Interneurons play a critical role in the proper coordination of limbs in neonatal mice. Restricted; Files Only
Mistretta, Olivia (Spring 2025)
Abstract
Proper limb coordination during locomotion is fundamental for effective movement and relies on the precise timing of motoneuron activation by spinal interneurons. Among these, the V1 class of inhibitory interneurons plays a critical role in shaping motor output. The V1 class is highly heterogeneous and the respective contributions of each sub clade is still poorly understood. This dissertation investigates the function of Foxp2-expressing V1 (Foxp2-V1) interneurons in the maturation of interlimb and intralimb coordination in neonatal mice. Using an intersectional genetic approach, we chronically and acutely silenced Foxp2-V1 interneurons to determine their impact on locomotor behavior during early postnatal development.
We further developed and employed the L-DOPA-induced air-stepping paradigm, as a means of evaluating rhythmic stepping in neonates, combined with high-resolution kinematic and electromyographic (EMG) analyses. Chronic silencing of Foxp2-V1 interneurons resulted in significant disruptions in flexor-extensor alternation, increased variability in interlimb coordination, and impaired step rhythmicity. Mice exhibited prolonged stance phases, irregular joint kinematics, and aberrant activation patterns of tibialis anterior and lateral gastrocnemius muscles, implicating Foxp2-V1 interneurons in the fine-tuning of flexor-extensor timing. Moreover, acute silencing after normal motor development induced pronounced impairments in rhythmic air-stepping, suggesting that Foxp2-V1 interneurons remain essential for the execution of mature locomotor patterns.
Our findings demonstrate that Foxp2-V1 interneurons are required for the development of coordinated limb movements by ensuring precise inhibitory control over motoneuron activity. These results provide novel insights into the role of spinal inhibitory circuits in locomotor maturation and have important implications for understanding motor dysfunction in conditions such as cerebral palsy and amyotrophic lateral sclerosis, where disruptions in inhibitory interneuron networks have been implicated. This work advances our understanding of spinal circuit development and highlights Foxp2-V1 interneurons as a critical component of the neural control of movement.
Table of Contents
TABLE OF CONTNETS
FIGURE LIST
CHAPTER 1: Introduction
1.1 General Introduction: development of motor function in newborns
1.2 Air-stepping in neonatal rodents: a window into the development of limb coordination.
1.3 Generation of locomotion; what is required.
1.4 Inhibitory networks and their early development
1.5 Embryonic origins of spinal interneurons; the V code
1.6 The largest class of inhibitory interneurons in the spinal cord ventral horn; V1s
1.7 Foxp2-V1 interneurons: current understanding and implications in disease states
1.8 Animal models
1.9 Goals of the study and Hypothesis
1.10 References
CHAPTER 2: Air-stepping in the neonatal mouse: a powerful tool for analyzing early stages of rhythmic limb movement development.
2.1 Abstract
2.2 Introduction
2.3 Materials and Methods
2.3.1. Animals
2.3.2. Apparatus
2.3.3. Procedure
2.3.4. Video Collection
2.3.5. Video Analysis
2.3.6. EMG Recordings
2.3.7. EMG Analysis
2.3.8. Statistics
2.4 Results
2.4.1. Air – stepping formal description and quantification.
2.4.2. Interlimb Coordination: Ipsilateral Forelimb and Hindlimb
2.4.3. Interlimb Coordination: Left and Right Hindlimbs
2.4.4. Interlimb Coordination: Diagonal Limbs and gait analysis
2.4.5. Intralimb Coordination
2.4.6. Electromyographic Recordings from Tibialis Anterior and Lateral Gastrocnemius
2.5 Discussion
2.6 References
CHAPTER 3: Materials and Methods
3.2 Ethical Standards
3.3 Animals
3.4 Air-stepping protocol
3.5 Video and Electromyography Collection
3.6 Video Analysis
3.7 Intralimb Joint Angle Analysis
3.8 Interlimb Coordination Analysis
3.9 EMG Analysis
3.10 Acute Silencing
3.11 Statistics
3.12 References
CHAPTER 4: Spinal V1 inhibitory interneurons expressing the transcription Foxp2 are necessary for the maturation of limb coordination in neonates.
4.1 Results
4.1.1. Chronically silencing spinal Foxp2-V1 interneurons diminishes L-DOPA-induced air-stepping responses, slows step durations and induces a loss of rhythmicity.
4.1.2. Silencing of Foxp2-V1 interneurons results in joint flexion-extension alterations.
4.1.3. Foxp2-V1 interneurons are necessary for accurate timing of tibialis anterior and lateral gastrocnemius activation during step cycle.
4.1.4. Alternation of ipsilateral limbs during air stepping is highly variable in animals with chronically silenced Foxp2-V1 interneurons.
4.1.5. Foxp2-V1 interneurons play roles in left-right limb coordination.
4.1.6. Foxp2-V1 interneurons are necessary for generating consistent gaits during rhythmic stepping.
4.2 Supplemental Tables
4.3 References
CHAPTER 5: Spinal V1 inhibitory interneurons expressing the transcription Foxp2 are necessary for the maturation of limb coordination in neonates; further elucidated by an acute silencing technique.
5.1 Results
5.1.1. Acute silencing of Foxp2-V1 interneurons after proper development results in a large reduction in L-DOPA induced air-stepping.
5.1.2. Acute silencing of Foxp2-V1 interneurons disrupts hindlimb joint kinematics and ankle flexor and extensor activations.
5.1.3. Acute Silencing Impairs Limb Coordination
5.2 Supplemental Figures
5.3 Supplemental Tables
5.4 References
CHAPTER 6: Discussion
6.1 Discussion
6.2 Hyperflexion
6.3 Interlimb Discoordination
6.4 Stepping Speed
6.5 Conclusions and Future Directions
6.6 References
CHAPTER 7: Conclusions and Future Directions
7.1 Conclusions
7.1.1 Development of a Technique to Study Limb Coordination During Maturation
7.1.2 Foxp2-V1 Interneurons are Critical for Limb Coordination
7.1.3 Differences in Flexor and Extensor Muscle Activation
7.1.4 Acute Versus Chronic Silencing of Foxp2-V1 Interneurons
7.2 Future Direction
7.2.1 Investigating the Connections of Chronically Silenced Animals
7.2.2 Investigating the Connections of Foxp2-V1 Interneurons Between the Forelimb and Hindlimb
7.2.3 Understanding the Heterogeneity of Foxp2-V1 Interneurons and Its Relevance to Other V1 Subpopulations
7.3 Final Words
7.4 References
About this Dissertation
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File download under embargo until 22 May 2026 | 2025-04-10 16:58:05 -0400 | File download under embargo until 22 May 2026 |
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