Anatomical distribution of cocaine- and amphetamine-regulated transcript (CART) peptide in the human spinal cord

Anatomical distribution of cocaine- and amphetamine-regulated transcript (CART) peptide in the human spinal cord | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anatomical distribution of cocaine- and amphetamine-regulated transcript (CART) peptide in the human spinal cord Öykü Deniz Kanat, Esra Candar, Ibrahim Demircubuk, Ferihan Çetin, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6297031/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The cocaine- and amphetamine-regulated transcript (CART) peptide, a neuropeptide highly expressed in the central nervous system, is involved in various physiological processes, including pain modulation, reward, learning, and memory. While previous studies have examined the distribution of CART peptides in the rat and human brain, no data exist on their distribution in the human spinal cord. Therefore, we investigated the localization of CART peptides in the human spinal cord using immunohistochemistry. Our analysis revealed dense CART-immunoreactive fibers and varicosities in the anterior, lateral, and dorsal funiculi of the white matter along the entire spinal cord. CART-immunoreactive neurons were identified in the Rexed’s laminae (strong immunoreactivity in laminae I-II and IX, and moderate immunoreactivity in laminae III-VIII and X. Strong CART immunoreactivity was observed in the dorsal (Clarke), intermediolateral and sacral parasympathetic nuclei, and moderate in the internal basilar, lateral spinal and lateral cervical, central cervical, and lumbar and sacral precerebellar nuclei. The widespread but regionally varied immunoreactivity suggests a potential role for CART in modulating spinal cord functions, including pain processing, autonomic regulation, and motor control. CART peptide chemoarchitecture neurochemistry neuropeptide spinal cord Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The cocaine- and amphetamine-regulated transcript (CART) was first identified as a messenger RNA (mRNA) that responds to cocaine and amphetamine exposure in the rat striatum (Douglas et al. 1995). Later, Thim and colleagues ( 1999 ) isolated different CART peptides from various regions of the body: CART(1–89) and CART(10–89) from the adrenal gland, CART(42–89) and CART(49–89) from the hypothalamus and nucleus accumbens in rats. Over time, research has linked CART peptides to a wide range of physiological processes, including pain modulation, reward processing, sleep regulation, learning and memory, and autonomic nervous system control (Subhedar et al. 2014 ). However, despite these associations, CART’s precise physiological role remains unclear, largely due to the lack of well-defined, specific CART receptors (Stanek 2006 ; Ahmadian-Moghadam et al. 2018 ). Early immunohistochemical and in situ hybridization studies revealed that CART peptides are widely distributed throughout both rat and human brains. Using five polyclonal antisera, Koylu et al. ( 1997 ) detected CART peptides in key endocrine regions of the rat, including the hypothalamus, pituitary, and adrenal glands. Building on these initial findings, the same research team conducted a broader immunohistochemical study to map CART peptide distribution across the rat brain. Their results (Koylu et al. 1998 ) showed strong CART immunoreactivity in areas such as the nucleus accumbens, centromedial amygdaloid nuclei, parabrachial nucleus, and principal inferior olivary nucleus, while regions like the entorhinal cortex and nucleus ambiguus displayed moderate immunoreactivity. Additionally, they identified CART-immunoreactive fibers and varicosities within the spinal cord, particularly in laminae I-II, the dorsolateral fasciculus, and the lateral spinal nucleus (LSp), as well as immunoreactive neurons in the intermediolateral nucleus (IML) and lamina X. Expanding beyond rodent models, Hurd and Fagergren ( 2000 ) examined CART mRNA expression in five human post-mortem brains using in situ hybridization. Their findings indicated CART mRNA presence in several key brain regions, including the dorsolateral prefrontal cortex, nucleus accumbens, bed nucleus of the stria terminalis, locus coeruleus, hypothalamus, and thalamus. Meanwhile, Dun et al. ( 2000 ) observed CART- immunoreactive fibers, but not cell bodies, in the sacral parasympathetic nucleus of the rat spinal cord. Interestingly, while CART mRNA is absent in the rat cerebellum (Koylu et al. 1998 ), CART peptides have been detected in climbing fibers (Press and Wall 2008 ). Further supporting the peripheral presence of CART, Kozsurek et al. ( 2007 ) identified CART-immunoreactive cell bodies and axons in the rat dorsal root ganglia. These studies collectively highlight the widespread distribution of CART peptides and suggest their involvement in diverse neural processes across different species. The CART peptides are known to coexist with several neurotransmitters in the mammalian nervous system. In the rat brain, CART peptides are found alongside γ-aminobutyric acid (GABA) and dynorphin within the substantia nigra and ventral tegmental area (Dallvechia-Adams et al. 2002 ). Lambert et al. ( 1998 ) observed neuropeptide Y (NPY) immunoreactive varicosities surrounding CART-immunoreactive cells in the paraventricular hypothalamic nucleus. Additionally, CART peptides colocalize with calcitonin gene-related peptide (CGRP), substance P, and somatostatin (SOM) in fibers within the laminae I and II of the spinal cord, hinting at a role in nociception and other sensory functions (Kozsurek et al. 2007 ). An ultrastructural study by Smith et al. ( 1999 ) demonstrated synaptic interactions between CART-immunoreactive dendrites and tyrosine hydroxylase-positive axon terminals in the monkey nucleus accumbens. Using light microscopy with double labeling, Couceyro et al. ( 1998 ) showed that a subset of choline acetyltransferase (ChAT) neurons in the rat myenteric plexus also contain CART peptides. In the chinchilla claustrum, CART peptides coexist with parvalbumin (Pv), SOM, and NPY (Szalak et al. 2023 ). Moreover, Miller et al. ( 2024 ) reported that approximately half of the melanin-concentrating hormone cells in the medial hypothalamus contain CART peptides. While previous studies have explored the presence of CART peptides in rat and human brains, their distribution in the human spinal cord remains largely unknown. Understanding where these peptides are located within the spinal cord could provide valuable insights into their potential roles in the nervous system. In this study, we aimed to map the anatomical distribution of the CART peptides in the human spinal cord using immunohistochemistry. Additionally, we sought to classify the neuronal populations that exhibit CART immunoreactivity. Materials and Methods Tissue preparation For this study, we used five human spinal cords with no known neurological conditions, obtained from the cadaver collection at Ege University School of Medicine, Izmir, Turkiye. To prepare the specimen, the vertebral pedicles and dura mater were carefully removed to expose the spinal cord which was then immersed in a 10% formalin solution for three days. After fixation, the spinal cord was divided into 31 segments under a Leica M651 surgical operating microscope (Leica Microsystems AG, Heerbrugg, Switzerland). The segments were then placed in a 30% sucrose solution for cryoprotection before being sectioned at a thickness of 30 µm using a Leica CM1950 cryostat (Leica Microsystems, Wetzlar, Germany). This study was approved by the Medical Research Ethics Committee at Ege University, Izmir, Turkiye (approval number 23 − 2.1/T/22) and all procedures were conducted in accordance with the principles of the Declaration of Helsinki. Immunohistochemistry To visualize the anatomical distribution of CART peptides in the human spinal cord, we performed free-floating immunohistochemical staining on the tissue sections. First, the sections were incubated in 0.3% H 2 O 2 for 30 minutes to block endogenous peroxidase activity. This was followed by a 2-hour incubation in a blocking solution containing 10% normal goat serum (NGS; Vector Laboratories, S-1000-20) at room temperature. For CART detection, the sections were incubated for 48 hours at 4°C with a primary antibody against CART (Phoenix Pharmaceuticals, H-003-62, dilution 1:5000) prepared in a solution of 3% NGS, 0.3% Triton X-100, and 0.01 M PBS. Next, they were treated with a corresponding secondary antibody (Sigma-Aldrich, AP124B, dilution 1:100) in 3% NGS, and 0.3% Triton X-100 in 0.01 M PBS for 2 hours. The sections were then incubated with streptavidin peroxidase (Thermo Scientific, TS-125-HR) for 10 minutes. Finally, the reaction was developed in 3,3’-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich, D5905) and the stained sections were coverslipped with Entellan. To assess antibody specificity, immunohistochemical stainings were conducted on several spinal cord sections by excluding either the primary or secondary antibody, and no immunoreactivity was detected in these control sections. Image processing and data analysis The sections were scanned at 10x magnification using the Aperio Microscope Slide Scanner (Aperio AT2, Leica Biosystems, France). Image adjustments, such as brightness and contrast, were performed using Adobe Photoshop (Adobe Photoshop 7.0, Adobe Systems Incorporated, CA, USA). To determine the anatomical divisions of the spinal cord, we referenced the human spinal cord atlases by Sengul et al. ( 2013 ) and Sengul ( 2021 ). CART immunoreactivity in each section was evaluated semiquantitatively employing a four-tiered scale: absent (-), weak (+), moderate (++), and strong (+++). Across the analyzed sections, the median semiquantitative score was used to represent the average staining intensity. CART-immunoreactive neurons were classified based on their morphological types. Multipolar neurons were identified by their extensive dendritic arborization, featuring at least three dendrites of varying thickness. Fusiform neurons exhibited spindle-shaped somata with primary dendrites extending from opposite poles. Ovoid neurons had round somata with no distinct dendritic branching pattern. Triangular neurons displayed a characteristic soma. Results In this study, we examined the anatomical distribution of CART peptides in the human spinal cord. Our analysis identified a diverse range of CART-immunoreactive neurons across all Rexed’s laminae, each exhibiting distinct morphological characteristics. The intensity of CART immunoreactivity was consistent across all laminae in the various spinal cord segments. Additionally, CART-immunoreactive fibers were widely distributed throughout the white matter. A summary of our findings, including the levels of CART immunoreactivity—categorized as weak (+), moderate (++), and strong (+++)—is presented in Table 1 . Laminae I and II Laminae I and II displayed strong CART immunoreactivity, with significantly higher levels compared to laminae III-IV (Figs. 1 – 4 ). No consistent pattern of immunoreactivity variation was observed across different spinal cord segments in these laminae. In lamina I, CART-immunoreactive cells were aligned parallel to the dorsal curvature of the dorsal horn. A subset of immunoreactive dendrites followed the contour of the dorsal horn, while others extended ventrally. In lamina II, the predominant CART-immunoreactive cell types were ovoid and fusiform neurons. Laminae III and IV Immunohistochemical analysis revealed a population of CART-immunoreactive neurons and fibers within laminae III and IV (Figs. 1 – 4 ). The density of CART immunoreactivity in these laminae was noticeably lower than in laminae I and II. The immunoreactive neurons exhibited a range of morphological characteristics, predominantly fusiform and ovoid shapes. Laminae V-VIII Moderate CART immunoreactivity was detected in laminae V-VIII (Figs. 1 – 4 ). The neurons in these laminae displayed diverse morphological characteristics, with four distinct morphotypes identified: multipolar, ovoid, fusiform, and triangular. Notably, at the limb enlargements, CART-immunoreactive neurons were identified in lamina VI. Bundles of CART-immunoreactive fibers were observed extending medially from the lamina VII toward the border of lamina X. Laminae IX and X As shown in Figs. 1 – 4 , a large number of CART-immunoreactive neurons and fibers were observed in lamina IX, exhibiting strong staining intensity, with multipolar neurons being the predominant cell type. The neurons in this region were arranged in a longitudinal pattern, forming motor columns. Lamina X neurons exhibited moderate CART immunoreactivity. Nuclei of the human spinal cord Immunohistochemical analysis revealed CART-immunoreactive neurons and fibers in both Clarke’s nucleus (dorsal nucleus, D) and the IML. D formed a longitudinal column extending from T1 to L2 segments, with ovoid and multipolar neurons as the principal cell type (Fig. 2 ). Within the IML, CART-immunoreactive neurons were distinctly aligned, separating this nucleus from the adjacent gray matter in the T1-L2 segments (Fig. 2 ). Additionally, CART-immunoreactive neurons and fibers were observed in the LSp across all spinal cord segments (Figs. 1 – 4 ), with no significant differences in immunoreactivity between segments. In the C1–C4 segments, CART-immunoreactive neurons and fibers were also detected in the lateral cervical nucleus (LatC) (Fig. 1 ). However, immunoreactivity was more pronounced in the lateral spinal nucleus (LSp) than in the LatC. Additionally, CART immunoreactivity was observed in the internal basilar nucleus (IB) and the central cervical nucleus (CeCv) (Fig. 1 ). Moderate CART immunoreactivity was detected in the lumbar precerebellar nucleus (LPrCb) within the L1–L5 segments (Fig. 3 ) and in the sacral precerebellar nucleus (Stilling’s sacral nucleus, SPrCb) within the S1–Co1 segments (Fig. 4 ). Furthermore, CART-immunoreactive neurons and fibers were identified in the sacral parasympathetic nucleus (SPSy) of the S1–S5 segments (Fig. 4 ). Discussion CART peptides are widely expressed across various brain regions (Ahmadian-Moghadam et al. 2018 ). The goal of this study was to determine their localization within the human spinal cord. We examined all spinal cord segments using CART immunohistochemistry, which revealed a distinct topographical organization with varying levels of immunoreactivity, ranging from weak to strong. While previous studies have mapped the distribution of CART peptides in both human and rat brains (Koylu et al. 1998 ; Hurd and Fagergren 2000 ), no data has been available regarding their distribution in the human spinal cord. Our findings identified CART-immunoreactive neurons and fibers in multiple anatomical regions associated with motor, sensory, and autonomic functions. Laminae I and II In the dorsal horn, laminae I and II serve as primary termination sites -for A-delta and C-fibers (Heise and Kayalioglu 2009 ). GABAergic neurons have been identified in these laminae (Barber et al. 1982 ; Todd and Sullivan 1990 ), with lamina II containing a substantial population of interneurons involved in modulating and transmitting nociceptive signals (Yasaka et al. 2010 ). Upadhya et al. ( 2011 ) reported an increased density of CART-immunoreactive fibers in the ventrolateral periaqueductal gray matter (PAG), the dorsal subdivision of the dorsal raphe nucleus and the locus coeruleus following neuropathic pain. Behavioral studies using intrathecal injections of the CART peptide (55–102) demonstrated reduced paw withdrawal latency, suggesting a role in pain modulation (Ohsawa et al. 2000 ). Additionally, Damaj et al. ( 2006 ) investigated the effects of CART(55–102) in neuropathic and inflammatory pain models, providing evidence that CART peptides can attenuate hyperalgesia associated with chronic pain. According to Schoenen and Faull ( 2004 ), human lamina I contains fusiform and multipolar neurons, while lamina II houses four distinct neuronal types—stellate, curly, filamentous, and islet—based on their dendritic and axonal morphology. In the present study, we defined three types of CART-immunoreactive neurons in laminae I and II: fusiform, ovoid, and multipolar. Laminae III and IV Neurons in laminae III-V are capable of responding to C-fiber input, although C fibers primarily terminate in the superficial laminae of the spinal cord (Naim et al. 1997 ). A previous retrograde labeling study demonstrated that neurons in laminae I, III, and IV expressing the neurokinin 1 receptor project to the PAG, and lateral parabrachial nucleus (Al-Khater and Todd 2009 ). Additionally, GABA and CGRP immunoreactivities have been reported in human laminae III and IV (Waldvogel et al. 1990 ; Jakab et al. 1990 ). Human lamina III consists of two distinct neuronal populations: one characterized by an asymmetrical dendritic tree and the other by a radial dendritic tree (Schoenen and Faull 2004 ). A distinguishing feature of lamina IV is the presence of antenna-like cells (Szentágothai and Rethelyi 1973; Schoenen and Faull 2004 ). In our study, we identified four morphotypes of CART-immunoreactive neurons: multipolar, fusiform, ovoid, and triangular. Laminae V-VIII Lamina V is more cellularly heterogeneous than lamina IV (Sengul and Watson 2011 ). Ritz and Greenspan ( 1985 ) studied the morphological characteristics of neurons in lamina V of the cat sacrocaudal spinal cord, identifying multireceptive cells that respond to innocuous and noxious mechanical stimuli. These neurons had large somata with extensive dendritic arborizations in all directions, and their axons crossed into the contralateral ventral white matter. Lamina VI is found only in the cervical and lumbosacral enlargements of the spinal cord in marmoset monkeys, rhesus monkeys (Sengul et al. 2013 ), rats (Molander et al. 1984 ; 1989 ), and humans (Sengul 2021 ). Propriospinal neurons play a crucial role in coordinating movements across different body regions by linking motor pathways (Laliberte et al. 2019 ). Ni et al. ( 2014 ) studied proprioceptive neurons in the mouse spinal cord using a recombinant rabies virus-based approach. Their findings identified proprioceptive neurons within laminae VII-VIII in the cervical segments and laminae IV-VIII in the thoracic segments. Additionally, Flynn et al. ( 2017 ) examined the anatomical and molecular characteristics of long descending propriospinal neurons in mice, detecting them predominantly in laminae VII and VIII. Lamina VII primarily consists of premotor interneurons that project to motor neuron pools (Sengul and Watson 2011 ). While its neuronal population shares similarities with those in laminae VI and VIII, lamina VII is distinguished by a higher number of horizontal or oblique dendrites (Schoenen and Faull 2004 ). In our study, we observed numerous CART-immunoreactive fibers within lamina VII. Additionally, lamina VII contains Renshaw cells, which establish inhibitory connections with alpha motor neurons (Rea 2016 ). Renshaw cells have been shown to express both gephyrin and calbindin (Cb) in rat lamina VII (Carr et al. 1998 ). A detailed analysis of CART and Cb colocalization could provide further insight into CART’s potential role in motor regulation. Laminae IX and X Unlike other laminae, lamina IX consists of a cluster of motor neuron columns embedded within laminae VII and VIII (Sengul and Watson 2011 ). In the rat spinal cord, motor neurons contain both ChAT and dopamine (Barber et al. 1984 ; Holstege et al. 1996 ). Moffett et al. ( 2006 ) demonstrated that CART knock-out mice exhibited reduced locomotor activity compared to wild-type mice in an open-field test, suggesting a potential role of CART in motor action. More recently, Eleftheriadis et al. (2022) reported colocalization of CART and ChAT within C-bouton synapses, further highlighting CART’s involvement in motor control. While Couceyro et al. ( 1997 ) found no CART-immunoreactive cells in the lumbar spinal cord of rats, our findings compared the presence of CART immunoreactive neurons in lamina X of the human lumbar spinal cord. Nuclei of the human spinal cord The D was first described by British physician Jacob Augustus Lockhart Clarke in 1851 (Clarke 1851 ; Demircubuk et al. 2023 ). Due to its location in the dorsal region of the spinal cord, German anatomist Benedict Stilling later proposed the term “dorsal nucleus” (Stilling 1859 ; Demircubuk et al. 2023 ). In the human spinal cord, D is located within the medial part of laminae VII, spanning segments C8 to L2 segments (Sengul and Watson 2011 ). Immunoreactivity for substance P, methionine-enkephalin, calbindin (Cb), calretinin (Cr), parvalbumin (Pv), choline acetyltransferase (ChAT), glutamic acid decarboxylase 65/67 (GAD 65/67), and vesicular glutamate transporter 1 (VGLUT1) has been detected in neurons of the dorsal nucleus (D) (Demircubuk et al. 2025 ). The neurons in D give rise to axons that form the dorsal spinocerebellar tract, which plays a key role in proprioceptive signal transmission to the cerebellum (Heise and Kayalioglu 2009 ). Additionally, D neurons contribute to local spinal corollary circuits involved in motor planning (Hantman and Jessell 2010 ). The IML, which houses the preganglionic sympathetic neurons, extends from C8 to L1 (Sengul and Watson 2011 ). In the pig spinal cord, colocalization of ChAT and nitric oxide synthase has been observed within the IML. Similarly, studies in the cat lumbar spinal cord reported Cr-immunoreactive neurons in the IML (Veshchitskii et al. 2021 ). Our findings indicate that both D and IML contain CART-immunoreactive neurons. Notably, in Parkinson’s disease, Lewy bodies and neuronal loss have been documented within the IML. Further research is needed to explore potential alterations in the D and IML in neurodegenerative diseases. The LSp extends throughout the entire length of the spinal cord, whereas the LatC is restricted to upper cervical spinal cord segments (Sengul 2021 ). Numerous studies have investigated these nuclei in various species, including rats, mice, and humans (Demircubuk et al. 2024 ). Animal studies suggest that LSp and LatC play a role in nociceptive signal transmission (Giesler Jr. et al. 1979 ; Olave and Maxwell 2004 ). In our study, we identified CART-immunoreactive fibers within both LSp and LatC, supporting previous hypotheses that CART peptides contribute to pain processing. In the human spinal cord, the lumbar precerebellar nucleus (LPrCb) is located within the L1–L5 segments, while the sacral precerebellar nucleus (SPrCb) is found within the S1–Co1 segments (Sengul 2021 ). As shown in our previous study (Demircubuk et al. 2025 ), neurons and fibers immunoreactive for calbindin (Cb), calretinin (Cr), parvalbumin (Pv), choline acetyltransferase (ChAT), glutamic acid decarboxylase 65/67 (GAD 65/67), and vesicular glutamate transporter 1 (VGLUT1) are present in both LPrCb and SPrCb. In the current study, CART-immunoreactive neurons and fibers were also identified in these nuclei. Limitations of the study This study primarily relies on immunohistochemistry, which provides anatomical localization but does not offer direct insights into dynamic physiological processes. Future studies using complementary approaches, such as in vivo imaging or functional assays, could provide a deeper understanding of CART’s real-time activity in the spinal cord. Conclusions This study offers the first detailed characterization of CART peptide distribution in the human spinal cord, highlighting its presence in key motor, sensory, and autonomic regions. The widespread but regionally distinct immunoreactivity suggests a potential role for CART in modulating spinal cord functions, including pain processing, autonomic regulation, and motor control. Further studies are needed to elucidate the functional significance of CART in these spinal circuits and its potential relevance to neurological disorders of the spinal cord. Declarations Conflicts of interest On behalf of all authors, the corresponding author states that there is no conflict of interest. Author Contribution O.D. K. for Methodology, formal analysis, investigation, writing – original draft; E.C. for methodology, formal analysis, investigation, writing – review and editing; I.D. for methodology, formal analysis, investigation, writing – original draft; F.C. for conceptualization, methodology, review and editing; G.S. for conceptualization, methodology, formal analysis, writing – review and editing, supervision. Acknowledgments The authors sincerely thank those who donated their bodies to science so that anatomical research could be performed. Results from such research can potentially increase mankind's overall knowledge that can then improve patient care. 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Academic Press, London Smith Y, Kieval J, Couceyro PR, Kuhar MJ (1999) CART peptide-immunoreactive neurones in the nucleus accumbens in monkeys: ultrastructural analysis, colocalization studies, and synaptic interactions with dopaminergic afferents. J Comp Neurol 407:491-511. https://doi.org/10.1002/(sici)1096-9861(19990517)407:43.0.co;2-0 Stanek LM (2006) Cocaine- and amphetamine related transcript (CART) and anxiety. Peptides 27:2005–2011. https://doi.org/10.1016/j.peptides.2006.01.027 Stilling B (1859) Neue untersuchungen ueber den bau des rückenmarks. Heinrich Hotop, Cassel Subhedar NK, Nakhate KT, Upadhya MA, Kokare DM (2014) CART in the brain of vertebrates: circuits, functions and evolution. Peptides 54:108-130. https://doi.org/10.1016/j.peptides.2014.01.004 Szalak R, Matysek M, Mozel S, Arciszewski MB (2023) Cocaine- and amphetamine-regulated transcript (CART) peptide is co-expressed with parvalbumin, neuropeptide Y and somatostatin in the claustrum of the chinchilla. Animals (Basel) 13:2177. https://doi.org/10.3390/ani13132177 Szentágothai J, Réthelyi M (1973) Cyto- and neuropil architecture of the spinal cord. In: Desmedt JE (ed) Human reflexes, pathophysiology of motor systems, methodology of human reflexes. Karger, Basel, pp 20-37 Thim L, Kristensen P, Nielsen PF, Wulff BS, Clausen JT (1999) Tissue-specific processing of cocaine- and amphetamine-regulated transcript peptides in the rat. Proc Natl Acad Sci USA 96:2722-2727. https://doi.org/10.1073/pnas.96.6.2722 Todd AJ, Sullivan AC (1990) Light microscope study of the coexistence of GABA-like and glycine-like immunoreactivities in the spinal cord of the rat. J Comp Neurol 296:496-505. https://doi.org/10.1002/cne.902960312 Upadhya MA, Dandekar MP, Kokare DM, Singru PS, Subhedar NK (2011) Evidence for the participation of cocaine- and amphetamine-regulated transcript peptide (CART) in the fluoxetine-induced anti-hyperalgesia in neuropathic rats. Peptides 32:317-326. https://doi.org/10.1016/j.peptides.2010.09.030 Veshchitskii AA, Musienko PE, Merkulyeva NS (2021) Distribution of calretinin-immunopositive neurons in the cat lumbar spinal cord. J Evol Biochem Phys 57:817–834. https://doi.org/10.1134/S0022093021040074 Waldvogel HJ, Faull RL, Jansen KL, Dragunow M, Richards JG, Mohler H, Streit P (1990) GABA, GABA receptors and benzodiazepine receptors in the human spinal cord: an autoradiographic and immunohistochemical study at the light and electron microscopic levels. Neuroscience 39:361-385. https://doi.org/10.1016/0306-4522(90)90274-8 Yasaka T, Tiong SYX, Hughes DI, Riddell JS, Todd AJ (2010) Populations of inhibitory and excitatory interneurons in lamina II of the adult rat spinal dorsal horn revealed by a combined electrophysiological and anatomical approach. Pain 151:475-488. https://doi.org/10.1016/j.pain.2010.08.008 Table 1 Table 1 Median values of the four-tiered semiquantitative evaluation of CART immunoreactivity in the human spinal cord: -, absent; +, weak; ++, moderate; +++, strong Cervical Thoracic Lumbar Sacral Coccygeal Laminae I-II +++ +++ +++ +++ +++ Laminae III-IV ++ ++ ++ ++ ++ Laminae V-VIII ++ ++ ++ ++ ++ Lamina IX +++ +++ +++ +++ +++ Lamina X ++ ++ ++ ++ ++ CeCv ++ IB ++ LatC + LSp ++ ++ ++ ++ ++ D +++ +++ IML +++ +++ LPrCb ++ SPrCb ++ ++ SPSy +++ CeCv: Central cervical nucleus, D: dorsal nucleus; IB: internal basilar nucleus; IML: intermediolateral nucleus; LatC: lateral cervical nucleus; LPrCb: lumbar precerebellar nucleus; LSp: lateral spinal nucleus; SPrCb: sacral precerebellar nucleus; SPSy: sacral parasympathetic nucleus. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6297031","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":441228484,"identity":"3a408bd7-b59c-4c90-81ef-f0c9a4709b75","order_by":0,"name":"Öykü Deniz Kanat","email":"","orcid":"","institution":"Istanbul Medeniyet University","correspondingAuthor":false,"prefix":"","firstName":"Öykü","middleName":"Deniz","lastName":"Kanat","suffix":""},{"id":441228486,"identity":"3c4cefd2-35d8-4afa-8e25-4db66f3c121a","order_by":1,"name":"Esra Candar","email":"","orcid":"","institution":"Ege University","correspondingAuthor":false,"prefix":"","firstName":"Esra","middleName":"","lastName":"Candar","suffix":""},{"id":441228490,"identity":"a7583c77-04f9-4d8a-8404-4b5612bb34be","order_by":2,"name":"Ibrahim Demircubuk","email":"","orcid":"","institution":"Ege University","correspondingAuthor":false,"prefix":"","firstName":"Ibrahim","middleName":"","lastName":"Demircubuk","suffix":""},{"id":441228493,"identity":"ebe741b8-618c-4cfe-a353-981c48cdcf5e","order_by":3,"name":"Ferihan Çetin","email":"","orcid":"","institution":"Istanbul Medeniyet University","correspondingAuthor":false,"prefix":"","firstName":"Ferihan","middleName":"","lastName":"Çetin","suffix":""},{"id":441228496,"identity":"6630c7f8-2421-46ef-b824-6df6f9327221","order_by":4,"name":"Gulgun Sengul","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYFAC5gYE+0EFhJbAr4URSUvCGbgWAyK1JLYRoUU+IrH5Ne8OBnt+6fZrEonzbOz6GZgP3uZh+JOPS4vhjcQ2a94zDIkz55wpk0jclpY8s4Et2ZqHwcCyAZeWGYltxrxtDAkGN3LSgFoOJxsc4DGTBmrB6TKYFnt7sJY5/5PtD/B/w6tFXiKx+TFQC+MGifRjEokNB+wMGHjY8Gox4HnYxji3TSJxxo0cZouEY8kJEofZjC3nGBjjtqU9+fCHt2029vwz0h/e+FBjZ8/f3vzwxpsKOdy2HGBgk4BENw9YUWIDM1gclwagLQ0MzB8gTPYHINIet9pRMApGwSgYqQAAkntQk+M49m0AAAAASUVORK5CYII=","orcid":"","institution":"Ege University","correspondingAuthor":true,"prefix":"","firstName":"Gulgun","middleName":"","lastName":"Sengul","suffix":""}],"badges":[],"createdAt":"2025-03-24 15:53:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6297031/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6297031/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80385018,"identity":"1842fcaa-1972-4f9d-8d24-e90f39c76d8c","added_by":"auto","created_at":"2025-04-11 09:54:07","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1508529,"visible":true,"origin":"","legend":"\u003cp\u003eTransverse section of the human spinal cord showing the distribution of CART immunoreactivity in C4 segment (left side). The diagram of cytoarchitectonic subdivisions of the C4 human spinal cord section, prepared by Sengul (2021) (right side). Scale bar indicates 100 μm. 1Sp: Rexed’s lamina I; 2SpO: Rexed’s lamina II outer part; 2SpI: Rexed’s lamina II inner part; 3Sp: Rexed’s lamina III; 4Sp: Rexed’s lamina IV; 5SpM: Rexed’s lamina V medial part; 5SpL: Rexed’s lamina V lateral part; 7Sp: Rexed’s lamina VII; 8Sp: Rexed’s lamina VIII; 10Sp: Rexed’s area X; Ax9: axial motor neurons; Bi9: biceps motoneurons of lamina IX; De9: deltoid motoneurons of lamina IX; LatC: lateral cervical nucleus; LSp: lateral spinal nucleus; Ph9: phrenic motoneurons of lamina IX; Rh9: rhomboid muscle motoneurons of lamina IX; TzSM: trapezius and sternomastoid motoneurons of lamina IX.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6297031/v1/c8c7deaa363d8aa4accb6b00.jpeg"},{"id":80385456,"identity":"0c75ae28-41f2-4f66-8172-c8eba97a24c1","added_by":"auto","created_at":"2025-04-11 10:02:07","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1635616,"visible":true,"origin":"","legend":"\u003cp\u003eTransverse section of the human spinal cord showing the distribution of CART immunoreactivity in T2 segment (left side). The diagram of cytoarchitectonic subdivisions of the C4 human spinal cord section, prepared by Sengul (2021) (right side). Scale bar indicates 100 μm. 1Sp: Rexed’s lamina I; 2SpO: Rexed’s lamina II outer part; 2SpI: Rexed’s lamina II inner part; 3Sp: Rexed’s lamina III; 4Sp: Rexed’s lamina IV; 5SpM: Rexed’s lamina V medial part; 5SpL: Rexed’s lamina V lateral part; 7Sp: Rexed’s lamina VII; 8Sp: Rexed’s lamina VIII; 10Sp: Rexed’s area X; Ax9: axial motor neurons; D: dorsal nucleus; ICl: intercalated nucleus; ICo9: intercostal muscle motoneurons of lamina IX; IML: intermediolateral nucleus; LSp: lateral spinal nucleus.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6297031/v1/d6fee36a77a16013a1159793.jpeg"},{"id":80385022,"identity":"2ea0cb07-07d1-4d35-9e56-b64d324b3364","added_by":"auto","created_at":"2025-04-11 09:54:07","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1398680,"visible":true,"origin":"","legend":"\u003cp\u003eTransverse section of the human spinal cord showing the distribution of CART immunoreactivity in L3 segment (left side). The diagram of cytoarchitectonic subdivisions of the C4 human spinal cord section, prepared by Sengul (2021) (right side). Scale bar indicates 100 μm. 1Sp: Rexed’s lamina I; 2SpO: Rexed’s lamina II outer part; 2SpI: Rexed’s lamina II inner part; 3Sp: Rexed’s lamina III; 4Sp: Rexed’s lamina IV; 5SpM: Rexed’s lamina V medial part; 5SpL: Rexed’s lamina V lateral part; 6SpM: Rexed’s lamina VI medial part; 6SpL: Rexed’s lamina VI lateral part; 7Sp: Rexed’s lamina VII; 8Sp: Rexed’s lamina VIII; 10Sp: Rexed’s area X; Ad9: adductor motoneurons of lamina IX; Ax9: axial motor neurons; IMM: intermediomedial nucleus; LSp: lateral spinal nucleus; Ps9: psoas motoneurons of lamina IX; Q9: quadriceps motoneurons of lamina IX.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6297031/v1/22f9569d4f28eb63312ecbc8.jpeg"},{"id":80385021,"identity":"dddcc6fe-ab04-4976-9329-99f92a9cb904","added_by":"auto","created_at":"2025-04-11 09:54:07","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":986757,"visible":true,"origin":"","legend":"\u003cp\u003eTransverse section of the human spinal cord showing the distribution of CART immunoreactivity in S5 segment (left side). The diagram of cytoarchitectonic subdivisions of the C4 human spinal cord section, prepared by Sengul (2021) (right side). Scale bar indicates 100 μm. 1Sp: Rexed’s lamina I; 2SpO: Rexed’s lamina II outer part; 2SpI: Rexed’s lamina II inner part; 3Sp: Rexed’s lamina III; 4Sp: Rexed’s lamina IV; 5SpM: Rexed’s lamina V medial part; 5SpL: Rexed’s lamina V lateral part; 7Sp: Rexed’s lamina VII; 8Sp: Rexed’s lamina VIII; 10Sp: Rexed’s area X; Ax9: axial motor neurons; ExA9: external anal sphincter motor neurons of lamina IX; ExU9: external urethral sphincter motoneurons of lamina IX; Gl9: gluteal motor neurons of lamina IX; LSp: lateral spinal nucleus; Pes9: pes motor neurons of lamina IX; SPSy: sacral parasympathetic nucleus.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6297031/v1/cffc6ce6a6007e4cc1311440.jpeg"},{"id":81404951,"identity":"a95b3aff-8f36-4560-9444-c83564d8ed6d","added_by":"auto","created_at":"2025-04-25 17:31:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6148685,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6297031/v1/1829bd6a-eb36-4da2-ad9e-9dcc1837c293.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anatomical distribution of cocaine- and amphetamine-regulated transcript (CART) peptide in the human spinal cord","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe cocaine- and amphetamine-regulated transcript (CART) was first identified as a messenger RNA (mRNA) that responds to cocaine and amphetamine exposure in the rat striatum (Douglas et al. 1995). Later, Thim and colleagues (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) isolated different CART peptides from various regions of the body: CART(1\u0026ndash;89) and CART(10\u0026ndash;89) from the adrenal gland, CART(42\u0026ndash;89) and CART(49\u0026ndash;89) from the hypothalamus and nucleus accumbens in rats. Over time, research has linked CART peptides to a wide range of physiological processes, including pain modulation, reward processing, sleep regulation, learning and memory, and autonomic nervous system control (Subhedar et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, despite these associations, CART\u0026rsquo;s precise physiological role remains unclear, largely due to the lack of well-defined, specific CART receptors (Stanek \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Ahmadian-Moghadam et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEarly immunohistochemical and in situ hybridization studies revealed that CART peptides are widely distributed throughout both rat and human brains. Using five polyclonal antisera, Koylu et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) detected CART peptides in key endocrine regions of the rat, including the hypothalamus, pituitary, and adrenal glands. Building on these initial findings, the same research team conducted a broader immunohistochemical study to map CART peptide distribution across the rat brain. Their results (Koylu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) showed strong CART immunoreactivity in areas such as the nucleus accumbens, centromedial amygdaloid nuclei, parabrachial nucleus, and principal inferior olivary nucleus, while regions like the entorhinal cortex and nucleus ambiguus displayed moderate immunoreactivity. Additionally, they identified CART-immunoreactive fibers and varicosities within the spinal cord, particularly in laminae I-II, the dorsolateral fasciculus, and the lateral spinal nucleus (LSp), as well as immunoreactive neurons in the intermediolateral nucleus (IML) and lamina X.\u003c/p\u003e \u003cp\u003eExpanding beyond rodent models, Hurd and Fagergren (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) examined CART mRNA expression in five human post-mortem brains using in situ hybridization. Their findings indicated CART mRNA presence in several key brain regions, including the dorsolateral prefrontal cortex, nucleus accumbens, bed nucleus of the stria terminalis, locus coeruleus, hypothalamus, and thalamus. Meanwhile, Dun et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) observed CART- immunoreactive fibers, but not cell bodies, in the sacral parasympathetic nucleus of the rat spinal cord.\u003c/p\u003e \u003cp\u003eInterestingly, while CART mRNA is absent in the rat cerebellum (Koylu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), CART peptides have been detected in climbing fibers (Press and Wall \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Further supporting the peripheral presence of CART, Kozsurek et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) identified CART-immunoreactive cell bodies and axons in the rat dorsal root ganglia. These studies collectively highlight the widespread distribution of CART peptides and suggest their involvement in diverse neural processes across different species.\u003c/p\u003e \u003cp\u003eThe CART peptides are known to coexist with several neurotransmitters in the mammalian nervous system. In the rat brain, CART peptides are found alongside γ-aminobutyric acid (GABA) and dynorphin within the substantia nigra and ventral tegmental area (Dallvechia-Adams et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Lambert et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) observed neuropeptide Y (NPY) immunoreactive varicosities surrounding CART-immunoreactive cells in the paraventricular hypothalamic nucleus. Additionally, CART peptides colocalize with calcitonin gene-related peptide (CGRP), substance P, and somatostatin (SOM) in fibers within the laminae I and II of the spinal cord, hinting at a role in nociception and other sensory functions (Kozsurek et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAn ultrastructural study by Smith et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) demonstrated synaptic interactions between CART-immunoreactive dendrites and tyrosine hydroxylase-positive axon terminals in the monkey nucleus accumbens. Using light microscopy with double labeling, Couceyro et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) showed that a subset of choline acetyltransferase (ChAT) neurons in the rat myenteric plexus also contain CART peptides. In the chinchilla claustrum, CART peptides coexist with parvalbumin (Pv), SOM, and NPY (Szalak et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, Miller et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported that approximately half of the melanin-concentrating hormone cells in the medial hypothalamus contain CART peptides.\u003c/p\u003e \u003cp\u003eWhile previous studies have explored the presence of CART peptides in rat and human brains, their distribution in the human spinal cord remains largely unknown. Understanding where these peptides are located within the spinal cord could provide valuable insights into their potential roles in the nervous system. In this study, we aimed to map the anatomical distribution of the CART peptides in the human spinal cord using immunohistochemistry. Additionally, we sought to classify the neuronal populations that exhibit CART immunoreactivity.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTissue preparation\u003c/h2\u003e \u003cp\u003eFor this study, we used five human spinal cords with no known neurological conditions, obtained from the cadaver collection at Ege University School of Medicine, Izmir, Turkiye. To prepare the specimen, the vertebral pedicles and dura mater were carefully removed to expose the spinal cord which was then immersed in a 10% formalin solution for three days. After fixation, the spinal cord was divided into 31 segments under a Leica M651 surgical operating microscope (Leica Microsystems AG, Heerbrugg, Switzerland). The segments were then placed in a 30% sucrose solution for cryoprotection before being sectioned at a thickness of 30 \u0026micro;m using a Leica CM1950 cryostat (Leica Microsystems, Wetzlar, Germany). This study was approved by the Medical Research Ethics Committee at Ege University, Izmir, Turkiye (approval number 23\u0026thinsp;\u0026minus;\u0026thinsp;2.1/T/22) and all procedures were conducted in accordance with the principles of the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eTo visualize the anatomical distribution of CART peptides in the human spinal cord, we performed free-floating immunohistochemical staining on the tissue sections. First, the sections were incubated in 0.3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 30 minutes to block endogenous peroxidase activity. This was followed by a 2-hour incubation in a blocking solution containing 10% normal goat serum (NGS; Vector Laboratories, S-1000-20) at room temperature. For CART detection, the sections were incubated for 48 hours at 4\u0026deg;C with a primary antibody against CART (Phoenix Pharmaceuticals, H-003-62, dilution 1:5000) prepared in a solution of 3% NGS, 0.3% Triton X-100, and 0.01 M PBS. Next, they were treated with a corresponding secondary antibody (Sigma-Aldrich, AP124B, dilution 1:100) in 3% NGS, and 0.3% Triton X-100 in 0.01 M PBS for 2 hours. The sections were then incubated with streptavidin peroxidase (Thermo Scientific, TS-125-HR) for 10 minutes. Finally, the reaction was developed in 3,3\u0026rsquo;-diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich, D5905) and the stained sections were coverslipped with Entellan. To assess antibody specificity, immunohistochemical stainings were conducted on several spinal cord sections by excluding either the primary or secondary antibody, and no immunoreactivity was detected in these control sections.\u003c/p\u003e\n\u003ch3\u003eImage processing and data analysis\u003c/h3\u003e\n\u003cp\u003eThe sections were scanned at 10x magnification using the Aperio Microscope Slide Scanner (Aperio AT2, Leica Biosystems, France). Image adjustments, such as brightness and contrast, were performed using Adobe Photoshop (Adobe Photoshop 7.0, Adobe Systems Incorporated, CA, USA). To determine the anatomical divisions of the spinal cord, we referenced the human spinal cord atlases by Sengul et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and Sengul (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). CART immunoreactivity in each section was evaluated semiquantitatively employing a four-tiered scale: absent (-), weak (+), moderate (++), and strong (+++). Across the analyzed sections, the median semiquantitative score was used to represent the average staining intensity. CART-immunoreactive neurons were classified based on their morphological types. Multipolar neurons were identified by their extensive dendritic arborization, featuring at least three dendrites of varying thickness. Fusiform neurons exhibited spindle-shaped somata with primary dendrites extending from opposite poles. Ovoid neurons had round somata with no distinct dendritic branching pattern. Triangular neurons displayed a characteristic soma.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn this study, we examined the anatomical distribution of CART peptides in the human spinal cord. Our analysis identified a diverse range of CART-immunoreactive neurons across all Rexed\u0026rsquo;s laminae, each exhibiting distinct morphological characteristics. The intensity of CART immunoreactivity was consistent across all laminae in the various spinal cord segments. Additionally, CART-immunoreactive fibers were widely distributed throughout the white matter. A summary of our findings, including the levels of CART immunoreactivity\u0026mdash;categorized as weak (+), moderate (++), and strong (+++)\u0026mdash;is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eLaminae I and II\u003c/h3\u003e\n\u003cp\u003eLaminae I and II displayed strong CART immunoreactivity, with significantly higher levels compared to laminae III-IV (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e ). No consistent pattern of immunoreactivity variation was observed across different spinal cord segments in these laminae. In lamina I, CART-immunoreactive cells were aligned parallel to the dorsal curvature of the dorsal horn. A subset of immunoreactive dendrites followed the contour of the dorsal horn, while others extended ventrally. In lamina II, the predominant CART-immunoreactive cell types were ovoid and fusiform neurons.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLaminae III and IV\u003c/h2\u003e \u003cp\u003eImmunohistochemical analysis revealed a population of CART-immunoreactive neurons and fibers within laminae III and IV (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The density of CART immunoreactivity in these laminae was noticeably lower than in laminae I and II. The immunoreactive neurons exhibited a range of morphological characteristics, predominantly fusiform and ovoid shapes.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLaminae V-VIII\u003c/h3\u003e\n\u003cp\u003eModerate CART immunoreactivity was detected in laminae V-VIII (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The neurons in these laminae displayed diverse morphological characteristics, with four distinct morphotypes identified: multipolar, ovoid, fusiform, and triangular. Notably, at the limb enlargements, CART-immunoreactive neurons were identified in lamina VI. Bundles of CART-immunoreactive fibers were observed extending medially from the lamina VII toward the border of lamina X.\u003c/p\u003e\n\u003ch3\u003eLaminae IX and X\u003c/h3\u003e\n\u003cp\u003eAs shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, a large number of CART-immunoreactive neurons and fibers were observed in lamina IX, exhibiting strong staining intensity, with multipolar neurons being the predominant cell type. The neurons in this region were arranged in a longitudinal pattern, forming motor columns. Lamina X neurons exhibited moderate CART immunoreactivity.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eNuclei of the human spinal cord\u003c/h2\u003e \u003cp\u003eImmunohistochemical analysis revealed CART-immunoreactive neurons and fibers in both Clarke\u0026rsquo;s nucleus (dorsal nucleus, D) and the IML. D formed a longitudinal column extending from T1 to L2 segments, with ovoid and multipolar neurons as the principal cell type (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Within the IML, CART-immunoreactive neurons were distinctly aligned, separating this nucleus from the adjacent gray matter in the T1-L2 segments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, CART-immunoreactive neurons and fibers were observed in the LSp across all spinal cord segments (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), with no significant differences in immunoreactivity between segments. In the C1\u0026ndash;C4 segments, CART-immunoreactive neurons and fibers were also detected in the lateral cervical nucleus (LatC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, immunoreactivity was more pronounced in the lateral spinal nucleus (LSp) than in the LatC. Additionally, CART immunoreactivity was observed in the internal basilar nucleus (IB) and the central cervical nucleus (CeCv) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Moderate CART immunoreactivity was detected in the lumbar precerebellar nucleus (LPrCb) within the L1\u0026ndash;L5 segments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and in the sacral precerebellar nucleus (Stilling\u0026rsquo;s sacral nucleus, SPrCb) within the S1\u0026ndash;Co1 segments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, CART-immunoreactive neurons and fibers were identified in the sacral parasympathetic nucleus (SPSy) of the S1\u0026ndash;S5 segments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCART peptides are widely expressed across various brain regions (Ahmadian-Moghadam et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The goal of this study was to determine their localization within the human spinal cord. We examined all spinal cord segments using CART immunohistochemistry, which revealed a distinct topographical organization with varying levels of immunoreactivity, ranging from weak to strong. While previous studies have mapped the distribution of CART peptides in both human and rat brains (Koylu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Hurd and Fagergren \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), no data has been available regarding their distribution in the human spinal cord. Our findings identified CART-immunoreactive neurons and fibers in multiple anatomical regions associated with motor, sensory, and autonomic functions.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLaminae I and II\u003c/h2\u003e \u003cp\u003eIn the dorsal horn, laminae I and II serve as primary termination sites -for A-delta and C-fibers (Heise and Kayalioglu \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). GABAergic neurons have been identified in these laminae (Barber et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Todd and Sullivan \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), with lamina II containing a substantial population of interneurons involved in modulating and transmitting nociceptive signals (Yasaka et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Upadhya et al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) reported an increased density of CART-immunoreactive fibers in the ventrolateral periaqueductal gray matter (PAG), the dorsal subdivision of the dorsal raphe nucleus and the locus coeruleus following neuropathic pain. Behavioral studies using intrathecal injections of the CART peptide (55\u0026ndash;102) demonstrated reduced paw withdrawal latency, suggesting a role in pain modulation (Ohsawa et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Additionally, Damaj et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) investigated the effects of CART(55\u0026ndash;102) in neuropathic and inflammatory pain models, providing evidence that CART peptides can attenuate hyperalgesia associated with chronic pain. According to Schoenen and Faull (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), human lamina I contains fusiform and multipolar neurons, while lamina II houses four distinct neuronal types\u0026mdash;stellate, curly, filamentous, and islet\u0026mdash;based on their dendritic and axonal morphology. In the present study, we defined three types of CART-immunoreactive neurons in laminae I and II: fusiform, ovoid, and multipolar.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLaminae III and IV\u003c/h2\u003e \u003cp\u003eNeurons in laminae III-V are capable of responding to C-fiber input, although C fibers primarily terminate in the superficial laminae of the spinal cord (Naim et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). A previous retrograde labeling study demonstrated that neurons in laminae I, III, and IV expressing the neurokinin 1 receptor project to the PAG, and lateral parabrachial nucleus (Al-Khater and Todd \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Additionally, GABA and CGRP immunoreactivities have been reported in human laminae III and IV (Waldvogel et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Jakab et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Human lamina III consists of two distinct neuronal populations: one characterized by an asymmetrical dendritic tree and the other by a radial dendritic tree (Schoenen and Faull \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). A distinguishing feature of lamina IV is the presence of antenna-like cells (Szent\u0026aacute;gothai and Rethelyi 1973; Schoenen and Faull \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In our study, we identified four morphotypes of CART-immunoreactive neurons: multipolar, fusiform, ovoid, and triangular.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLaminae V-VIII\u003c/h2\u003e \u003cp\u003eLamina V is more cellularly heterogeneous than lamina IV (Sengul and Watson \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Ritz and Greenspan (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1985\u003c/span\u003e) studied the morphological characteristics of neurons in lamina V of the cat sacrocaudal spinal cord, identifying multireceptive cells that respond to innocuous and noxious mechanical stimuli. These neurons had large somata with extensive dendritic arborizations in all directions, and their axons crossed into the contralateral ventral white matter. Lamina VI is found only in the cervical and lumbosacral enlargements of the spinal cord in marmoset monkeys, rhesus monkeys (Sengul et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), rats (Molander et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), and humans (Sengul \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePropriospinal neurons play a crucial role in coordinating movements across different body regions by linking motor pathways (Laliberte et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Ni et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) studied proprioceptive neurons in the mouse spinal cord using a recombinant rabies virus-based approach. Their findings identified proprioceptive neurons within laminae VII-VIII in the cervical segments and laminae IV-VIII in the thoracic segments. Additionally, Flynn et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) examined the anatomical and molecular characteristics of long descending propriospinal neurons in mice, detecting them predominantly in laminae VII and VIII. Lamina VII primarily consists of premotor interneurons that project to motor neuron pools (Sengul and Watson \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). While its neuronal population shares similarities with those in laminae VI and VIII, lamina VII is distinguished by a higher number of horizontal or oblique dendrites (Schoenen and Faull \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In our study, we observed numerous CART-immunoreactive fibers within lamina VII. Additionally, lamina VII contains Renshaw cells, which establish inhibitory connections with alpha motor neurons (Rea \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Renshaw cells have been shown to express both gephyrin and calbindin (Cb) in rat lamina VII (Carr et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). A detailed analysis of CART and Cb colocalization could provide further insight into CART\u0026rsquo;s potential role in motor regulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLaminae IX and X\u003c/h2\u003e \u003cp\u003eUnlike other laminae, lamina IX consists of a cluster of motor neuron columns embedded within laminae VII and VIII (Sengul and Watson \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In the rat spinal cord, motor neurons contain both ChAT and dopamine (Barber et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Holstege et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Moffett et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) demonstrated that CART knock-out mice exhibited reduced locomotor activity compared to wild-type mice in an open-field test, suggesting a potential role of CART in motor action. More recently, Eleftheriadis et al. (2022) reported colocalization of CART and ChAT within C-bouton synapses, further highlighting CART\u0026rsquo;s involvement in motor control. While Couceyro et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) found no CART-immunoreactive cells in the lumbar spinal cord of rats, our findings compared the presence of CART immunoreactive neurons in lamina X of the human lumbar spinal cord.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eNuclei of the human spinal cord\u003c/h2\u003e \u003cp\u003eThe D was first described by British physician Jacob Augustus Lockhart Clarke in 1851 (Clarke \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1851\u003c/span\u003e; Demircubuk et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Due to its location in the dorsal region of the spinal cord, German anatomist Benedict Stilling later proposed the term \u0026ldquo;dorsal nucleus\u0026rdquo; (Stilling \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1859\u003c/span\u003e; Demircubuk et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the human spinal cord, D is located within the medial part of laminae VII, spanning segments C8 to L2 segments (Sengul and Watson \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Immunoreactivity for substance P, methionine-enkephalin, calbindin (Cb), calretinin (Cr), parvalbumin (Pv), choline acetyltransferase (ChAT), glutamic acid decarboxylase 65/67 (GAD 65/67), and vesicular glutamate transporter 1 (VGLUT1) has been detected in neurons of the dorsal nucleus (D) (Demircubuk et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The neurons in D give rise to axons that form the dorsal spinocerebellar tract, which plays a key role in proprioceptive signal transmission to the cerebellum (Heise and Kayalioglu \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Additionally, D neurons contribute to local spinal corollary circuits involved in motor planning (Hantman and Jessell \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The IML, which houses the preganglionic sympathetic neurons, extends from C8 to L1 (Sengul and Watson \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In the pig spinal cord, colocalization of ChAT and nitric oxide synthase has been observed within the IML. Similarly, studies in the cat lumbar spinal cord reported Cr-immunoreactive neurons in the IML (Veshchitskii et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Our findings indicate that both D and IML contain CART-immunoreactive neurons. Notably, in Parkinson\u0026rsquo;s disease, Lewy bodies and neuronal loss have been documented within the IML. Further research is needed to explore potential alterations in the D and IML in neurodegenerative diseases.\u003c/p\u003e \u003cp\u003eThe LSp extends throughout the entire length of the spinal cord, whereas the LatC is restricted to upper cervical spinal cord segments (Sengul \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Numerous studies have investigated these nuclei in various species, including rats, mice, and humans (Demircubuk et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Animal studies suggest that LSp and LatC play a role in nociceptive signal transmission (Giesler Jr. et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1979\u003c/span\u003e; Olave and Maxwell \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In our study, we identified CART-immunoreactive fibers within both LSp and LatC, supporting previous hypotheses that CART peptides contribute to pain processing.\u003c/p\u003e \u003cp\u003eIn the human spinal cord, the lumbar precerebellar nucleus (LPrCb) is located within the L1\u0026ndash;L5 segments, while the sacral precerebellar nucleus (SPrCb) is found within the S1\u0026ndash;Co1 segments (Sengul \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As shown in our previous study (Demircubuk et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), neurons and fibers immunoreactive for calbindin (Cb), calretinin (Cr), parvalbumin (Pv), choline acetyltransferase (ChAT), glutamic acid decarboxylase 65/67 (GAD 65/67), and vesicular glutamate transporter 1 (VGLUT1) are present in both LPrCb and SPrCb. In the current study, CART-immunoreactive neurons and fibers were also identified in these nuclei.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eLimitations of the study\u003c/h2\u003e \u003cp\u003eThis study primarily relies on immunohistochemistry, which provides anatomical localization but does not offer direct insights into dynamic physiological processes. Future studies using complementary approaches, such as in vivo imaging or functional assays, could provide a deeper understanding of CART\u0026rsquo;s real-time activity in the spinal cord.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study offers the first detailed characterization of CART peptide distribution in the human spinal cord, highlighting its presence in key motor, sensory, and autonomic regions. The widespread but regionally distinct immunoreactivity suggests a potential role for CART in modulating spinal cord functions, including pain processing, autonomic regulation, and motor control. Further studies are needed to elucidate the functional significance of CART in these spinal circuits and its potential relevance to neurological disorders of the spinal cord.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of interest\u003c/h2\u003e \u003cp\u003eOn behalf of all authors, the corresponding author states that there is no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eO.D. K. for Methodology, formal analysis, investigation, writing \u0026ndash; original draft; E.C. for methodology, formal analysis, investigation, writing \u0026ndash; review and editing; I.D. for methodology, formal analysis, investigation, writing \u0026ndash; original draft; F.C. for conceptualization, methodology, review and editing; G.S. for conceptualization, methodology, formal analysis, writing \u0026ndash; review and editing, supervision.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe authors sincerely thank those who donated their bodies to science so that anatomical research could be performed. Results from such research can potentially increase mankind's overall knowledge that can then improve patient care. Therefore, these donors and their families deserve our highest gratitude (Iwanaga et al. 2021). The authors would like to thank the Council of Higher Education (CoHE) of Turkiye for supporting Esra Candar under \u0026ldquo;100/2000 CoHE Ph.D. Scholarship Program\u0026rdquo; in the field of \u0026ldquo;Translational Medicine\u0026rdquo;, and The Scientific and Technological Research Council of Turkey for their support of Ibrahim Demircubuk through \u0026ldquo;National Ph.D. Scholarship Program\u0026rdquo;.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmadian-Moghadam H, Sadat-Shirazi MS, Zarrindast MR (2018) Cocaine- and amphetamine-regulated transcript (CART): a multifaceted neuropeptide. Peptides 110:56-77. https://doi.org/10.1016/j.peptides.2018.10.008\u003c/li\u003e\n\u003cli\u003eAl-Khater KM, Todd AJ (2009) Collateral projections of neurons in laminae I, III, and IV of rat spinal cord to thalamus, periaqueductal gray matter, and lateral parabrachial area. J Comp Neurol 515:629-646. https://doi.org/10.1002/cne.22081\u003c/li\u003e\n\u003cli\u003eBarber RP, Phelps PE, Houser CR, Crawford GD, Salvaterra PM, Vaughn JE (1984) The morphology and distribution of neurons containing choline acetyltransferase in the adult rat spinal cord: an immunocytochemical study. J Comp Neurol 229:329-346. https://doi.org/10.1002/cne.902290305\u003c/li\u003e\n\u003cli\u003eBarber RP, Vaughn JE, Roberts E (1982) The cytoarchitecture of GABAergic neurons in rat spinal cord. Brain Res 238:305-328. https://doi.org/10.1016/0006-8993(82)90107-X\u003c/li\u003e\n\u003cli\u003eCarr PA, Alvarez FJ, Leman EA, Fyffe RE (1998) Calbindin D28k expression in immunohistochemically identified Renshaw cells. Neuroreport 9:2657-2661. https://doi.org/10.1097/00001756-199808030-00043\u003c/li\u003e\n\u003cli\u003eClarke JAL (1851) XXVI. Researches into the structure of the spinal chord. Phil Trans R Soc 141:607\u0026ndash;621. https://doi.org/10.1098/rstl.1851.0029\u003c/li\u003e\n\u003cli\u003eCouceyro P, Paquet M, Koylu E, Kuhar MJ, Smith Y (1998) Cocaine- and amphetamine-regulated transcript (CART) peptide immunoreactivity in myenteric plexus neurons of the rat ileum and co-localization with choline acetyltransferase. Synapse 30:1-8. https://doi.org/10.1002/(SICI)1098-2396(199809)30:1\u0026lt;1::AID-SYN1\u0026gt;3.0.CO;2-7\u003c/li\u003e\n\u003cli\u003eCouceyro PR, Koylu EO, Kuhar MJ (1997) Further studies on the anatomical distribution of CART by in situ hybridization. J Chem Neuroanat 12:229-241. https://doi.org/10.1016/s0891-0618(97)00212-3\u003c/li\u003e\n\u003cli\u003eDallvechia-Adams S, Kuhar MJ, Smith Y (2002) Cocaine- and amphetamine-regulated transcript peptide projections in the ventral midbrain: colocalization with gamma-aminobutyric acid, melanin-concentrating hormone, dynorphin, and synaptic interactions with dopamine neurons. J Comp Neurol 448:360-372. https://doi.org/10.1002/cne.10268\u003c/li\u003e\n\u003cli\u003eDamaj MI, Zheng J, Martin BR, Kuhar MJ (2006) Intrathecal CART (55-102) attenuates hyperlagesia and allodynia in a mouse model of neuropathic but not inflammatory pain. Peptides 27:2019-2023. https://doi.org/10.1016/j.peptides.2005.09.019\u003c/li\u003e\n\u003cli\u003ede Lanerolle NC, LaMotte CC (1982) The human spinal cord: substance P and methionine-enkephalin immunoreactivity. J Neurosci 2:1369\u0026ndash;1386. https://doi.org/10.1523/JNEUROSCI.02-10-01369.1982\u003c/li\u003e\n\u003cli\u003eDemircubuk I, Candar E, Sengul G (2023) Jacob Augustus Lockhart Clarke (1817-1880). J Neurol 270:592-593. https://doi.org/10.1007/s00415-022-11384-5\u003c/li\u003e\n\u003cli\u003eDemircubuk I, Candar E, Sengul G (2023) The seminal contributions of Benedict Stilling (1810-1879) to neuroanatomy. Childs Nerv Syst 39:1985-1994. https://doi.org/10.1007/s00381-022-05512-9\u003c/li\u003e\n\u003cli\u003eDemircubuk I, Candar E, Sengul G (2024) The historical evolution of topographical mapping and nomenclature of the lateral cervical and lateral spinal nuclei. World Neurosurg 186:62-67. https://doi.org/10.1016/j.wneu.2024.03.079\u003c/li\u003e\n\u003cli\u003eDemircubuk I, Candar E, Sengul G (2025) Anatomical and neurochemical organization of the dorsal, lumbar precerebellar and sacral precerebellar nuclei in the human spinal cord. Ann Anat 259:152390. https://doi.org/10.1016/j.aanat.2025.152390\u003c/li\u003e\n\u003cli\u003eDouglass J, McKinzie AA, Couceyro P (1995) PCR differential display identifies a rat brain mRNA that is transcriptionally regulated by cocaine and amphetamine. J Neurosci 15:2471-2481. https://doi.org/10.1523/JNEUROSCI.15-03-02471.1995\u003c/li\u003e\n\u003cli\u003eDun SL, Chianca DA Jr, Dun NJ, Yang J, Chang JK (2000) Differential expression of cocaine- and amphetamine-regulated transcript-immunoreactivity in the rat spinal preganglionic nuclei. Neurosci Lett 294:143-146. https://doi.org/10.1016/s0304-3940(00)01575-5\u003c/li\u003e\n\u003cli\u003eEleftheriadis PE, Pothakos K, Sharples SA, Apostolou PE, Mina M, Tetringa E, Tsape E, Miles GB, Zagoraiou L (2023) Peptidergic modulation of motor neuron output via CART signaling at C bouton synapses. Proc Natl Acad Sci USA 120:e2300348120. https://doi.org/10.1073/pnas.2300348120\u003c/li\u003e\n\u003cli\u003eFlynn JR, Conn VL, Boyle KA, Hughes DI, Watanabe M, Velasquez T, Goulding MD, Callister RJ, Graham BA (2017) Anatomical and molecular properties of long descending propriospinal neurons in mice. 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J Neuroendocrinol 9:823-833. https://doi.org/10.1046/j.1365-2826.1997.00651.x\u003c/li\u003e\n\u003cli\u003eKozsurek M, Luk\u0026aacute;csi E, Fekete C, Wittmann G, R\u0026eacute;thelyi M, Pusk\u0026aacute;r Z (2007) Cocaine- and amphetamine-regulated transcript peptide (CART) is present in peptidergic C primary afferents and axons of excitatory interneurons with a possible role in nociception in the superficial laminae of the rat spinal cord. Eur J Neurosci 26: 1624-1631. https://doi.org/10.1111/j.1460-9568.2007.05789.x\u003c/li\u003e\n\u003cli\u003eLaliberte AM, Goltash S, Lalonde NR, Bui TV (2019) Propriospinal neurons: essential elements of locomotor control in the intact and possibly the injured spinal cord. Front Cell Neurosci 13:512. https://doi.org/10.3389/fncel.2019.00512\u003c/li\u003e\n\u003cli\u003eLambert PD, Couceyro PR, McGirr KM, Dall Vechia SE, Smith Y, Kuhar MJ (1998) CART peptides in the central control of feeding and interactions with neuropeptide Y. Synapse 29:293-298. https://doi.org/10.1002/(SICI)1098-2396(199808)29:4\u0026lt;293::AID-SYN1\u0026gt;3.0.CO;2-0\u003c/li\u003e\n\u003cli\u003eMiller PA, Williams-Ikhenoba JG, Sankhe AS, Hoffe BH, Chee MJ (2024) Neuroanatomical, electrophysiological, and morphological characterization of melanin-concentrating hormone cells coexpressing cocaine- and amphetamine-regulated transcript. J Comp Neurol 532:e25588. https://doi.org/10.1002/cne.25588\u003c/li\u003e\n\u003cli\u003eMoffett M, Stanek L, Harley J, Rogge G, Asnicar M, Hsiung H, Kuhar M (2006) Studies of cocaine- and amphetamine-regulated transcript (CART) knockout mice. Peptides 27:2037-2045. https://doi.org/10.1016/j.peptides.2006.03.035\u003c/li\u003e\n\u003cli\u003eMolander C, Xu Q, Grant G (1984) The cytoarchitectonic organization of the spinal cord in the rat. I. The lower thoracic and lumbosacral cord. 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J Neurosci 34:9404-9417. https://doi.org/10.1523/JNEUROSCI.1771-14.2014\u003c/li\u003e\n\u003cli\u003eOhsawa M, Dun SL, Tseng LF, Chang J, Dun NJ (2000) Decrease of hindpaw withdrawal latency by cocaine- and amphetamine-regulated transcript peptide to the mouse spinal cord. Eur J Pharmacol 399:165-169. https://doi.org/10.1016/s0014-2999(00)00374-5\u003c/li\u003e\n\u003cli\u003eOlave MJ, Maxwell DJ (2004) Axon terminals possessing alpha2C-adrenergic receptors densely innervate neurons in the rat lateral spinal nucleus which respond to noxious stimulation. Neuroscience 126:391-403. https://doi.org/10.1016/j.neuroscience.2004.03.049\u003c/li\u003e\n\u003cli\u003ePress DA, Wall MJ (2008) Expression of cocaine- and amphetamine-regulated transcript (CART) peptides at climbing fibre-Purkinje cell synapses in the rat vestibular cerebellum. Neuropeptides 42:39-46. https://doi.org/10.1016/j.npep.2007.11.001\u003c/li\u003e\n\u003cli\u003eRea P (2016) Chapter 1 - Overview of the nervous system. 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Peptides 32:317-326. https://doi.org/10.1016/j.peptides.2010.09.030\u003c/li\u003e\n\u003cli\u003eVeshchitskii AA, Musienko PE, Merkulyeva NS (2021) Distribution of calretinin-immunopositive neurons in the cat lumbar spinal cord. J Evol Biochem Phys 57:817\u0026ndash;834. https://doi.org/10.1134/S0022093021040074\u003c/li\u003e\n\u003cli\u003eWaldvogel HJ, Faull RL, Jansen KL, Dragunow M, Richards JG, Mohler H, Streit P (1990) GABA, GABA receptors and benzodiazepine receptors in the human spinal cord: an autoradiographic and immunohistochemical study at the light and electron microscopic levels. Neuroscience 39:361-385. https://doi.org/10.1016/0306-4522(90)90274-8\u003c/li\u003e\n\u003cli\u003eYasaka T, Tiong SYX, Hughes DI, Riddell JS, Todd AJ (2010) Populations of inhibitory and excitatory interneurons in lamina II of the adult rat spinal dorsal horn revealed by a combined electrophysiological and anatomical approach. Pain 151:475-488. https://doi.org/10.1016/j.pain.2010.08.008\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMedian values of the four-tiered semiquantitative evaluation of CART immunoreactivity in the human spinal cord: -, absent; +, weak; ++, moderate; +++, strong\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCervical\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThoracic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLumbar\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSacral\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCoccygeal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLaminae I-II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLaminae III-IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLaminae V-VIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLamina IX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+++\u003c/p\u003e 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\u003cp\u003eLPrCb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSPrCb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSPSy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eCeCv: Central cervical nucleus, D: dorsal nucleus; IB: internal basilar nucleus; IML: intermediolateral nucleus; LatC: lateral cervical nucleus; LPrCb: lumbar precerebellar nucleus; LSp: lateral spinal nucleus; SPrCb: sacral precerebellar nucleus; SPSy: sacral parasympathetic nucleus.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CART peptide, chemoarchitecture, neurochemistry, neuropeptide, spinal cord","lastPublishedDoi":"10.21203/rs.3.rs-6297031/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6297031/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe cocaine- and amphetamine-regulated transcript (CART) peptide, a neuropeptide highly expressed in the central nervous system, is involved in various physiological processes, including pain modulation, reward, learning, and memory. While previous studies have examined the distribution of CART peptides in the rat and human brain, no data exist on their distribution in the human spinal cord. Therefore, we investigated the localization of CART peptides in the human spinal cord using immunohistochemistry. Our analysis revealed dense CART-immunoreactive fibers and varicosities in the anterior, lateral, and dorsal funiculi of the white matter along the entire spinal cord. CART-immunoreactive neurons were identified in the Rexed\u0026rsquo;s laminae (strong immunoreactivity in laminae I-II and IX, and moderate immunoreactivity in laminae III-VIII and X. Strong CART immunoreactivity was observed in the dorsal (Clarke), intermediolateral and sacral parasympathetic nuclei, and moderate in the internal basilar, lateral spinal and lateral cervical, central cervical, and lumbar and sacral precerebellar nuclei. The widespread but regionally varied immunoreactivity suggests a potential role for CART in modulating spinal cord functions, including pain processing, autonomic regulation, and motor control.\u003c/p\u003e","manuscriptTitle":"Anatomical distribution of cocaine- and amphetamine-regulated transcript (CART) peptide in the human spinal cord","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-11 09:54:02","doi":"10.21203/rs.3.rs-6297031/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fcfe8173-0c41-499e-a887-18a1441fd67e","owner":[],"postedDate":"April 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-25T17:23:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-11 09:54:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6297031","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6297031","identity":"rs-6297031","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}