William N. Frost

Intrinsic neuromodulation: altering neuronal circuits from within

There are two sources of neuromodulation for neuronal circuits: extrinsic inputs and intrinsic components of the circuits themselves. Extrinsic neuromodulation is known to be pervasive in nervous systems, but intrinsic neuromodulation is less recognized, despite the fact that it has now been demonstrated in sensory and neuromuscular circuits and in central pattern generators. By its nature, intrinsic neuromodulation produces local changes in neuronal computation, whereas extrinsic neuromodulation can cause global changes, often affecting many circuits simultaneously. Studies in a number of systems are defining the different properties of these two forms of neuromodulation.

A Cellular Mechanism for Prepulse Inhibition

In prepulse inhibition (PPI), startle responses to sudden, unexpected stimuli are markedly attenuated if immediately preceded by a weak stimulus of almost any modality. This experimental paradigm exposes a potent inhibitory process, present in nervous systems from invertebrates to humans, that is widely considered to play an important role in reducing distraction during the processing of sensory input. The neural mechanisms mediating PPI are of considerable interest given evidence linking PPI deficits with some of the cognitive disorders of schizophrenia. Here, in the marine mollusk Tritonia diomedea, we describe a detailed cellular mechanism for PPI--a combination of presynaptic inhibition of startle afferent neurons together with distributed postsynaptic inhibition of several downstream interneuronal sites in the startle circuit.

Swim Initiation Neurons in Tritonia diomedea)

SYNOPSIS. Two groups of interneurons, Tr1 and DRI, have been identified in the escape swim circuit of the marine mollusc Tritonia diomedea that have important roles in behavioral initiation. DRI functions as a command neuron, receiving direct excitatory input from the afferent neurons, and in turn directly exciting the DSI neurons of the central pattern generator. DRI fires throughout the swim motor program, and activity in DRI is both necessary and sufficient for sensory input to elicit the swim motor program. Tr1 is an excitatory interneuron that fires briefly in response to sensory input and then remains silent during the motor program. Tr1 excites DRI with an excitatory connection that has fast and slow components and thus appears to have a role in converting brief afferent neuron activity to long-lasting firing in downstream circuit elements. These neurons complete the description of a continuous synaptic pathway from afferent to flexion neurons in the Tritonia swim circuit. Their identification should facilitate studies of motor program initiation, as well as of how various forms of experience, including simple forms of learning, act to influence neuronal decision-making processes.

Modular Deconstruction Reveals the Dynamical and Physical Building Blocks of a Locomotion Motor Program

The neural substrates of motor programs are only well understood for small, dedicated circuits. Here we investigate how a motor program is constructed within a large network. We imaged populations of neurons in the Aplysia pedal ganglion during execution of a locomotion motor program. We found that the program was built from a very small number of dynamical building blocks, including both neural ensembles and low-dimensional rotational dynamics. These map onto physically discrete regions of the ganglion, so that the motor program has a corresponding modular organization in both dynamical and physical space. Using this dynamic map, we identify the population potentially implementing the rhythmic pattern generator and find that its activity physically traces a looped trajectory, recapitulating its low-dimensional rotational dynamics. Our results suggest that, even in simple invertebrates, neural motor programs are implemented by large, distributed networks containing multiple dynamical systems.

Variable Neuronal Participation in Stereotypic Motor Programs

To what extent are motor networks underlying rhythmic behaviors rigidly hard-wired versus fluid and dynamic entities? Do the members of motor networks change from moment-to-moment or from motor program episode-to-episode? These are questions that can only be addressed in systems where it is possible to monitor the spiking activity of networks of neurons during the production of motor programs. We used large-scale voltage-sensitive dye (VSD) imaging followed by Independent Component Analysis spike-sorting to examine the extent to which the neuronal network underlying the escape swim behavior of Tritonia diomedea is hard-wired versus fluid from a moment-to-moment perspective. We found that while most neurons were dedicated to the swim network, a small but significant proportion of neurons participated in a surprisingly variable manner. These neurons joined the swim motor program late, left early, burst only on some cycles or skipped cycles of the motor program. We confirmed that this variable neuronal participation was not due to effects of the VSD by finding such neurons with intracellular recording in dye-free saline. Further, these neurons markedly varied their level of participation in the network from swim episode-to-episode. The generality of such unreliably bursting neurons was confirmed by their presence in the rhythmic escape networks of two other molluscan species, Tritonia festiva and Aplysia californica. Our observations support a view that neuronal networks, even those underlying rhythmic and stereotyped motor programs, may be more variable in structure than widely appreciated.

Validation of Independent Component Analysis for Rapid Spike Sorting of Optical Recording Data

Independent component analysis (ICA) is a technique that can be used to extract the source signals from sets of signal mixtures where the sources themselves are unknown. The analysis of optical recordings of invertebrate neuronal networks with fast voltage-sensitive dyes could benefit greatly from ICA. These experiments can generate hundreds of voltage traces containing both redundant and mixed recordings of action potentials originating from unknown numbers of neurons. ICA can be used as a method for converting such complex data sets into single-neuron traces, but its accuracy for doing so has never been empirically evaluated. Here, we tested the accuracy of ICA for such blind source separation by simultaneously performing sharp electrode intracellular recording and fast voltage-sensitive dye imaging of neurons located in the central ganglia of Tritonia diomedea and Aplysia californica, using a 464-element photodiode array. After running ICA on the optical data sets, we found that in 34 of 34 cases the intracellularly recorded action potentials corresponded 100% to the spiking activity of one of the independent components returned by ICA. We also show that ICA can accurately sort action potentials into single neuron traces from a series of optical data files obtained at different times from the same preparation, allowing one to monitor the network participation of large numbers of individually identifiable neurons over several recording episodes. Our validation of the accuracy of ICA for extracting the neural activity of many individual neurons from noisy, mixed, and redundant optical recording data sets should enable the use of this powerful large-scale imaging approach for studies of invertebrate and suitable vertebrate neuronal networks.

A stereo-compound hybrid microscope for combined intracellular and optical recording of invertebrate neural network activity.

Optical recording studies of invertebrate neural networks with voltage-sensitive dyes seldom employ conventional intracellular electrodes. This may in part be due to the traditional reliance on compound microscopes for such work. While such microscopes have high light-gathering power, they do not provide depth of field, making working with sharp electrodes difficult. Here we describe a hybrid microscope design, with switchable compound and stereo objectives, that eases the use of conventional intracellular electrodes in optical recording experiments. We use it, in combination with a voltage-sensitive dye and photodiode array, to identify neurons participating in the swim motor program of the marine mollusk Tritonia. This microscope design should be applicable to optical recording studies in many preparations.

Memory Formation in Tritonia via Recruitment of Variably Committed Neurons

Prior studies have found that functional networks can rapidly add neurons as they build short-term memories, yet little is known about the principles underlying this process. Using voltage-sensitive dye imaging, we found that short-term sensitization of Tritonias swim motor program involves rapid expansion of the number of participating neurons. Tracking neurons across trials revealed that this involves the conversion of recently discovered variably participating neurons to reliable status. Further, we identify a candidate serotonergic cellular mechanism mediating this process. Our findings reveal a new mechanism for memory formation, involving recruitment of pre-positioned, variably committed neurons into memory networks. This represents a shift from the fields long-term focus on synaptic plasticity, toward a view that certain neurons have characteristics that predispose them to join networks with learning.

Long-Term Habituation in the Marine Mollusc Tritonia diomedea

Tritonia diomedea is one of several gastropod molluscs used to study cellular mechanisms of learning and memory. Previous studies in this organism have focused on short-term habituation and sensitization. This report presents the first detailed description of long-term habituation in Tritonia. Experimental animals were given 11 swim sessions, each consisting of 10 trials, over 6 days, during which they typically displayed an initial sensitization, followed by short-term, within-session habituation. Responses were compared to controls, which were given a single stimulus per day. Cycle number habituation steadily accumulated over the days of training, and then persisted for at least 2 days after the end of training. These findings will permit comparative studies of the cellular mechanisms of short- and long-term memory in this highly tractable model system.

Recent developments in VSD imaging of small neuronal networks

Voltage-sensitive dye (VSD) imaging is a powerful technique that can provide, in single experiments, a large-scale view of network activity unobtainable with traditional sharp electrode recording methods. Here we review recent work using VSDs to study small networks and highlight several results from this approach. Topics covered include circuit mapping, network multifunctionality, the network basis of decision making, and the presence of variably participating neurons in networks. Analytical tools being developed and applied to large-scale VSD imaging data sets are discussed, and the future prospects for this exciting field are considered.