424 Neurobiology : Frost and Katz A 1 Normal Saline 2 High Divalents B 1 DSI and C 2 Hyperpolarized 2 DRI , DSI and C

While there are many instances of single neurons that can drive rhythmic stimulus-elicited motor programs, such neurons have seldom been found to be necessary for motor program function. In the isolated central nervous system of the marine mollusc Tritonia diomedea, brief stimulation (1 sec) of a peripheral nerve activates an interneuronal central pattern generator that produces the longlasting (-30-60 sec) motor program underlying the animal's rhythmic escape swim. Here, we identify a single interneuron, DRI (for dorsal ramp interneuron), that (i) conveys the sensory information from this stimulus to the swim central pattern generator, (ii) elicits the swim motor program when driven with intracellular stimulation, and (iii) blocks the depolarizing "ramp" input to the central pattern generator, and consequently the motor program itself, when hyperpolarized during the nerve stimulus. Because most of the sensory information appears to be funneled through this one neuron as it enters the pattern generator, DRI presents a striking example of single neuron control over a complex motor circuit. Over 30 yr ago, Wiersma and Ikeda (1) introduced the term "command neuron" to describe single interneurons in the crayfish that could drive coordinated movements of the animal's swimmerets. In its most restricted form, a command neuron is currently defined as a single interneuron, situated between sensory neurons and the motor pattern-generating circuitry, whose activity is both necessary and sufficient for sensory activation of the motor program (2). Many cells have now been described that can drive stimulus-elicited motor programs (3-13), but only rarely have such neurons been shown to also be necessary for circuit operation. Instead, in most cases these neurons have been found to operate in parallel with other circuit elements that fill-in when the cell in question is removed from the network (7-13). These and other findings have given rise in recent years to the view that, in most systems, command properties are distributed across broad interneuronal networks, with single neurons having only minor roles. We here describe a newly found interneuron in the Tritonia escape swim neural circuit that fulfills the strict definition of a command neuron (see also Discussion). The properties of this neuron, the dorsal ramp interneuron (DRI), provide further support for the original idea that the command function can be highly localized within a circuit, in this case, by funneling sensory information to the swim central pattern generator (CPG) and thereby controlling whether or not the swim motor program will be activated. When the marine mollusc Tritonia diomedea encounters the tube feet of certain predatory sea stars, it responds with a vigorous rhythmic escape swim, consisting of a series of alternating ventral and dorsal whole-body flexions (14, 15). The neural circuit generating this behavior has been wellstudied and consists of identified populations of afferent neurons (16, 17), CPG neurons (18-24), and efferent neurons The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 422 (25, 26). The Tritonia swim CPG is a network oscillator; its rhythmic output (Fig. 1C) arises entirely from the synaptic connectivity of the neurons, with no cells having intrinsic bursting properties of their own (27). This network also operates with little or no need for sensory feedback and thus can be studied in isolated brain preparations (14). A key missing element in the Tritonia swim network has been the hypothesized interneurons that transform the brief activity of the sensory neurons into the long-lasting, declining "ramp" depolarization in the dorsal swim interneurons (DSIs) of the CPG (28, 29). This depolarization serves as the main extrinsic excitatory drive for the swim motor program. Because of the strategic position of these neurons in the swim circuit, both in terms of their role in driving the motor program as well as their potential role as storage sites for learned information (30), we sought to locate them. We here identify a "ramp" interneuron in the Tritonia swim circuit, DRI, and find it to have an unusually prominent role for an individual neuron in the activation of a complex motor program. MATERIALS AND METHODS All experiments used an isolated central nervous system preparation, consisting of the left and right cerebral, pleural, and pedal ganglia. After removal from the animal the ganglia were pinned dorsal side up on the Sylgard floor of a recording chamber perfused with normal saline at 2°C. The connective tissue over the cerebral and pleural ganglia was then dissected away to expose the underlying neurons. Suction electrodes for extracellular recording and stimulation were made from polyethylene tubing and attached to the left and right pedal nerve 3 (PdN3), two of the many nerves that can be used to elicit the swim motor program (see Fig. 1A and ref. 25 for nomenclature). The preparation was then warmed to 10°C and rested for a minimum of 3 hr before beginning recordings. Swim motor programs were elicited with a brief stimulation of PdN3 (2-msec pulses, 10 Hz, 1 sec). This stimulus typically elicited a four to seven cycle swim motor program lasting 30-60 sec. Intracellular recordings were made with glass microelectrodes (10-40 Mfl) filled with either 3 M potassium chloride or 4 M potassium acetate. The CPG neurons were identified on the basis of soma location and coloration, synaptic interactions, and activity pattern during the swim motor program (20-23). DRI was labeled by iontophoretic injection of 5% carboxyfluorescein (Molecular Probes) in 0.1 M potassium acetate. Normal saline composition was as follows: 420 mM NaCl, 10 mM KCl, 10 mM CaC12, 50mM MgCl2, 10 mM Hepes (pH 7.6), and 11 mM D-glucose. High divalent-cation saline composition was as follows: 285 mM NaCl, 10 mM KCl, 25 mM CaC12, 125 mM MgCl2, 10 mM Hepes, (pH 7.6), and 11 mM D-glucose. Animals were collected from Bellingham Bay, Washington. Abbreviations: CPG, central pattern generator; DRI, dorsal ramp interneuron; PdN3, pedal nerve 3; DSI, dorsal swim interneuron; EPSP, excitatory postsynaptic potential. *To whom reprint requests should be addressed. Proc. Natl. Acad. Sci. USA 93 (1996) 423