Article Figures & Data
Figures
- Fig. 1.
CBI-2 drives both ingestive- and egestive-like motor programs in an isolated CNS. Constant-current injection into CBI-2 induced cyclic activity of the protraction phase interneuron B20 and the retraction phase neuron B4/5. Rhythmic activity was also induced in the I2 and RNs. The protraction phase (open bar) is defined by activity in the I2, which contains axons of the protraction phase motoneurons B31/32 and B61/62. The retraction phase (filled bar) is defined by sustained depolarization of B4/5 and the hyperpolarization of B20 that follow the protraction phase. Radula closure activity is monitored on the basis of activity in the RN, which contains axons of the radula closure motoneurons B8. A, RN activity primarily occurred during the retraction phase; thus the motor program was ingestive.B, RN activity primarily occurred during the protraction phase; thus the motor program was egestive. Notice that inB, neurons B20 and B4/5 displayed higher levels of activity than in A.
- Fig. 2.
B20 drives egestive motor programs. DC current injection into neuron B20 induced a feeding motor program. Rhythmic activity was recorded in B20, B4/5, B19, the I2 nerve, and RNs. Radula closure activity (RN) occurred only during protraction (see activity in the I2 nerve;open bar), not during retraction (filled bar), thus indicating that the motor program was egestive. Note that similar to egestive motor programs induced by CBI-2, B4/5 fired strongly in B20-elicited egestive motor programs.
- Fig. 3.
Protraction phase interneuron B20 is necessary for expression of CBI-2-elicited egestive motor programs. A, C, Stimulation of CBI-2 with DC current injection induced egestive motor programs, because the radula closure motoneuron B8 was active only during protraction (open bar), not during retraction (filled bar). Note that both the ipsilateral B20 and c-B20 are strongly active. B, When B20 neurons were bilaterally hyperpolarized (bars underB20 recordings), the radula closure motoneuron B8 became predominantly active during the retraction phase, thus indicating an ingestive motor program. BN2, Buccal nerve 2.
- Fig. 4.
Multiaction retraction phase neuron B4/5 suppresses the activity of the ipsilateral radula closure motoneurons B8 and B16 during the retraction phase of CBI-2-elicited ingestive programs. One cycle of a feeding motor program was elicited by stimulation of CBI-2 with brief current pulses at 12 Hz (A) or 16 Hz (B). In the motor programs elicited by CBI-2 (A, 1, 3, B, 1, 3), the RN activity primarily occurred during the retraction phase (defined as sustained depolarization in B4/5; filled bar) that followed the protraction phase (defined as bursting in the I2 nerve; open bar), thus suggesting that these motor programs were ingestive. Consistently, radula closing motoneurons B8 (A, 1, 3) and B16 (B, 1, 3) fired more spikes (average firing rates: B8, ∼8.9 Hz; B16, ∼9.2 Hz), and B4/5 fired fewer spikes during the retraction phase. However, when B4/5 was depolarized to fire (A, 2, B, 2, bars) during the retraction phase, the activity of the ipsilateral B8 (A, 2) and B16 (B, 2) was mostly eliminated. Note that RN contains axons from both ipsilateral and contralateral B8 neurons, and because B4/5 inhibits only ipsilateral B8 neurons, some activity remained in RN during retraction when B4/5 was stimulated.
- Fig. 5.
Protraction phase interneuron B20 is synaptically excited by CBI-2 and by other buccal protraction phase interneurons. A, CBI-2 elicited a long-latency slow EPSP in B20 in high-divalent saline, but unitary EPSPs that followed one-for-one presynaptic spikes were not observed. B, B34 elicited EPSPs in c-B20. These EPSPs followed presynaptic spikes one-for-one (1, 2), and persisted in high-divalent saline (2), thus suggesting that these connections were monosynaptic. C, B63 elicited EPSPs in c-B20 in normal saline (1) and in high-divalent saline (2, 3). C, 2, 3, B63 was stimulated with brief current pulses at 5 Hz (2) or 20 Hz (3). The EPSPs in c-B20 followed presynaptic spikes one-for-one. Notice that the EPSPs from B63 outlasted the fast EPSPs (C, 2, 3), thus indicating the presence of a slow component. D, B65 elicited EPSPs in c-B20 in normal saline (1) and high-divalent saline (2). The EPSPs followed presynaptic spikes one-for-one.
- Fig. 6.
Electrical coupling between B20 and its contralateral homolog and other buccal protraction phase neurons.A, The bilaterally symmetrical neurons left B20 (LB20) and right B20 (RB20) are electrically coupled. B, B20 is electrically coupled to the contralateral B31 (c-B31). The hyperpolarization in the contralateral B34 (c-B34) was very small, barely visible, suggesting that there was very weak electrical coupling between B20 and c-B34. C, D, B20 is electrically coupled to B63 (C) and B65 (D) contralaterally. A–C, Recordings were from high-divalent saline. D, Recordings were made in ASW.
- Fig. 7.
Synaptic connections of B20 with the retraction phase interneuron B64. A, B64 elicited IPSPs in B20 in normal saline (1). These IPSPs persisted in high-divalent saline (2). The IPSPs in B20 followed B64 presynaptic spikes one-for-one. A, 3, Inhibitory connections from B64 were functional, because B64 suppressed the firing of c-B20 in normal saline. B20 is not spontaneously active; therefore, we elicited spiking in B20 by a constant intracellular current injection. B, Neuron B20 elicited a mixed response (early excitation followed by slow inhibition) in B64 in normal saline. This response is probably polysynaptic, because it was not present in high-divalent saline (data not shown).
- Fig. 8.
B20 excites the radula-closing motoneuron B8.A, B20-elicited EPSPs in B8 followed B20 spikes one-for-one. B8 was hyperpolarized by 10 mV (1). When B8 was at its resting potential (2), firing of B20 induced B8 firing. B, B20 enhanced the excitability of neuron B8. Current test pulses in B8 (3 sec duration) were applied every 30 sec to induce regular firing in B8 (16 spikes in1, 15 spikes in 3). When B20 was fired (2), B8 fired a larger number of spikes (24 spikes).
- Fig. 9.
B4/5 reduces the excitability of the radula-closing motoneuron B8. Test pulses in B8 (3 sec duration) were applied every 30 sec to induce regular firing in B8 (28 spikes inA and C). When B4/5 was fired (B), B8 fired fewer action potentials (10 spikes).
- Fig. 10.
B20 synaptically excites neuron B4/5.A, B20 elicited EPSPs in the contralateral B4/5 (c-B4/5) and ipsilateral B4/5. This excitation had a fast and a slow component (1). The fast EPSPs followed presynaptic spikes one-for-one (2, expanded records of 1, between arrowheads). This experiment was performed in high-divalent saline. B, B20 enhanced the excitability of neuron B4/5. Depolarizing current pulses (3 sec duration) were injected into B4/5 every 30 sec (13 spikes in1, 8 spikes in 3). When B20 was fired before current was injected into B4/5 (2), B4/5 fired a larger number of spikes (28 spikes).
- Fig. 11.
CBI-3 converts CBI-2-elicited egestive programs to ingestive ones by suppressing activity in B20.A, Stimulation of CBI-2 with short current pulses at 10 Hz elicited a single cycle of an egestive motor program, because high-frequency activity in the RN occurred only during protraction (defined by bursting in the I2 nerves; open bar). The retraction phase (filled bar) is defined by periods of hyperpolarization of B20 after the protraction phase ended.B, DC current stimulation of the ipsilateral CBI-3 and the contralateral CBI-3 (c-CBI-3) during CBI-2 stimulation switched the motor program to an ingestive one (high-frequency activity in the RN was now observed during retraction). Coincidentally, B20 activity was suppressed. C, The same stimulation was applied on CBI-2 and CBI-3s as in B, but in addition, B20 was fired strongly by DC current injection. The motor program reverted to an egestive mode. D, Another cycle of an egestive motor program was elicited by CBI-2 stimulation after that shown in C. This cycle of the motor program was similar to the cycle shown in A.
- Fig. 12.
Activity of the protraction phase interneuron B34 (A) and B63 (B) during single cycles of CBI-2-elicited motor programs. A, CBI-2 was stimulated by a brief current pulse (10 msec) at 15 Hz. When firing of CBI-3 was not experimentally manipulated, motor programs induced by CBI-2 stimulation were egestive (1, 3), because the high-frequency activity in RN occurred during protraction (open bar), not during retraction (filled bar). When CBI-3 was depolarized, the motor program induced by CBI-2 became ingestive (2), because the high-frequency activity in RN now occurred during retraction. In both types of programs, B34 and the protraction phase motoneuron B61 were strongly active. B, CBI-2 was stimulated by DC current. B, 1, 3, Without controlled firing of CBI-3, motor programs induced by CBI-2 were egestive in 1; i.e., RN activity primarily occurred during the protraction phase (open bar); but they were mixed in 3; i.e., RN activity occurred in both protraction and retraction phases (filled bar). B, 2, When ipsilateral CBI-3 and contralateral CBI-3 (c-CBI-3) were depolarized, the motor program induced by CBI-2 was ingestive. In all types of motor programs, B63 was strongly active.
- Fig. 13.
CBI-3 monosynaptically inhibits B20.A, Both the ipsilateral CBI-3 and the contralateral CBI-3 (c-CBI-3) suppressed B20 firing (1, 2) in normal saline. B20 is not spontaneously active; therefore, its spiking was induced by a small DC current injection. The IPSPs elicited by CBI-3 followed presynaptic spikes one-for-one (3, 4). A, 3 and4 are expanded records of 1 and2 (between arrows) respectively. Spikes in B20 were clipped. B, In high-divalent saline (HD), stimulation of CBI-3 with brief current pulses at 12 Hz did not induce visible IPSPs when B20 was at its resting potential (1), but it induced one-for-one facilitating IPSPs in B20 when B20 was depolarized by 20 mV (2). Note that in B, 2, B20 was not spiking even with 20 mV depolarization, because high-divalent saline raised the spike threshold of B20.
- Fig. 14.
CBI-3 reduces the excitability of B20 and B4/5 in a frequency-dependent manner. Current pulses (3 sec duration) were injected into B20 (A) and B4/5 (B) every 30 sec. CBI-3 was stimulated by brief current pulses at 5, 10, or 20 Hz. A, CBI-3 was stimulated before and during the injection of current pulses into B20. B20 firing was reduced in a frequency-dependent manner (fromleft to right, 29, 26, 11, 0, and 28 spikes). Recordings were obtained from high-divalent saline. B, CBI-3 was stimulated for 8 sec before injection of current pulses into B4/5. B4/5 firing was also reduced in a frequency-dependent manner (from left to right, 22, 15, 8, 1, and 20 spikes). Recordings were obtained from normal saline.
- Fig. 15.
APGWamide decreases the excitability of B20 and B4/5 in a concentration-dependent manner. Depolarizing current pulses (3 sec duration) were injected into B20 (A) and B4/5 (B) every 60 sec. APGWamide was applied in ascending concentrations (from 10−7 to 10−5m).A, Examples from experiments that were performed in normal saline (1) and in high-divalent saline (2). The application of APGWamide reduced the excitability of B20 in a concentration-dependent manner (fromleft to right, 40, 24, 12, 0, and 33 spikes in1; 28, 22, 12, 0, and 19 spikes in 2). B20 spiking showed almost complete recovery in normal saline (1) and only partial recovery in high-divalent saline (2) after peptide washout.B, B4/5 excitability was reduced by APGWamide in a concentration-dependent manner (from left toright, 24, 18, 6, 0, and 24 spikes). B4/5 spiking recovered completely after peptide washout. Experiments were performed in normal saline.
- Fig. 16.
APGWamide occludes the CBI-3-induced inhibition of B4/5 excitability. A, Test pulses in B4/5 (3 sec duration) were applied every 30 sec to induce regular firing in B4/5. CBI-3 was stimulated by brief current pulses at 10 Hz for 8 sec before injection of current pulses into B4/5. Both in control conditions and after APGWamide washout, CBI-3 suppressed B4/5 spiking (compare Fig.14B). However, in the presence of 10−4m APGWamide, CBI-3 no longer suppressed B4/5 spiking. In fact, B4/5 activity was increased. Note that APGWamide by itself also reduced B4/5 excitability (Fig.15B). The size of current pulses injected into B4/5 during bath application of APGWamide was increased to induce B4/5 firing comparable with that in control. B, Plot of group data of the effect of APGWamide on CBI-3 inhibition of B4/5 excitability (n = 4). Error bars indicate SEM.
- Fig. 17.
Functional configurations of theAplysia feeding CPG in the egestive (A) and ingestive (B) modes. Each cycle of buccal motor programs consists of two phases: radula protraction (P) and retraction (R), which are mediated respectively by protraction phase interneurons (PI) [i.e., B34, B63 (Hurwitz et al., 1997), and B40] and retraction phase interneurons (RI) [e.g., B64 (Hurwitz and Susswein, 1996)]. B40 is a newly identified buccal–cerebral interneuron (Jing and Weiss, 2001). The protraction phase is initiated by CBI-2, which drives PIs. Mutual inhibition between PIs and RIs ensures that protraction and retraction phases alternate with respect to each other. The switch from protraction to retraction is presumed to be mediated by a hypothesizedz cell, which receives excitation from PIs and in turn activates the RIs (Baxter et al., 1997). The main criterion for distinguishing between ingestive and egestive motor programs is the timing of activity in radula closure motoneurons B8 and B16 (Cropper et al., 1990; Morton and Chiel, 1993a,b; Church and Lloyd, 1994). During ingestion, radula closure occurs primarily during retraction (B). During egestion, radula closure occurs during protraction (A). A, In the absence of CBI-3 activity, CBI-2 drives B20 through direct and indirect (via PIs) pathways. B20 in turn enhances B4/5 firing. Strong activity in B20 induces B8/B16 firing during protraction; thus the motor programs are egestive. In the protraction phase, the weak inhibitory connections from PIs to B8 are overridden by excitatory actions of B20 (compare Fig. 11C). In the retraction phase, the excitatory connections from RIs and PIs to B8/B16 are overridden by B4/5 (compare Fig. 4A, 2, B, 2).B, When CBI-3 is coactive with CBI-2, ingestive programs are generated. Fast and slow inhibitory actions of CBI-3 functionally remove B20 and B4/5 from the circuit. In the absence of excitation from B20 during protraction, fast inhibition of B8 by the PIs suppresses B8 firing. During the retraction phase, the inhibitory input from B4/5 is no longer present; therefore, a combination of slow and fast excitation, originating respectively from the PIs (B34 and B40) and the RIs, induces B8 firing. Thus, B8 and B16 fire during retraction, and the motor programs become ingestive. Note that the inhibitory connection from B64 to B20 is omitted for clarity. Inactive neurons and their synaptic outputs are shown in gray.
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