The basic motor signals that direct limb movements during walking (locomotion) are generated by circuits of neurons called central pattern generators (CPGs) which are located within the spinal cord. Networks of interneurons control the activity of motor neurons which send signals that control muscles that produce flexion and extension movements in both sides of the body to generate the normal alternating gait seen during locomotion. The identity and contribution of defined interneuron populations to mammalian locomotor behaviors is poorly understood. It has been suggested that ipsilateral excitatory interneurons (IINs), those whose nerve fibers (axons) do not cross the spinal cord midline, are involved in the generation and maintenance of locomotion. This research project will use the neonatal mouse spinal cord preparation to identify IINs which are believed to initiate the locomotor pattern (termed pacemaker neurons). The timing and pattern of the firing properties of IINs will be electrophysiologically recorded and assessed during a drug-induced locomotor-like motor pattern using serotonin (5-HT) and glutamate (known to be essential for producing locomotion in mammals). Additionally, pacemaker-like properties in the IINs will be identified based on the effects of 5-HT and glutamate on the IINs intrinsic membrane properties. Finally, the effects of 5-HT and glutamate on the flow of calcium (essential for the release of neurotransmitters during behaviors such as locomotion) through voltage-activated calcium channels of the IINs will be assessed using fluorescent dyes sensitive to calcium and visualized using fast confocal microscopy. This study will provide a better understanding of important general principles for how neuronal populations and their membrane properties interact to shape motor behaviors such as locomotion. The research projects conducted will provide training opportunities to undergraduate and graduate-level students from under-represented minority groups by engaging and exposing them to state-of-the-art anatomical, physiological and biophysical research techniques using the mouse spinal cord as their research model.
The consumption of known natural or artificial psychostimulants, such as caffeine, by animals or humans can have a significant impact on the short and long-term performance of cognitive and/or motor skills. We assessed the effects of caffeine to the locomotor output produced by a circuit of neurons known as a central pattern generator (CPG) which is located within the spinal cord of mammals. A locomotor rhythm was obtained by adding a combination of serotonin (5-HT), N-methyl-D-Aspartate (NMDA; glutamate analog) and dopamine (all known to be necessary for eliciting locomotion in vertebrates) to the recording chamber and an alternating locomotor-like rhythm was confirmed by recording motor activity using suction electrodes on lumbar ventral roots (see Figure). Addition of caffeine to a chamber containing the spinal cord preparation significantly decreased the cycle period of the ongoing locomotor-like rhythm, while decreasing burst duration in most preparations in a reversible manner (see figure). The application of an A1 but not of an A2a adenosine receptor antagonist mimicked the effects produced by caffeine of accelerating the locomotor rhythm by decreasing the step cycle period and burst duration. Application of an A1 receptor agonist decelerated the locomotor rhythm while an agonist for A2a receptors had no effect, suggesting the role of A1 adenosine receptors as the primary target of caffeine. These results support the stimulant effects of caffeine onto adenosine receptors located within the spinal network controlling walking, acting mostly through the inhibition of A1 adenosine receptors. By studying the effects of psychostimulants on relevant behaviors such as locomotion we can develop a better understanding of how our nervous system deals at the cellular level with this and other kinds of perturbations. There is high debate regarding the actual existence of a sole population of neurons which function as the actual pacemaking circuit controlling walking located within the upper lumbar region of the spinal cord of mammals. We conducted recordings from identified ipsilateral (axons on same side of the spinal cord) and commissural (axons cross the midline through the ventral commissure of the spinal cord) interneurons by way of in vivo retrograde labeling using fluorescent markers. We have recorded from neurons belonging to either of these anatomical groups in the presence of blockers for synaptic transmission in order to study them in isolation from other neighboring neurons and looked at the effects of applying serotonin (5-HT), the glutamate analog NMDA and dopamine (DA; all known to be important in eliciting locomotion in mammals) on neural excitability and possible bursting (rhythmic) behavior. We found that the application of 5-HT/NMDA/DA depolarizes the membrane potential, reduces the threshold for action potential generation, increased neuronal excitability and reduces the action potential after-hyperpolarization in the majority of the neurons recorded (see figure). Additionally, we found that in the presence 5-HT/NMDA/DA, most ipsilateral interneurons presented rhytmic oscillations which are voltage-dependent with hyperpolarization leading to an increase in oscillation frequency, indicating that the frequency of oscillations is determined by intrinsic neuronal properties rather than by the network. The reduction in frequency with depolarization primarily results from a prolongation of the depolarized phase of the oscillation. We also studied potential ionic conductances which could be mediating the above described responses using the patch clamp technique in voltage clamp mode. We have recorded from a total of 9 ipsilateral interneurons in the presence of blockers of potassium and sodium conductances to focus on calcium conductances. We found that the reduction of the action potential after-hyperpolarization is at least partly mediated by a voltage-dependent calcium channel since the overall calcium current was reduced in the presence of 5-HT/NMDA/DA in most cells recorded so far (12/19). We began assessing the effects on a calcium-activated potassium channel (IKCa) and found that the application of 5-HT/NMDA/DA reduced IKCa in most preparations in a reversible manner and additional found that the application of apamin, an IKCa channel blocker, mimicked the effects of the drug cocktail supporting specificity of the observed effects. We will continue to explore other potential conductances including potassium and sodium conductances through further voltage clamp studies. The understanding of the basic neural organization of circuits such as the spinal circuitry controlling locomotion could have significant impact on other fields such as Medicine, Engineering and Mathematics due to its relevance in applying basic connectivity and functional data to the development of new therapeutical, structural and modeling strategies. These new strategies could translate into better ways we can provide medical services, develop better infrastructural projects or formulate better theories that could help us understanding higher order neural processes. These studies exposed three Hispanic women working in these projects to advanced instrumentation and experimental techniques. Furthering the scientific career of underrepresented groups in science is of high priority in our research laboratory and aligned to the programmatic mission of NSF. Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4
