Spatio-molecular analysis of primate subthalamus defines anatomical domains relevant to Parkinson’s disease and neuropsychiatry

Para-subthalamic nucleus adjoins subthalamic nucleus and medial forebrain bundle, major DBS-targets in Parkinson’s disease, OCD, and depression | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Para-subthalamic nucleus adjoins subthalamic nucleus and medial forebrain bundle, major DBS-targets in Parkinson’s disease, OCD, and depression View ORCID Profile Sylvie Dumas , View ORCID Profile Chantal Francois , View ORCID Profile Marie Biviano Rosenkilde , View ORCID Profile Urmas Roostalu , View ORCID Profile Carine Karachi , Åsa Wallén-Mackenzie doi: https://doi.org/10.1101/2025.05.21.655268 Sylvie Dumas 1 Oramacell, Paris , 75006 Paris, France PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sylvie Dumas Chantal Francois 2 Sorbonne Université, Institut de Cerveau, Paris Brain Institute , ICM, Inserm, CNRS, AP-HP, Hôpital de le Pitié Salpêtrière, 75013 Paris, France PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Chantal Francois Marie Biviano Rosenkilde 3 Gubra, Hørsholm Kongevej 11B , 2970 Hørsholm, Denmark MSc Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Marie Biviano Rosenkilde Urmas Roostalu 3 Gubra, Hørsholm Kongevej 11B , 2970 Hørsholm, Denmark PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Urmas Roostalu Carine Karachi 2 Sorbonne Université, Institut de Cerveau, Paris Brain Institute , ICM, Inserm, CNRS, AP-HP, Hôpital de le Pitié Salpêtrière, 75013 Paris, France PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Carine Karachi Åsa Wallén-Mackenzie 4 Lund University, Department of Experimental Medical Science , Molecular Neuroanatomy Lab, Sölvegatan 19, 223 62 Lund, Sweden 5 Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network , Chevy Chase, MD 20815 PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: asa.mackenzie{at}med.lu.se Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract The subthalamic nucleus (STN) is a deep brain stimulation (DBS) treatment target for several disorders of vastly different symptoms, including Parkinson’s disease (PD) and obsessive compulsive disorder (OCD). The STN is located in close proximity to the superolateral branch of the medial forebrain bundle (slMFB), a recent DBS target for treatment-resistant depression. Despite clinical application, spatio-molecular understanding of the STN and slMFB has remained elusive. To help solving this, transcriptome data generated in mice was here implemented in comparative analysis with macaque and human. Fluorescent in situ hybridization (FISH) analysis was performed on multiple serial brain sections covering the whole STN area in mice and macaque. Selected markers were further analysed in human sections and in whole mouse brains to allow spatial 3D analysis by light-sheet microscopy. Contrary to available brain atlases, we can now show that the hypothalamic structure known as the para-STN, a critical hub for interoception and emotional regulation, aligns with the STN, and is partly embedded within the MFB. Thus, para-STN occupies considerable space around several major DBS sites. Spatio-molecular data expose STN, MFB and para-STN as highly heterogeneous, including gradients of serotonin receptor subtype 2C (HTR2C). Molecularly reinforced anatomical maps of STN, para-STN, and MFB reveal unexpected details, and may inform spatial precision in neuromodulation strategies while also providing openings for molecular drug discovery. Introduction The subthalamic nucleus (STN), an excitatory brain structure 1 – 3 , has attracted attention in the Parkinson’s disease (PD) community for a long time due to its aberrant firing pattern 4 – 6 , a core pathophysiological symptom of the disease, and due to its susceptibility to modulation via deep brain stimulation (DBS) which successfully treats cardinal PD motor symptoms 7 – 11 . The STN is also a DBS target for treatment of obsessive-compulsive disorder (OCD), Tourette syndrome (TS), and dystonia (DYT) 12 – 22 . Based on recent mapping analyses of frontal circuits upon STN-DBS in each of these disorders, clinicians and scientists have forwarded the idea of the STN as a network node in which these divergent dysfunctions converge 13 , 23 . Optimal stimulation sites, so called sweet spots 24 – 26 , for PD, TS, and DYT are located in distinct positions within the dorsal (dorsolateral, dl) territory of the STN, often referred to as the sensorimotor STN territory. In anatomical contrast, the optimal stimulation site for OCD has been defined as the anteromedial or ventromedial (vm) STN, often denoted as the limbic or associative STN 13 , 25 . Ventral and dorsal STN have long been ascribed distinct roles 27 . Based on anatomy and connectivity, as well as experimental and clinical results of lesioning and DBS, the primate STN has also been described as tripartite structure in which limbic, associative and motor functions indeed are anatomically segregated 1 , 2 , 4 , 20 , 22 , 28 – 36 . However, this tripartite model has been questioned and debated 28 , 37 – 39 and the internal organization of the STN remains to fully understand. Further, the success rate for STN-DBS varies between disorders 13 , 23 , 26 , 40 , and is far lower for OCD than PD 20 , arguing for a need for better understanding of neuroanatomical underpinnings of discrete target areas within the STN. In addition to the long-standing implementation of the STN as a major DBS site in PD, the superolateral branch of the medial forebrain bundle (slMFB) has emerged as a promising white-matter DBS target for the treatment of treatment-resistant depression (TRD) and OCD 41 – 45 . TRD and OCD are two psychiatric disorders for which selective serotonin reuptake inhibitors (SSRIs) are first choice pharmacological treatment, albeit at different application regimes 46 . The MFB is a diffuse and loosely textured mixed monoaminergic (primarily serotonergic and dopaminergic) and glutamatergic fiber bundle of ascending and descending axons connecting the forebrain, brainstem and limbic system. The slMFB conceptually comprises an assembly of glutamatergic pathways descending from the prefrontal cortex and projecting onto dopamine pathways originating in the ventral tegmental area (VTA) 42 , 43 , 45 , 47 , thus forming an important part of the brain reward system 48 . Descending axons of the MFB pass the medial aspect of the STN, occupying the space that separates the STN from zona incerta on their way toward the prefrontal and orbital cortices. The anatomical position for slMFB-DBS in TRD is located between the red nucleus, substantia nigra, mammillothalamic tract, and the STN; in close apposition to anteromedial STN 42 , 44 , 45 , 49 . Several studies over the past years have shown that slMFB-DBS results in rapid and long-lasting anti-depressant effects in TRD42- 45 , 49 . Notably, depression is a main psychiatric symptom of PD alongside anxiety and apathy 50 – 54 , psychiatric conditions for which emerging evidence support the application of DBS within or nearby the STN as treatment option 52 , 54 . Further, dopamine-based medication stand at risk of causing compulsions in PD as part of impulse control disorders (ICDs) 55 . Psychiatric and cognitive symptoms of PD severly affect the quality of life for PD patients 52 , 56 . For these reasons, slMFB-DBS might be relevant as treatment of consideration for reward-related dysfunction in PD. Further complexity of the brain area surrounding the STN and slMFB area is given by the para-STN (pSTN), described as part of the posterior hypothalamus and of importance to autonomic and interoceptive functions 57 – 62 . In current brain atlases of both the macaque and mouse, pSTN is outlined as a small drop-like structure medial to the ventromedial tip of the STN 63 , 64 . No distinct anatomical border or landmark separates the STN from pSTN, and the pSTN is even omitted in some anatomical literature 62 , 63 , 65 – 67 . In light of recent advancements in therapeutical applications of the STN and MFB as containing several important DBS targets, and based on recent single nuclei RNA-sequencing (snRNASeq) data through which we described spatio-molecular profiles within the STN and pSTN in mice 68 , we here set out to explore if differential gene expression profiles in mice translate across species and thereby enables the identification of molecular profiles also in the primate. Applying fluorescent in situ hybridization (FISH) across mouse and macaque monkey brain sections, with selected data validated in human brain sections, the results visualize the spatial relationship between, and the internal organization of, the STN, pSTN and MFB; FISH data which is summarized in molecularly reinforced anatomical brain maps. Materials and Methods Availability Statement Key lab materials used in this study are listed in a Key Resource Table (KRT) alongside their persistent identifiers and all data: https://zenodo.org/records/20007922 Mice All experimental procedures using mice followed Swedish Legislation (Animal Welfare Act SFS 1998:56) and European Union Legislation (Convention ETS 123 and Directive 2010/63/EU). 16 wildtype mice (C5BL/6NTac from Taconic) were used for this study; 8 male and 8 female mice, all at the age of postnatal day 28 (P28). Brains were quickly dissected from mice euthanized by cervical dislocation, snap-frozen in cold (−30°C to - 35°C) 2-methylbutane (R99%, Honeywell) and stored at - 80°C until usage. Coronal serial sections were prepared on a Leica CM1950 Cryostat at 16 μm thickness and placed onto Superfrost glass slides in series of 8 slides (Thermo Fisher). The whole STN bregma (mm) −1.70 to −2.45 was sectioned in each mouse, in series of 8 slides. Each slide contained 6 sections, thus in total 48 sections per mouse. Each section contained both left and right STN; thus n=96 STN per mouse, and in total (16 mice), n=1536 STN sections were analysed. The MFB above the STN and the whole pSTN are included within these sections. Section (S) S1-S4, referring to bregma (mm) −1.91 (S1), −2.15 (S2), −2.27 (S3), −2.45 (S4) were selected for illustrations. Macaque and human brain samples Two types of primate brain samples were analyzed: 1) Non-human primate brain (Macaca mulatta, also known as rhesus macaque monkey, a non-human primate with high similarity in brain anatomy to the human brain, and 2) human brain biopses. Animal care was carried out in strict accordance with the European Union Directive of 2010 (Council Directive 2010/63/EU) for care and use of laboratory animals. Brain was removed from the skull and divided into several blocks 20 . No animal was sacrificed for the present study, instead only left-over brain material from a previous study 20 was used. One male macaque monkey, age 10 years; and one female macaque monkey, age 17 years. For the present study, blocks containing the STN area was cut on a cryostat at 16 μm. Over 300 sections including the whole STN (bregma – 9.45; bregma – 13.50) were collected, one section per slide. Storage at - 80°C until use. Section thickness 16 μm. n=197 sections from the male macaque and n=90 sections from the female macaque were analysed. The MFB above the STN and the whole pSTN are included within these sections. Section (S) S1-S4, referring to bregma (mm) −10.80 (S1), −11.70 (S2), −12.60 (S3), −13.00 (S4) were selected for illustrations. Human STN sections (n=3) 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 research 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 y, post-mortem interval 24 h. Fluorescent in situ hybridization (FISH) and immunohistochemistry on brain sections Sequence and primer information Complete list in KRT. Full names of genes/mRNAs (with abbreviations in italics in brackets): Paired-like homodomain 2 ( Pitx2 ), Vesicular glutamate transporter 1 ( Slc17a7 / Vglut1 ), Vesicular glutamate transporter 2 ( Slc17a6 / Vglut2 ), Vesicular glutamate transporter 3 ( Slc17a8 / Vglut3 ), 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 ), Calbindin 2 ( Calb2 ), BAI1 Associated Protein 3 ( Baiap3 ), Glutamic acid decarboxylase subtype 1 ( Gad1 ), Glutamic acid decarboxylase subtype 2 ( Gad2 ), Vesicular inhibitory amino acid transporter ( Slc32a1 / Viaat ). Primate gene counterparts and mRNA in capital letters roman and italics: VGLUT1 , VGLUT2 , VGLUT3 , PITX2 , NXPH1 , NXPH4 , KCNAB3 , PVALB , COL24A1 , TAC1 , CALB2 , BAIAP3 , GAD1 , GAD2 , VIAAT . When comparing mouse and macaque in the main text, nomenclature for primate mRNA is primarily used (capital lettering, italics) for ease of reading, while species specific nomenclature is used in all figures. FISH experimental procedure The FISH protocol originally described by Dumas and Wallén-Mackenzie (“sdFISH” 69 ) and described in a step-by-step protocol referenced in the KRT was implemented. Cryo-sections were air-dried and fixed in 4% paraformaldehyde, after which they were acetylated in 0.25% acetic anhydride/100mM triethanolamine (pH 8) and washed in PBS. Sections were hybridized for 18 h at 65°C in 100 μl of hybridization formamide-buffer containing 1 μg/ml digoxigenin (DIG) labeled and 1 μg/ml fluorescein labeled probes for detection of mRNA. Sections were washed at 65°C with SSC buffers (5x and 0.2x), followed by blocking with 20% heat-inactivated fetal bovine serum and 1% blocking solution (Roche #11096176001) in maleic acid buffer. Sections were next incubated with horse radish peroxidase (HRP)-conjugated anti-DIG Fab fragments (Sigma-Aldrich #11207733910) at a dilution of 1:2500 in the same blocking buffer). Signal detection was obtained using Cy3-tyramide at a dilution of 1:100. Fluorescein epitopes were then detected with HRP conjugated anti-fluorescein Fab fragments (Sigma-Aldrich #11426346910) at a dilution of 1:5000 in PBST and revealed using Cy2-tyramide at a dilution of 1:250. Cy2-tyramide and Cy3-tyramide were synthesized as previously described. DAPI was used for nuclear staining. Histology and immunofluorescence on brain sections Selected sections were stained with cresyl violet (0.5%, C0775, Merck Millipore, MA, USA) for histological analysis, or analyzed by immunofluorescence post-FISH according to the step-by-step protocol referenced in the KRT. Tyrosine hydroxylase (TH) immunoreactivity was assessed using anti-TH antibody (MAB318, diluted 1:250, Merck Millipore, MA, USA) in FISH blocking buffer overnight at RT. After washes in maleic buffer, 0.1% MABT and PBS, sections were incubated with biotinylated goat anti-mouse igG antibody (Vector Laboratories #BP-9200) diluted 1:400 in PBS, 1 h at RT. Sections were next washed in PBS and incubated in streptavidin-HRP (Vector Laboratories #SA-5004) in PBS for 1 hr. Revelation of signals was performed using Cy2-tyramide at a dilution of 1:250. Slide scanning and analysis All slides were scanned at 20x magnification on a NanoZoomer 2.0-HT (Hamamatsu). The Ndp2.view software (Hamamatsu) was employed for viewing the images. Published atlases were used for guidance of anatomical structures, primarily references 63 , 64 . The subthalamic area was delineated by the STN laterally, ZI dorsally, ventral tegmental area ventrally, posterior hypothalamus anteriorly, and the wall of the third ventricle medially. Cell countings Cell countings were performed manually using the NDP.view2 and QuPath softwares as referenced in the KRT. Whole mouse brain in situ hybridization and 3D imaging by lightsheet microscopy Whole mouse brain in situ hybridization was performed by GUBRA according to previously published protocols 70 . HCR™ RNA probes targeting mRNA encoded by 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 Vglut2 signals were mapped to the LSFM reference brain atlas 71 together with data from TH whole brain immunolabelling previously generated 72 . Results Mouse and primate STN positive for VGLUT2 and PITX2 with complete overlap The molecular composition of distinct STN neurons in the primate remains to fully uncover 39 . To address this, gene expression patterns at the mRNA level were here compared between mouse and the non-human primate (NHP) macaque monkey, based on our recent molecular data originating in the mouse 68 . Serial coronal sections covering the whole STN from anterior to posterior STN (S1-S4) in mice and macaques were prepared ( Figure 1a ) and structures visualized with cresyl violet staining to ascertain the location of STN and adjacent brain areas ( Figure 1b ). These 4 section levels were selected for illustration ( Figure 1a , b ) and applied throughout the study. A battery of tests by fluorescent in situ hybridization (FISH) were subsequently performed, comparing NHP with mouse. Selected results were validated in human brain sections. Download figure Open in new tab Figure 1. Mouse and primate STN positive for VGLUT2 and PITX2 mRNAs with complete overlap. (a) Section (S) levels applied throughout the study (S1-S4) for mouse and macaque brains covering the STN structure across the anteroposterior (AP) and dorsoventral (DV) axes; anatomical axes indicated. (b) Cresyl violet histological staining throughout S1-S4 (corresponding bregma level indicated) in mouse (top panel) and macaque (bottom panel) brains. (c) Vglut2 FISH in mouse subthalamic nucleus (STN) (S2 level), corresponding VGLUT2 FISH in macaque STN (S3 level), positive cells also detected in thalamus (Thal) and dorsal lateral geniculate (DLG). Insets display anatomical brain atlas images for guidance. (d-e) co-FISH for glutamatergic ( Vglut1 , Vglut2 , Vglut3 ) and GABAergic ( Gad1 , Gad2 , Viaat ) mRNA markers. (f) co-FISH, Pitx2 and Vglut2 mRNAs. Single labeling in red or green, co-localization in yellow, see close-ups for high-resolution detail. Mouse brain atlas reference 64 . Abbreviations. FISH, fluorescent in situ hybridization. Scale bars indicate magnification. The majority of STN cells was confirmed as positive for the glutamatergic marker Vesicular glutamate transporter 2 in both mouse ( Vglut2 ) and macaque ( VGLUT2 ) ( Figure 1c ), identifying a higher intensity of the labeling in STN and thalamus of mouse than of macaque. In contrast to VGLUT2 ( Vglut2 ), VGLUT1 ( Vglut1 ) and VGLUT3 ( Vglut3 ) were not detected in the STN of either species ( Figure 1d ). Further, STN cells were negative (or very rarely positive) for inhibitory markers GAD1 ( Gad1 ), GAD2 ( Gad2 ), and VIAAT ( Viaat ) mRNAs ( Figure 1e ). Similar to original reports of the mouse STN in which the mRNA encoding the homeobox transcription factor Pitx2 73 , 74 has been shown to overlap to 100% with Vglut2 61 , 69 , 75 , 76 , macaque STN was here demonstrated as positive for PITX2 , overlapping 100% with VGLUT2 ( Figure 1f ). Thus, the glutamatergic phenotype of the STN was molecularly verified by VGLUT2 in both mouse and macaque monkey, with indication of sparse presence of inhibitory neurons. STN neurons in mouse and macaque monkey thereby display similar molecular compostion for excitatory and inhibitory neurons. Further, PITX2 , the primate counterpart of Pitx2 mRNA, an established STN molecular marker in mice 60 , 69 , 73 – 75 , was identified as STN marker also in the macaque, in both species completely overlapping with VGLUT2 . Para-STN aligns with posterior STN along its dorsolateral axis in mouse To next assess if molecular profiles (mRNA patterns) described in the mouse STN are useful to molecularly describe the internal organization also of the primate STN, a battery of mRNA markers originating from our original snRNASeq experiment 68 was next applied in comparative analysis between mouse and macaque monkey. Several unexpected observations were made on molecular (mRNA) and anatomical levels in both species, presented step-wise below ( Figures 2 - 7 and Videos 1-5 ) and with findings summarized in molecularly supported anatomical maps of the STN, pSTN, and slMFB ( Figure 8 ). Download figure Open in new tab Figure 2. pSTN aligns with posterior STN along its dorsolateral axis in mouse. Fluorescent mRNA-directed probes detecting mRNA distribution by FISH in the STN and pSTN of the mouse. (a) From left to right: Pitx2 , Vglut2 , Htr2c , Pvalb , Col24a1 , Tac1 , Calb2 , Baiap3 mRNAs across STN and pSTN. (b) Reference mouse brain atlas, section levels S1-S4 with bregma levels indicated. (c) Section levels S1-S4 (anterior to posterior), from left to right, co-FISH: Vglut2 / Pitx2 , Htr2c / Vglut2 , Pvalb / Pitx2 , Col24a1 / Pitx2 , Tac1 / Pitx2 , Calb2 / Pitx2 , Baiap3 / Pitx2 mRNAs; schematic illustration of results in far-right panel with legend at bottom. (d) From left to right, section level S3: Pitx2 , Nxph4 / Htr2c , Nxph1 / Htr2c , Nxph4 / Nxph1 , Col24a1 / Pvalb , Baiap3 / Pitx2 , Baiap3 / Kcnab3 mRNAs. Single labeling in red or green, co-localization in yellow. Full mRNA names and bregma levels, see main text. Mouse brain atlas reference: 64 First, FISH labeling supported previous conclusions that Pitx2 , Vglut2 , Htr2c mRNAs are ubiquitously distributed across the mouse STN, and that Pvalb and Col24a1 are locally distributed in the STN in an opposite gradient manner ( Pvalb : dorsal-to-ventral; Col24a1 : ventral-to-dorsal), confirming the molecular distinction of the STN into three major spatio-molecular internal STN domains in mice 68 : STN a Pvalb - / Col24a1 + (ventral STN); STN b Pvalb + / Col24a1 + (central STN); STN c Pvalb + / Col24a1 - (dorsal STN) ( Figure 2a ). Further, FISH labeling supported our previous conclusions 68 that Tac1 mRNA is a selective marker for the adjacent pSTN structure, excluded from the STN and surrounding hypothalamus; that Calb2 mRNA is present in a cluster within the ventral aspect of the mouse STN but primarily distributed to the pSTN and adjacent hypothalamus; and that Baiap3 mRNA is a marker for both pSTN and adjacent hypothalamus while excluded from the STN (single labelings, Figure 2a-d ). By next comparing multiple combinations of these STN and pSTN markers by co-labeling-FISH (co-FISH) in serial sections throughout the entire area (S1-S4, mouse atlas 64 )( Figure 2b ), a striking difference in the overall shape of both STN and pSTN across the antero-posterior and dorso-ventral axes was observed, not recapitulated in published mouse atlases 64 , and therefore addressed in detail to clarify ( Figure 2b-d ) . While preserving their relative distribution throughout the STN structure, the molecularly defined internal STN domains occupied different amount of space across the STN (bregma levels (mm) −9.90 (S1), −11.70 (S2), −12.60 (S3), −13.50 (S4)) ( Figure 2c,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 only, while 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 2c,d ). By comparing spatial distribution of the above listed STN mRNA markers ( Pitx2 , Pvalb , Col24a1 ) as well as previously described pan-STN markers Nxph1 , Nxph4 , and Kcnab3 with pSTN markers Tac1 , Calb2 and Baiap3 mRNAs and with Vglut2 (which labels both STN and pSTN in mice) in multiple co-FISH experiments, the spatial relationship between STN and pSTN was revealed as more complex than illustrated in current brain atlases ( Figure 2c,d ). In fact, the roundish shape at the medial tip of the STN most commonly used in publications to denote the pSTN structure ( Figure 2b ) was present at the S2 level only, while anteriorly (S1), no pSTN at all was detected, and posteriorly (S3, S4), the shape and spatial relationship STN vs. pSTN was dramatically different. At S3, the level at which the STN was detected as a narrow slice, the pSTN was observed as an equally narrow and elongated structure which was completely 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 the classically illustrated STN shape 64 , but which when analyzing serial sections is actually only observed at S2 ( Figure 2c,d ) . Thus, at this bregma level, the cellular density resembling the shape commonly illustrated as the STN, is composed of both STN and pSTN. At S4, the most posterior position of the STN (STN a ), the pSTN is at its largest and wraps around the entire STN, with an outline that strikingly resembles the classical STN structure at S2 ( Figure 2c,d ). The whole STN structure in mice was sectioned for these analyses, and the pSTN structure was present in 80% of all sections, absent only at anterior STN, thus verifying the histological findings that the pSTN is substantially larger in size than illustrated in current brain atlases. The molecularly reinforced anatomical details were next validated using 3D light-sheet imaging of the intact mouse brain, applying fluorescent probes directed at Pitx2 and Tac1 mRNAs to visualize the spatial distribution of STN and pSTN, relative to each other in space. In coronal fly-through videos (set at slower speed at the level of STN and pSTN to clearly outline the spatial distribution of the markers), analysis of Pitx2 ( Supplementary Video 1 ), Tac1 ( Supplementary Video 2 ), and Pitx2 / Tac1 combined ( Supplementary Video 3 ) allowed the outlining of the structural relationship between STN and pSTN across all axes. This 3D imaging validated the finding in serial sections of a substantially larger pSTN than outlined in mouse brain atlases, and that pSTN aligns the STN, in particular at the posterior level. Further, not only STN but also pSTN showed molecularly heterogeneity. 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 in the pSTN, suggesting they represent different pSTN subpopulations ( Figure 2a-d ). In summary, 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. Para-STN aligns with anterior STN in the macaque brain Next, co-FISH experiments were performed in the macaque STN, analyzing the same battery of differentially expressed genes to ascertain the translational value of these from mouse and NHP, and also to assess the spatial relationship between STN and pSTN in the primate brain. FISH probes detecting the primate counterpart mRNAs were applied onto serial sections (S1-S4) covering the whole macaque monkey STN. As we have recently described, TAC1 mRNA identifies pSTN cells not only in the mouse but also in the macaque brain 61 , 68 . In light of the unexpected finding that the pSTN in the mouse is substantially larger than outlined in any brain atlases, and that is aligns with the STN structure, it was of interest to study if the primate STN and pSTN show a similar spatial relationship, and to ascertain the full extent of the primate pSTN. By addressing VGLUT2 and TAC1 probes in serial macaque brain sections, TAC1 -positive pSTN cells were identified as lining the anterior STN (S1, S2), but not posterior STN (S3, S4). At the most anterior STN (S1), a distinct cluster of TAC1 + cells were identified which allowed the position of pSTN be defined at the medial aspect of the ventromedial STN tip. At S2, the TAC1 + pSTN density was of substantial size and aligned the STN medially. At S3 and S4, no TAC1 + cells were detected ( Figure 3a ). Thus, the macaque pSTN covers a major area directly associated with anteromedial STN, but not posterior STN (the opposite to what was found in the mouse brain). Download figure Open in new tab Figure 3. pSTN aligns with anterior STN in the macaque brain. Fluorescent mRNA-directed probes detecting mRNA distribution by FISH in the STN and pSTN of the macaque monkey. (a) TAC1 / VGLUT2 across S1-S4 (anterior to posterior), bregma levels indicated. (b) From left to right, top panel: VGLUT2 , NXPH4 , NXPH1 , COL24A1 , KCNAB3 , BAIAP3 mRNAs. Middle panel: NXPH4 / VGLUT2 , NXPH1 / VGLUT2 , NXPH4 / NXPH1 . Bottom panel: COL24A1 / VGLUT2 , KCNAB3 / VGLUT2 , BAIAP3 / VGLUT2 . Single labeling in red or green, co-localization in yellow; see close-ups for high-resolution detail. The whole STN structure in the macaque brain was sectioned for these analyses, and the pSTN structure was present in 17% of all sections, absent at posterior STN, verifying the findings that pSTN is present in the macaque brain and substantially larger in size than illustrated in current brain atlases. Similar as we previously reported for the mouse, NXPH4 , NXPH1 , and KCNAB3 mRNAs were identified as ubiquitously expressed across the STN (same as PITX2 and VGLUT2 ), co-localizing fully with VGLUT2 ; thus all these markers fully co-localizing with each other across the STN of the macaque ( Figure 3b ). In macaque, but not in mouse, COL24A1 was detected across the STN structure, detecting a putative molecular difference between the two species ( Figure 3a ). As for TAC1 , also BAIAP3 mRNA was excluded from the STN in the macaque brain, but was present in the pSTN. In contrast to TAC1 , BAIAP3 was not exclusive for pSTN but detected also in surrounding hypothalamus, i.e. similar to the mouse ( Figure 3b ). Finally, we addressed the pan-STN markers NXPH4 , NXPH1 , and KCNAB3 across the whole pSTN structure, molecularly outlined with TAC1 and BAIAP3 . Within the TAC1 / BAIAP3 -positive pSTN structure, NXPH4 , NXPH1 , and KCNAB3 mRNAs could be detected, but only in rare cells. Thus, these markers are selective for the STN and do not specifically label the pSTN in the NHP brain. In summary, in the NHP brain, the pSTN ( TAC1 + ) is present primarily at the anterior aspect of the STN (primarily at bregma −11,70mm (S2)) while it is absent from the posterior STN. Thus, the pSTN aligns the STN in both mouse and macaque, but their relative position is different with pSTN at the posterior aspect of the STN in mouse and at the anterior aspect in the primate. Further, similar as in the mouse where the NXPH4 , NXPH1 , and KCNAB3 were orginially identified as pan-STN markers, also the macaque STN is positive for all three mRNAs. Anterior ventromedial STN defined by a distinct HTR2C profile in macaque Another interesting difference between mouse and macaque was displayed by HTR2C mRNA, encoding the serotonin receptor subtype 2C protein (5-HTR2C). In the mouse STN, we previously reported that Htr2c mRNA is detected across the STN structure, in a similar patterns as all identified pan-STN markers shown here, a finding confirmed above ( Figure 2 and reference 68 ). In contrast, in the present analysis of the macaque STN in serial sections (S1-S4), FISH detecting the primate counterpart HTR2C mRNA showed a clear gradient distribution profile as it was most prominently detected in the anterior ventromedial (vm) aspect of the STN (vmSTN) structure in the macaque while largely absent from the dorsal STN (dSTN) ( Figure 4a ). Download figure Open in new tab Figure 4. Anterior ventromedial STN defined by a distinct HTR2C profile in macaque and human brains. Fluorescent mRNA-directed probes detecting mRNA distribution by FISH in the STN of the macaque monkey and human. (a) HTR2C mRNA in macaque STN (counterstaining with DAPI) (b) HTR2C mRNA in human STN (counterstaining with DAPI). (c-d) Comparative analysis of PVALB (b) and HTR2C (c) across anteroposterior axis; bregma levels indicated. (e) Schematic illustrations of the findings in c-d, outlining ventromedial (vm) STN and dorsal (d) STN as defined by spatial anatomy and expression profiles of PVALB and HTR2C mRNAs. (f) HTR2C / VGLUT2 co-FISH; closeups to right indicate patterns of expression and level of co-expression within 3 selected squares at S2 to visualize ventral to dorsal STN, vmSTN and dSTN (corresponding to squares 1-3). (g) HTR2C / PVALB co-FISH, single and double labelings; closeups indicate level of co-expression (none) at far medial vmSTN in macaque brain. (h) Quantifications of HTR2C / PVALB overlap across S1-S4 in macaque brain. (i) HTR2C / PVALB co-FISH, single and double labelings; closeups indicate level of co-expression (none) at far medial vmSTN in human brain. (j) Quantification of HTR2C / PVALB overlap in human brain biopsy sample. All quantifications and corresponding pie charts are shown in higher magnification in Supplementary Figure 1, raw data in KRT. Analysis of human brain section showed the same ventral high -dorsal low HTR2C mRNA profile ( Figure 4b ). Thus, HTR2C mRNA allowed the subdivision of NHP and human STN into a HTR2C -positive vmSTN domain and a HTR2C- negative dSTN domain. In the primate (NHP and human) and rat, the parvalbumin protein (PV) and its corresponding mRNA ( PVALB ) have been shown in several studies as a marker of dorsal and lateral STN 65 , 77 , 78 (reviewed in 39 ), and we recently described a similar distribution of PVALB ( Pvalb ) mRNA applying FISH analysis in both macaque and mouse 61 . Considering the ventral distribution of HTR2C , we next investigated if PVALB and HTR2C mRNAs form overlapping or discrete profiles throughout the STN by co-FISH comparing the distribution pattern of HTR2C and PVALB mRNAs in the macaque monkey ( Figure 4c,d ). 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 4c,d ). PVALB mRNA was the most dominant of the two mRNAs overall but both mRNAs 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 mRNA covered more than half of the STN at S1 and was most prominent in vmSTN STN. HTR2C decreased posteriorly, but was always found located in a strip of vmSTN (S2-S4) ( Figure 4c,d ). 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 4c,d ). PVALB was more dominant posteriorly, while HTR2C dominated anteriorly ( Figure 4c,d ). Thus, apart from limited overlap, PVALB and HTR2C were mutually exclusive in the STN, and both mRNAs showed rather sharp borders ( Figure 4c,d , close-ups of borders). By summarizing the molecular profiles displayed by PVALB and HTR2C , distinct internal STN domains were visualized that differed across the antero-posterior axis, with a clear HTR2C -positive vmSTN tip at all bregma levels (S1-S4) ( Figure 4e ). The intensity level of the VGLUT2 FISH signal in the macaque brain was higher in the HTR2C -positive domain of vmSTN ( Figure 4f ). However, both PVALB and HTR2C overlapped extensively with VGLUT2 mRNA, within their regional distribution areas ( Figure 4f ). Using VGLUT2 as pan-STN glutamatergic marker, quantification of cells positive for PVALB vs. VGLUT2 , and HTR2C vs. VGLUT2 , and direct comparison PVALB vs. HTR2C confirmed the observation by visual inspection above of more abundant HTR2C than PVALB mRNA labeling anteriorly and ventromedially, and vice versa posteriorly and laterally. In a total STN count (S2), PVALB + / HTR2C - was the dominating combination of STN neurons (77%), with similar amounts of cells positive for both PVALB and HTR2C (PVALB + /HTR2C + , 13%) as those selective for HTR2C ( PVALB + / HTR2C + , 13%; and PVALB - / HTR2C + , 10%), and with opposing numerical gradients across S1-S4 ( Figure 4g,h and Supplementary Figure 1 ). Next, a similar opposite gradient distribution of PVALB and HTR2C mRNA was found in the human STN, with a PVALB gradient dorsal high -ventral low meeting the opposite ventral high -dorsal low HTR2C mRNA profile, and with similar relative percentage of distribution as in the macaque STN (72% PVALB + / HTR2C - ; PVALB - / HTR2C + , 23%; PVALB + / HTR2C + , 5%) ( Figure 4i,j ). In summary, the primate STN contains opposing gradients of the previously established STN marker PVALB and the here identified HTR2C mRNA, with PVALB dominating posteriorly and dorsally, and HTR2C mRNA most prominent anteriorly and ventrally, and with HTR2C exclusively expressed in the tip of vmSTN across all section levels. HTR2C mRNA gradient allows distinction of pSTN into two domains: pSTN core and pSTN shell In light of the striking localized pattern of HTR2C mRNA in vmSTN of macaque and human brains, the presence of HTR2C mRNA was next explored in the pSTN. HTR2C mRNA was assessed in the pSTN by comparison with the pSTN marker TAC1 . HTR2C and TAC1 mRNAs were analyzed across the STN-pSTN area in mouse ( Figure 5a,b ) and macaque ( Figure 5c ). Download figure Open in new tab Figure 5. HTR2C mRNA gradient allows distinction of pSTN into two domains: pSTN core and STN shell. (a) Tac1 / Htr2c co-FISH (top panel), Tac1 single labeling (middle panel), Htr2c single labeling (bottom panel) across STN and pSTN at S1-S4 levels in mouse, bregma levels indicated. (b) Left panel: Tac1 , pSTN marker; Htr2c , primarily STN marker, but also detected in pSTN, overlap Tac1 / Htr2c in pSTN. Right panel: Calb2 / Tac1 single and double labelings, Calb2 in both STN and pSTN, co-localization in pSTN. (c) HTR2C / VGLUT2 and TAC1 / CALB2 FISH across STN and pSTN in macaque brain. (d) Schematic illustration: Htr2c / HTR2C decreasing gradient laterally in Tac1 / TAC1 -positive pSTN allows definition of pSTN core (pSTNCo) closest to STN, and pSTN shell (pSTNSh) further laterally in both mouse and macaque brains. Single labeling in red or green, co-localization in yellow; see close-ups for high-resolution detail. 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 2 , Htr2c mRNA was 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 mRNA 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 ). Further, Calb2 mRNA was detected throughout pSTN and and regionally within vmSTN ( Figure 5b ) Next addressing the macaque pSTN, and comparing HTR2C in the pSTN with VGLUT2 and TAC1 , HTR2C mRNA allowed the subdivision of pSTN into a core (pSTN Co ) and shell (pSTN Sh ) area also in this species, and many pSTN cells were identified as positive for CALB2 mRNA ( Figure 5c ). In summary, based on mRNA profiles of Tac1 and Htr2c in mouse, and corresponding primate transcripts TAC1 and HTR2C in macaque, a gradient of HTR2C expression levels allows the subdivision of the newly described pSTN structure into two anatomical subdivisions; the core and shell area (illustrated in Figure 5d ). pSTN cells embedded in dopaminergic nerve bundles of MFB With the current identification of molecular profiles enabling the full extent of pSTN neurons in the macaque, their complete anatomical distribution can now be visualized. At its dorsal aspect, the STN borders to the monoaminergic projection of MFB. With the observation that pSTN aligns with the dorsal STN at its anterior aspect in the macaque brain, it was of interest to address if pSTN and MFB might co-localize above the STN. The MFB, which largely consists of dopaminergic projections from the VTA in the midbrain, has been shown to contain certain cell groups of various identity, including neuropeptidergic and hypothalamic cells 79 , 80 . However, pSTN neurons, which we only recently described molecularly ( TAC1 + ) in the macaque brain 61 and investigated further in the present study, 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 within the MFB, molecular profiles were addressed in co-localization analysis on serial macaque sections using tyrosine hydroxylase (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 ( 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 ). Download figure Open in new tab Figure 6. TAC1 + pSTN cells embedded in dopaminergic nerve bundles of the MFB. Fluorescent mRNA-directed probes ( VGLUT2 and TAC1 ) and fluorescent labeling of the tyrosine hydroxylase protein (TH, in bold) in serial sections. (a) Antero-posterior (AP) axis, left to right; bregma (mm) – 9.90, −11.30, −11.70, −12.40, −13.50. Overview of sections: TH immunofluorescent labeling detects dopaminergic fibers in the medial forebrain bundle (MFB) originating from TH-positive cell bodies in substantia nigra pars compacta (SNC), dorsal and ventral tiers (SNCD, SNCV) while VGLUT2 mRNA detects glutamatergic cell bodies. (b) TH/ VGLUT2 , and ( c ) TH/ TAC1 double immunofluorescent and FISH analysis at bregma −11.30 mm; STN ( VGLUT2 + ) and pSTN ( TAC1 + / VGLUT2 + ); pSTN aligned with the MFB at the medial aspect of the STN (pSTN_MFB), ventromedial pSTN not aligned with MFB (pSTN_not MFB), but located at the ventromedial (vm) STN at this anterior level. Close-ups; VGLUT2 + and TAC1 + cells bodies within the TH-positive dopaminergic MFB nerve bundle at the dorsal STN aspect, identifying TAC1 + pSTN cells embedded in the MFB. ( d ) Schematic illustration at the anterior STN (bregma −11.30 mm), indicating pSTN and MFB as partly intermingled structures. Fluorophore indicated by red and green color, while co-localization is in yellow and DAPI nuclear staining in blue. Reference atlas 63 . Download figure Open in new tab Figure 7. Molecular heterogeneity detected in several DBS sites. Top panel, schematic illustration of the STN, pSTN, MFB triad in the primate with approximate DBS sites indicated (MFB DBS, vmSTN-DBS, dlSTN-DBS). FISH data originating from figures above, here placed in DBS context for sake of illustration. Middle panel: MFB-DBS site in molecular markers: TH (tyrosine hydroxylase protein; dopaminergic projections of the MFB), TAC1 , CALB2 , GAD1 , GAD2 , BAIAP3 . Bottom panel: vmSTN-DBS site in molecular markers: VGLUT2 , HTR2C (+), PVALB (-). dlSTN-DBS in site in molecular markers: VGLUT2 , HTR2C (-), PVALB (+). Abbreviations: dl, dorsolateral; vm, ventromedial. Download figure Open in new tab Figure 8. Moleculary supported anatomical maps of the subthalamic area. Schematic illustrations comparing current brain atlases of the mouse (a) and macaque (b) 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 as presented throughout this study, allowing the identification of transcriptome-enhanced details in anatomical maps in mouse and macaque. Full mRNA names in main text. Mouse and monkey brain atlas references 63 , 64 . 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 ) . Thus, pSTN neurons are indeed embedded in the MFB at the dorsal aspect of anterior STN. This surprising finding was confirmed in the mouse. 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 fly-through videos allowed the visualization of STN and pSTN distribution along the TH-positive MFB. This validated the presence of Tac1 + , but not Pitx2 + , cells within the MFB at the dorsal aspect of STN in the mouse. In summary, pSTN cells are embedded within the MFB above the STN, thus identifying a new molecularly defined cell population within the dopaminergic axons of this fiber bundle. Molecular heterogeneity detected in several DBS sites Based on coordinates for DBS lead implantation sites for motor symptoms in PD in dorsolateral (dl) STN 13 , for OCD symptoms in anterior vmSTN 20 , and for depression in slMFB 44 , for sake of illustration of the molecular heterogeneity identified throughout this study, we grossly reconstructed these sites by overlaying approximate DBS positions with our spatio-molecular FISH data in macaque monkey. Here, we identified that MFB, a DBS site for depression, may contain pSTN neurons and that these have a a molecular profile of TAC1 + / BAIAP3 + / HTR2C gradient ( Figure 7 ). Further, in addition to pan-STN markers VGLUT2 , PITX2 , NXPH1 , NXPH4 , KCNAB3 , dlSTN, containing the DBS site for PD motor symptoms, shows a PVALB + / HTR2C - molecular profile, while anterior vmSTN, a DBS site for OCD, shows a unique HTR2C + / PVALB - profile ( Figure 7 ). Depending on exact DBS lead position across the axes in the diseased human brain undergoing treatment, these molecular profiles may vary. Molecularly supported anatomical maps of the subthalamic area Based on the presented molecular profiles detected by FISH, the data was summarized in table format ( Table 1 ) , and anatomical brain maps were created throughout the STN area, in which pSTN and MFB were added to visualize the spatial distribution of STN, pSTN, and MFB relative to each other, and with molecular profiles within the STN and pSTN as described throughout this study, outlined schematically ( Fig. 8 ) . View this table: View inline View popup Download powerpoint Table 1. Summary of data from FISH analyses using multiple mRNA markers throughout the subthalamic nucleus (STN) and para-STN (pSTN) in mouse and primate. pSTN subdivided into pSTN Co and pSTN Sh based on Htr2c ( HTR2C ) mRNA gradient. STNa, STNb, STNc refers to internal STN domains originally described in Wallén-Mackenzie et al 68 . vmSTN refers to ventromedial STN, dlSTN to dorsolateral STN. 0 means no FISH signal, +, ++, +++: increasing FISH signal. Discussion Recent advances in neuromodulation, including deep brain stimulation (DBS), focused ultrasound, and repetitive transcranial magnetic stimulation, have expanded therapeutic options for the treatment of neurological and neuropsychatric disorders, such as PD, OCD, and major depression (reviewed in 23 , 26 , 81 – 83 ). These developments have been paralleled by improvements in device engineering, stimulation paradigms, and neuroimaging 84 . However, the efficacy of all neuromodulation strategies remains critically dependent on anatomical precision. Technological sophistication cannot compensate for inaccurate targeting, underscoring the need for refined brain maps that capture biologically meaningful subdivisions of heterogeneous structures. In this study, we address this need by applying transcriptome-based anatomical mapping to the subthalamic region. High-resolution molecular profiling enables the identification of spatially defined neuronal subpopulations based on combinatorial gene expression, providing a level of anatomical resolution not achievable with conventional cytoarchitectonics 85 . This approach is particularly relevant in anatomically compact and heterogeneous regions, where functionally distinct nuclei are closely intermingled. In addition to reinforcing the level of spatial detail in brain anatomy, transcriptome analysis may also provide clues towards molecular pathways and reveal signaling cascades downstream of neurotransmitter–receptor interactions in discrete neurons. Such molecular insight may help uncover the neurobiological underpinnings of neuromodulatory stimulation and, in turn, inform the development of pharmacological interventions. Together, these features position transcriptome mapping as a powerful framework for bridging anatomical definition with mechanistic and therapeutic insight. We focus on the STN, the pSTN, and the MFB, a tightly organized triad located at the midbrain–thalamus–hypothalamus interface. This region contains several clinically relevant DBS targets but remains incompletely resolved spatio-molecularly. While the STN 13 and slMFB 45 are established treatment targets, the pSTN has received comparatively little attention, particularly in primates. Our data support the view that the pSTN constitutes a distinct but anatomically integrated component of this region, with potential relevance for neuromodulation outcomes. A principal finding is the close spatial integration of the STN, pSTN, and MFB. In primates, we find that the pSTN is positioned predominantly at the anterior aspect of the STN and is partially embedded within dopaminergic projections of the MFB, whereas in mice pSTN is located more posteriorly. Notably, the proximity of the pSTN to the anteromedial STN, commonly targeted in DBS for obsessive–compulsive disorder 16 – 18 , and to the slMFB, a new DBS target for TRD 45 , suggests that pSTN neurons are likely to be co-modulated during stimulation. Such effects may contribute to both therapeutic efficacy and side effects. Our analysis further demonstrates pronounced molecular heterogeneity within the STN. In primates, we identify three major domains defined by the expression of HTR2C and PVALB mRNAs, which exhibit opposing gradients across both anteroposterior and dorsoventral axes. The ventromedial STN, in particular anteriorly, is characterized by enrichment of HTR2C expression, whereas dorsolateral regions, as established in literature (reviewed in 39 , 60 ), are dominated by PVALB -positive neurons. This organization, building upon oppsite gradients, provides a molecular framework for established functional subdivisions of the STN and supports the concept of spatially segregated but partially overlapping motor and non-motor territories. The PVALB -positive domain corresponds to dorsal and posterior STN regions and is conserved across species. These neurons are glutamatergic projection neurons, consistent with previous reports 39 , and are likely to contribute to sensorimotor processing via projections to basal ganglia output nuclei 1 – 3 . In contrast, the HTR2C -enriched ventromedial domain appears to be a primate-specific feature, as Htr2c expression is more uniformly distributed in the mouse STN. Although further studies are required to confirm this species-selective distinction, the observed pattern tentatively suggests an evolutionary specialization of serotonergic signaling within the primate STN. The functional implications of this organization are considerable. 5-HT, and in particular its action via 5-HTR2C, has well-established roles in mood, anxiety, and emotional regulation 86 – 89 , raising the possibility that specific neurons within vmSTN that show a HTR2C high profile correspond to limbic and associative function. This interpretation is consistent with the localization of DBS targets for psychiatric indications within the anteromedial STN and with known connectivity to limbic structures, including the ventral pallidum, as well as its serotonergic innervation pattern 27 , 90 , 91 . Our findings therefore provide a molecular correlate for clinically defined functional subdivisions of the STN. Interestingly, such patterns are less defined in non-primate species, reinforcing the notion of species-specific organization 1 , 2 , 90 , 92 – 94 . The identification of a ventralized HTR2C high mRNA profile in the primate STN extends this literature by providing a molecular signature for serotonergic specialization within the STN. Recent findings in parkinsonian monkeys have described reduced serotonergic innervation, suggested to represent compensatory mechanisms to attenuate the hyperexcitabiliy of STN neurons 95 . Given the central role of serotonergic dysfunction in depression, with high prevalence of depression in PD 50 , 51 , 53 , 96 , our molecular findings are of particular clinical relevance. The potential involvement of vmSTN in affective processing is further supported by recent circuit-level studies 13 , 23 . Projections from the STN to the ventral pallidum and lateral habenula have recently been implicated in aversive and avoidance-related behaviors 61 . Whether the HTR2C -enriched neuronal population preferentially contributes to these circuits remains to be determined. However, the present data provide a framework for hypothesis-driven investigations linking molecular identity to circuit function and behavior. In parallel, our findings highlight the importance of the pSTN as a candidate modulator of neuromodulatory interventions. Primarily characterized in rodents, where it has been linked to feeding, arousal, and behavioral responses to novelty 57 – 60 , 62 , 97 – 101 , the hypothalamic substructure pSTN remains poorly defined in primates. In a recent study, we identified TAC1 + pSTN neurons in the macaque brain 61 . Based on this recent molecular identification, the present study demonstrates that in the primate brain, the pSTN ( TAC1 + ) occupies a strategic position at the interface of the STN and slMFB, and even extends into the fiber tract itself. This anatomical arrangement suggests that pSTN neurons are likely to be engaged during stimulation of either structure. Given the functional diversity attributed to the pSTN, such engagement may have complex and context-dependent effects on clinical outcomes. Recent clinical studies have identified optimal stimulation sites and networks within the STN and associated frontal circuitry, often described as sweet spots or sweet networks 24 , 25 . However, these models are typically based on structural imaging and connectivity analyses, without incorporating molecular information. The integration of transcriptomic maps with functional imaging and electrophysiological data may therefore represent an important next step toward precision neuromodulation. In this context, our data suggest that molecularly defined domains within the STN could refine targeting strategies. For example, whereas the dorsolateral ( PVALB -positive) STN domain is already prioritized for motor symptoms, ventromedial HTR2C -enriched cellular hotspots within vmSTN may represent a candidate substrate for modulating affective symptoms with advanced spatial precision. Although speculative, this framework provides a testable basis for linking cellular composition to clinical response. Future studies combining molecular mapping with patient-specific outcome data will be required to validate this approach. In summary, we provide a transcriptome-based anatomical framework for the subthalamic region that reveals previously unrecognized molecular heterogeneity and spatial organization in both mouse and primate brains. These findings refine current models of STN organization, identify the pSTN as a relevant but underappreciated structure, and highlight the close anatomical and functional integration of the STN - pSTN - MFB complex. By linking molecular identity to anatomical location, this work contributes to a more precise understanding of neuromodulation targets and provides a foundation for future studies aimed at improving therapeutic specificity. Ultimately, incorporating molecular information into anatomical and functional models may inform drug discovery and also enhance precision and predictability of neuromodulation therapies for both motor and non-motor symptoms of PD, as well as several additional brain disorders for which the STN and slMFB already are validated treatment targets. Limitations to the study. Several limitations of the present study should be considered. The analysis is based on a substantial amount of mouse brain sections and validated in whole brain preparations, but with a limited number of primate and human samples. The findings are primarily descriptive. Functional interpretations of gene expression patterns remain to be established through complementary approaches, including electrophysiology, circuit tracing, and behavioral analysis. In addition, potential confounding factors such as age and tissue variability cannot be excluded. Further studies will be needed to match spatio-molecular profiles with precise DBS sites. Despite these limitations, the consistency of our findings with existing anatomical and functional data supports their validity. Author Contributions Conceptualization, Å.W-M; Methodology, visualization, analysis, and figure preparation, S.D (all section analyses), M.B.R, U.R (whole brain analysis, light sheet microscopy); Methodology, analysis (macaque): C.F, C.K; Contribution of biological material; Å.W-M, S.D, C.F, M.B.R, U.R, C.K; Writing, original draft, review and editing, Å.W-M; Writing, review and editing, S.D, C.F; Project administration and funding acquisition, Å.W-M. All authors approved the submitted version of the manuscript. Competing Interests S.D is the owner of Oramacell, France. M.B.R and U.R are employed at Gubra, Denmark. All other authors declare no competing financial or non-financial interest. Download figure Open in new tab Supplementary Figure 1. Quantification data presented in pie charts in Figure 4 , here in higher magnification. Raw data in KRT. 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. 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. 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. 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. 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. Acknowledgments We thank Dr Anders Björklund, Lund University, Dr Volker Coenen, Freiburg University, Dr Candice Contet, The Scripps Research Institute, San Diego, CA, Dr Nick Hollon, University of California San Diego, CA, and Dr Jérôme Baufreton, Université de Bordeaux, for constructive feedback. We thank Sabrina Leclerc-Turband, Manager at Biobanque Neuro-CEB APHP, France, for human brain tissue biopsy samples, and Nermine Saidi from Institut de la Vision, Paris, France, for slide scanning. MackenzieLab staff is thanked for constructive feedback. This research was funded in part by research funding grants from the Swedish Research Council (Vetenskapsrådet), Swedish Brain Foundation (Hjärnfonden), the Swedish Parkinson Foundation (Parkinsonfonden), Åhlénstiftelsen, and Hållsten Research Foundation (to ÅWM). This research was funded in part by Aligning Science Across Parkinson’s [ASAP-020600] through the Michael J. Fox Foundation for Parkinson’s Research (MJFF). For the purpose of open access, the author has applied a CC BY public copyright license to all Author Accepted Manuscripts arising from this submission. Funder Information Declared Aligning Science Across Parkinson's , ASAP-020600 , ASAP-027214 Swedish Research Council, https://ror.org/03zttf063 Hjärnfonden Parkinsonfonden Hållsten Research Foundation Footnotes This revision contains the addition of new data from mouse brains (both sections and whole brains), the clarification of section angles, the addition of mRNA profiles in examples of DBS sites, and revised illustration of brain maps. The text has been substantially revised in response to reviewer comments and feedback from experts in the field. https://zenodo.org/records/20007922 References 1. ↵ Parent , A. & Hazrati , L. N . Functional anatomy of the basal ganglia . I. 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