Cross-species transcriptome analysis of the subthalamic and para-subthalamic nuclei reveal mRNA patterns unique to primate and relevant to neuropsychiatry

doi:10.21203/rs.3.rs-4669811/v1

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Cross-species transcriptome analysis of the subthalamic and para-subthalamic nuclei reveal mRNA patterns unique to primate and relevant to neuropsychiatry

https://doi.org/10.21203/rs.3.rs-4669811/v1

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The ventromedial subthalamic nucleus (STN) is associated with affective function via limbic brain networks. Clinical data indicate the anterior aspect of ventromedial STN as an important target in Deep brain stimulation (DBS) intervention of psychiatric disorder, including Obsessive-compulsive disorder (OCD), for which Selective serotonin reuptake inhibitors (SSRIs) are prescribed. However, far from all patients respond to either SSRIs or DBS. Despite clinical relevance, the primate STN is lacking in molecular and anatomical detail. Here, transcriptome data generated in mice was implemented in comparative analysis with macaque and human. Remarkable species differences were observed. Serotonin receptor 2c (HTR2C) mRNA was identified in a distinct ventromedial STN profile in macaque and human, non-paralleled in mouse. This included the medial tip and anteromedial STN, where HTR2C was distributed in patterns matching reports of serotonergic innervation. In primates, but not mice, a previously undescribed far-anterior STN brain area was uncovered, and the anterior, but not posterior, STN was found to border the hypothalamic para-STN. In both mice and primates, gradients of HTR2C mRNA allowed para-STN to be subdivided into core and shell domains. Dorsal to STN, para-STN was partly embedded in the Medial forebrain bundle (MFB), a DBS target in OCD and depression. Transcriptome data visualized in serial sections and 3D brain fly-through videos challenge current atlases of STN, pSTN, and MFB anatomy in mice and primates. Differential mRNA patterns in the primate, including unique HTR2C profiles, may provide openings for drug discovery and enhanced anatomical precision, and uncover neurobiological underpinnings of psychiatric disorders.

Biological sciences/Neuroscience/Molecular neuroscience

Health sciences/Biomarkers/Predictive markers

The subthalamic nucleus (STN) is best known for its role in motor control, and its dysregulation is a key feature of Parkinson´s disease, an aberration which can be ameliorated with Deep brain stimulation (DBS) in the dorsal territory of the STN (1–6). The STN is also an important DBS target in psychiatric disorders, including Obsessive-compulsive disorder (OCD), for which the anteromedial STN is a validated stimulation target (7–10). In addition to neuropsychatric DBS targets within the STN structure, the Medial forebrain bundle (MFB), which passes the dorsal aspect of the STN, is a recent DBS target for the treatment of depression and OCD (11–14), the two disorders sharing their primary medical treatment by Selective serotonin reuptake inhibitors (SSRIs) (15–17). The MFB primarily consists of dopaminergic and serotonergic projections reaching from midbrain to forebrain via the Fields of Forel (18,19), but also contains cell groups of different character, including hypothalamic neurons (20,21). Another structure close to the STN is the para-STN (pSTN), a hypothalamic structure engaged in autonomic and interoceptive functions (22). No distinct border separates STN and pSTN, and pSTN is even overlooked in some anatomical literature covering the primate brain (23–26); instead, the pSTN has been mostly studied in rodents (22,27,28). Together, the STN, pSTN and MFB form a triad of anatomically neighbouring structures at the midbrain-thalamus-hypothalamus intersection, an anatomically and functionally heterogeneous brain area of major significance to human health, and of great clinical importance.

The application of DBS for the treatment of neuropsychiatric disorders has led to some positive results and is consequently gaining interest also beyond OCD and depression, such as for example addiction (7,9,11,12,14,29–33). However, more than half of psychiatry patients either respond poorly to DBS, or do not respond at all (7,9). For the application of DBS in the STN, one reason for lack of predictability and higher success rate is likely the elusive organization within this structure. The STN has been described as anatomically and functionally complex, engaging in several aspects of limbic, associative/cognitive and motor functions (34–42). Based on anatomy and connectivity, as well as experimental and clinical results of lesioning and DBS (43–48), the primate STN has been described as tripartite structure in which limbic, associative and motor functions are anatomically segregated (37,44,49–52). However, the anatomical-functional tripartite model has also been questioned and debated (44,53,54). In accordance with several reports of the past couple of years, one recent study of DBS-treated patients from several different groups of disorders (including OCD, Parkinson´s disease, Tourette syndrome, dystonia), identified optimal neural stimulation positions, so called sweet spots, within the STN, supporting distinctly targetable connectivity and function for differently located neuronal populations within the STN structure (55). Based on the urgent need for improved treatment prospects in neuropsychiatry, one key step forward could now be the revelation of higher level of anatomical detail in the brain maps covering the STN and neighbouring structures, including molecular profiles that can aid in the subdivision of the STN into molecular-anatomical domains. Molecularly reinforced anatomical brain maps with high resolution across the STN and associated structures should be therapeutically informative in DBS, and also beyond surgical intervention strategies, for example to improve understanding of cellular subtypes, which in turn may help uncover pathophysiological mechanisms useful for drug discovery.

Transcriptomics, referring to gene expression analysis on the RNA level, has shown great potential in enriching knowledge of differential mRNA patterns within heterogeneous brain structures (56). Applying single nuclei RNA-sequencing (snRNASeq) on STN neurons from mice, we recently identified clusters of differentially expressed genes that upon brain section analysis of corresponding mRNA profiles using fluorescent in situ hybridization (FISH) enabled the subdivision of the mouse STN into three major spatio-molecular domains (57) (reviewed in (58)). In a follow-up study, taking advantage of additional snRNASeq-derived data, FISH analysis of distinct mRNA patterns allowed us to segregate the STN vs. pSTN in the brain of both mouse and macaque monkey (28) (reviewed in (59)). Further, in a collaborative effort addressing non-human primate and human STN with magnetic resonance imaging (MRI) and FISH, we recently identified selective dopaminergic and serotonergic innervation in the anteromedial STN (60).

Here, we set out to test if transcripts encoding differentially expressed genes in the STN of mice could be used as tools to visualize the internal organization of the STN in the rhesus macaque monkey (Macaca mulatta), a non-human primate with high similarity in brain anatomy to the human brain. The findings identify a remarkable spatial distribution pattern of the mRNA encoding Serotonin receptor 2c (5-HT2CR) in the anterior and ventromedial STN, uniquely in the primate brain. The current data may be of importance to neuropsychiatry.

Fluorescent in situ hybridization (FISH) on brain sections

Sections from whole mouse brain, and from macaque and human brain tissue blocks, were analyzed by FISH in accordance with our previously published protocols (28,60). All sequence and primer information is listed in Supplementary Table 1. Full names of genes and references (abbreviations in brackets): Paired-like homodomain 2 (Pitx2) (61,62), Vesicular glutamate transporter (Vglut2) (63,64); Vglut2 and Pitx2 previously shown to overlap 100% in STN of mice (65–67), Serotonin receptor subtype 2c (Htr2c), Neurexophilin 1 (Nxph1), Neurexophilin 4 (Nxph4), Potassium voltage-gated channel subfamily A regulatory beta subunit 3 (Kcnab3), Parvalbumin (Pvalb), Collagen type XXIV Alpha 1 chain (Col24a1), Tachykinin 1 (Tac1), Calbindin2 (Calb2), BAI1 Associated Protein 3 (Baiap3) (57), and Glutamic acid decarboxylase subtypes 1 (Gad1), Glutamic acid decarboxylase subtypes 2 (Gad2), Vesicular inhibitory amino acid transporter (Viaat), GABA transporter 1 (Gat1), Excitatory amino acid transporter (aka Glutamate aspartate transporter 1) (Eaat1/Glast1), CxC3 motif chemokine receptor 1 (Cx3cr1). Primate gene counterparts in capital letters and mRNA in roman (non-italics).

Selected sections were stained with cresyl violet (0.5%, C0775, Merck Millipore, MA, USA), or analyzed for Tyrosine hydroxylase (TH) immunoreactivity using standardized immunofluorescence protocol (anti-TH antibody, MAB318, diluted 1:250, Merck Millipore, MA, USA; detection with biotinylated goat anti-mouse IgG antibody, diluted 1:400, Vector Laboratories, Streptavidin-HRP, SA50141, Vector Laboratories).

Species: Mice were housed in accordance with local and European regulations; brain sections prepared as described (26). Ethical approval was obtained from the Uppsala Ethical Committee: Dnr 5.8.18-18646/2020. Macaque brain sections were obtained from tissue blocks derived from two adult Macaca Mulatta, described in (60), including ethical statement. Human STN samples were obtained from brains collected in a Brain Donation Program of the Brain Bank “GIE NeuroCEB” (GIE Neuro-CEB BB-0033-00011) run by a consortium of Patients Associations: ARSEP (Association for esearch on multiple sclerosis), CSC (Cerebellar ataxias), France Alzheimer, and France Parkinson. Consents were signed by patients themselves or their next of kin in their name, in accordance with the French Bioethical Laws. The Brain Bank GIE NeuroCEB has been declared at the Ministry of Higher Education and Research and has received approval to distribute samples (agreement AC-2013-1887), France. ID: 7582, 74 ys, DPM 24 hs.

Viral-genetic cell tracing in transgenic mice

Pitx2-Cre (courtesy of James Martin, University of Texas, USA) and Tac1-Cre (Jackson Laboratories, B6;129S-Tac1tm1.1(cre)Hze/J; Jax #021877) transgenic mice, validated to express Cre recombinase in STN vs. pSTN, were injected with rAAV2/EF1a-DIO-eYFP virus (UNC Vector Core, Chapel Hill, NC, USA) into the STN and pSTN area, in accordance with our previous protocol (28) with the exception of injection volume (2x150nl). Tac1-Cre mice were also bred to floxed CAG-Sun1/sfGFP reporter mice (CAG-Sun1/sfGFP (Jackson Laboratories, B6;129-Gt(ROSA)26Sor^{tm5(CAG-Sun1/sfGFP)Nat}/J Strain #:021039) to visualize nuclear envelope in cells positive for Tac1-Cre recombinase (pSTN). Immunofluorescence was performed as described (28) with the addition of TH detection (rabbit anti-TH, AB512, Merck Millipore; detection by A594 donkey anti-rabbit 1:1000, AB150068, Abcam).

Whole mouse brain in situ hybridization and 3D imaging

Whole mouse brain in situ hybridization was performed according to previous protocol (68). HCR™ RNA probes targeting Tac1 (Gene ID: 21333), Pitx2 (Gene ID: 18741), Vglut2 (Gene ID: 140919) were designed to target all splice variants using a custom probe design proprietary to Molecular Instruments. Brains were imaged on a Luxendo LCS-SPIM (Bruker) light-sheet microscope at 4.4x magnification with 10µm z-stack intervals. Imaris (Bitplane) software was used for 3D rendering and for generating movies and images. Tac1, Pitx2, and Vglut2signals were mapped to the LSFM reference brain atlas (69) together with data from TH whole brain immunolabelling previously generated (70).

Relative position and size of STN vs. pSTN differ across the anteroposterior (AP) axis

To assess if mRNA profiles originally identified by transcriptomics of the STN and adjacently located pSTN in the mouse are applicable for the study of corresponding structures in the primate STN, the battery of mRNA markers originating from our transcriptomics study (57) was here applied in serial sections covering the AP axis across STN and pSTN in both mouse and macaque, allowing their comparison (Figure 1 and Figure 2). Selected mRNA markers were subsequently analyzed using light-sheet microscopy to validate findings from the section analysis using 3D imaging of the intact mouse brain (Supplementary Videos 1-3).

FISH labeling supported previous conclusions that Pitx2, Vglut2, Htr2c, Kcnab3, Nxph1, Nxph4 mRNAs are ubiquitously distributed across the STN, so called pan-STN markers, and that Pvalb and Col24a1 are locally distributed in the STN in a gradient manner (Pvalb: dorsal-to-ventral; Col24a1: ventral-to-dorsal), confirming the molecular distinction of the STN into three major domains (STN^a Pvalb^-/Col24a1⁺; STN^b Pvalb⁺/Col24a1⁺; STN^c Pvalb⁺/Col24a1^-) (57). Further, Tac1 mRNA was confirmed as a selective pSTN marker, Baiap3 as present in pSTN and adjacent hypothalamus but not STN, and Calb2 as present in ventral STN but primarily in pSTN and adjacent hypothalamus (57) (Figure 1a-d).

By now comparing multiple combinations of these markers by co-FISH in serial sections throughout the entire area, a striking difference in the overall shape of both STN and pSTN was observed across the AP axis, not recapitulated in published mouse atlases (71) (Figure 1b-d). In serial section FISH analysis at section levels (S) S1-S4, referring to bregma (mm) -1.91 (S1), -2.15 (S2), -2.27 (S3), -2.45 (S4), the anatomical appearance of the STN and pSTN was different in each of the four selected section levels. Thus, while preserving their relative distribution throughout the STN structure, the molecularly defined internal STN domains occupied different amount of space across S1-S4 (Figure 1c,d, with illustrations summarizing the results to the right). At S1 (anterior), only the molecular profile of STN^a was seen; at S2, the STN shape was the classical almond shape commonly used to visualize the STN in literature (STN^a, STN^b, STN^c); at S3, the STN was a narrow slice but still positive for markers allowing the subdivision into the three domains; at S4 (posterior), again, only the molecular profile of STN^a was seen (Figure 1c,d).

Also the pSTN showed an unexpected spatial distribution. Tac1 mRNA compared with Calb2 and Baiap3 as well as the STN markers clearly showed that the pSTN covers substantially larger space than previously described (Figure 1c,d). In fact, the roundish shape at the medial tip of the STN most commonly used in publications to denote pSTN (Figure 1b) was present at S2 only, while anteriorly (S1), no pSTN was detected, and posteriorly (S3, S4), the shape and spatial relationship STN vs. pSTN was dramatically different. At S3, the pSTN was observed as a narrow but elongated structure, which was aligned with the entire dorsal aspect of the STN (aligning STN^a, STN^b, STN^c). Thus, at S3, analysis of several markers clearly showed that the cellular densities of STN and pSTN together form a shape which is very similar to the characteristic STN shape observed at S2 (and illustrated in most atlases, (71)) (Figure 1c,d). Finally, at S4, the most posterior position of the STN (STN^a), the pSTN is at its largest and wraps around the STN, with an outline that resembles the almond-like STN structure at S2 (Figure 1c,d). Further, not only STN but also pSTN showed molecularly heterogeneity; rare Pitx2-positive cells were observed in pSTN which all co-localized with Vglut2, but not all Vglut2-positive pSTN cells were positive for Pitx2; further, Tac1 and Calb2 mRNAs did not show complete overlap, suggesting they represent different pSTN subpopulations (Figure 1a-d).

These molecularly enhanced anatomical details were firmly confirmed using 3D light-sheet imaging of the intact mouse brain, applying fluorescent probes directed at Pitx2 and Tac1 to visualize the spatial distribution of STN and pSTN, respectively. In coronal fly-through (set at slower speed at the level of STN and pSTN to clearly outline the spatial distribution of the markers), Pitx2(Supplementary Video 1), Tac1 (Supplementary Video 2), and Pitx2/Tac1 combined (Supplementary Video 3) allowed the entire STN/pSTN complex be spatially revealed within the brain, outlining the structural relationship between STN and pSTN across all axes.

In conclusion, mRNA markers enabled the visualization of anatomical features that have not previously been described for the mouse brain, such as a parallel alignment of STN and pSTN as pSTN gradually increases in size relative to STN posteriorly.

Ventromedial STN defined by a distinct Serotonin receptor 2c profile in macaque

The macaque brain was subsequently analyzed. First, the question of neurotransmitter identity was addressed. The STN is well known as primarily excitatory in nature (72–76), however, the presence of inhibitory neurons and glia cells has remained less explored. By co-FISH analysis, the vast majority of STN cells were identified as positive for the glutamatergic marker Vglut2 in both mouse and macaque, while Vglut1 and Vglut3 were not detected, and only rare cells were positive for inhibitory markers Gad1, Gad2, and, Viaat mRNAs. Similar to mouse STN, the macaque STN (mRNAs in capital lettering) was also positive for PITX2, overlapping completely with VGLUT2. In contrast to rare inhibitory markers, almost all STN neurons in mouse and macaque were positive for Gat1 mRNA, a marker for cells with capacity for transport of extracellular GABA (77). Finally, glial markers Eaat1/Glast1 or Cx3cr1 mRNAs were detected in only few cells mouse and macaque STN (Supplementary Figure 1). Thus, the strong glutamatergic phenotype of the STN was verified in both mouse and monkey, with indication of sparse presence of inhibitory neurons and glia cells. Further, PITX2, the primate counterpart of Pitx2, a well-described STN marker in mice (61,62,67), was identified as marker for VGLUT2⁺ neurons throughout the STN also in the macaque.

Next, co-FISH was performed, analyzing the differentially expressed genes originating from snRNASeq in mice (57). Serial section across the entire STN area in the macaque brain identified NXPH4, NXPH1, KCNAB3 mRNAs as pan-STN markers, present across the STN in a similar manner to PITX2, and also in this species, co-localizing 100% with VGLUT2 (Figure 2a,b). The pSTN and several adjacent structures were positive for BAIAP3, a marker excluded from the STN in both species (Figure 2a,b). The pSTN in macaque was also positive for TAC1, as recently described (28); further analyzed below.

Apart from these similarities between mouse and macaque, one striking difference was detected. COL24A1 mRNA, in the mouse a distinct marker for the ventromedial STN (shown in Figure 1), was present throughout the whole STN structure in the macaque in a ubiquitous manner, overlapping with VGLUT2 (Figure 2c). In contrast, HTR2C mRNA, encoding the serotonin receptor subtype 2c protein (5-HTR2c), which in the mouse was ubiquitously present throughout the STN, was restricted to the ventromedial aspect of the STN in the macaque (Figure 2c). Thus, the ventromedial STN is defined by Col24a1 mRNA in mouse, but by HTR2C mRNA in macaque; these two mRNA showing differential localization in the STN of mouse vs. macaque (Figure 2b-d).

Serotonin receptor 2c mRNA defines a ventromedial domain in macaque STN particularly prominent in its anterior aspect

The striking pattern of Htr2C mRNA in the ventromedial STN of the macaque was of particular interest to address further, not least from the perspective of the importance of the serotonergic system in pharmacology-based medical treatments of neuropsychiatric disorders, including OCD and depression. Indeed, serotonergic innervation of the STN structure has been shown in several studies (19,40,41,78–80), including a recent study in which we identified serotonergic innervation selectively in the anteromedial STN (60).

In the primate (monkey and human) and rat, the parvalbumin protein (PV) has been shown in several studies as a marker of dorsal and lateral STN (77,81,82), and we recently described a similar distribution of its corresponding mRNA (PVALB) in macaque (28). To now investigate if PVALB and HTR2C mRNAs form overlapping or discrete profiles throughout the STN, the distribution pattern of Htr2C mRNA was next compared to PVALB in the monkey (Figure 3).

At section levels S1-S4 (bregma (mm) -10.35 (S1), -11.70 (S2), -12.60 (S3), -13.50 (S4), with S1 representing the anterior STN), PVALB and HTR2C mRNAs showed both overlapping and distinct distribution, depending on section level (Figure 3a,b). PVALB mRNA was the most dominant of the two mRNAs but both showed regional distribution across the STN. Anteriorly (S1), only a small medial STN area was positive for PVALB, while posteriorly (S2-S4), the PVALB⁺ area covered the majority (but not all) of the STN. In contrast, HTR2C mRNA was most prominent anteriorly (S1). HTR2C covered more than half of the STN at S1 and was most prominent in the ventromedial STN. HTR2C decreased posteriorly, but was always found located in a ventromedial strip of the STN (S2-S4), positive in the medial tip throughout the STN (Figure 3a,b). HTR2C/PVALB overlap was observed in ventral STN but never in the medial tip, due to the fact that PVALB mRNA was consistently excluded from this STN area in which HTR2C was exclusive at all levels (Figure 3a). PVALB was more dominant posteriorly, while HTR2C dominated anteriorly (Figure 3a,b). Thus, apart from limited overlap, PVALB and HTR2C were mutually exclusive in the STN, and both mRNAs showed rather sharp borders (Figure 3a-c, close-ups of borders).

The intensity level of the VGLUT2 FISH signal in the macaque brain was higher medially and ventromedially than laterally (Figure 3e).However, both PVALB and HTR2C overlapped extensively with VGLUT2 mRNA, within their regional distribution areas (Figure 3e). Using VGLUT2 as pan-STN glutamatergic marker, quantification of cells positive for PVALB vs. VGLUT2, or HTR2C vs. VGLUT2, and direct comparison PVALB vs. HTR2C confirmed the observation by visual inspection above of more abundant HTR2C than PVALB labeling anteriorly and ventromedially, and vice versa posteriorly and laterally. In a total STN count (not taking precise position into account), PVALB⁺/HTR2C^- was the dominating combination of STN neurons (77%), with similar amounts of cells positive for both PVALB and HTR2C, or selective for HTR2C (PVALB⁺/HTR2C⁺, 13%; and PVALB^-/HTR2C⁺, 10%) (Figure 3d). Next, to assess if these patterns are reproduced in the human STN, biopsy sections were addressed, revealing a similar distribution of PVALB and HTR2C mRNA in human STN as in the macaque STN (Figure 3f,g).

Concluding these findings, along the axes of the macaque and human STN, three domains can be identified, each reflecting the spatial distribution of PVALB and HTR2C: STN^{PVALB+/HTR2C-}, STN^{PVALB+/HTR2C+}, STN^{PVALB-/HTR2C+}. The STN^{PVALB-/HTR2C+} molecular phenotype is most abundant in the ventromedial STN, and includes the medial STN tip while also dominating in the anterior STN (illustrated in Figure 3c,g).

Unique molecular profile identifies a far-anterior STN domain in the primate

While addressing the spatial distribution of HTR2C in macaque STN, positive cells were observed anterior to the area outlined in atlases as the STN. This was curious and called for more careful assessment. Thus, to further investigate macaque brain anatomy, the proximity of the posterior STN to substantia nigra (SN), and of the anterior STN to hypothalamic structures serial sections were analyzed by histological cresyl violet staining (Figure 4a) and FISH for TH, VGLUT2 AND GAD1 (Figure 4b,c). At the AP level of posterior hypothalamus, a cellular density was found in connection with STN, but which does not appear described in primate brain atlases (Figure 4a-c). To analyze if this might be an overlooked STN or pSTN domain, the molecular profile of this density was analyzed using the same battery of STN and pSTN mRNA markers as implemented above.

First, VGLUT2/GAD1 co-localization analysis showed that this anterior density of cells was enriched in both VGLUT2 and GAD1, but not their overlap. More cells were positive for VGLUT2 than GAD11, but quantification revealed a stronger GAD1-presence in this far-anterior cellular density than in the regular STN (35% vs. 1% GAD1-positive cells in far-anterior vs. regular STN; 7% in pSTN) (Figure 4d). This far-anterior aspect, rich in both VGLUT2 and GAD1, was given the working nick-name vgrR (VGLUT2/GAD1-rich Region) (Figure 4b-d).

Next, to determine if molecular profiles could help determine if this far-anterior structure was of STN or pSTN identity, further mRNA analysis was performed (Figure 4e-g). Results showed that vgrR was negative for PVALB, the marker of dorsal STN, and positive for HTR2C, the marker of anterior and medial STN, described above. Further, vgrR was positive for macaque pan-STN markers PITX2, KCNAB3, and NXPH4 (which all co-localised to 100% with VGLUT2), while completely negative for the pSTN marker TAC1, and almost completely negative for CALB2 and BAIAP3 (<5%), the markers most positive in pSTN and posterior hypothalamus (Figure 4e-g).

In summary, apart from GAD1-positive cells, the vgrR area shared its molecular phenotype with that of the anterior STN, including the PVALB^-/HTR2C⁺ profile. However, the combined VGLUT2/GAD1 positive molecular phenotype distinguished vgrR from the remaining STN. This mixed neurotransmitter phenotype might reflect the location far-anteriorly, and based on its molecular profile, vgrR will here be referred to as the “far-anterior extension of the STN” (STN^ext) (illustration in Figure 4h). Notably, STN^ext could not be detected in the mouse, and is thus a primate feature.

Serotonin receptor 2c mRNA allows distinction of pSTN into two domains: pSTN core and pSTN shell

In light of the striking localized pattern of HTR2C mRNA in the ventromedial STN of macaque and human brains, and given the possibility to use this marker to help define the previously undescribed STN^ext area of the macaque brain, the presence and putative distribution of HTR2C mRNA was next explored in the pSTN, revealed above as a more spatious structure than recognized, and to partially align the posterior STN in mice (Figure 1). Similar to the STN, serotonergic innervation of the pSTN has been shown (83,84). Now, it was of interest to detect if pSTN might be selectively positive for HTR2C in macaque, in a manner similar as revealed above for STN (Figure 2,3).

HTR2C was next assessed in the pSTN by comparison with the pSTN marker TAC1, originally described as marker for distinct pSTN neurons in mice (57) and recently identified as selective for a subpopulation of pSTN neurons also in macaque (28). HTR2C and TAC1 mRNAs were analyzed across the STN-pSTN area in mouse (Figure 5a,b) and macaque (Figure 5c,d).

First applying co-FISH analysis across S1-S4 levels in the mouse, and paying attention to the structural differences of STN and pSTN across the AP axis described in Figure 1, Htrc2 mRNA was confirmed as strongly positive across STN while pSTN was selectively positive for Tac1 (Figure 5a,b). However, Htrc2 and Tac1 mRNAs were not mutually exclusive. Instead, Htr2c was could be detected also in pSTN, albeit at much lower detection levels than in the STN (Figure 5a,b). Tac1⁺/Htr2c⁺double-positive pSTN cells (with moderate Htr2c labeling compared to STN cells) were found primarily in a domain bordering to the STN, while Tac1⁺/Htr2c^-cells (low or zero Htr2c labeling) were identified more laterally. This difference in Htr2c between medial and lateral pSTN allowed the subdivision of pSTN into a core (Co) and shell (Sh) area: pSTN^Co(pSTN^Co;Tac1⁺/Htr2c^medium) closest to the STN, and pSTN^Sh (pSTN^Sh; Tac1⁺/Htr2c^weak), lining the pSTN core laterally (Figure 5a,b). In contrast to the sharp borders of Tac1 and Htr2c, Calb2 was detected throughout pSTN (Figure 5b).

Across the AP axis in the macaque, and by using VGLUT2 as reference, TAC1 mRNA was identified in pSTN at section levels S1 and S2, but not S3 and S4; macaque pSTN thereby lining the STN anteriorly, but not posteriorly. At S1, TAC1 cells were observed close to the anterior aspect of the STN (medial STN tip and STN^ext), while at S2, TAC1-positive cells covered a substantially larger area lining the dorsal STN (Figure 5c). Analysis of HTR2C and TAC1 thus demonstrated that the macaque pSTN covers a major area directly associated with anterior STN. Further, HTR2c allowed the subdivision of pSTN into a core and shell area in similar manner as for mouse, and many pSTN cells positive for Calb2 mRNA also in macaque (Figure 5d).

Concluding, based on mRNA profiles of TAC1 and HTR2C, the pSTN is anatomically associated with anterior STN in the primate, and with posterior STN in the mouse. In both species, the differential levels of serotonin receptor 2c mRNA allows the subdivision of pSTN into a core and shell area (summarized in Figure 5e).

pSTN, but not STN, cells embedded in dopaminergic nerve bundles of MFB in both mouse and macaque.

At its dorsal aspect, the STN borders to the monoaminergic projections of the MBF. With the present observation that pSTN aligns with the dorsal STN, it was of interest to address if pSTN and MFB co-localize above the STN. The MFB, which is a projection bundle, has been shown to contain certain cell groups of various identity, including hypothalamic cells (20,21), however, pSTN cells have not previously been associated with the MFB.

To now address if these three aligning brain structures (STN, pSTN, MFB) were overlapping such that STN or pSTN cells are embedded in the MFB, molecular profiles were addressed in co-localization analysis on serial macaque sections using TH immunoreactivity as a marker for the dopaminergic identity of the MFB. Indeed, TH-positive projections within the MFB align with both the STN and pSTN, including the far-anterior STN^ext (Figure 6a). TH-positive fibers contained ball-like globular structures, positive for TH, and DAPI staining showed presence of cell nuclei in the MFB (Figure 6b,c). In combined immunofluorescent/FISH analysis addressing TH protein and either VGLUT2 or TAC1 mRNAs (Figure 6b, TH/VGLUT2; Figure 6c, TH/TAC1), MFB (TH⁺) was seen to directly border the dorsal aspect of the STN and pSTN. Further, VGLUT2⁺/TAC1⁺cells were discovered within the TH-positive fiber bundles (Figure 6b,c; illustration in Figure 6d).

To further assess the observation of TAC1⁺neurons embedded within the MFB, and exclude that this cellular feature was a tissue-processing anomaly, cellular tracing using viral-genetics methodology in recombinase-expressing mice was next implemented. In contrast to the monkey, no globular ball-like TH-positive structures were observed in the mouse MFB (Figure 6e,f). However, upon injection of adeno-associated virus (AAV) containing a double-floxed reporter gene construct encoding the Yellow fluorescent protein (YFP) into the STN and pSTN of Pitx2-Cre and Tac1-Cre transgenic mice, and comparison of YFP-positive cells with TH immunofluorescence, YFP-positive cells were amply detected within the mouse MFB. Importantly, this was only observed in the Tac1-Cre mouse line, reflecting pSTN neurons, and not in the Pitx2-Cre mouse line, showing STN neurons. Thus, pSTN, but not STN, neurons are embedded in the MFB in both mouse and primate (Figure 6e,f; Supplementary Figures 2,3).

Finally, using light sheet-microscopy for whole mouse brain 3D imaging analyzing Pitx2 (STN) combined with TH immunofluorescence (Supplementary Video 5) and Tac1 (pSTN) combined with TH immunofluorescence (Supplementary Video 6), coronal flythrough allowed the visualization the of STN and pSTN distribution along the TH-positive MFB, and, while weakly detected, also validated the presence of Tac1⁺, but not Pitx2⁺, cells within the MFB at the dorsal aspect of STN.

High-resolution molecular profiling of fluorescently labeled probes selective for differentially expressed gene products within heterogeneous brain structures offers the advantage of visualizing anatomy independently of preconceptions of anatomical boundaries, by instead bringing unbiased focus to the spatial distribution of mRNA patterns. Several studies have addressed gene expression patterns within the STN (reviewed in (58)), and here we focused primarily on those identified in snRNASeq in mice (57). Comparative analysis of mouse, macaque and human brains now reveals several features of the primate STN, not replicated in the mouse. Of particular importance to human health and neuropsychiatry is the identification of HTR2C mRNA selectively in the anterior and ventromedial STN of the primate, including the anteromedial STN which is an important target area for DBS in OCD. This primate-unique profile of HTR2C mRNA is highly interesting, both from the experimental setting, by challenging the application of the mouse brain as model for the human brain, and from the medical setting, considering the great importance of the serotonergic system in mood and mental disorder. Thus, with the presented identification of HTR2C mRNA selectively in the anterior and ventromedial STN of the human and macaque brains, two current treatment strategies for OCD, namely SSRI and DBS, may converge at the anteromedial aspect of the STN.

The clinical relevance of serotonergic neurotransmission in the STN is immediately evident from the pharmacological perspective, considering the prescription of SSRIs as primary mediciation for both OCD and depression (15,16,85); with SSRIs even described as the only effective substances for the treatment of OCD (86,87). The correlation between the serotonergic system and the STN has been amply studied and reported (88,89). However, the highly localized HTR2C mRNA distribution pattern in the anterior and ventromedial STN in the macaque and human brain, identified in this current study, has not been previously recognized. Based on our original finding in mouse transcriptomics of Htr2c as a pan-STN marker, the present identification of highly selective HTR2C mRNA in macaque and human STN was therefore unexpected. Considering our small cohorts (tissue blocks from two monkey brains and one human), we consulted the recent data presented in the human brain in situ hybridization (ISH) database in Allen Brain Map (https://human.brain-map.org) for validation. Indeed, despite low detection levels in this material, similar results were available for at least two patients ((125560979 experiment (160039351);126848758 experiment (160507038)). No other serotonin receptor subtype (from 5-Htr1 to 5-Htr7 subtypes) shows a strong ISH-positive signal in either mouse or human, according to our assessment of Allen Brain Map databases. However, protein studies have identified the most abundant 5-HTR subtypes as 1A, 1B, 2C, and 4 (reviewed in (88)).

An indirect, but functionally important, confirmation of the current HTR2C data is the long-standing demonstration of serotonergic innervation of the ventromedial STN. Indeed, when consulting early mapping papers using antibodies to serotonin itself, it was evident that a similar pattern as observed in our study with HTR2C mRNA was found in the STN or macaque in the 1980’s (79). In addition, it was demonstrated that selective serotonergic innervation in the ventromedial STN strip was unique to macaque, not present in rat or cat. These findings were subsequently verified in several studies, albeit with some discrepancy regarding the actual location of the densest innervation in the STN (40,41,78–80). Considering previous species selective patterns of serotonergic fibers innervating the STN, our findings of similar HTR2C mRNA distribution, it seems evident that the anterior and ventromedial STN is regulated differently in the primate compared to the STN in mouse, rat, and cat. Further, in a collaborative study addressing the STN in human and macaque, sharing the same monkey brains as used in the current study, we recently identified a distinct serotonergic innervation pattern in the anteromedial STN (60). In conclusion, in the primate, serotonergic projections selectively reach the anterior and ventromedial STN, where neurons evidently express the gene encoding 5-HT2CR. Thus, the ventromedial STN, and specifically, the anteromedial aspect, is clearly a primate-unique hotspot for serotonergic regulation.

HTR2C mRNA was less intense immediately outside the STN, but its distribution in the adjacent pSTN allowed us to subdivide this far less studied brain area into two substructures, the core (closest to the STN) and the shell. This subdivision of pSTN was evident in both mouse and primate, tentatively suggesting that the pSTN, which is of hypothalamic identity, is evolutionary more conserved across species than the STN. However, similar to the STN, the pSTN has been shown to receive serotonergic innervation (83,84); it would be interesting to address how this distributes in the newly described domains, pSTN^Co and pSTN^Sh.

The pSTN has been functionally characterized mainly using experimental rodents, while it remains an understudied region in humans and non-human primates. Results in rodents have implicated the pSTN in a wide range of functions including feeding, aversion, and novelty response, in addition to its most well-known role in the regulation of autonomic and interoceptive functions (22,59). Here, we find that pSTN neurons are embedded within the monoaminergic MFB at its dorsal border towards the STN. The MFB, anatomically described in (18–21,90), is a key structure of the reward-seeking circuit mediating experience of pleasure and motivated behavior (91,92). The superolateral MFB branch, which passes the STN and internal capsule, has been clinically tested as a new DBS target in the treatment of severe depression (12,93,94), and more recently also OCD (11,13). Finding that MFB is intermixed with Tac1⁺ pSTN neuronal cell bodies might be clinically urgent considering the functional roles ascribed to this nucleus (22), and which present themselves in the DBS context as either positively or negatively contributing to treatment outcome. While the presence of pSTN (Tac1⁺) neurons in the MFB was present in both mouse and primate, no STN (Pitx2⁺) cells were apparent in the MFB; instead the Pitx2-positive area formed a rather distinct border to the MFB.

While the current study is histological, the spatial precision of promoter activities in primates may guide towards a new level in the understanding of the anatomical-functional organization of the primate STN that could be applicable therapeutically. For example, already today, anteromedial STN is a main DBS target area in OCD (while for example dorsolateral STN is the preferred DBS target in Parkinson´s disease). In the context of precision-targeting within the STN structure, future studies could aim to superimpose the kind of histological data presented here, in which we outline distinct molecular profiles within the STN structure, with clinically evaluated optimal target site, the sweet spot, for alleviation of disabling symptoms (55,95–99). Strictly hypothetically, the “sweetest spot” for DBS in OCD might be in the anterior STN^{PVALB-/HTR2C+} STN domain presented here, and/or affecting the newly identified STN^ext, or perhaps even in the vicinity of the pSTN, shown here to occupy considerable space directly at the border to anterior STN. Further, one might speculate that efficient symptom alleviation, or lack of it, may be a direct consequence of the position of the stimulation lead to such a level of precision that molecularly distinct STN subtypes are implicated in the outcome.

Concluding, we uncovered remarkable gene expression patterns in mouse, macaque and human brains that may open up new opportunities in understanding the STN and adjacent structures in mental health and its disorder. A limitation to this descriptive study is that any functional correlations to the observed mRNA patterns remain to be tested. However, we reason that already the observed proximity between STN, pSTN and MFB, their striking molecular heterogeneity and neuropharmacological properties, may be important to consider when aiming to clinically target this brain area, either pharmacologically or by invasive surgey for neuromodulation. Therefore, updating current anatomical atlases with transcriptome-informed data should be of particular interest. To this end, we provide such molecularly enhanced anatomical maps covering STN, pSTN and MFB, and compare them with current brain atlases (Supplementary Figure 4). Any manipulation at the thalamus-hypothalamus-midbrain intersection warrants caution, but better molecular and anatomical understanding of the STN and its neighbours pSTN and MFB holds promise towards new treatment opportunities in neuropsychiatry.

Acknowledgments: We thank Candice Contet, The Scripps Research Institute (La Jolla, CA) for constructive feedback on the manuscript, Sabrina Leclerc-Turband, Manager at Biobanque Neuro-CEB APHP, for human brain tissue biopsy samples, and Nermine Saidi from Institut de la Vision for slide scanning. This work was supported by Uppsala University and research funding grants from the Swedish Research Council (Vetenskapsrådet), Swedish Brain Foundation (Hjärnfonden), Swedish Parkinson Foundation (Parkinsonfonden), Åhlénstiftelsen, and Hållsten Research Foundation (to Å.W-M).

Competing Interests: S.D is the owner of Oramacell, France. M.B.R is employed at Gubra, Denmark. All other authors declare no competing financial and non-financial interest.

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Sylvie Dumas is the owner of Oramacell. All other authors declar no conflict of interest.

SupplementaryFigure1Dumasetal.jpg
Supplementary Figure 1 Both mouse and macaque subthalamic nucleus (STN) show primarily a glutamatergic neurotransmitter phenotype detected by Vglut2 (but not Vglut1 and Vglut3), with sparse detection of inhibitory neurons or glia cells.Fluorescent mRNA-directed probes detecting distribution of the subthalamic nucleus (STN) in mouse vs. macaque brain. mRNAs detected (in the order presented): (a,b) Vglut2 (overview, mouse (a), macaque (b); (c, d) Vglut2/Vglut1, Vglut2/Vglut3 in STN of mouse (c) and macaque (d) with closeups to right in each panel, and additional areas in bottom panel to visualize cells positive for Vglut1 and Vglut3, respectively, as positive controls (as these markers are negative in STN); (e,f) Pitx2, Gad1, Viaat/Gad2, Gat1/Vglut2, Gat1/Glast, Gat1/Cx3cr1 in mouse STN (e); PITX2/VGLUT2, GAD1/VGLUT2, GAT1/VGLUT2 in macaque STN (f). Blue signal, DAPI nuclear dye. Full mRNA names in main text. Mouse and monkey brain atlas references: (25,71). Abbreviations: Thal, thalamus; DLG, dorso lateral geniculate; Hip, hippocampus; Cd, caudate nucleus; Cx, cerebral cortex.
SupplementaryFigure2Dumasetal.jpg
Supplementary Figure 2. Pitx2-Cre mice transduced with AAV2-eYFP visualizing YFP-positive cells in the subthalamic nucleus (STN), and TH-positive dopaminergic fibers of the medial forebrain bundle (MFB) throughout the extent of the STN. CPu, caudate putamen; EP, entopeduncular nucleus; SNR, substantia nigra pars reticulata, SNC, substantia nigra pars compacta; VTA, ventral tegmental area.
SupplementaryFigure3Dumasetal.jpg
Supplementary Figure 3. Tac1-Cre mice transduced with AAV2-eYFP visualizing YFP-positive cells in the para-subthalamic nucleus (pSTN), and TH-positive dopaminergic fibers of the medial forebrain bundle (MFB) throughout the extent of the subthalamic nucleus (STN) and pSTN, followed by Tac1-Cre/Sun mice detecting nuclear envelope of Tac1-Cre positive cells. CPu, caudate putamen; EP, entopeduncular nucleus; SNR, substantia nigra pars reticulata, SNC, substantia nigra pars compacta; VTA, ventral tegmental area.
SupplementaryFigure4Dumasetal.jpg
Supplementary Figure 4. Schematic illustrations comparing current brain atlases of the mouse (a) and macaque (c) brains at the levels of subthalamic nucleus (STN) and para-subthalamic nucleus (pSTN) as indicated by bregma levels; mRNA patterns representing STN and pSTN including internal domains in each of these structures (STN^a, STN^b, STN^c, pSTN^Co, pSTN^Sh) as presented throughout this study, allowing the identification of transcriptome-enhanced details in anatomical maps in mouse (b) and macaque (d). Full mRNA names in main text. Mouse and monkey brain atlas references: (25,71).
SupplementaryTable1DumasetalSubmit.xlsx
Supplementary Table 1. NM sequence and primer sequence for construction of fluorescent in situ hybridization (FISH) probes selective for mouse, macaque, and human mRNA.
SupplementaryVideo1DumasetalPitx2rawflythrough.mp4
Supplementary Video 1. Fluorescent light-sheet microscopy imaging of whole mouse brain in 3D visualizing the detection and spatial distribution of Pitx2 mRNA. Raw data coronal fly-though. Abbreviations: STN, subthalamic nucleus; pSTN, para-subthalamic nucleus.
SupplementaryVideo2DumasetalTac1rawflythrough.mp4
Supplementary Video 2. Fluorescent light-sheet microscopy imaging of whole mouse brain in 3D visualizing the detection and spatial distribution of Tac1 mRNA. Raw data coronal fly-though. Abbreviations: STN, subthalamic nucleus; pSTN, para-subthalamic nucleus.
SupplementaryVideo3DumasetalTac1Pitx2rawflythrough.mp4
Supplementary Video 3. Fluorescent light-sheet microscopy imaging of whole mouse brain in 3D visualizing the detection and spatial distribution of Pitx2 and Tac1 mRNAs. Raw data coronal fly-though. Abbreviations: STN, subthalamic nucleus; pSTN, para-subthalamic nucleus.
SupplementaryVideo4DumasetalPitx2THmappedflythrough.mp4
Supplementary Video 4. Fluorescent light-sheet microscopy imaging of whole mouse brain in 3D visualizing the detection and spatial distribution of Pitx2 mRNA, mapped to TH immunofluorescence for visualization of dopaminergic cell bodies and projections, including the medial forebrain bundle (MFB). Coronal fly-though. Abbreviations: STN, subthalamic nucleus; pSTN, para-subthalamic nucleus, TH, Tyrosine hydroxylase.
SupplementaryViedo5DumasetalTac1THmappedflythrough.mp4
Supplementary Video 5. Fluorescent light-sheet microscopy imaging of whole mouse brain in 3D visualizing the detection and spatial distribution of Tac1 mRNA, mapped to TH immunofluorescence for visualization of dopaminergic cell bodies and projections, including the medial forebrain bundle (MFB). Coronal fly-though. Abbreviations: STN, subthalamic nucleus; pSTN, para-subthalamic nucleus, TH, Tyrosine hydroxylase.

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Cross-species transcriptome analysis of the subthalamic and para-subthalamic nuclei reveal mRNA patterns unique to primate and relevant to neuropsychiatry

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