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Neuromodulation: Letting Sources of messenger pathways [5,6]. In larval zebrafish, for example, dopamine can Spinal Dopamine Speak for promote locomotion via activation of lower-affinity D1 or higher-affinity D4 Themselves receptors, and inhibit locomotion via higher-affinity D2 or D3 receptors [7,8]. To begin to place these observations A recent study of dopaminergic neurons in the brain of larval zebrafish has in a behavioral context, Jay et al. [1] important implications for interpreting the natural actions of neuromodulators focused on an evolutionarily in the . conserved group of dopaminergic diencephalospinal neurons (DDNs) in Sandeep Kishore head on, as it were, using the zebrafish the , which provide the sole and David L. McLean* model system. source of spinal dopamine not only in One of the earliest demonstrations zebrafish [9,10], but also in mammals The surest way to get reliable that aminergic neuromodulators play a [11]. The authors took advantage of an information is to go to the source. In critical role in facilitating vertebrate enhancer trap transgenic line of vertebrates, key sources of aminergic locomotion was provided by zebrafish, Tg(ETvmat2:GFP), in which neuromodulators that help produce experiments in the 1960s, where these neurons are labeled by green locomotion are located in the brain. systemic application of a precursor to fluorescent protein (GFP) [12]. The However, much of our understanding catecholamine synthesis, L-DOPA, relatively large size and location of the of during locomotion rescued walking movements in cells made it possible to monitor not has come from studies where the spinalized cats [2]. This led to a focus on only their activity patterns, but also spinal cord is isolated, drugs are bath the actions of amines such as their excitatory and inhibitory synaptic applied, and changes in locomotor , noradrenaline and dopamine inputs, using patch-clamp recordings output due to changes in spinal neuron within the spinal cord, which contains in intact, chemically-immobilized fish excitability and connectivity are the circuitry necessary for executing capable of generating ‘fictive’ measured. Because of the difficulty of locomotion [3]. A body of work too large swimming (Figure 1A). By including recording from sources of to adequately cover here has fluorescent dye in the patch pipette, or neuromodulators in the brain of intact, subsequently described myriad actions by using a post-hoc stain, the authors locomoting animals, the behavioral in numerous species [4]. Of particular confirmed that their recordings were relevance of pharmacological relevance to the Jay et al. [1] study is the from spinal projecting neurons. manipulations in the spinal cord is still fact that dopamine can exert different Unexpectedly, they also found that unclear. In this issue of Current effects via receptor subtypes with DDN axons exited the central nervous Biology, Jay et al. [1] tackle this issue different affinities and second system and targeted auditory and Dispatch R147

somatosensory structures in the swimming could occur in the absence of A 4-day-old larval zebrafish periphery (Figure 1A). So what might bursting and vice versa. Consistent with Spinal cord these unusual neurons be doing? this idea, they found no relationship The authors first investigated the between the occurrence of a DDN burst firing behavior of DDNs and the and the duration of a swim bout, LL underlying synaptic drive. They suggesting spinal dopamine is unlikely OC identified two firing regimes that to play a role in patterning spontaneous occurred spontaneously within the locomotor output. Critically, however, Motor neurons same fish, namely asynchronous ‘tonic’ when combined with the patch-clamp or muscle fibers and synchronous ‘burst’ activity. The observations, these data reveal for the Dopaminergic diencephalospinal neurons (DDNs) tonic pattern was characterized by a first time that DDNs receive a bolus of relatively low, steady sequence of firing excitatory drive concomitant with B Tonic DDN activity (frequencies less than 6 Hz). In excitation to the spinal circuits mammalian dopamine responsible for locomotion. neurons, this form of tonic firing is The work described thus far is No motor activity largely a product of their intrinsic already a remarkable achievement, as electrical properties [13]. Using few studies have monitored the natural antibiotic-based perforated patch- firing patterns of spinal-projecting Burst DDN activity clamp methods that preserve the modulatory populations during integrity of the intracellular milieu and locomotion [16], and none have sequential pharmacological blocks of examined the synaptic inputs excitatory glutamate and inhibitory responsible for driving these patterns. Episodic motor activity GABA receptors, Jay et al. [1] However, Jay et al. [1] followed this up demonstrated that DDN neurons also by answering a major open question: have the inherent ability to spike what happens if you selectively remove tonically, and that variability in this the principal source of spinal C DDN intact DDN ablated pattern of firing is a product of dopamine? The authors took intermittent glutamate and GABA input. advantage of the fact that the DDNs are Dish The burst behavior, on the other the first dopamine neurons to develop hand, was characterized by higher in zebrafish [17] and are thus more incidences of firing (frequencies up to easily targeted. Using intense Larva 40 Hz), which resulted from a ultraviolet light to selectively ablate synchronous volley of glutamate input. GFP-positive DDNs in one-day-old Normal swim Reduced swim Again, behaviorally relevant switches ETvmat2:GFP embryos (the Current Biology between tonic and burst modes of effectiveness and selectivity of which firing have been reported in mammalian were confirmed using immunolabeling Figure 1. Anatomy, activity and ablation of dopamine neurons [14]. So what, if any, in four-day-old larvae), they then spinal sources of dopamine in larval zebra- is the behavioral distinction between assessed the behavioral consequences fish. tonic and burst firing in zebrafish? via semi-automated tracking of real (A) Schematic of the front half of a four-day- At this point, the advantage of swimming in four-day-old larvae within old larval zebrafish (head is to the left) conducting such experiments in an a multiwell dish (Figure 1C). In control illustrating the experimental set up for patch-clamp recordings. Projections from animal capable of autonomously and DDN ablated fish, the characteristic DDNs (in red) innervate the spinal cord by generating fictive locomotion becomes episodic locomotor pattern was still way of the midbrain and hindbrain (light apparent. Using whole-cell patch- observed, yet in the absence of DDNs blue), and also exit the central nervous sys- clamp recordings from either the the larvae tended to cover less tem (dashed lines), where they innervate the muscles that produce swimming distance. More detailed analysis auditory otic capsule (OC), somatosensory movements or the motor neurons that revealed that this was due to the total lateral line (LL) neuromasts, and cranial neu- romasts (not shown). To facilitate access to drive the muscles, Jay et al. [1] revealed time spent swimming, rather than the the DDNs, the left eye (dashed grey circle) that the burst firing pattern, and not speed or duration of episodic was removed. (B) When larvae are not the tonic firing pattern, is associated swimming bouts. The picture that attempting to move, DDN neurons fire with the production of locomotion emerges is that one of the natural sporadically at low rates (tonic activity; (Figure 1A,B). Swimming in larval functions of dopamine release in red vertical lines); however, episodic bouts zebrafish is episodic, characterized by zebrafish spinal cord is to maintain of swimming activity (black bars) are associated with more temporally clustered, a ‘beat and glide’ movement pattern some level of excitatory tone, which higher frequency firing behavior in DDNs [15]. During tonic firing, no motor ultimately facilitates the production of (burst activity). (C) Semi-automated behav- activity is observed, but bursts within locomotion, as it does in mammals [4]. ioral analysis was performed from videos DDN neurons largely coincide with This function seems best suited to captured from above tracking the move- the episodic bouts of swimming tonic DDN firing, presumably through ments of freely swimming larvae (in red) in a (Figure 1B). The authors are careful to higher affinity dopamine receptors, but multiwell dish (grey circles). Selective abla- tion of DDNs decreases the overall distance point out that, while DDN bursts and what about burst firing? Do sudden traveled in a 10-minute period, but the locomotor activity were largely increases in spinal dopamine activate episodic pattern of locomotion (dashed black coincidental, burst activity in DDN lower affinity receptors and have the lines) is preserved. neurons is unlikely to be necessary or opposite effect? As Jay et al. [1] point sufficient to drive locomotion, because out, modeling studies of mammalian Current Biology Vol 25 No 4 R148 dopamine neurons suggest that tonic genes that drive optogenetic actuators single-neuron integration of zebrafish ascending and descending dopaminergic versus burst firing would result in to activate or silence DDNs [19], and systems. Nat. Commun. 2, 171. differences in the relative occupancy of the development of closed loop 10. McLean, D.L., and Fetcho, J.R. (2004). receptors with different affinities [18].In systems that drive more complex larval Relationship of tyrosine hydroxylase and serotonin immunoreactivity to sensorimotor support, pharmacological experiments behaviors [20], make it likely that circuitry in larval zebrafish. J. Comp. Neurol. in zebrafish have demonstrated that answers to these questions are not far 480, 57–71. 11. Koblinger, K., Fuzesi, T., Ejdrygiewicz, J., dopamine can shorten the duration of off. Given the conserved genetic Krajacic, A., Bains, J.S., and Whelan, P.J. drug-evoked episodic swimming bouts origins of DDNs and the similarity in (2014). Characterization of A11 neurons in spinalized larvae, something that their activity patterns to mammalian projecting to the spinal cord of mice. PLoS One 9, e109636. is attributed to the progressive dopamine neurons, the zebrafish 12. Wen, L., Wei, W., Gu, W., Huang, P., Ren, X., innervation of the spinal cord by DDNs model system will surely be a reliable Zhang, Z., Zhu, Z., Lin, S., and Zhang, B. (2008). Visualization of monoaminergic neurons and [7]. However, this observation is source for principles underlying the neurotoxicity of MPTP in live transgenic difficult to reconcile with the fact that modulation of circuits and behavior by zebrafish. Dev. Biol. 314, 84–92. early ablation of DDNs produced dopamine in years to come. 13. Surmeier, D.J., Mercer, J.N., and Chan, C.S. (2005). Autonomous pacemakers in the basal no effects on the patterning of ganglia: who needs excitatory synapses spontaneous, real swimming. While References anyway? Curr. Opin. Neurobiol. 15, 312–318. the authors suggest possible 1. Jay, M., De Faveri, F., and McDearmid, J.R. 14. Tsai, H.C., Zhang, F., Adamantidis, A., Stuber, (2015). Firing dynamics and modulatory actions G.D., Bonci, A., de Lecea, L., and Deisseroth, K. explanations for this discrepancy, of supraspinal dopaminergic neurons during (2009). Phasic firing in dopaminergic neurons is including potential off-target effects of zebrafish locomotor behavior. Curr. Biol. 25, sufficient for behavioral conditioning. Science 324, 1080–1084. the pharmacological manipulations, it 435–444. 2. Jankowska, E., Jukes, M.G., Lund, S., and 15. Buss, R.R., and Drapeau, P. (2001). Synaptic remains to be seen exactly what DDN Lundberg, A. (1967). The effect of DOPA on the drive to motoneurons during fictive swimming bursting contributes to zebrafish spinal cord. 5. Reciprocal organization of in the developing zebrafish. J. Neurophysiol. pathways transmitting excitatory action to 86, 197–210. locomotion. alpha motoneurones of flexors and extensors. 16. Veasey, S.C., Fornal, C.A., Metzler, C.W., and In this sense, the work by Jay et al. [1] Acta Physiol. Scand. 70, 369–388. Jacobs, B.L. (1995). Response of serotonergic caudal raphe neurons in relation to specific achieves the goal of all high quality 3. Grillner, S., and Jessell, T.M. (2009). Measured motion: searching for simplicity in spinal motor activities in freely moving cats. studies, in that it generates more locomotor networks. Curr. Opin. Neurobiol. 19, J. Neurosci. 15, 5346–5359. questions than it answers. The 572–586. 17. McLean, D.L., and Fetcho, J.R. (2004). 4. Miles, G.B., and Sillar, K.T. (2011). Ontogeny and innervation patterns of description of different modes of firing Neuromodulation of vertebrate locomotor dopaminergic, noradrenergic, and serotonergic not only helps put pharmacological control networks. Physiology 26, 393–411. neurons in larval zebrafish. J. Comp. Neurol. 480, 38–56. observations in a proper context, but 5. Missale, C., Nash, S.R., Robinson, S.W., Jaber, M., and Caron, M.G. (1998). Dopamine 18. Dreyer, J.K., Herrik, K.F., Berg, R.W., and also provides a framework for receptors: from structure to function. Physiol. Hounsgaard, J.D. (2010). Influence of phasic investigating how dynamic changes in Rev. 78, 189–225. and tonic dopamine release on receptor 6. Clemens, S., Belin-Rauscent, A., Simmers, J., activation. J. Neurosci. 30, 14273–14283. dopamine levels in the spinal cord and and Combes, D. (2012). Opposing modulatory 19. Auer, T.O., Duroure, K., Concordet, J.P., and elsewhere may exert differential effects effects of D1- and D2-like receptor activation Del Bene, F. (2014). CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines on locomotor behavior. Are different on a spinal central pattern generator. J. Neurophysiol. 107, 2250–2259. in zebrafish. Nat. Protoc. 9, 2823–2840. dopamine receptor subtypes located 7. Lambert, A.M., Bonkowsky, J.L., and Masino, 20. Engert, F. (2012). Fish in the matrix: motor on the same or different spinal circuit M.A. (2012). The conserved dopaminergic learning in a virtual world. Front. Neural Circuits diencephalospinal tract mediates vertebrate 6, 125. elements? How about targets in the locomotor development in zebrafish larvae. or the periphery? Does the J. Neurosci. 32, 13488–13500. transition from tonic to burst firing 8. Thirumalai, V., and Cline, H.T. (2008). Department of Neurobiology, Northwestern Endogenous dopamine suppresses initiation of University, Evanston, IL 60208, USA. orchestrate a common behavioral goal swimming in prefeeding zebrafish larvae. *E-mail: [email protected] via these distributed targets? If so, J. Neurophysiol. 100, 1635–1648. 9. Tay, T.L., Ronneberger, O., Ryu, S., Nitschke, what is this behavior? The ability to R., and Driever, W. (2011). Comprehensive replace GFP in ETvmat2:GFP fish with catecholaminergic projectome analysis reveals http://dx.doi.org/10.1016/j.cub.2015.01.001

Photoreceptor Evolution: Ancient new animal forms ushered in the Cambrian epoch, and at its end, a ‘Cones’ Turn Out to Be Rods little more than 500 million years ago, the earliest true vertebrates appeared. These were the so-called Vertebrate rod photoreceptors are thought to have evolved from cone jawless fishes, or Agnathans, of photoreceptors only after the divergence of the jawed and jawless fishes, but which only two lineages survive until this idea is questioned by new evidence that the short ‘cones’ of jawless sea the present day, the hagfishes and lampreys are physiologically equivalent to rods. the lampreys. From the Agnatha evolved the jawed fishes, or Eric J. Warrant began: in the space of just 20 million Gnathostomes, and from these years — a blink of an eye in geological arose all the vertebrate lineages we About 540 million years ago one of terms — many of our familiar modern are familiar with today, including our the most spectacular events in the animal lineages suddenly appeared own. The eyes of these early jawed history of the evolution of animal life on the Earth. This explosion of fishes were probably very much like