Course Structure and Modules

In this module, students use a variety of complementary experimental techniques to study sensory processing and behavior in the nematode Caenorhabditis elegans. This module continues the theme of using state-of-the art approaches to study and manipulate the function of neurons within the brains of intact, behaving animals. C. elegans is an excellent preparation for this task, with a complete neural wiring diagram, genetic access to each neuron, optogenetic tools for neural stimulation and activity recording by calcium imaging, detailed behavioral analysis, and an extensive library of genetic mutants. Information flow can be traced from sensory stimulus to neural signals to behavioral output, in living and behaving animals.

In the first few days of the modules, all students are trained in pairs to perform a series of complementary experiments in recording and analysis of nematode behavior, neural stimulation and imaging, and C. elegans culture. Behavioral assays are performed using machine vision cameras and MATLAB- or LabView-based quantification systems. Three distinct sensory modalities are typically used for behavioral and neural activity analyses: olfactory, mechanical, and thermal stimulation. Precise chemical stimulation is performed using microfluidic arenas and chambers. Mechanical stimulation is delivered through an Arduino controlled tapper, thermal stimuli are precisely regulated using peltier-based systems. Behavior and neural imaging systems are capable of optogenetic activation or silencing of specific neurons via multicolor LEDs. In the first few days, students are taught how to run many experiments to give them a sense for which types of experiments are best for different project questions. Students are tasked with replicating real, published experiments, to gain confidence with each system. Because these systems are well-established and their use is relatively simple, students can become facile at the core techniques within the first few days. Students continue to build their expertise with the use of MATLAB for designing stimuli, running experiments, and performing analysis. We also use the first few days of the module to familiarize students with the basics of C. elegans neurobiology and genetics, and provide mini ‘boot camps’ on genetic crosses, RNAi, molecular biology, and other relevant topics.

By the fourth day of the first week, the students are sufficiently competent to plan their own projects. The focus of these projects changes from year-to-year as the faculty endeavor to compile a list of ideas based on current research in the field as well as the interests of the students. One consistent theme over the last several years has been imaging neural activity in intact animals during sensory stimulation and behavior using genetically encoded calcium indicators. Students have worked on projects investigating sensory adaptation, synaptic plasticity, and the interplay between sensory and interneuron activity. For instance, one project in compared sensory adaptation between natural sensory stimuli and optogenetic manipulations. Another project investigated the functional importance of a plastic synaptic connection by bypassing this plasticity with a genetically encoded synthetic gap junction to artificially re-wire the C. elegans connectome. In 2018, one project assessed the role of interneurons in behavioral decision-making by tracking activity within interneurons of freely moving animals during directed migration on a thermal gradient. One of the major outcomes of this module is that students come to realize the power of molecular biology and genetics to address questions with precision and clarity. The well-established imaging and behavioral systems, in conjunction with the ability of faculty to bring a wide variety of new worm strains each year, provides a powerful foundation for students to explore discovery-driven projects based on current interest and state-of-the-art genetic tools.

Sensory systems provide a wealth of information about the environment and the body to the animal. From this wealth of information, the brain extracts information that is relevant for making behavioural decisions, e.g. in the context of foraging or communication with conspecifics. In the electric fish cycle, we are going to investigate how neurons in the hindbrain and midbrain become more selective to behaviorally relevant sensory stimuli.

Weakly electric fish generate an electric field around their body (electric organ discharge: EOD) and sense perturbations of the self-generated field caused by the presence of nearby objects, such as prey, or by the EODs of conspecifics. Depending on the properties of the conspecific EOD a given fish may modulate its EOD in certain stereotyped fashion.

On the second day (i.e. after the demo) of the electric fish cycle, you will take a quantitative look at some of the animal’s electrical behaviors. Over the next few days, you will learn how to record extracellularly from pyramidal cells in the hindbrain as well as performing in vivo blind whole cell patch clamp recordings from midbrain neurons. You will be introduced to a range of analysis methods including information-theoretical methods.

Projects for the second weak may ask questions about the role of specific feedback pathways for sensory processing or about the role of specific ion channels for the response properties of central neurons. Other options include the processing of information on moving objects, which is crucial for prey detection and localization, linear versus nonlinear sensory processing, and electrosensory processing in species that have not been studied yet.

Our module will focus on the fruit fly, Drosophila melanogaster. Students will learn how to pose and answer questions regarding sensorimotor physiology using this model. Methodologically, we will employ modern techniques for quantitative behavior, whole-cell patch-clamp electrophysiology, 2-photon calcium imaging, and genetic methods for manipulating neural activity.

In the first days of the module, as an introduction to fly behavior, students will examine visually guided steering of tethered, flying flies in a virtual reality flight simulator. These arenas can be operated in two modes: ‘open-loop’, in which the experimenter presents a visual stimulus to which the flies responds, and 2) ‘closed-loop’ in which the flies themselves control a panoramic visual display by modulating their wing motion (fly virtual-reality). By performing experiments in open- and closed-loop, it is possible to study the functional architecture of the visual and flight control systems of intact, behaving animals. This exercise will expose students to the art of handling Drosophila and performing quantitative behavioral experiments on individual flies, alongside data analysis in Matlab/Python.

Following these initial behavioral studies, students will design and implement independent experiments, in groups of two, that focus on behavioral and physiological measurements to inform computations related to vision, proprioception, audition, and aspects of sensorimotor integration in Drosophila.  These individual projects will again focus on quantitative, behavioral measurements alongside the use of two-photon calcium imaging and whole-cell patch clamping in some groups to form more direct, cellular-level insights.

Together, these exercises and experiments will familiarize students with how to pose and answer questions in the Drosophila system. While relevant methods will be taught, emphasis will be placed on major questions in systems neuroscience and how these are being tackled in the fly system in the modern era.

In Cycle 1, all of the students work on the leech as an experimental system. Students learn neurophysiology and neuroanatomical techniques. Techniques include intracellular electrophysiological recordings in current clamp and voltage clamp configuration, extracellular recordings, and intracellular dye injection, and MATLAB instruction. The goal is to identify neurons by their electrophysiological properties and learn about their electrical and chemical interactions. Towards the end of the cycle students work in pairs on individual research projects that they design.

This module focuses on the dynamics of neural activity in the cortex and hippocampus of behaving mice. Both of these brain structures exhibit rich, highly organized activity, including distinct brain states and predictable transitions between them, oscillations at multiple interrelated spatial and temporal scales, and even spontaneous “replay” of previous experiences. These neural dynamics interact with neuromodulators to implement fundamental processes such as gain control and state-dependence that ultimately underlie complex behaviors. 

Depending on your interests, in the first few days of the module you will be introduced to (1) wide-field calcium imaging of inhibitory and excitatory activity across the cortex of head-fixed behaving mice; (2) neural ensemble recording in hippocampus of freely moving mice using silicon probes (field potentials and units), and (3) fiber photometry to measure dopamine levels, separately or simultaneously with (2). Whatever you choose, the data sets you will collect will be large and complex, and the majority of your time in the second half of the module will be devoted to data processing and analysis.

In head-fixed mice, possible projects include 1) characterizing the state-dependent changes in long-range cortical circuits during spontaneous behaviors (assessed by pupil, facial motion, locomotion, grooming, sleep, etc), 2) comparisons of the spatiotemporal patterns of excitatory and inhibitory activity across the cortex, and 3) the cortical impact of pharmacological manipulations of cholinergic or noradrenergic signaling.

In freely moving mice, possible projects include characterizing (1) the relationship between the activity of “place cells” in the hippocampus to performance on the task, (2) the relationship between extracellular DA and behavior, and (3) the connection between hippocampal activity and DA.

In this cycle we will explore sensory coding in the rodent vibrissa (whisker) tactile sensory system through a broad range of techniques and experimental designs.  Two rigs will focus on activity in primary somatosensory (“barrel”) cortex of anesthetized rats during vibrissa or cortical electrical stimulation, using in vivo whole cell recordings of synaptic and spiking responses in rat.  Two other rigs will investigate neural activity in the thalamus and cortex using whole-cell and juxtacellular recording approaches.

Understanding how neuronal networks enable animals, including humans, to make coordinated movements is a continuing goal of neuroscience research. The stomatogastric nervous system of decapod crustaceans and particularly the networks of the stomatogastric ganglion (STG), which control feeding functions, have significantly contributed to our present understanding of general principles underlying rhythmic motor circuit operation at the cellular level, and has shed light on the mechanisms of network homeostasis and plasticity.

Rhythmic behaviors include all motor acts that, at their core, involve a rhythmic repeating set of movements (e.g., locomotion, breathing, chewing and scratching). The circuits underlying such rhythmic behaviors, central pattern generators (CPGs), operate on the same general principles across all nervous systems. These networks can generate rhythmic output in the completely isolated nervous system, even in the absence of any rhythmic neuronal input, including feedback from sensory systems. Although the details differ in each circuit, all CPGs use the same set of cellular-level mechanisms for circuit construction. More importantly, CPG circuits are usually not dedicated to producing a single neuronal activity pattern. This flexibility results largely from the ability of many different neuromodulators to change the cellular and synaptic properties of individual circuit neurons. When the properties of circuit components are changed, the output of the circuit itself is modified.

The STG contains a set of distinct but interacting motor circuits. The value of this system has resulted from its experimental accessibility, owing to the small number of large and individually identifiable neurons and the use of several innovative techniques. Because of the many similarities between vertebrate and invertebrate systems, especially with regards to basic principles of neuronal function, invertebrate model systems such as the crustacean stomatogastric nervous system continue to provide key insight into how neural circuits operate in the numerically larger and less accessible vertebrate CNS.

The stomatogastric cycle (STG cycle) examines mechanisms of generation, regulation and plasticity of rhythmic neural activity produced by CPG networks. The STG cycle exercises highlight fundamental features of the cellular basis of motor pattern generation and the characterization of dynamical neural systems. Students obtain hands-on experience with the principle that rhythmically active networks can continue to generate rhythmic motor patterns in the isolated CNS, a defining feature of CPGs. We also focus on the fact that anatomically hard-wired circuits remain functionally flexible. All neural networks are malleable through the action of neuromodulatory inputs and intrinsic homeostatic mechanisms, which modify time and voltage-dependent properties of intrinsic membrane properties, and functional synaptic connectivity. Additionally, several key aspects of neuronal communication are studied, including properties of spike-mediated and graded synaptic transmission, short-term synaptic plasticity and the input/output properties of electrical synapses.

We build on skills developed during Cycle I, emphasizing electrophysiological analysis of neural network activity, and its underlying ionic mechanisms, in an isolated nervous system. We emphasize modern experimental tools and paradigms: intracellular recordings of multiple identified neurons; extracellular recordings of identified neurons; single-electrode discontinuous current clamp methods for current injection and recording; synaptic pharmacology defined with pharmacological agonists and antagonists; superfusion of neuromodulators and their release by identified projection neurons; study of graded transmitter release, the dynamic clamp technique for determining the functional impact of intrinsic and synaptic currents in network function, and quantitative analysis of electrophysiological recordings.

Students spend the first week building their understanding of STG networks. For example, students will learn dynamical system techniques to describe activity network parameters, such as phase analysis, resetting, entrainment and phase response curves (PRCs). The two-electrode voltage clamp technique is introduced to quantify time and voltage dependent properties of membrane channels and to clarify how ionic currents (which will be characterized mathematically using the Hodgkin-Huxley framework) contribute to neuronal and network function. These ionic currents will be manipulated using the dynamic clamp technique to understand the functional significance of mathematically defined ionic conductances. The dynamic clamp technique will also be used to examine the role of synapses in the network, by artificially adding or removing synapses in the biological network.

The second week of the STG Cycle is dedicated to independent projects that reflect contemporary research issues asking questions about mechanisms underlying neural network activity, and address specific principles of motor network function common to all animals. These projects are expected, with guidance from faculty mentors, to gather new, previously unpublished data, much as preliminary experiments would accomplish in a research lab. Almost all projects in the STG cycle address unknown conceptual questions and many projects in recent years have led to novel results.

This cycle will investigate the brain circuitry that subserves the complex, learned vocalizations of songbirds. We will investigate how and where auditory and motor information about song is encoded in the brain using in vivo extracellular electrophysiology and calcium imaging in adult zebra finches. Experiments will be performed in male birds, who learn to sing a complex motor sequence, and female birds, who do not sing but do attend to the spectro-temporal properties of male songs when identifying individuals and evaluating potential mates.

In the first week, students will characterize auditory responses in multiple song nuclei in anesthetized birds, and then record singing-related activity and auditory responses in awake, behaving birds. Students will also be introduced to calcium-imaging techniques to record the spatiotemporal activity of many neurons simultaneously using miniature head-mounted microscopes in awake-behaving birds. In the second week, students will design and implement independent projects to investigate a specific question regarding the sensory and/or motor coding of a complex, procedurally-learned behavior.