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University of Graz Bothe, Maximilian, PhD Research
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Research

Neuroanatomy of the spinal cord

backfill of dorsal and ventral root in the rattlesnake
Double staining of motoneurons (magenta) and sensory terminals (green) backfilled via whole nerve stainings of ventral and dorsal roots of the western diamondback rattlesnake (Crotalus atrox).

Semi-intact, in-vitro tissue can be used to perform staining of whole spinal nerves. Such staining allows to trace neurons that project through the respective nerves, including their dendritic and terminal fields. The data can be used to describe the connectivity and distribution e.g., of sensory input- and motor output layers in the spinal cord. The displayed image shows a staining of the sensory input- and the output layers of the spinal cord of a rattlesnake. Through tracer application on ventral and dorsal roots, motoneurons (magenta) and terminals of sensory neurons (green) were visualized.

Pectoral motor pool organization in teleosts

Injections of neural tracers into specific muscles can be used for targeted backfills of motoneurons that innervate them. In combination with investigations of motoneuron electrophysiology, this gives important insights into the anatomical and physiological organization of motor pools within the spinal cord. One of my research interests lies in the comparative analysis of such data recorded from different teleosts that perform specialized pectoral motor behaviors. The insight gained from such comparative studies can provide important learnings on the organizational principles of motor pools that allow for the targeted activtion of specific muscles by the central nervous system to execute different motor behaviors.

zebrafish abductor motoneurons
Motoneurons backfilled via tracer injection into the pectoral abductor muscle of an adult zebrafish (Danio rerio).

µCT and MRI imaging of teleost musculoskeletal systems

prelim_µCT_mudskipper
Preliminary µCT scan, depicting skeletal elements of an indian mudskipper (Periophthalmus novemradiatus). Image aquisition: T. Jonsson.
prelim_µCT_mudskipper_iodine
Preliminary µCT scan, depicting muscle and other soft tissue of an indian mudskipper (Periophthalmus novemradiatus). Image aquisition: T. Jonsson.
prelim_musculoskeletal reconstruction_PN
Threshold-based volumetric reconstruction of a preliminary scan of an indian mudskipper (Periophthalmus novemradiatus) stained with iodine solution.

The University of Graz offers fascilities and experienced staff to perform micro-computed tomography (µCT) and magnetic resonance imaging (MRI) of many types of samples. Combining µCT and MRI enables detailed reconstructions of musculoskeletal systems of fish. Unstained specimen can be scanned by means of µCT to provide images of the skeleton. Incubation of specimen in iodine-solutions such as Lugol’s, allows for detailed µCT scans of soft tissue. This is due to the incorporation of iodine into soft tissues which greatly enhances tissue contrast for µCT scans. MR imaging provides additional information, for example, on muscular structure by methods like diffusion tensor imaging (DTI). Posthoc aligment allows for an integrated analysis of the different scans and musculoskeletal modelling. Open source software packages like 3D slicer can be used to extract the 3D volume of individual structures of interest from scans by means of different approaches, including manual and threshold-based segmentation.

zebrafish pectoral MRI
MRI scan of the pectoral muscles and fins of a juvenile zebrafish (Danio rerio). Image aquistion: C. Diwoky.

Behavioral tracking

fin tip tracking marbled hatchet fish
Video of two marbled hatchet fish with automatically tracked fin tips. Tracking was performed using DeepLabCut.

Markerless motion tracking

To allow for a thorough interpretation of experimental results from investigations of neural networks that underlie locomotor behavior of animals, it is important to integrate data on neurophysiological and anatomical characteristics of motor networks with data on the actual execution of the motor behaviors they control. In recent years, great progress has been made in the development of markerless motion tracking: a ready to use software package for such tracking analysis is DeepLabCut. It provides a ready to use interface that allows researchers to train neural networks to perform reliable and fast tracking of predefined body parts from video recordings. Tracking data is afterwards available for further analysis.

To analyze the kinematics of motor behaviors, I record freely moving animals and use DeepLabCut for automated tracking of bodyparts involved in the execution of locomotor patterns. The integrated analysis of motor behavior kinematics, associated musculoskeletal systems and anatomical and physiological characteristics of neural networks underlying the execution of motor behaviors, provides great chances to get deeper insights into the adaptability of motor programs and which anatomical and physiological organization principles are associated with it.

To the right, you can see data from a preliminary analysis of locomotor behaviors of an african mudskipper while swimming under water (left panel) and while performing crutching locomotion on land (right panel). For swimming, the amplitude of motion of tracked bodyparts increases the further caudal they lie. Also note the phase shift of undulatory motion along the rostrocaudal body axis, indicating sequencial muscle activation. The right panel shows the execution of one "step" of the animal on land. Here, markers are set to track the pectoral and pelvic fin, as well as a point on the upper back of the animal.

ms_prelim_tracking
An african mudskipper (Periophthalmus barbarus) tracked during swimming (left panel) and walking on land (right panel). Click on image to enlarge.

Electrophysiology

Using patch clamp recordings of motoneurons allows for detailed investigations of their electrophysiological characteristics. Manipulation e.g., by means of pharmacologically blocking specific types of ion channels, allows for insights into the underlying molecular basis of motoneuron electrophysiological properties.

rattlesnake_spinal_MNs
Precision of action potential generation in motoneurons from rattlesnake spinal cord transmitting CPG patterns for locomotion and rattling is significantly different.
Bothe, M. S., et al. (2024). Timing and precision of rattlesnake spinal motoneurons are determined by the KV72/3 potassium channel. Current Biology, 34(2), 286-297.e5., licence: CC BY 4.0
rattlesnake_pharma
Pharmacological manipulation of spinal motorneurons controlling locomotion and rattling in rattlesnakes reveals a major role of KV72/3 potassium channels in the precise generation of action potentials in these motoneurons.
Bothe, M. S., et al. (2024). Timing and precision of rattlesnake spinal motoneurons are determined by the KV72/3 potassium channel. Current Biology, 34(2), 286-297.e5., licence: CC BY 4.0
hatchetfish_MNs
Hatchet fish pectoral motoneurons can be divided in two classes depending on their electrophysiological properties: small "integrator" neurons which fire action potential at lower rheobase and with less precise timing and bigger "onsetter" neurons that show higher rheobase values and fire action potential with higher temporal precision. Both neuron types are likely important to control kinematically very different types of motor behaviors used by the hatchet fish.
*Gutjahr, R., *Bothe, M. S., et al. (2024). Diversification of pectoral control through motor pool extension. Proceedings of the National Academy of Sciences, 121(49). *Equal contribution. Licence: CC BY-NC-ND 4.0

Contact

Maximilian S. Bothe PhD

maximilian.bothe(at)uni-graz.at

Department of Biology
Universitätsplatz 2
8010 Graz - Austria

ORCID: 0009-0008-1628-7727

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