William B. Kristan

Multifunctional pattern-generating circuits.

The ability of distinct anatomical circuits to generate multiple behavioral patterns is widespread among vertebrate and invertebrate species. These multifunctional neuronal circuits are the result of multistable neural dynamics and modular organization. The evidence suggests multifunctional circuits can be classified by distinct architectures, yet the activity patterns of individual neurons involved in more than one behavior can vary dramatically. Several mechanisms, including sensory input, the parallel activity of projection neurons, neuromodulation, and biomechanics, are responsible for the switching between patterns. Recent advances in both analytical and experimental tools have aided the study of these complex circuits.

Photochemical control of endogenous ion channels and cellular excitability

Light-activated ion channels provide a precise and noninvasive optical means for controlling action potential firing, but the genes encoding these channels must first be delivered and expressed in target cells. Here we describe a method for bestowing light sensitivity onto endogenous ion channels that does not rely on exogenous gene expression. The method uses a synthetic photoisomerizable small molecule, or photoswitchable affinity label (PAL), that specifically targets K+ channels. PALs contain a reactive electrophile, enabling covalent attachment of the photoswitch to naturally occurring nucleophiles in K+ channels. Ion flow through PAL-modified channels is turned on or off by photoisomerizing PAL with different wavelengths of light. We showed that PAL treatment confers light sensitivity onto endogenous K+ channels in isolated rat neurons and in intact neural structures from rat and leech, allowing rapid optical regulation of excitability without genetic modification.

Optically monitoring voltage in neurons by photo-induced electron transfer through molecular wires

Fluorescence imaging is an attractive method for monitoring neuronal activity. A key challenge for optically monitoring voltage is development of sensors that can give large and fast responses to changes in transmembrane potential. We now present fluorescent sensors that detect voltage changes in neurons by modulation of photo-induced electron transfer (PeT) from an electron donor through a synthetic molecular wire to a fluorophore. These dyes give bigger responses to voltage than electrochromic dyes, yet have much faster kinetics and much less added capacitance than existing sensors based on hydrophobic anions or voltage-sensitive ion channels. These features enable single-trial detection of synaptic and action potentials in cultured hippocampal neurons and intact leech ganglia. Voltage-dependent PeT should be amenable to much further optimization, but the existing probes are already valuable indicators of neuronal activity.

Distributed processing of sensory information in the leech. II. Identification of interneurons contributing to the local bending reflex

Isolated midbody ganglia of the leech Hirudo medicinalis were surveyed for interneurons contributing to the dorsal component of the local bending reflex, i.e., to the excitation of dorsal excitatory motor neurons that follows stimulation of dorsal mechanoreceptors responsive to pressure (P cells). Nine types of local bending interneuron could be distinguished on physiological and morphological grounds--8 paired and 1 unpaired cell per ganglion. Synaptic latencies from sensory neurons to interneurons were consistent with a direct or possibly disynaptic pathway. Connections between interneurons appeared to be rare and hyperpolarization of individual interneurons during local bending produced small but reliable decrements in motor neuron response, suggesting that multiple parallel pathways contribute to the behavior. Paradoxically, most interneurons received substantial inputs from ventral as well as dorsal mechanoreceptors, indicating that interneurons that were distinguished by their contribution to dorsal local bending were, in fact, active in ventral and lateral bends as well. The capacity to detect a particular stimulus and produce the appropriate response cannot be localized to particular types of interneuron; rather, it appears to be a distributed property of the entire local bending network.

A neuronal network for computing population vectors in the leech

The correlation of neuronal activity with sensory input and behavioural output has revealed that information is often encoded in the activity of many neurons across a population, that is, a neural population code is used,. The possible algorithms that downstream networks use to read out this population code have been studied by manipulating the activity of a few neurons in a population,. We have used this approach to study population coding in a small network underlying the leech local bend, a body bend directed away from a touch stimulus. Because of the small size of this network we are able to monitor and manipulate the complete set of sensory inputs to the network. We show here that the population vector formed by the spike counts of the active mechanosensory neurons is well correlated with bend direction. A model based on the known connectivity of the identified neurons in the local bend network can account for our experimental results, and is suitable for reading out the neural population vector. Thus, for the first time to our knowledge, it is possible to link a proposed algorithm for neural population coding with synaptic and network mechanisms in an experimental system.

Imaging Dedicated and Multifunctional Neural Circuits Generating Distinct Behaviors

Central pattern generators (CPGs) control both swimming and crawling in the medicinal leech. To investigate whether the neurons comprising these two CPGs are dedicated or multifunctional, we used voltage-sensitive dye imaging to record from ∼80% of the ∼400 neurons in a segmental ganglion. By eliciting swimming and crawling in the same preparation, we were able to identify neurons that participated in either of the two rhythms, or both. More than twice as many cells oscillated in-phase with crawling (188) compared with swimming (90). Surprisingly, 84 of the cells (93%) that oscillated with swimming also oscillated with crawling. We then characterized two previously unidentified interneurons, cells 255 and 257, that had interesting activity patterns based on the imaging results. Cell 255 proved to be a multifunctional interneuron that oscillates with and can perturb both rhythms, whereas cell 257 is an interneuron dedicated to crawling. These results show that the swimming and crawling networks are driven by both multifunctional and dedicated circuitry.

Spatial pattern of risk of common raven predation on desert tortoises

Common Ravens (Corvus corax) in the Mojave Desert of California, USA are subsidized by anthropogenic resources. Large numbers of nonbreeding ravens are attracted to human developments and thus are spatially restricted, whereas breeding ravens are distributed more evenly throughout the area. We investigated whether the spatial distribution of risk of predation by ravens to juveniles of the threatened desert tortoise (Gopherus agassizii) was determined by the spatial distribution of (1) nonbreeding ravens at human developments (leading to “spillover” predation) or (2) breeding individuals throughout developed and undeveloped areas (leading to “hyperpredation”). Predation risk, measured using styrofoam models of juvenile desert tortoises, was high near places attracting large numbers of nonbreeding ravens, near successful nests, and far from successful nests when large numbers of nonbreeding ravens were present. Patterns consistent with both “spillover” predation and “hyperpredation” were thus observed, attr...

Identification of Neural Circuits by Imaging Coherent Electrical Activity with FRET-Based Dyes

We show that neurons that underlie rhythmic patterns of electrical output may be identified by optical imaging and frequency-domain analysis. Our contrast agent is a two-component dye system in which changes in membrane potential modulate the relative emission between a pair of fluorophores. We demonstrate our methods with the circuit responsible for fictive swimming in the isolated leech nerve cord. The output of a motor neuron provides a reference signal for the phase-sensitive detection of changes in fluorescence from individual neurons in a ganglion. We identify known and possibly novel neurons that participate in the swim rhythm and determine their phases within a cycle. A variant of this approach is used to identify the postsynaptic followers of intracellularly stimulated neurons.

Behavioral choice by presynaptic inhibition of tactile sensory terminals

When presented with multiple stimuli, animals generally choose to respond only to one input. The neuronal mechanisms determining such behavioral choices are poorly understood. We found that the medicinal leech had greatly diminished responses to moderate mechanosensory input as it fed. Feeding dominated other responses by suppressing transmitter release from mechanosensory neurons onto all of their neuronal targets. The effects of feeding on synaptic transmission could be mimicked by serotonin. Furthermore, the serotonin antagonist mianserin blocked feeding-induced decreases in synaptic transmission. These results indicate that feeding predominates behaviors by using serotonin at an early stage of sensory processing, namely on presynaptic terminals of mechanosensory neurons.

Neuronal Decision-Making Circuits

Studying the neural basis of decision-making has largely taken one of two paths: one has involved cell-by-cell characterization of neuronal circuits in invertebrates; and the other, single-unit studies of monkeys performing cognitive tasks. Here I shall attempt to bring these two disparate approaches together.