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First published online December 3, 2004
Journal of Experimental Biology 207, 4535-4542 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01315

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A cephalic projection neuron involved in locomotion is dye coupled to the dopaminergic neural network in the medicinal leech

Kevin M. Crisp¹ and Karen A. Mesce^1,2,*

¹ Graduate Program in Neuroscience, University of Minnesota, 219 Hodson Hall, 1980 Folwell Avenue, St Paul, MN 55108, USA
² Departments of Neuroscience and Entomology, University of Minnesota, 219 Hodson Hall, 1980 Folwell Avenue, St Paul, MN 55108, USA

View larger version (109K): [in a new window]   Fig. 1. Confocal photomicrographs showing Neurobiotin transfer from cell Tr2, in the subesophageal ganglion (SEG), to neurons identified as synthesizing DA. (A) Shown are Tr2 and Neurobiotin-containing neuronal populations visualized with Cy3-streptavidin. The soma of only one Tr2 neuron was iontophoretically injected with Neurobiotin using 500 ms pulses of positive current (1.5 nA) at a rate of 1 Hz for about 30 min. Cell Tr2 is shown crossing the midline and descending out of the SEG. (B) Same sample as in (A) showing Cy5 fluorescence from the network of dopaminergic neurons stained with an antibody against tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of dopamine (DA). (C) Merged two-color fluorescence showing neurons (yellow) that contain both Neurobiotin (green) and TH (red). In this sample, arrows indicate the presence of four TH-immunoreactive (TH-ir) cells that are dye coupled to Tr2. (D) In another sample, the processes and projection patterns of the TH-ir neurons are displayed. Scale bars, 100 µm.

View larger version (13K): [in a new window]   Fig. 2. Dopamine inhibits the expression of fictive swimming. Robust swimming was induced by a combination of a 30 min bath application of the amine mixture (50 µmol l–1 serotonin and 50 µmol l–1 octopamine) administered to the brain and nerve cord, followed by a 30 min saline `washout' (Mesce et al., 2001). At the end of washout, 50 µmol l–1 DA was applied to the brain and nerve cord for 30 min, thus terminating the expression of fictive swimming. The entire last 9 min of washout (labeled min 1, to arrow) and first 6 min of DA application (min 10 to end of record) are depicted here. Black boxes depict entire swim episodes, defined as a series of uninterrupted swim motor neuron (DE-3) bursts in the dorsal posterior (DP) nerve. Swim inhibition by DA occurred within 1 min, and swimming was never observed in any preparations treated with DA (N=5).

View larger version (52K): [in a new window]   Fig. 3. Fictive crawling is not inhibited by bath application of 50 µmol l–1 DA. The fictive crawling motor rhythm is characterized by alternating bursts of action potentials in the dorsal longitudinal muscle excitor DE-3 (the largest unit in the DP nerve) and the circular muscle contractor CV in ganglion 9, with a cycle period of 7–20 s (Eisenhart et al., 2000). (A) Spontaneous fictive crawling activity after a 10 min bath application of 50 µmol l–1 DA. (B) Spontaneous fictive crawling activity in saline without the amine.

View larger version (33K): [in a new window]   Fig. 4. Descending brain interneuron Tr2 receives rhythmic inhibition in phase with the elongation phase of the fictive crawling motor rhythm. The CV neuron shows bursting during the elongation phase of crawling and the DE-3 motor neuron (DP nerve) bursts during the contraction phase. (A) At a resting membrane potential of –68 mV, inhibitory post-synaptic potentials (IPSPs) ranging from 3–5 mV in amplitude are apparent during the peak of CV burst activity during each crawl cycle. (B) When the membrane potential of Tr2 is hyperpolarized to –98 mV, by an injection of –0.2 nA of constant current, the IPSPs are evident as positive deflections of the membrane potential, ranging in amplitude from 2-4 mV. (C) Between crawl episodes, synchronous activity is observed between DE-3 (the largest unit in the DP nerve), circular contractor motor neuron CV, and Tr2, suggesting a shared synaptic input between these three neurons. During this recording, Tr2 is once again hyperpolarized to –98 mV with a constant current injection of –0.2 nA. (D) Portions of the CV and Tr2 traces shown in C (see rectangle in C) are expanded in time to resolve whether individual PSPs are 1:1. Although the summed potentials are clearly phase locked, unitary PSPs are not.