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Show Neurocircuitry underlying vocal production of the African clawed frog, Xenopus laevis! Joseph Perry1, Kristy Lawton1, Todd Appleby2, Ayako Yamaguchi2, Erik Zornik1 1Reed College, Portland, OR 97202 2University of Utah, Salt Lake City UT 84103 Xenopus vocal behaviors 500 ms fast trill slow trill Generating vocal rhythms: fast trill neurons (FTNs) Can FTNs generate fast trill rhythms without inputs from n.IX-X? Click Sound Nerve Compound ac5on poten5al (CAP) 1s Nerve recording Sound recording (hydrophone) African clawed frogs, Xenopus laevis, are fully aquatic and call underwater during courtship. Calls consist of clicks produced by the larynx at various temporal patterns. This work focuses on the male advertisement call (see oscillogram above); this call is defined by alternating fast (~60 Hz) and slow (~30 Hz) click trills. Nerve recordings from awake, calling frogs (depicted above, bottom trace), revealed that a single compound action potential (CAP) precedes every vocal click (above left; Yamaguchi and Kelley, 2000). This means that vocal patterns are produced by the brain, and that nerve activity provides a direct readout of the behavior. The isolated brain The vocal CPG remains viable in vitro. Serotonin application elicits nerve activity that is similar to what has been recorded during in vivo calling. We are using the "fictively" calling isolated brain to discover how vocal patterns are generated. We use an ex vivo brain to record multiple regions. The photo shows a local field potential (LFP) electrode (left DTAM), whole-cell patch electrode (right DTAM), and a suction electrode on the vocal nerve. Above: a nerve recording during "fic5ve vocaliza5on". Compound ac5on poten5als occur in dis5nct, alterna5ng, fast and slow trills. fast trill slow trill LFP Whole-cell electrode Nerve electrode • Nerve recordings: fictive advertisement calls (bottom trace) consist of nerve CAPs that occur at advertisement call rates. • LFP recordings: slow baseline waves are correlated with fast trills; higher frequency activity is phase-locked to nerve CAPs (middle trace). • Whole-cell recordings: "fast trill neurons" (FTNs; top) produce spikes phase-locked to nerve CAPs near the fast trill rate of 60 Hz (spike histogram; Zornik and Yamaguchi, 2012). • When the connections between DTAM and n.IX-X are cut, LFP waves still occur with 5-HT application (lower trace). • Unlike intact recordings, no fast trill rhythms occur during the wave (lower trace). • FTNs depolarize and spike throughout each wave (top trace). • Instantaneous spike rates of this cell were faster than FTN spike rates in the intact CPG (spike histogram). A model of the vocal CPG Updated model: 1. Motor neurons provide an efference copy of their activity to interneurons in n.IX-X. 2. n.IX-X interneurons are activated by motor neurons via a cholinergic synapse. 3. Feedback inhibition entrains and synchronizes FTN spiking, resulting in fast trill rhythm generation. 4. Without feedback inhibition, FTNs spike at faster and broader range of rates (as observed following transection, motor neuron silencing, and blockade of nicotinic receptors) 5. Motor activity also induces an excitatory feedback signal to contralateral FTNs that requires gap junctions. Premotor (DTAM) Motor (n.IX-X) n.IX-X inputs synchronize FTNs • In the intact CPG: FTNs largely spike at fast trill rates of ~50 - 60 Hz (example shown in dotted black line). • After transections, FTN spike frequencies are more broadly tuned (each solid line represents the spike rates of one FTN). Conclusion 1: Feedback inputs from n.IX-X to DTAM entrain and synchronize FTN firing, ensuring proper production of fast trill rhythms Vocal central pattern generator (CPG) Dorsal tegmental area of the medulla (DTAM) n. IX-‐X (vocal motor nucleus) N.IX-‐X (laryngeal nerve) Hindbrain Midbrain Forebrain Two brainstem nuclei comprise the vocal CPG: a premotor nucleus (DTAM) and the laryngeal motor nucleus (n.IX-X). DTAM and n.IX-X are connected by reciprocal projections. Premotor (DTAM) Motor (n.IX-X) Is motor neuron activity involved in generating the feedback signal? Experiment: block nicotinic receptors with tubocurarine LFP Nerve 500 ms Whole cell -‐60 mV 20 mV LFP Whole-cell LFP Whole-cell 500 ms 20 mV Whole-cell LFP 0 5 10 15 20 25 0 50 100 150 200 250 Percent of spike intervals Instantaneous spike frequency (Hz) 0 2 4 6 8 10 0 50 100 150 200 250 Percent of spike intervals Instantaneous spike frequency (Hz) 0 5 10 15 20 25 0 50 100 150 200 250 Percent of spike intervals Instantaneous spike rate (Hz) ? Experiment: silence motor neurons by backfilling motor nerve with QX-314 (and intracellular Na+ channel blocker) LFP 20 mV 500 ms Whole-‐cell 0 5 10 15 20 25 0 50 100 150 200 250 Percent of spike intervals Instantaneous spike rate (Hz) MN silenced Intact How do motor neurons activate feedback pathway? Identifying the feedback circuit LFP Whole-‐cell Nerve 20 mV 500 ms tubocurarine (20 μm) 0 5 10 15 20 25 0 50 100 150 200 250 Percent of spike intervals Instantaneous spike rate (Hz) Tubocurarine Control Cholinergic synapse? • Silencing motor neurons disrupts fast trill rhythms in FTNs. • Blockade of nicotinic acetylcholine receptors induces LFP waves, but 60 Hz fast trill rhythms are lost. • FTNs spike faster than in the intact circuit, similar to transection experiments. Does the feedback signal synchronize FTNs via inhibition? 5 mV 100 ms S5mulus ar5fact Whole-cell FTN recording IPSP latency: ~10 ms GABAergic synapse? Experiment 1: stimulate motor nerve during whole-cell FTN recording • Nerve stimulation results in short-latency IPSPs in FTNs Experiment 2: block GABAergic synapses in DTAM (gabazine injection [500 μM] followed by 5-HT application). 500 ms LFP Nerve • Blocking GABA-A receptors in DTAM results in abnormal fictive vocal behaviors. • Nerve and LFP activity lack correlates of synchronous fast trill rhythms. • This supports the hypothesis that synchronous premotor activity requires inhibitory feedback inputs. Conclusion 2: FTNs are entrained by feedback inhibition. These signals arise as an efference copy of motor output, and are transduced through an intervening inhibitory interneuron. muscles motor neurons CPG CPG motor neurons muscles Previous model: 1. FTNs directly activate vocal motor neurons (Zornik and Kelley, 2007). 2. n.IX-X interneurons project to DTAM (Zornik and Kelley, 2008), transmitter unknown 3. FTNs receive phasic IPSPs, but the source is unknown (Zornik and Yamaguchi, 2012). A novel vertebrate CPG circuit Canonical vertebrate CPG X. laevis vocal CPG Vertebrate CPGs are thought to function in a top-down manner: CPG output activates motor neurons that, in turn, induce muscle activity. Motor neurons play little, if any, role in generating behavioral rhythms. The Xenopus vocal CPG is an exception to this top-down rule. Motor neurons act as a critical component of the vocal CPG by entraining the activity of premotor neurons. Experiment 1: block electrical synapses with carbenoxolone while stimulating the vocal nerve. • Stimulating the motor nerve at fast trill rates (60 Hz) induces EPSP summation; blockade of gap junctions eliminates these EPSPs. • Blockade of electrical synapses abolishes fictive advertisement calls and induces fast FTN spiking. • The results support the hypothesis that ascending excitatory feedback from n.IX-X to DTAM mediated by electrical synapses are critical for the generation of advertisement calls. Do feedback signals include excitatory inputs? Experiment 2: block electrical synapses with carbenoxolone and apply serotonin. 2 mV 200 ms 10 mV 200 ms carbenoxolone + serotonin Whole-‐cell Whole-‐cell Whole-‐cell Control Carbenoxolone Stimulus artifacts Nerve Excitatory synapses? Electrical synapses? Motor (n.IX-X) Premotor (DTAM) |
