Multimodal sensory information is represented by a combinatorial code in a sensorimotor system.

A ubiquitous feature of the nervous system is the processing of simultaneously arriving sensory inputs from different modalities. Yet, because of the difficulties of monitoring large populations of neurons with the single resolution required to determine their sensory responses, the cellular mechanisms of how populations of neurons encode different sensory modalities often remain enigmatic. We studied multimodal information encoding in a small sensorimotor system of the crustacean stomatogastric nervous system that drives rhythmic motor activity for the processing of food. This system is experimentally advantageous, as it produces a fictive behavioral output in vitro, and distinct sensory modalities can be selectively activated. It has the additional advantage that all sensory information is routed through a hub ganglion, the commissural ganglion, a structure with fewer than 220 neurons. Using optical imaging of a population of commissural neurons to track each individual neuron's response across sensory modalities, we provide evidence that multimodal information is encoded via a combinatorial code of recruited neurons. By selectively stimulating chemosensory and mechanosensory inputs that are functionally important for processing of food, we find that these two modalities were processed in a distributed network comprising the majority of commissural neurons imaged. In a total of 12 commissural ganglia, we show that 98% of all imaged neurons were involved in sensory processing, with the two modalities being processed by a highly overlapping set of neurons. Of these, 80% were multimodal, 18% were unimodal, and only 2% of the neurons did not respond to either modality. Differences between modalities were represented by the identities of the neurons participating in each sensory condition and by differences in response sign (excitation versus inhibition), with 46% changing their responses in the other modality. Consistent with the hypothesis that the commissural network encodes different sensory conditions in the combination of activated neurons, a new combination of excitation and inhibition was found when both pathways were activated simultaneously. The responses to this bimodal condition were distinct from either unimodal condition, and for 30% of the neurons, they were not predictive from the individual unimodal responses. Thus, in a sensorimotor network, different sensory modalities are encoded using a combinatorial code of neurons that are activated or inhibited. This provides motor networks with the ability to differentially respond to categorically different sensory conditions and may serve as a model to understand higher-level processing of multimodal information.

Spatial distribution of intermingling pools of projection neurons with distinct targets: A 3D analysis of the commissural ganglia in Cancer borealis.

Projection neurons play a key role in carrying long-distance information between spatially distant areas of the nervous system and in controlling motor circuits. Little is known about how projection neurons with distinct anatomical targets are organized, and few studies have addressed their spatial organization at the level of individual cells. In the paired commissural ganglia (CoGs) of the stomatogastric nervous system of the crab Cancer borealis, projection neurons convey sensory, motor, and modulatory information to several distinct anatomical regions. While the functions of descending projection neurons (dPNs) which control downstream motor circuits in the stomatogastric ganglion are well characterized, their anatomical distribution as well as that of neurons projecting to the labrum, brain, and thoracic ganglion have received less attention. Using cell membrane staining, we investigated the spatial distribution of CoG projection neurons in relation to all CoG neurons. Retrograde tracing revealed that somata associated with different axonal projection pathways were not completely spatially segregated, but had distinct preferences within the ganglion. Identified dPNs had diameters larger than 70% of CoG somata and were restricted to the most medial and anterior 25% of the ganglion. They were contained within a cluster of motor neurons projecting through the same nerve to innervate the labrum, indicating that soma position was independent of function and target area. Rather, our findings suggest that CoG neurons projecting to a variety of locations follow a generalized rule: for all nerve pathway origins, the soma cluster centroids in closest proximity are those whose axons project down that pathway.

Optical imaging of neuronal activity and visualization of fine neural structures in non-desheathed nervous systems.

Locating circuit neurons and recording from them with single-cell resolution is a prerequisite for studying neural circuits. Determining neuron location can be challenging even in small nervous systems because neurons are densely packed, found in different layers, and are often covered by ganglion and nerve sheaths that impede access for recording electrodes and neuronal markers. We revisited the voltage-sensitive dye RH795 for its ability to stain and record neurons through the ganglion sheath. Bath-application of RH795 stained neuronal membranes in cricket, earthworm and crab ganglia without removing the ganglion sheath, revealing neuron cell body locations in different ganglion layers. Using the pyloric and gastric mill central pattern generating neurons in the stomatogastric ganglion (STG) of the crab, Cancer borealis, we found that RH795 permeated the ganglion without major residue in the sheath and brightly stained somatic, axonal and dendritic membranes. Visibility improved significantly in comparison to unstained ganglia, allowing the identification of somata location and number of most STG neurons. RH795 also stained axons and varicosities in non-desheathed nerves, and it revealed the location of sensory cell bodies in peripheral nerves. Importantly, the spike activity of the sensory neuron AGR, which influences the STG motor patterns, remained unaffected by RH795, while desheathing caused significant changes in AGR activity. With respect to recording neural activity, RH795 allowed us to optically record membrane potential changes of sub-sheath neuronal membranes without impairing sensory activity. The signal-to-noise ratio was comparable with that previously observed in desheathed preparations and sufficiently high to identify neurons in single-sweep recordings and synaptic events after spike-triggered averaging. In conclusion, RH795 enabled staining and optical recording of neurons through the ganglion sheath and is therefore both a good anatomical marker for living neural tissue and a promising tool for studying neural activity of an entire network with single-cell resolution.