Functional Mapping of the Molluscan Brain Guided by Synchrotron X-Ray Tomography

Neurology Life Sciences Bioengineering Medicine Biophysics
synchrotron X-ray tomography molluscan neural circuit brain atlas

Academic Background

The field of neuroscience has long been dedicated to understanding how neural circuits generate and control complex behaviors. Although simple model organisms, such as mollusks, crustaceans, and annelids, have provided valuable models for research due to their accessible nervous systems and large characteristic neurons, in many cases, the understanding of neural circuits is constrained by the lack of detailed brain maps. Particularly for mollusks, despite their brains being composed of morphologically consistent and functionally interrogable neurons, the total number of neurons, organizational principles, and detailed neuron-level maps in their nervous systems remain unclear. These issues limit systematic studies of neural circuit functions.

To address this challenge, this study utilized Synchrotron X-ray Tomography (SXRT) to perform high-resolution imaging of the brain of the classic molluscan model Lymnaea stagnalis, constructing a detailed 3D atlas of its feeding circuit and using this atlas to guide the identification and functional characterization of key neuron types. This study not only provides new insights into the neural circuits of mollusks but also offers a generalizable method for central nervous system (CNS) atlas construction in other model organisms.

Source of the Paper

This research was jointly conducted by Michael Crossley, Anna Simon, Shashidhara Marathe, Christoph Rau, Arnd Roth, Vincenzo Marra, and Kevin Staras, with the research team hailing from the University of Sussex (UK), University College London, and the UK National Synchrotron Light Source Facility (Diamond Light Source). The paper was published on February 27, 2025, in PNAS (Proceedings of the National Academy of Sciences), titled “Functional mapping of the molluscan brain guided by synchrotron x-ray tomography”.

Research Workflow

1. Synchrotron X-ray Tomography Imaging

The research team first isolated intact brains from adult Lymnaea stagnalis and embedded them in resin. The samples were then sent to the UK National Synchrotron Light Source Facility and imaged using the I13-2 beamline. Through this method, the research team obtained 3D image stacks of the entire CNS, with resolution sufficient to display individual neuron cell bodies and the arrangement of their major nerve bundles.

2. 3D Reconstruction and Neuron Annotation

To reveal the 3D structure of the Lymnaea brain, the research team selected representative samples for image segmentation, labeling the 11 independent ganglia and their major nerve tracts. Subsequently, the team focused on the buccal ganglia (cheek ganglia) where the feeding circuit resides, using semi-automatic segmentation techniques to annotate cell bodies and construct a complete 3D model of the cheek ganglia. Through this detailed annotation, the research team accurately estimated the total number of neurons in the cheek ganglia, finding that they contain 1,099 neurons—a figure three times higher than previous estimates.

3. Functional Characterization of Neurons

Based on the constructed 3D atlas, the research team performed functional characterization of key neurons. Using intracellular recording and fluorescent dye filling, the team identified and characterized three key neuron types: - Dine: A command-like neuron located inside the cheek ganglia, capable of driving feeding behavior by activating the feeding command center in the brain. - N2M: A bipolar neuron located on the ventral side of the cheek ganglia, playing a key role in the feeding central pattern generator (CPG), able to modulate motor neuron activity via electrical coupling. - B12: A motor neuron located on the posterior side of the cheek ganglia, activated only during ingestion behavior and inhibited during egestion.

4. Construction of a Functional CNS Atlas

The research team used SXRT imaging and electrophysiological data to construct a functional CNS atlas of Lymnaea, annotating the positions, morphologies, and functional properties of 36 major feeding circuit neurons. This atlas provides the research community with a scalable resource that can help researchers more accurately identify and record specific neuron types.

Main Results

  1. Total Number of Neurons and Organizational Structure: The study found that the cheek ganglia contain 1,099 neurons, three times higher than previous estimates. Additionally, the study revealed a relationship between neuron volume and its position within the ganglion: larger cell bodies tend to be located on the dorsal surface of the ganglion, while smaller cell bodies are located inside the ganglion, with cell body volume gradually decreasing with increasing depth.

  2. Function of Dine Neurons: Dine neurons were identified as command-like neurons capable of driving feeding behavior by activating the feeding command center in the brain. Dine neurons are closely related to sensory input and can initiate the feeding circuit upon detecting food.

  3. Function of N2M Neurons: N2M neurons play a key role in the feeding CPG and can modulate motor neuron activity through electrical coupling. The study also found that subthreshold depolarization of N2M neurons is sufficient to drive action potentials in motor neurons.

  4. Function of B12 Neurons: B12 neurons are activated only during ingestion behavior and are inhibited during egestion. The discovery of this behavior-specific motor neuron indicates that certain neuron types in the feeding circuit are recruited differently in different behaviors.

  5. Functional CNS Atlas: The research team constructed a functional CNS atlas of Lymnaea, annotating the positions, morphologies, and functional properties of 36 major feeding circuit neurons. This atlas provides the research community with a scalable resource that can help researchers more accurately identify and record specific neuron types.

Conclusions and Significance

This study successfully constructed a 3D atlas of the Lymnaea stagnalis brain using synchrotron X-ray tomography and identified and characterized three key neuron types based on this atlas. These findings not only update our understanding of the feeding circuit’s function but also provide a generalizable method for CNS atlas construction in other model organisms. Additionally, the organizational principles of neurons revealed in the study offer new insights into the evolution of brain design.

Research Highlights

  1. Innovative Imaging Technology: This study is the first to apply synchrotron X-ray tomography to 3D imaging of molluscan brains, providing a new method for rapidly constructing cellular atlases of large-scale nervous systems.
  2. Revision of Total Neuron Count: The study revealed that the cheek ganglia contain 1,099 neurons, three times higher than previous estimates, indicating that the complexity of these structures has been greatly underestimated.
  3. Identification of Key Neurons: The study identified and characterized three key neuron types, including the command-like neuron Dine, the CPG interneuron N2M, and the behavior-specific motor neuron B12, updating our understanding of the feeding circuit.
  4. Construction of a Functional CNS Atlas: The research team constructed a functional CNS atlas of Lymnaea, providing the research community with a scalable resource that can help researchers more accurately identify and record specific neuron types.

Other Valuable Information

The research team also developed an image-sharing system based on the WebKnossos platform, allowing researchers to quickly browse and annotate 3D image stacks, further promoting the sharing and comparative study of brain atlases. Additionally, the organizational principles of neurons revealed in the study provide new insights into the evolution of brain design, suggesting that the relationship between neuron volume and its position within the ganglion may have operational advantages, minimizing wiring length and brain volume.