In vivo experiments are performed in the live mouse. In our lab, we utilize two main forms of behavioral in vivo recording: kinematics and muscle electromyogram (EMG) recordings.
We collect kinematic data by placing reflective markers on multiple joints and segments (pelvis, hip, knee, ankle, and toe) in the hindlimb of the mouse. Infrared cameras detect these markers in our motion capture system (Vicon Motion Systems Ltd.), in which the software records the movement of these markers in three dimensions. Thus, as the mouse takes steps, we are able to retroactively reconstruct and analyze leg movements.
Muscle EMG Data
We collect EMG data by building fine wire electrodes that can be temporarily inserted into an animal’s muscle. The electrodes pick up electrical signals caused by muscle activation, and by using data acquisition and analysis software (Spike2, CED), we can record and analyze these muscle activities. We can also chronically implant electrodes in up to 8 muscles in order to measure and record EMG data over time in the same mouse (PMID: 23369875)
We use these techniques in studying a variety of different behaviors. For example, mice are run on a treadmill with both the reflective markers and the EMG electrodes in place. Thus, simultaneous data can be collected and compared to maximize our ability to quantify and characterize gross motor function in these animals. We also use these techniques to quantify behaviors such as motor unit recordings (PMID: 24805075) or evoked spasms tested through with our flexor withdrawal method (see above).
Gross Behavioral Recovery Scores
In addition to the physiological data, we measure behavioral movement scores using two established and standardized scales, the Basso Mouse Scale (BMS) (PMID: 16689667) and the Basso, Beattie, and Bresnahan (BBB) scale modified for the mouse (PMID: 11893021). These scales allow us to quantify how much volitional movement recovery the animal has gained since its SCI.
Paralysis is a well-known result of spinal cord injury, but most people with SCI additionally develop involuntary muscle contractions that further prohibit mobility. After SCI, modifications occur such that a sensory input to the limb can be amplified within the spinal cord and result in an abnormal motor output. These involuntary contractions, or spasms, are one of the main foci of our research. We can test these spasms by utilizing the flexor withdrawal reflex. A sensory input is administered to the bottom of a mouse’s foot which elicits a flexor withdrawal reflex and results in a prolonged hindlimb muscle contraction. This motor output is measured using the EMG electrode recording technique mentioned above.
In vitro experiments are performed outside of a normal biological context; in this case, our experiments occur outside of a physiologically intact animal. We utilize three main forms of in vitro recording: sacral cord root recording, extracellular interneuron recording, and intracellular motor neuron recording.
Sacral Cord Reflex Testing
After doing our in vivo behavioral testing, we remove the sacral part of the spinal cord with dorsal and ventral roots and nerves attached. This spinal section is kept artificially alive by perfusing with oxygenated artificial cerebrospinal fluid. The cord is mounted onto a circuit board with the nerve roots attached to metal electrodes. These electrodes deliver electrical stimulation to the dorsal, sensory roots, which elicits a reflex and produces a motor output from the ventral roots. Additionally, we can include different experimental drugs in the perfusion solution. This enables us to test their effects on motor neuron output.
Extracellular Interneuron Recording
In the same sacral cord prep, we are able to use sharp glass electrodes for extracellular recordings. This enables us to characterize the changes in electrical properties in the extracellular space as interneurons fire. Interneurons can regulate among sensory signals, supraspinal signals, and output motor signals, so understanding their regulatory roles is important for understanding cellular changes in the spinal cord. (PMID: 27486104 PMCID: PMC5144700 [Available on 2017-10-01])
Intracellular Motoneuron Recording
In the sacral cord preparation, we are able to do intracellular recordings in motoneurons. The motoneuron integrates inputs from many different sources and makes the final motor output decisions. We again use sharp glass electrodes in order to control and record from motoneurons. Intracellular recordings measure both passive electrical properties of a single cell, and electrical changes within the cell when given stimulation.