The zebrafish has significant advantages for studying the morphological development of the brain. However, little is known about the functional development of the zebrafish brain. We used patch clamp electrophysiology in live animals to investigate the emergence of excitability in cerebellar Purkinje cells, functional maturation of the cerebellar circuit, and establishment of sensory input to the cerebellum. Purkinje cells are born at 3 days post-fertilization (dpf). By 4 dpf, Purkinje cells spontaneously fired action potentials in an irregular pattern. By 5 dpf, the frequency and regularity of tonic firing had increased significantly and most cells fired complex spikes in response to climbing fiber activation. Our data suggest that, as in mammals, Purkinje cells are initially innervated by multiple climbing fibers that are winnowed to a single input. To probe the development of functional sensory input to the cerebellum, we investigated the response of Purkinje cells to a visual stimulus consisting of a rapid change in light intensity. At 4 dpf, sudden darkness increased the rate of tonic firing, suggesting that afferent pathways carrying visual information are already active by this stage. By 5 dpf, visual stimuli also activated climbing fibers, increasing the frequency of complex spiking. Our results indicate that the electrical properties of zebrafish and mammalian Purkinje cells are highly conserved and suggest that the same ion channels, Nav1.6 and Kv3.3, underlie spontaneous pacemaking activity. Interestingly, functional development of the cerebellum is temporally correlated with the emergence of complex, visually-guided behaviors such as prey capture. Because of the rapid formation of an electrically-active cerebellum, optical transparency, and ease of genetic manipulation, the zebrafish has great potential for functionally mapping cerebellar afferent and efferent pathways and for investigating cerebellar control of motor behavior.

Whether changes in neuronal excitability can cause neurodegenerative disease in the absence of other factors such as protein aggregation is unknown. Mutations in the Kv3.3 voltage-gated K+ channel cause spinocerebellar ataxia type-13 (SCA13), a human autosomal dominant disease characterized by locomotor impairment and the death of cerebellar neurons. Kv3.3 channels facilitate repetitive, high-frequency firing of action potentials, suggesting that pathogenesis in SCA13 is triggered by changes in electrical activity in neurons. To investigate whether SCA13 mutations alter excitability in vivo, we expressed the human dominant negative R420H mutant subunit in zebrafish. The disease-causing mutation specifically suppressed the excitability of Kv3.3-expressing, fast-spiking motor neurons during evoked firing and fictive swimming and, in parallel, decreased the precision and amplitude of the startle response. The dominant negative effect of the mutant subunit on K+ current amplitude was directly responsible for the reduced excitability and locomotor phenotype. Our data provide strong evidence that changes in excitability initiate pathogenesis in SCA13 and establish zebrafish as an excellent model system for investigating how changes in neuronal activity impair locomotor control and cause cell death.

SUMMARYExamining neuronal network activity in freely behaving animals is advantageous for probing the function of the vertebrate central nervous system. Here, we describe a simple, robust technique for monitoring the activity of neural circuits in unfettered, freely behaving zebrafish (Danio rerio). Zebrafish respond to unexpected tactile stimuli with short-or long-latency escape behaviors, which are mediated by distinct neural circuits. Using dipole electrodes immersed in the aquarium, we measured electric field potentials generated in muscle during short-and long-latency escapes. We found that activation of the underlying neural circuits produced unique field potential signatures that are easily recognized and can be repeatedly monitored. In conjunction with behavioral analysis, we used this technique to track changes in the pattern of circuit activation during the first week of development in animals whose trigeminal sensory neurons were unilaterally ablated. One day post-ablation, the frequency of short-and long-latency responses was significantly lower on the ablated side than on the intact side. Three days post-ablation, a significant fraction of escapes evoked by stimuli on the ablated side was improperly executed, with the animal turning towards rather than away from the stimulus. However, the overall response rate remained low. Seven days post-ablation, the frequency of escapes increased dramatically and the percentage of improperly executed escapes declined. Our results demonstrate that trigeminal ablation results in rapid reconfiguration of the escape circuitry, with reinnervation by new sensory neurons and adaptive changes in behavior. This technique is valuable for probing the activity, development, plasticity and regeneration of neural circuits under natural conditions.

Creation of a dominance hierarchy within a population of animals typically involves a period of agonistic activity in which winning and losing decide relative positions in the hierarchy. Among crayfish, fighting between size-matched animals leads to an abrupt change of behavior as the new subordinate retreats and escapes from the attacks and approaches of the dominant (Issa et al., 1999). We used high-speed videography and electrical recordings of aquarium field potentials to monitor the release of aggressive and defensive behavior, including the activation of neural circuits for four different tail-flip behaviors. We found that the sequence of tail-flip circuit excitation traced the development of their dominance hierarchy. Offensive tail flipping, attacks, and approaches by both animals were followed by a sharp rise in the frequency of nongiant and medial giant escape tail flips and a fall in the frequency of offensive tail flips of the new subordinate. These changes suggest that sudden, coordinated changes in the excitability of a set of neural circuits in one animal produce the changes in behavior that mark its transition to subordinate status.

Fifty years of study of the nervous system and behavior of crayfish have revealed neural circuits for movements that are similar to those seen during formation of a dominance hierarchy. Given this background, it is of interest to ask what is understood about the neural substrates of dominance hierarchy formation. Here we will consider the social behavior that crayfish display in the wild and in the laboratory, and its relationship to movements released by activation of specific neural circuits. We will consider how these movements might be knit together to produce the behavior patterns that are characteristic of dominant and subordinate animals.