A clarifying method that improves imaging of Aplysia ganglia.

Improvements in the imaging of neural circuits are essential for studies of network function in both invertebrates and vertebrates. Therefore, CLARITY, a new imaging enhancement technique developed for mouse brains has attracted broad interest from researchers working on other species. We studied the potential of a modified version of CLARITY to enhance the imaging of ganglia in an invertebrate Aplysia. For example, we have modified the hydrogel solution and designed a small container for the Aplysia ganglia. The ganglia were first processed for immunohistochemistry, and then for CLARITY. We examined the compatibility of these techniques and the extent to which the imaging of fluorescence improved using confocal microscopy. We found that CLARITY did indeed enhance the imaging of CP2 immunopositive neurons in Aplysia ganglia. For example, it improved visualization of small, weak immunoreactive neurons deep in the ganglia. Our modifications of CLARITY make this new method suitable for future use in Aplysia experiments. Furthermore, our techniques are likely to facilitate imaging in other invertebrate ganglia.

Complementary interactions between command-like interneurons that function to activate and specify motor programs.

Motor activity is often initiated by a population of command-like interneurons. Command-like interneurons that reliably drive programs have received the most attention, so little is known about how less reliable command-like interneurons may contribute to program generation. We study two electrically coupled interneurons, cerebral-buccal interneuron-2 (CBI-2) and CBI-11, which activate feeding motor programs in the mollusk Aplysia californica. Earlier work indicated that, in rested preparations, CBI-2, a powerful activator of programs, can trigger ingestive and egestive programs. CBI-2 reliably generated ingestive patterns only when it was repeatedly stimulated. The ability of CBI-2 to trigger motor activity has been attributed to the two program-promoting peptides it contains, FCAP and CP2. Here, we show that CBI-11 differs from CBI-2 in that it contains FCAP but not CP2. Furthermore, it is weak in its ability to drive programs. On its own, CBI-11 is therefore less effective as a program activator. When it is successful, however, CBI-11 is an effective specifier of motor activity; that is, it drives mostly ingestive programs. Importantly, we found that CBI-2 and CBI-11 complement each other's actions. First, prestimulation of CBI-2 enhanced the ability of CBI-11 to drive programs. This effect appears to be partly mediated by CP2. Second, coactivation of CBI-11 with CBI-2 makes CBI-2 programs immediately ingestive. This effect may be mediated by specific actions that CBI-11 exerts on pattern-generating interneurons. Therefore, different classes of command-like neurons in a motor network may make distinct, but potentially complementary, contributions as either activators or specifiers of motor activity.

A Single Central Pattern Generator for the Control of a Locomotor Rolling Wave in Mollusc Aplysia.

Locomotion in mollusc Aplysia is implemented by a pedal rolling wave, a type of axial locomotion. Well-studied examples of axial locomotion (pedal waves in Drosophila larvae and body waves in leech, lamprey, and fish) are generated in a segmented nervous system via activation of multiple coupled central pattern generators (CPGs). Pedal waves in molluscs, however, are generated by a single pedal ganglion, and it is unknown whether there are single or multiple CPGs that generate rhythmic activity and phase shifts between different body parts. During locomotion in intact Aplysia, bursting activity in the parapedal commissural nerve (PPCN) was found to occur during tail contraction. A cluster of 20 to 30 P1 root neurons (P1Ns) on the ventral surface of the pedal ganglion, active during the pedal wave, were identified. Computational cluster analysis revealed that there are 2 phases to the motor program: phase I (centered around 168°) and phase II (centered around 357°). PPCN activity occurs during phase II. The majority of P1Ns are motoneurons. Coactive P1Ns tend to be electrically coupled. Two classes of pedal interneurons (PIs) were characterized. Class 1 (PI1 and PI2) is active during phase I. Their axons make a loop within the pedal ganglion and contribute to locomotor pattern generation. They are electrically coupled to P1Ns that fire during phase I. Class 2 (PI3) is active during phase II and innervates the contralateral pedal ganglion. PI3 may contribute to bilateral coordination. Overall, our findings support the idea that Aplysia pedal waves are generated by a single CPG.