Sitting at the bottom of the brain is a thumb-sized structure called the brainstem that connects the organ to the spinal cord.1 It has crucial functions in regulating breathing, heart rate, consciousness, and sleep, and it relays information between the brain regions and the body.
“It’s a huge controller of the brain,” said Mihail Bota, a neuroanatomist at the Indian Institute of Technology, Madras (IITM). “Without it, it’s impossible to live.”
Indeed, damage to the brainstem is linked with conditions like Parkinson’s and Alzheimer’s diseases.2 Moreover, researchers hypothesize that neuronal loss in the brainstem during aging could contribute to neurofunctional decline.3
However, despite the vital role of the brainstem, scientists do not fully understand its complete anatomy. One of the hurdles to this has been technical, said Mohanasankar Sivaprakasam, a researcher focused on healthcare technology at IITM.
“The brainstem is very heterogeneous and very densely packed,” said Sivaprakasam. “To do [immunohistochemistry] reliably across the entire brain stem, image it properly, digitize it, and then put it all back together…is a pretty tough technological and engineering challenge,” he explained. But, a few years ago, Sivaprakasam, Bota, and their colleagues decided to tackle it.
Recently, in a non-peer reviewed preprint, their team introduced a three-dimensional (3D) human brainstem atlas that integrates data from magnetic resonance imaging (MRI), histology, and immunohistochemistry.4 The freely available online platform, called the Atlas of Neurochemical Characterization of the Human Brainstem with 3D Reconstruction (ANCHOR), offers a glimpse into the prenatal, childhood, and adult brainstem, providing a resource for research and clinical purposes.
“[This] is a nice contribution towards a better knowledge of the brainstem,” said Martin Parent, a neuroscientist at the CERVO Brain Research Center, who was not involved in the work. “It's…really important work [since] we don't know that much about the brainstem.”
Quanxin Wang, a neuroanatomist at Allen Institute, who was not involved in developing ANCHOR, agreed. “[It is] quite a milestone of work for the field,” he said.
The researchers constructed ANCHOR using brainstem sections showing the block-face image, Nissl staining, immunohistochemistry, and anatomical annotations.
Sudha Gopalakrishnan Brain Centre, IIT Madras
Mapping an Overlooked Brain Structure
The project began when Sivaprakasam, Bota, and their colleagues started mapping human brain structures to develop a brain atlas.5 “While we were doing that, we noticed that the brainstem as a structure has been ignored; it's not been studied deeply despite being so critical,” said Sivaprakasam, which prompted them to examine this structure across the human lifespan.
They obtained three postmortem brain samples—one each from a 25-week-old fetus, a nine-year-old child, and a 54-year-old adult—from hospitals in South India. They processed the samples as per a protocol they had previously validated and subjected the specimens to MRIs.6
To dig deeper, the researchers sliced the brainstem samples into thin sections to peek at them under the microscope using histological and immunohistochemical staining. Overlaying images obtained via the different approaches revealed the organization of the brainstem at resolutions ranging from millimeters to micrometers.
Finally, the researchers annotated their atlas by spatially mapping cell types into the brainstem’s 3D structure. “[Annotation is] basically creating maps for each section,” said Bota, explaining that it is comparable to cutting a globe into thin slices to identify country borders and reconstruct geographical features like mountains or rivers in each section.
Since their atlas examines the brainstem at a high resolution for the first time, they had to annotate the different cell types manually using the scans and microscopy images because “AI has not seen this before,” said Sivaprakasam. Of the 800 sections they obtained, the researchers annotated about 200 manually.
This process allowed them to observe more than 200 clusters of neuronal cell bodies in the brainstem. Peeking into the structure also helped them identify a bundle of nerve fibers that had previously been observed only in the African wild dog. The atlas also offered important clues about the distribution of catecholaminergic neurons that synthesize and release catecholamine neurotransmitters.
Looking at the fruits of this painstaking process was “more relief and excitement rather than [just] excitement,” said Sivaprakasam.
ANCHOR online viewer enables users to transition from gross brain structures in the MRI to features at the cellular level.
Sudha Gopalakrishnan Brain Centre, IIT Madras
ANCHOR Atlas: Implications and Future Work
While other scientists have developed atlases of the brainstem, combining multiple modalities through MRI and microscopy “is the strength of the paper,” said Wang. “[They] have very rich information.” He added that the new atlas is a valuable tool for both scientific researchers and doctors: In addition to offering important insights about the brainstem’s neurochemical architecture, it could also guide treatments.
“It's really important because people do surgery in the brainstem; you don't want to hit some important structure when you do surgery,” agreed Parent. He noted that this atlas could also provide information about the exact location electrodes could be implanted for deep brain stimulation to treat specific conditions.
Wang noted that annotating more sections would be valuable since it would offer more information on how the brainstem develops and changes during aging. Parent added that at the moment, the atlas has only one sample of each age group and that having more age- and sex-matched samples would give a clearer picture of the brainstem.
However, both Wang and Sivaprakasam said that one sample is a good representative of age-matched brainstems. “The heterogeneity is within the brainstem and across age groups, not necessarily across different brains in the same age group,” explained Sivaprakasam. While they plan to image more samples, these will focus on different brain-related conditions now that they have established an atlas with neurotypical cases.
Even as they continue to improve the resolution of the atlas to include omics data to develop molecular maps along with the neurochemical map, Bota and Sivaprakasam said that they are excited to see how others in the field use this atlas. “There [are] so many discoveries waiting to happen,” said Sivaprakasam.
- Rushmore RJ, et al. 3D exploration of the brainstem in 50-micron resolution MRI. Front Neuroanat. 2020;14:40.
- Grinberg LT, et al. Brainstem: Neglected locus in neurodegenerative diseases. Front Neurol. 2011;2:42.
- Bouhrara M, et al. Maturation and degeneration of the human brainstem across the adult lifespan. Aging. 2021;13(11):14862-14891.
- Bota M, et al. ANCHOR: Atlas of neurochemical characterization of the human brainstem with 3d reconstruction. bioRxiv. 2026.06.03.727794
- Verma R, et al. DHARANI: A 3D developing human-brain atlas resource to advance neuroscience internationally integrated multimodal imaging and high-resolution histology of the second trimester. J Comp Neurol. 2025;533(2):e70006.
- James RI, et al. A standardized protocol for the safe retrieval of infectious postmortem human brain for studying whole-brain pathology. Am J Forensic Med Pathol. 2023;44(4):303-310.
