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First published online April 18, 2006
Journal of Experimental Biology 209, 1746-1756 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02204
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Beyond the central pattern generator: amine modulation of decision-making neural pathways descending from the brain of the medicinal leech

Kevin M. Crisp1,2 and Karen A. Mesce1,2,*

1 Graduate Program in Neuroscience, University of Minnesota, St Paul, MN 55108, USA
2 Departments of Entomology and Neuroscience, University of Minnesota, St Paul, MN 55108, USA


Figure 1
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  		Fig. 1. Distribution of 5-HT-immunoreactive (5-HT-ir) neurons and associated processes in the subesophageal (SEG) and supraesophageal (SPEG) ganglia comprising the leech head brain. The brain of an embryonic leech at stage embryonic day 20 (E20) is shown, which was immunostained and imaged in situ (N=6). (A) The neuropil of the SPEG is filled with a dense array of 5-HT-ir fibers, probably originating from within the CNS because no peripheral 5-HT-ir cell bodies were observed. The four neuromeres of the SEG (marked 1–4) contain sets of large 5-HT-ir somata, some of which project toward the SPEG, e.g. the large lateral (LL) cells in neuromere 1. Several 5-HT-ir fibers exit the brain, such as those projecting throughout the stomatogastric nerve ring (large arrow points to one half of nerve ring). The rectangular broken box outlines the image shown at higher magnification in B. (B) Punctate 5-HT-ir fibers were visible in a putative neurohemal region of the SPEG. The terminations of varicose 5-HT-ir fibers appear to be concentrated in the upper region of the image shown, which lies in the approximate location of a neurohemal release site (Webb, 1980Go). Optical sections/image, 39 (A); 9 (B). Section intervals, 2 µm (A); 1.5 µm (B). Scale bars, 100 µm.

 

Figure 2
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  		Fig. 2. Serotonergic neurons of the head brain were activated by swim-initiating stimuli and remained active throughout swimming. An electrical stimulus (100 ms, 3–5 V pulses delivered for 1 s) delivered to a posterior DP nerve has previously been shown to initiate swimming (Hashemzadeh-Gargari and Friesen, 1989Go). (A) Electrical shock (black bar) delivered to a DP(16) nerve excited the serotonergic LL cells of the first neuromere of the SEG (left). In addition, LL received rhythmic synaptic input correlating with individual swim-motor bursts during the expression of a spontaneous swim episode (right). (B) SEG Retzius cell (Rz) is excited by a swim-initiating electrical shock to a DP(16) nerve (left), and was persistently depolarized throughout the duration of the swim episode. During a spontaneous bout of swimming, the RZ fired above 1 Hz at the onset of swimming and remained active at a relatively high firing frequency (5 Hz or greater) (right). These data are consistent with 5-HT being secreted in the brain during both swim-initiation and swim episode maintenance via the somatic release of 5-HT (De-Miguel and Trueta, 2005Go).

 

Figure 3
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  		Fig. 3. Focal brain application of 5-HT indirectly inhibits the swim-gating neuron 204 (recorded in M11). Cell 204 becomes hyperpolarized and its tonic firing rate is suppressed in response to the brain application of 5-HT (5 µmol l–1) (middle), but not by octopamine (50 µmol l–1) (OA; bottom). During perfusion of amine-free saline (top), cell 204 fired action potentials at a constant rate of about 10 Hz. A schematic of the experimental preparation, cell 204 recording site, and placement of amine is shown to the left.

 

Figure 4
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  		Fig. 4. Brain application of 50 µmol l–1 5-HT hyperpolarized the swim-triggering neuron Tr1. 5-HT caused a gradual change of the resting membrane potential of Tr1, resulting in a 6 mV hyperpolarization after approximately 3 min of the application. At this hyperpolarized membrane potential, Tr1 ceased firing action potentials.

 

Figure 5
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  		Fig. 5. Amine modulation of command-like neurons known to activate or inhibit swimming. Bath application of a mixture of 5-HT and OA (50 µmol l–1) caused the inhibition of cell Tr1 (Brodfuehrer and Friesen, 1986bGo) and SIN1 (Brodfuehrer and Burns, 1995Go). (A) During perfusion of a saline baseline (left), swim-trigger neuron Tr1 fired a train of action potentials just before a swim episode (see swim motor neuron bursts in the DP nerve extracellular recording). After a 30-min application of the 5-HT/OA mixture (middle), Tr1 became hyperpolarized (5 mV) and fired no action potentials. Furthermore, Tr1 appeared to receive less synaptic activity (i.e. fewer small and rapid fluctuations in membrane potential) following mixture application. During washout of the mixture (with saline, right), Tr1 partially repolarized and synaptic inputs to Tr1 resumed. Tr1 spiked once preceding the onset of a swim bout. (B) During perfusion of a saline baseline (left), swim-inhibitory neuron SIN1 fired action potentials at a constant rate. After a 30-min application of the mixture (middle), the membrane potential of SIN1 became inhibited (18 mV), and large, rhythmic inhibitory inputs were observed. During washout of the mixture (right), SIN1 partially repolarized and rhythmic membrane potential fluctuations were phase-locked with swim-motor bursts in the DP nerve.

 

Figure 6
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  		Fig. 6. Application of the 5-HT/OA mixture (50 µmol l–1 for 30-min) or 5-HT (50 µmol l–1 for 30-min) changed the activity profile of fibers in the descending connectives (Conn) and altered the way in which this activity was recruited by brain command-like cells. (A) As is the case for cell Tr1 (Cellucci et al., 2000Go), intracellular depolarization of cell Tr2 triggered an increase in activity descending through a single hemiconnective (ca. 2800 fibers between M1 and M2) (Wilkinson and Coggeshall, 1975Go). This elevated activity long outlasted (by several seconds) the train of action potentials in cell Tr2, and appeared to represent the recruitment of other descending fibers in the brain that may also be involved in mediating the decision to swim or not to swim. (B) During treatment with the amine mixture, the basal activity in the hemiconnective increased. In addition, the delayed response of units in the connective to a spike train in cell Tr2 was no longer present. (C) A representative 1-s recording of descending activity from the brain in normal saline (left) and 1-s sampling of activity after 3 min of a 50 µmol l–1 5-HT exposure. In all preparations examined (N=5), there was a recruitment of larger amplitude fibers during 5-HT application.

 

Figure 7
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  		Fig. 7. Swim-gating neuron 204 (in M11) is indirectly hyperpolarized in response to a 30-min focal brain application of the 5-HT/OA mixture (50 µmol l–1). (A) A schematic of the experimental preparation, emphasizing that cell 204 was not directly exposed to the amine mixture. (B) During brain perfusion of the saline baseline (top), cell 204 fired action potentials at a constant rate. After a 30-min application of the mixture (middle), 204 became hyperpolarized (6 mV) and all action potentials ceased. This depression translated into a decrease in the excitatory drive to the swim CPG and no swimming was observed. During washout of the mixture (bottom), 204 partially repolarized and showed rhythmic depolarizations phase-locked with individual swim motor bursts in the DP nerve.

 

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© The Company of Biologists Ltd 2006