Structure and modulation of neural networks for rhythmic behaviors.
Our lab has spent the past 30 years studying the cellular and synaptic mechanisms by simple neural networks generate rhythmic behaviors, and how which neuromodulators such as dopamine and serotonin can reconfigure these small motor networks to generate variability in simple rhythmic behaviors. Our basic assumption is that relatively limited neural networks, called Central Pattern Generators (CPGs) drive the muscle contractions that evoke simple repetitive behaviors. The organization of these networks is for the most part unknown, and identifying the interneurons that comprise the CPG and their synaptic interactions that form the network is a major focus of our work. In order to allow CPG-evoked behaviors to be flexible rather than robotic, modulatory inputs, using transmitters such as serotonin and dopamine, alter the strengths of the synapses in the network, and also alter the intrinsic electrophysiological response properties of the component neurons. These actions essentially "rewire" the network in software, allowing a single anatomically defined network to generate a family of related behaviors. Our work is at the cellular and biophysical level, trying to understand how individual neurons and synapses are modulated.
The major tools we use include:
1) electrophysiological recordings from genetically identified neurons, studying their activity during behavior, their firing properties and responses to synaptic inputs; 2) voltage clamp analysis to study how neuromodulators alter the properties of identified ionic currents, which in turn alter the firing properties of the neurons and the release of transmitter from their terminals; 3) calcium imaging, in collaboration with Dr. Warren Zipfel (Biomedical Engineering, Cornell) to monitor calcium handling and grouped neuronal activity in complex tissues; 4) immunocytochemistry, to look for changes in expression of ion channels and receptors; 5) mathematical modeling, in collaboration with Dr. Ilya Rybak (Drexel University), to generate hypotheses about network function that can then be tested experimentally.
For many years we studied the CPG network that drives the pyloric motor rhythm in the stomatogastric ganglion of the spiny lobster. This 14-neuron pyloric network in the lobster stomatogastric ganglion. This is perhaps the best understood neural network at present; all of its neurons and their synaptic connections are known. We showed how the monoamines, dopamine, serotonin and octopamine can reconfigure this simple network to generate a different rhythmic movement of the pylorus in the lobster foregut. Our work has shown that network reconfiguration is very complex, involving modulation of a large majority of all possible connections and neurons. The mechanisms of these changes involve many ionic currents and synaptic mechanisms.
The CPG for locomotion in the mouse spinal cord
Our current major focus is to understand the function and modulation of the mouse spinal cord network for locomotion. While the brain send the commands to start and stop walking or to turn right or left, the spinal cord contains the CPG networks that determine the timing, phasing and intensity of the different muscle contractions that actually make walking possible. This locomotor CPG can be studied in the isolated spinal cord, where “fictive locomotion” can be evoked by bath application of neurotransmitters such as NMDA and serotonin, by electrical stimulation of caudal afferents from the tail, or by electrical stimulation of descending reticulospinal tracts from the brainstem. We are addressing a number of questions about this network, and its modulation by serotonin:
1) Which interneurons are components of the locomotor CPG, and what roles to they play in generating the rhythmic, alternating left/right and flexor/extensor motor pattern? We use transgenic mice that express fluorescent proteins driven by neuron-specific transcription factors to monitor the activity of identified interneurons either by electrophysiological studies (both whole cell and perforated patch recordings) or by calcium imaging. Other transgenic mice have been modified to delete certain neuron types, allowing us to study the consequences of the loss of these neurons. For example, deletion of the V2a interneurons, which express the Chx10 transcription factor, results in a very unique phenotype: the mice walk normally at low speeds, but as the speed of the treadmill is accelerated they become less coordinated and finally switch to a synchronous left-right bounding gait, which is never seen in normal mice. The V2a interneurons are rhythmically active during fictive locomotion, but the percentage of actively firing neurons and their rhythmicity increases as the locomotor speed increases. Thus, these neurons appear to play an important role in regulating left-right alternation, but mainly at higher speeds. We are also interested in the post-natal maturation of the locomotor CPG. Most studies have used neonatal (0-5-day old) spinal cords, because it is very hard to record activity from spinal interneurons at later ages. We have recently developed perforated patch methods to record activity of spinal interneurons in spinal cord slices at any age. This has allowed us to determine that the excitability of spinal interneurons grows with age, and they can show dramatically enhanced bistability upon addition of serotonin, which does not occur in the neonatal neurons. This research is supported by a Jacob Javits Neuroscience Investigator Award from the NIH. Recently, we began to study the consequences of spontaneous “errors” during fictive locomotion, where one or more of the spinal motor nerves show deletions, or a failure to fire at the time when they should. Detailed analysis of these deletions suggests that the spinal locomotor CPG has an asymmetric organization, with the rhythm dominated by flexor-related pathways. We were also able to monitor the activity of identified interneurons during these deletions, which helped us to define their position and function within a model of the locomotor network, in collaboration with Ilya Rybak (Drexel University). This work is being continued with support from a CRCNS computational neuroscience grant from the NIH.
2) How does serotonin modify the properties of locomotor CPG neurons? Serotonin (5-HT) plays an important role in preparing the locomotor network for action. In mice, if the serotonergic inputs to the spinal cord are blocked, it is difficult to evoke fictive locomotion. We hypothesize that 5-HT increases the excitability of the neurons and alters their synaptic strengths to allow rhythmic motor output. We have been studying the mechanisms by which 5-HT excites identified interneurons, including commissural interneurons, V2a interneurons and Hb9 interneurons. For example, 5-HT enhances the excitability of CINs in part by reducing the amplitude of the spike afterhyperpolarization; this allows the neuron to recover from the spike sooner and fire at higher frequencies. The mechanism is a reduction in a voltage-sensitive calcium current, which in turn reduces IK(Ca) to reduce the afterhyperpolarization. Our recent work on postnatal maturation of spinal interneurons shows that their responses to serotonin change with age. For example, serotonin excites neonatal commissural interneurons, which depolarize and fire tonically. This arises from a serotonergic reduction of N- and P/Q-type calcium currents, indirectly reducing the calcium-activated potassium current and exciting the neurons. In contrast, by two weeks after birth, serotonin evokes bistability and plateau potential firing in these neurons. It does so not by reducing calcium currents as in the neonatal neurons, but by increasing an L-type calcium current that underlies the prolonged depolarization of the plateau potential. These experiments combine whole cell recordings with calcium imaging using a two-photon microscope. This work is supported by a Jacob Javits Neuroscience Investigator Award from the NIH.
3) What are the cellular consequences of spinal cord injury (SCI)? Typically, spinal injuries occur in the cervical or thoracic regions of the spinal cord. The locomotor CPG, located in the lumbar cord, is not itself damaged by SCI, but it suffers from loss of both the descending glutamatergic and the modulatory inputs from the brain, including those releasing serotonin. The long-term consequences of this denervation are not clear. We have begun to analyze the SCI-induced changes in sensitivity to serotonin in identified locomotor-related interneurons. V2a interneurons do not change their intrinsic firing properties markedly, but become 100-1000 times more sensitive to serotonin one month after SCI, compared to sham controls. We combined electrophysiological and immunocytochemical approaches to study the mechanisms for this increase in sensitivity, showing that there is an increase in 5HT2A receptors and a class of calcium channels, and a decrease in the serotonin transporter. Future work will look at the effects of SCI on other interneurons, and on synaptic interactions between them. This work was supported by an ARRA grant from the NIH.