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REPORT
Department of Neuroscience, Mount Sinai School of Medicine, New York, New York
Submitted 27 August 2007; accepted in final form 2 October 2007
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ABSTRACT |
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INTRODUCTION |
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). Also different compartments of a neuron can receive different synaptic or hormonal inputs that in turn differentially shape the activity of these distinct compartments (Bucher et al. 2003; Coleman and Nusbaum 1994; Perrins and Weiss 1998; Westberg et al. 2000). Cerebral-buccal interneurons-5 and -6 (CBI-5/6) in Aplysia are examples of cells characterized by this type of compartmentalization. Previous work (Perrins and Weiss 1998) showed that there are two compartments: a cerebral compartment (CC) that contains the soma and a buccal compartment (BC) that is part of CBI-5/6 axon. In its BC, CBI-5/6 appears to utilize spike-dependent synaptic signaling. Action potentials originating in the BC of CBI-5/6 also propagate antidromically toward its CC and depolarize the soma but fail to elicit full-size action potentials. However, this depolarization can be transmitted to other cerebral neurons via electrical coupling.
Although CBI-5/6 makes synaptic connections in its BC, the functions of the BC in motor program generation are not understood. By systematically characterizing the synaptic outputs of CBI-5/6 and the consequences of CBI-5/6 activity, we provide evidence indicating that the BC of CBI-5/6 is an integral member of the buccal central pattern generator (CPG). Furthermore, it was shown that the CC of CBI-5/6 receives antidromic spikes from its BC during retraction and an excitatory input during protraction (Perrins and Weiss 1998; also this study). Here, we show that the unknown depolarizing inputs originate in identified buccal CPG elements. Thus the CPG may differentially control the phases in which this compartmentalized interneuron transmits information to its targets.
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METHODS |
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1.1 ml) perfused at 0.3–0.35 ml/min. The composition of normal artificial seawater (ASW) was (in mM) 460 NaCl, 10 KCl, 11CaCl2, 55 MgCl2, and 10 HEPES, at pH 7.6. The composition of a high-divalent-cation (Hi-Di) ASW was (in mM) 368 NaCl, 10 KCl, 13.8 CaCl2, 101 MgCl2, and 10 HEPES at pH 7.6. Electrophysiology
Intracellular recordings were made using single-barrel (4–10 M
) electrodes filled with 2 M KAc and 30 mM KCl. Intracellular signals were acquired using an AxoClamp 2B amplifier (Axon Instruments, Union City, CA) or Getting Model 5A amplifier (Getting Instruments, Iowa City, IA). Extracellular signals were acquired from polyethylene suction electrodes using a differential AC amplifier (Model 1700, A-M Systems, Carlsborg, WA). A Grass stimulator (Model S88, Grass Medical Instruments, Quincy, MA) was used for stimulation.
Neurons were identified as previously described (Borovikov et al. 2000; Church and Lloyd 1994; Evans and Cropper 1998; Gardner 1977; Hurwitz and Susswein 1996; Hurwitz et al. 1994, 1997; Jing and Weiss 2001; Jing et al. 2003, 2004; Kabotyanski et al. 1998; Perrins and Weiss 1998; Plummer and Kirk 1990; Rosen et al. 1991, 2000; Susswein and Byrne 1988; Teyke et al. 1993). CBI-5/6 was stimulated by combining a subthreshold depolarization with repetitive injections of current pulses (10–20 ms, 15–25 nA), each of which elicited a single spike.
Statistics
Data are presented as means ± SE. Data were analyzed using Student's t-test. Significance was set at P < 0.05.
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RESULTS |
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The feeding CPG generates two major classes of motor programs, ingestive and egestive. In both cases, protraction and retraction occur in a fixed sequence with protraction always preceding retraction. In contrast, the phasing of the activity in the closing circuitry is variable in that in ingestive programs, the closing circuitry is active during retraction, whereas in egestive programs, the closing circuitry is active during protraction. Regardless of whether motor programs are ingestive or egestive, CBI-5/6 spikes are generated during retraction (Perrins and Weiss 1998). This suggests that activity of CBI-5/6 may be relevant to the control of protraction/retraction movements. To determine whether this is the case, we examined synaptic connections from CBI-5/6 to motor neurons active during protraction and retraction. Although some of these connections were described previously (Perrins and Weiss 1998), the monosynapticity of previously described connections was not tested. CBI-5/6 inhibited the protraction-phase motoneurons B31/32 and B61/62 (Fig. 1A: left in ASW; right in a Hi-Di solution, n = 5–11), B13, B48, and B66 (Fig. 1B: n = 3–10). In contrast, CBI-5/6 excited all three retraction-phase motoneurons, B6, B9, and B44 (n = 3–7, Fig. 1C).
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; Wu et al. 2007). Consistent with the ability of CBI-5/6 to terminate the protraction phase (see Fig. 2, E and F), all of the inhibitory connections between CBI-5/6 and its followers were sufficiently strong to block generation of action potentials in the follower neurons.
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; Hurwitz et al. 1997; Jing and Weiss 2001, 2002; Jing et al. 2004; Kabotyanski et al. 1998), we performed a series of pharmacological experiments to determine whether CBI-5/6 is glutamatergic as is B64. Studies of B64 implicated glutamate as the transmitter for this neuron (Due et al. 2005). Here we focused on the transmission between CBI-5/6 and the protraction-phase motoneurons B61/62 and between CBI-5/6 and the retraction-phase interneuron B64 (synapses at which CBI-5/6 elicits opposite responses). First, a glutamate puff applied via pressure ejection on B61/62 (n = 3) elicited a hyperpolarizing response (Fig. 2A, top) and a glutamate puff on B64 (n = 4) elicited a depolarizing response (Fig. 2A, bottom). These responses were consistent with those evoked by stimulating CBI-5/6.
Second, we investigated whether CBI-5/6 connections are affected by the excitatory glutamate receptor antagonist CNQX. Bath superfusion of 10–5 M CNQX reduced the amplitude of CBI-5/6-to-B64 EPSPs by 62.0 ± 0.8% but had no effect on CBI-5/6-to-B61/62 inhibitory postsynaptic potentials (IPSPs, Fig. 2B; n = 4). Superfusion of a related antagonist, NBQX, at 10–4 M reduced CBI-5/6-to-B64 EPSPs by 72.3 ± 1.7% but had no effect on CBI-5/6-to-B61/62 IPSPs (n = 6, data not shown). Moreover, B64 was depolarized by superfusion of the glutamatergic agonists AMPA and kainate, whereas B61/62 was not affected (n = 3–10, data not shown). The ineffectiveness of CNQX/NBQX and AMPA/kainate on CBI-5/6-to-B61/62 IPSPs and the lack of an effect on the B61/62 resting potential prompted us to test the effects of two compounds, beta-alanin and hypotaurine that desensitize and block chloride-dependent glutamate responses (Kehoe and Vulfius 2000). Neither the B61/62 resting potential nor the CBI-5/6-to-B61/62 IPSPs were affected by these compounds (n = 4, data not shown). Furthermore, CBI-5/6-to-B61/62 IPSPs were not affected by chloride loading of B61/62 (n = 3, data not shown). We therefore examined the effects of quisqualate, a glutamate agonist that evokes a K+-dependent glutamate response (Katz and Levitan 1993; Kehoe 1994). Superfusion of 10–5 M quisqualate hyperpolarized B61/62 by –13.0 ± 1.3 mV (n = 8). CBI-5/6-to-B61/62 IPSPs were also reduced by 89.7 ± 3.0% by superfusion of 10–5 M quisqualate (Fig. 2C, n = 4), when the B61/62 membrane potential was held constant by injecting DC current through a second electrode to compensate for direct effects of the quisqualate on the membrane potential. In contrast, quisqualate had only a minor effect on CBI-5/6-to-B64 EPSPs (Fig. 2C, n = 5). Finally, glutamate superfusion occluded both CBI-5/6-to-B64 EPSPs and CBI-5/6-to-B61/62 IPSPs when the membrane potentials were kept constant through current injections (Fig. 2D, n = 4). Thus the pharmacological profile of CBI-5/6 suggests that this neuron, like B64, may be glutamatergic.
CBI-5/6 is a protraction terminator
The remarkable similarity of physiological characteristics of CBI-5/6 and B64 raised the possibility that they may fulfill similar functional roles in the generation of buccal motor programs. Stimulation of B64 before the end of the protraction phase terminates protraction and initiates retraction (Hurwitz and Susswein 1996; Wu et al. 2007). We found that in both ingestive programs (n = 11) elicited by stimulating CBI-2 (Fig. 2E) and egestive programs (n = 5) elicited by stimulating the esophageal nerve (Fig. 2F), early activation of CBI-5/6 advanced the phase transitions from protraction to retraction compared with controls. Thus CBI-5/6, like B64, functions as a protraction terminator.
Connections from the buccal CPG neurons to the CC of CBI-5/6
The preceding data suggest that CBI-5/6 functions as an element of the buccal CPG. However, the actions of CBI-5/6 are not limited to the buccal ganglion. Somatic recordings of CBI-5/6 during motor programs (Perrins and Weiss 1998) (Fig. 2, E and F) showed that before the soma is depolarized by the antidromically propagating action potentials during retraction, the CBI-5/6 soma was also strongly depolarized by synaptic inputs during protraction. Because the sustained depolarization of CBI-5/6 occurred during the protraction phase of both ingestive and egestive programs, we investigated the possibility that this depolarization is mediated by protraction-phase buccal-cerebral interneurons.
Indeed, the buccal-cerebral interneurons (BCIs) B34, B63, and B40, which project to the cerebral ganglion through the contralateral cerebral-buccal connective (Hurwitz et al. 1997; Jing et al. 2003), elicited fast EPSPs in the contralateral CBI-5/6 (Fig. 3A, n = 3–5) that persisted in a Hi-Di solution, suggesting that the connections were monosynaptic. B40 also induced a slow IPSP in the ipsilateral CBI-5/6 in Hi-Di (n = 2 of 3; data not shown), whereas no responses in ipsilateral CBI-5/6 from either B34 (n = 5) or B63 (n = 3) were observed. The EPSPs evoked by B34 and B63 appeared comparable in size to those observed during protraction. By hyperpolarizing B34 and B63, we sought to determine whether activity of these BCIs may account for the EPSPs that are recorded in CBI-5/6 during motor programs. Hyperpolarization (
10 nA) of B34 and B63 (Fig. 3B2) eliminated a major part of the CBI-5/6 depolarization that otherwise occurred during protraction (Fig. 3B1). Under control conditions, the somatic depolarization measured just before initiation of spiking during retraction was 24.3 ± 0.3 mV and became 4.7 mV ±1.9 mV when B34 and B63 were hyperpolarized (n = 3, P < 0.05). During B34 and B63 hyperpolarization, a small, remaining, depolarization might have been due to the activity of other BCIs including B40 which was not hyperpolarized (Fig. 3B2). These results suggest that the major component of the protraction-phase depolarization in CBI-5/6 is mediated by the activity of B34 and B63.
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DISCUSSION |
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). Here we characterized functional aspects of this compartmentalization in the context of feeding pattern generation. An important aspect of this compartmentalization is that the spike-dependent synaptic transmission from CBI-5/6 appears to be limited to the buccal ganglion, and that the phasic spike activity of CBI-5/6 begins at the time of the protraction-to-retraction transition and continues for the duration of retraction (Perrins and Weiss 1998) (Fig. 3). Importantly, we found that within the buccal ganglia, CBI-5/6 spikes inhibited protraction-phase interneurons and motoneurons but excited retraction-phase interneurons and motoneurons. Protraction-phase motoneurons and interneurons were inhibited independent of their function. Thus CBI-5/6 elicited IPSPs in protraction-movement generating motoneurons B31/32, B61/62 (Hurwitz et al. 1994), radula opener motoneurons B48 and B66 (Borovikov et al. 2000; Church and Lloyd 1994). Also protraction phase generating interneurons B63 and B34 (Hurwitz et al. 1997, 2005) as well as interneurons that are largely involved in the control of radula closing movements, B20 (Jing and Weiss 2001), B65 (Kabotyanski et al. 1998), B40 (Jing et al. 2003), and B30 (Jing et al. 2004) were hyperpolarized by CBI-5/6. Similarly, retraction phase neurons were excited independent of their function. Thus CBI-5/6 elicited EPSPs in the protraction terminator B64 (Hurwitz and Susswein 1996; Wu et al. 2007), afferent interneurons B21 (Rosen et al. 2000), hyper-retraction generating interneuron B51 (Evans and Cropper 1998; Jing et al. 2004), and multi-action neurons B4/5 (Evans et al. 1996; Jahan-Parwar et al. 1983). Overall, this pattern of synaptic action of CBI-5/6 could be seen as countering the activity of protraction-phase neurons and reinforcing the activity of retraction-phase neurons, independent of the specific function of these neurons. Consistent with this pattern of connectivity, CBI-5/6 activation could prematurely terminate protraction and advance the initiation of retraction. These findings, taken together with the phasic action of CBI-5/6, suggest that the BC of CBI-5/6 functions as an element of the buccal CPG. Notably, the fact that activation of CBI-5/6 can reliably terminate protraction with a short latency is consistent with the idea that CBI-5/6 acts as a CPG element rather than as a part of a modulatory network. Interestingly, the axon of a compartmentalized interganglionic interneuron in the crustacean stomatogastric nervous systems can also function as a CPG element separately from the soma (Coleman and Nusbaum 1994).
The physiological characteristics of CBI-5/6, as well as the pharmacological profile of its putative transmitter, are remarkably similar to those of B64, which is also considered to be an element of the buccal CPG, but unlike CBI-5/6, is anatomically restricted to buccal ganglia. Previous experimental and modeling studies suggested that, although B64 is a protraction terminator (Hurwitz and Susswein 1996; Jing et al. 2003), additional protraction terminators might exist (Baxter et al. 1997). Our finding suggests that within the BC, acting through locally generated spikes, CBI-5/6 may be one of the postulated additional protraction-terminators.
In contrast to the BC of CBI-5/6, its soma essentially does not display full-size action potentials in response to the large depolarization seen during protraction and retraction. Previous data (Perrins and Weiss 1998) suggested that the somatic depolarization of CBI-5/6 during protraction is synaptically driven, but the presynaptic neurons were not identified. We now identified these neurons as B34 and B63 that, importantly, are part of the buccal CPG. We found that the retraction-phase depolarization of CBI-5/6 soma was mediated by the antidromically propagating spikes from the BC of CBI-5/6 that we now show is an element of the CPG. Thus the somatic depolarization of CBI-5/6 during both phases of buccal programs is under the direct control of the buccal CPG (Fig. 3C). In turn, depolarization of CBI-5/6 within the cerebral ganglion may depolarize CPN-1via the electrical coupling between the two cells (Perrins and Weiss 1998).
In conclusion, the present findings in conjunction with previously published work indicate that in its buccal compartment, CBI-5/6 functions as an element of the CPG that generates action potentials and transmits information during a portion, i.e., the retraction phase of motor programs. In contrast, the cerebral compartment of CBI-5/6 integrates information of both the protraction and retraction phases of motor program. Thus this compartmentalization allows the CPG to directly control the program phases in which the two compartments of CBI-5/6 may transmit information to targets localized to different ganglia.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. R. Weiss, Dept. of Neuroscience, Mount Sinai School of Medicine, 1 Gustave Levy Place, New York, NY 10029 (E-mail: Klaudiusz.Weiss{at}mssm.edu)
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REFERENCES |
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ABSTRACT
INTRODUCTION
). B: 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX) reduced the CBI-5/6-elicited EPSPs in B64 but not the IPSPs in B61/62. C: quisqualate (QA) almost completely occluded IPSPs in B61/62 but only slightly reduced EPSPs in B64 elicited by CBI-5/6. D: glutamate occluded both IPSPs in B61/62 and EPSPs in B64 elicited by CBI-5/6 in a concentration-dependent manner. E: initiation of CBI-5/6 axon spikes through intracellular stimulation of CBI-5/6 soma terminates the protraction phase of motor programs prematurely. E1: ingestive program elicited by stimulating CBI-2 in the control condition. Protraction (
) is monitored by activity in I2 nerve. Retraction (
) is monitored by activity in B64. The program was ingestive because large units of radula nerve (RN) representing radula closure B8 activity were predominantly active during retraction. High levels of closure activity, in here and in F, are indicated by a series of squares that appear above the RN traces. E2: CBI-5/6 was activated through a positive DC injection (
) before the time at which the phase transition from protraction to retraction was expected to occur. Notice protraction in E2 is much shorter than that in E1. F1: egestive program elicited by stimulating the esophageal nerve in the control condition. The program was egestive because large units of RN were predominantly active during protraction. F2: CBI-5/6 was activated in a similar manner (
, denote the direction of information transmission.