial for cortical and striatal functions, yet careful characterization of BDNF neurons in corticostriatal circuitry is lacking. Here, using a combination of transgenic mouse lines together with viral-mediated gene delivery approaches, we mapped out BDNF-expressing cortical neurons that project to the dorsomedial striatum (DMS) and DLS.

Animals and breeding
Male C57BL/6J mice (six to eight weeks old at time of purchase) were obtained from The Jackson Laboratory. Male BDNF-Cre knock-in mice, which express Cre recombinase at the endogenous BDNF locus, were obtained from Zach Knight, UCSF (Tan et al., 2016). Ribotag mice (ROSA26CAGGFP-L10a), which express the ribosomal subunit RPL10a fused to EGFP (EGFP-L10a) in Cre-expressing cells (Zhou et al., 2013), were purchased from The Jackson Laboratory (B6;129S4-Gt (ROSA)26Sortm9(EGFP/ Rpl10a)Amc/J). Ribotag mice were crossed with BDNF-Cre mice allowing EGFP-L10a expression in BDNFexpressing cells. Mouse genotype was determined by PCR analysis of tail DNA.
Mice were individually housed on paper-chip bedding (Teklad #7084), under a reverse 12/12 h light/dark cycle (lights on 10 A.M. to 10 P.M.). Temperature and humidity were kept constant at 22 6 2°C, and relative humidity was maintained at 50 6 5%. Mice were allowed access to food (Teklad Global Diet #2918) and tap water ad libitum. All animal procedures were approved by the university's Institutional Animal Care and Use Committee and were conducted in agreement with the Association for Assessment and Accreditation of Laboratory Animal Care.

Immunohistochemistry, imaging, and quantification
Following intraperitoneal administration of euthasol (200 mg/kg), mice were transcardially perfused with phosphate buffered saline, followed by 4% paraformaldehyde (PFA), pH 7.4. Brains were quickly removed postperfusion and fixed for 24 h in 4% PFA before cryoprotection in 30% sucrose solution for 3 d at 4°C. Brains were then sectioned to 30 mm by cryostat (CM3050, Leica), collected serially and stored at À80°C. PFA-fixed sections were permeabilized and blocked in PBS containing 0.3% Triton X-100 and 5% donkey serum for 4 h at 4°C. Sections were then incubated for 18 h at 4°C on an orbital shaker with the primary antibodies anti-NeuN (1:500), anti-GFP (1:1000), or anti-VGlut1 (1:1000) diluted in 3% bovine serum albumin (BSA) in PBS. Next, sections were washed in PBS and incubated for 4 h at 4°C with Alexa Fluor 488-labeled donkey (1:500), Alexa Fluor 594-labeled donkey (1:500) antibodies in 3% BSA in PBS. After staining, sections were rinsed in PBS and were coverslipped using Prolong Gold mounting medium. Sections from rostral, rostrocaudal and caudal PFC were imaged on an Olympus Fluoview 3000 Confocal microscope (Olympus America) according to manufacture recommended filter configurations, using the same parameters across mice and images. Captured images were used to quantify the number of fluorescent cells in subregions of rostral, rostrocaudal, and caudal PFC using FIJI ImageJ (NIH; Schindelin et al., 2012). Specifically, the image of the PFC was precisely aligned to the anteroposterior (AP)-corresponding figure in the Paxinos atlas (Paxinos and Franklin, 2004), using the BigWarp interface included in FIJI (Bogovic, 2016). By placing corresponding landmarks on the image and atlas, the image and the AP figure were overlayed with precision. Next, regions of interest (ROIs) were traced on the overlayed image following the atlas delimitations using the polygon tool in FIJI. After adjusting the threshold (the same for each image), the number of positive neurons within an ROI was automatically quantified using FIJI counter plugin. To calculate the density of labeled neurons, the total number of labeled neurons in a region was divided by the surface of the region in mm 2 using Fiji (Schindelin et al., 2012).

Data analysis
GraphPad Prism 9 was used for statistical analysis. D'Agostino-Pearson normality test was used to verify the normal distribution of variables. Data were analyzed using one-way or two-way ANOVA where appropriate. One-way ANOVA was followed by Tukey's multiple comparisons test when appropriate. For two-way ANOVAs, significant main effects or interactions were calculated, followed by Sidak's multiple comparisons test; p value cutoff for statistical significance was set to 0.05.

Results
Experimental strategy for evaluating BDNFexpressing neurons distribution in the PFC The cerebral cortex expresses high levels of BDNF message (Hofer et al., 1990;Timmusk et al., 1993); however, a careful analysis of BDNF containing neurons in the PFC has not been conducted. Therefore, we first assessed the distribution of BDNF expression in cortical neurons. To do so, we used a BDNF-Cre transgenic mouse line allowing Cre-recombinase expression only in BDNF-expressing cells (Tan et al., 2016), which was crossed with a Ribotag mouse line expressing GFP-fused ribosomal subunit RPL10 in the presence of Cre-recombinase (Zhou et al., 2013;Fig. 1a). The presence of GFP-fused ribosomal subunit RPL10 (EGFP-L10a) enabled the visualization of BDNF-expressing cells.

Experimental strategy for mapping of prefrontal BDNF-positive neurons projecting to the DS
The cerebral cortex is the major source of BDNF in the striatum (Altar et al., 1997;Baquet et al., 2004). The striatum is divided into the ventral striatum and DS (Hunnicutt et al., 2016). We focused on the DS which is further divided into the lateral (DLS) and medial (DMS) regions (Hunnicutt et al., 2016), and assessed whether BDNF-expressing neurons in the PFC send projections to these two parts of the DS. To do so, we used a retrograde viral strategy and infected the anterior part of the DLS or the DMS of BDNF-Cre mice with a retrograde AAV-Nucflox-(mCherry)-EGFP viral construct (Bäck et al., 2019;Fig. 2a), which enables mCherry expression in BDNF-negative neurons and EGFP expression in BDNF-positive neurons (Bäck et al., 2019).

BDNF-expressing PFC neurons project to the DMS
First, we assessed whether BDNF-expressing cortical neurons project to the DMS by infecting the DMS of BDNF-Cre mice with retrograde AAV-Nuc-flox-(mCherry)-EGFP (Fig. 2a,b). As shown in Figure 2c, the DMS contains only cell nuclei labeled in red, corresponding to intrastriatal connections. The prefrontal regions show a high density of retrogradely EGFP-labeled neurons (Fig. 2c,g). Specifically, the mPFC including the PrL and Cg1 represents the major BDNF-positive output to the DMS, accounting for 42.9% of PFC-to-DMS projecting neurons (Fig. 2d,e). In addition, 26.3% of the BDNF-positive projections to the DMS are coming from M2 (Fig. 2d,e). The MO and ventral part of the OFC (VO) represent respectively 10.6% and 15.4% of the BDNF-positive PFC-to-DMS projecting neurons (Fig. 2d,e). Only 4.8% of total BDNF-positive neurons project from the LO, M1, and AI to the DMS (Fig. 2).

Density of BDNF-expressing neurons in PFC subregions that project to the DMS
To evaluate the density of the BDNF-positive PFC neurons projecting to the DMS within each PFC subregion, we analyzed the number of retrogradely labeled neurons per mm 2 (Fig. 2f). We found a significantly higher density of labeled BDNF-positive neurons in the Cg1 compared with the other regions, and in the PrL compared with LO, M1, and AI (one-way ANOVA, df = 7, F (7,32) = 22.72, p , 0.0001; Cg1 vs other regions: p , 0.0001, PrL vs LO: p = 0.0206, PrL vs AI: p = 0.0159, PrL vs M1: p = 0.0204, n = 5; Fig. 2f). In addition, the BDNF-positive PFC neurons project bilaterally, but predominantly in an ipsilateral manner, and the topographic pattern of PFC BDNF-expressing neurons is similar in the contralateral hemisphere (Extended Data Fig. 2-1).

BDNF-expressing PFC neurons project to the DLS
Next, we assessed whether BDNF-expressing cortical neurons project to the DLS by infecting the DLS of BDNF-Cre mice with retrograde AAV-Nuc-flox-(mCherry)-EGFP (Fig. 3a,b). Similar to the DMS, the DLS contained only red cell nuclei, corresponding to intrastriatal connections (Fig. 3c). Absence of EGFP-labeled neurons indicated once more a lack of BDNF message in the DLS. However, we found a high density of retrogradely EGFP-labeled neurons in the prefrontal regions (Fig. 3c,g). Specifically, the motor cortex (M1/M2), and the AI are the main regions that send BDNF-positive projection to the DLS, representing 80.9% and 12.6%, respectively, of the overall BDNFpositive projecting neurons (Fig. 3d,e). Fewer BDNF-positive neurons in the OFC (MO, VO, and LO) and mPFC (PrL and Cg1) project to the DLS, representing together only 6.4% of the overall BDNF-positive projecting neurons (Fig. 3d,e).

Comparison of topographical distribution of BDNFpositive PFC to DLS and DMS projecting neurons along the rostrocaudal axis
We performed a more detailed comparison of PFC topographical patterns of DMS and DLS projecting neurons along the rostrocaudal axis (Fig. 4). There was no significant difference between the PrL, VO, LO, and MO retrogradely labeled BDNF-positive neurons at the rostral position (Fig. 4a). Interestingly, rostrocaudal and caudal analysis revealed a specific AI-to-DLS circuit (Fig. 4b,c). In contrast with the rostral position, retrogradely labeled BDNF-positive neurons in the PrL/Cg1 projecting to the DMS or DLS exhibit an opposite distribution, with fewer DLS projecting neurons and an increased number of DMS projecting neurons along the rostrocaudal axis (Fig. 4b,c). We then focused on the OFC and compared the mediolateral distribution of BDNF-positive projecting neurons along the rostrocaudal axis (Fig. 4d). We found that labeled BDNF-expressing neurons in the caudal LO are projecting significantly more to the DLS than the DMS, whereas labeled BDNF-expressing neurons in the rostral and rostrocaudal parts of the VO are projecting significantly more to the DMS than the DLS (two-way ANOVA; interaction effect, F (8,66) = 6.398, p , 0.0001, main effect of rostrocaudal axis, F (8,66) = 3.177, p = 0.0042, main effect of OFC subregion, F (1,66) = 3.441, p = 0.0681, rostral VO: p = 0.0007, rostrocaudal VO: p = 0.011, caudal LO: p = 0.0028; n = 5; Fig. 4d).

The DS receives inputs from BDNF-positive OFC neurons
Next, we used an anterograde viral strategy to confirm the presence of a BDNF-specific circuit between the OFC and DS. To do so, we infected the OFC of BDNF-Cre mice with an AAV-Flex-GFP virus allowing visualization of projections extended by BDNF-positive OFC neurons (Fig.  5a-c). As shown in Figure 5d, BDNF-positive projections were localized in both the DLS and the DMS.
Finally, to determine whether the BDNF-positive OFC neurons form synapses with DLS neurons, we stained neurons with anti-vesicular glutamate transporter 1 (VGLUT1) antibodies labeling the cortical glutamatergic presynaptic compartment. GFP-positive projections and VGLUT1 were co-labeled in the DLS (Fig. 6), suggesting that BDNF-positive glutamatergic neurons from the OFC extend projections and form synapses with neurons in the DLS.

Discussion
In the present study, we mapped out cortical BDNFexpressing neurons projecting to the DLS and DMS. We found that BDNF-expressing neurons located in the mPFC project mainly to the DMS, whereas the motor cortex and the AI mainly project to the DLS. Interestingly, we observed an anatomic segregation along the rostrocaudal axis of OFC BDNF-positive neurons that project to the DLS and DMS. Thus, our data define novel BDNF neural circuits connecting the PFC to the DS.

Characterization of BDNF neurons in the PFC and the DS
In 1990, Hofer et al., used in situ hybridization to characterize BDNF message in the brain and reported that BDNF mRNA is abundant in the mouse cerebral cortex (Hofer et al., 1990). In contrast, BDNF protein was detected in the striatum, although BDNF mRNA was absent (Spires et al., 2004;Gharami et al., 2008). Altar et al., reported that BDNF protein found in the striatum is synthesized, anterogradely transported from the cell bodies located in the cerebral cortex and released presynaptically (Altar et al., 1997). Although more recent BDNF expression surveys have been conducted (Singer et al., 2018;Leschik et al., 2019;Wosnitzka et al., 2020), a careful mapping of BDNF-expressing neurons in cortical areas and their striatal projections has not been previously undertaken. Crossing a BDNF-Cre mouse line (Tan et al., 2016) with a Ribotag reporter line (Zhou et al., 2013) enabled labeling of BDNF-expressing neurons in the prefrontal cerebral cortex. Using this approach, which has superior accuracy compared with in situ hybridization and immunohistochemistry, we provide a definitive proof that a majority of neurons in prefrontal regions of the cortex express BDNF. Similar to what was previously reported (Hofer et al., 1990), we did not detect BDNF-expressing neurons in the striatum. Interestingly, although basal BDNF levels in the rodent striatum are negligible, a robust increase in BDNF message in the striatum is detected in response to behaviors such as voluntary alcohol intake (McGough et al., 2004;Jeanblanc et al., 2009) and cocaine administration (Liu et al., 2006), as well as in response to exercise (Marais et al., 2009) and stress (Miyanishi et al., 2021). Reconciling these potentially conflicting findings merits further investigation. One possibility is that these stimuli increase BDNF expression in cortical regions projecting to the striatum. Specifically, BDNF mRNA can be found in axon terminals (Lau et al., 2010), and recent studies show that presynaptic protein translation occurs within neuronal projections (Costa et al., 2019;Hafner et al., 2019). Thus, detectable BDNF in the striatum could be explained by the presence of BDNF mRNA within cortical neuron projections in the striatum.

Characterization of corticostriatal BDNF circuitries
Our viral tracing strategy enabled us to explore BDNFspecific circuitries. For instance, we uncovered a specific AI-to-DLS BDNF circuit. Interestingly, Haggerty and colleagues recently found that stimulation of the AI-to-DLS circuit decreases alcohol binge drinking in male mice which in turn reshapes glutamatergic synapses in the DLS from AI inputs (Haggerty et al., 2022). In addition, we previously found that activation of BDNF/TrkB signaling in the DLS keeps alcohol intake in moderation (Jeanblanc et al., 2009(Jeanblanc et al., , 2013. Thus, it is tempting to suggest a role for BDNF in AI-to-DLS synaptic adaptations that gate alcohol intake. We observed that the mPFC exhibits a large number of BDNF-positive neurons projecting to the DMS. These results are in accordance with previous behavioral studies showing that the DMS receives projections from associative cortices such as the PrL (Friedman et al., 2015;Hart et al., 2018;Shipman et al., 2019;Vicente et al., 2020). We also found that a high number of BDNF-positive neurons in the motor cortex extends projections to the DLS. The motor cortex is known for innervating the DLS (Yin and Knowlton, 2006), and Andreska and colleagues recently reported that BDNF in this corticostriatal circuit is essential for motor learning (Andreska et al., 2020).
We also show herein that OFC BDNF-positive neurons project to the DS. Interestingly these projections are organized in a mediolateral manner. Specifically, the DMS receives projections from BDNF-positive neurons located in the MO and VO, whereas BDNF-positive  neurons projecting to the DLS are located specifically in the LO. It is known that the OFC projects to the DS (Gremel and Costa, 2013;Gremel et al., 2016;Zimmermann et al., 2017), and previous studies showed that the DMS receives input from the MO Green et al., 2020). The DMS plays an important role for goal directed behavior (Vandaele et al., 2019), and BDNF in the MO is essential to sustain goal-sensitive action in mice . Specifically, Gourley and colleagues showed that BDNF knock-down in the MO decreases behavioral sensitivity to reinforcer devaluation. Thus, it is likely that this BDNF circuitry and perhaps BDNF released by MO neurons within the DMS regulate goal-directed action control.
We found that the majority of LO BDNF-positive neurons project to the DLS. The functional implication of this circuitry is unknown. Gourley and colleagues reported that BDNF in both VLO and MO participates in goal-directed behavior (Gourley et al., 2013Zimmermann et al., 2017), and Pitts and colleagues reported that inhibiting TrkB signaling in the DLS blocks habit formation (Pitts et al., 2018). In addition, mechanistic target of rapamycin complex 1 (mTORC1) in the VLO is involved in habitual alcohol seeking (Morisot et al., 2019). Putting these studies together, it is plausible that BDNF in the LO neurons projecting to the DLS plays a role in inflexible behavior.
Another possible role for BDNF in this corticostriatal circuit is to shape and modulate the synaptic output of striatal neurons. Using a two-neuron microcircuit approach in primary cortico-striatal neurons, Paraskevopoulou and colleagues showed that BDNF and glutamate coreleased from cortical projections are required to modulate inhibitory synaptic transmission of striatal neurons (Paraskevopoulou et al., 2019). Therefore, co-release of BDNF with glutamate in the DLS and DMS by PFC neurons may modulate inhibitory sensitivity and dendritic morphology of striatal neurons in these PFC-DS circuits.

BDNF-TrkB signaling in the DS
Most neurons in the striatum are MSN which are divided into two subpopulations, D1 MSN and dopamine D2 receptor (D2) expressing MSN (Gerfen et al., 1990). In the adult brain, both D1 and D2 MSN express TrkB receptors (Lobo et al., 2010). Engeln and colleagues showed that a subset of mice congenitally lacking TrkB receptor in D1-MSN in the DS exhibit repetitive circling behavior, suggesting a role of BDNF signaling in D1-MSN in this type of behavior (Engeln et al., 2020). Thus, it would be of great interest to map which dorsal striatal neuronal subtype receives inputs from cortical neuron. In addition, Nestler and colleagues provided data to suggest that activation of BDNF-TrkB signaling in D1 versus D2 MSN triggers in the nucleus accumbens (NAc) opposite effects on cocaine and morphine-dependent rewarding behaviors (Lobo et al., 2010;Koo et al., 2014). Additional studies are warranted to shed a light on the nature of cortical neurons projecting to the NAc. Furthermore, the DS is also organized into patch and matrix compartments which have distinct connectivity and genetic signature (Märtin et al., 2019). As both of patch and matrix neurons express TrkB (Costantini et al., 1999), more studies are required to decipher the contribution of BDNF/TrkB signaling in subpopulation of DS neurons.

Comparison between our findings and the literature
Using a retrograde viral tracing, we analyzed the spatial profiles of BDNF-positive projecting neurons in the PFC. We were able to confirm that these BDNF-positive circuits exhibit the same medial-lateral gradient of corticostriatal projections first reported in rats and primates (Selemon and Goldman-Rakic, 1988;Haber, 2003;Schilman et al., 2008). Although our data are generally consistent with the excitatory corticostriatal circuits described in the literature, several points might contribute to some discrepancies. For example, Balsters and colleagues compared corticostriatal circuits between human, nonhuman primates, and mice and found significant differences in cortical projections to the anterior putamen and caudate body (Balsters et al., 2020). Many previous studies have been done in other animal models such as rats and primates (Berendse et al., 1992;Haber et al., 2006;Schilman et al., 2008;Hoover and Vertes, 2011;Mailly et al., 2013). Neuroanatomical differences exist across species and detailed explorations of similarities and differences between mice and other species are of importance. Furthermore, in contrast with previous literature (Berendse et al., 1992;Hoover and Vertes, 2011), in our comparison of the topographical distribution of BDNF-positive neurons, we observed VO-to-DMS and mPFC to-DLS BDNF-positive projecting neurons. An explanation to some of the discrepancies between corticostriatal circuit mapping studies is the experimental strategy used by us and others. Specifically, unlike the majority of studies that used anterograde strategies to map corticostriatal circuits (Berendse et al., 1992;Haber et al., 2006;Schilman et al., 2008;Hoover and Vertes, 2011;Mailly et al., 2013), we used a retrograde strategy in which a retrograde AAV was used (Tervo et al., 2016). Retrograde AAV is known to enter at the postsynaptic compartment of neurons and then be retrogradely transported to the cell body (Tervo et al., 2016). Thus, unlike an anterograde tracer which labels the entire fibers, we specifically labeled neurons projecting and forming synapses at the site of infection. However, Tervo and colleagues have provided good evidence to support the entry of AAV retrograde at axonal terminals (Tervo et al., 2016), additional studies will be necessary to fully rule out its ability to infect axons of passage. Although our anterograde tracing confirmed both the sparse and dense BDNF-expressing projections, anterograde tracing is not appropriate for detection of sparse projections.
Pan and colleagues reported tracing data consistent with our data using a fluorescent latex microsphere retrograde injection in mice (Pan et al., 2010). The authors reported an intense retrograde labeling in the PrL-infralimbic cortex (IL) and Cg1-M2 and no labeling in the LO-AI when injecting the retrograde tracer in the anterior DMS (aDMS). Pan and colleagues further found an intense retrograde labeling in the LO-AI and Cg1-M2 when injecting the fluorescent latex microsphere in the anterior DLS (aDLS). In addition, using a retrograde viral strategy, Green and colleagues also reported neurons located in the Cg1/PrL and IL/MO projecting to the aDMS and neurons located in the AI and M1/M2 projecting to the aDLS (Green et al., 2020).
It is important to note that our study focused specifically on the anterior part of the DS. Interestingly, Pan et al., found no retrograde labeling in the PrL, IL, MO, LO, AI, Cg1, and M2 when they injected the retrograde tracer in posterior part of the DS (pDS; Pan et al., 2010). Since the pattern of projection in the striatum differs along the AP axis (Mailly et al., 2013;Hunnicutt et al., 2016), further work is needed to decipher PFC to pDS BDNF circuits in mice. In addition, using our BDNF-positive retrograde tracing strategy, the carefully mapped corticostrial circuities were restricted to the DMS and DLS and therefore lack dorsocentral inputs. Further tracing work is needed to describe the complete mediolateral PFC-to-DS circuitries.
In summary, in this study, we mapped out BDNF-expressing neurons in the PFC and deciphered BDNF-specific corticostriatal circuits. Furthermore, the discovery of LO-to-DLS BDNF microcircuitry highlights the importance for deciphering the function of BDNF in the context of microcircuits composed by very localized neuronal ensembles.