Invert Neurosci DOI 10.1007/s10158-009-0086-x
ORIGINAL PAPER
GABAA- and AMPA-like receptors modulate the activity of an identified neuron within the central pattern generator of the pond snail Lymnaea stagnalis Francesco Moccia Æ Carlo Di Cristo Æ William Winlow Æ Anna Di Cosmo
Received: 14 November 2008 / Accepted: 6 January 2009 Ó Springer-Verlag 2009
Abstract To examine the neurochemistry underlying the firing of the RPeD1 neuron in the respiratory central pattern generator of the pond snail, Lymnaea stagnalis, we examined electrophysiologically and pharmacologically either ‘‘active’’ or ‘‘silent’’ preparations by intracellular recording and pharmacology. GABA inhibited electrical firing by hyperpolarizing RPeD1, while picrotoxin, an antagonist of GABAA receptors, excited silent cells and reversed GABAinduced inhibition. Action potential activity was terminated by 1 mM glutamate (Glu) while silent cells were depolarized by the GluR agonists, AMPA, and NMDA. Kainate exerted a complex triphasic effect on membrane potential. However, only bath application of AMPA desensitized the firing. These data indicate that GABA inhibits RPeD1 via activation of GABAA receptors, while Glu stimulates the neuron by activating AMPA-sensitive GluRs. Keywords Lymnaea stagnalis GABA Glutamate AMPA RPeD1
F. Moccia A. Di Cosmo (&) Department of Structural and Functional Biology, University of Naples ‘‘Federico II’’ Complesso Universitario Monte S. Angelo, viale Cinthia, 80126 Naples, Italy e-mail:
[email protected] C. Di Cristo Department of Biological and Environmental Sciences, University of Sannio, 82100 Benevento, Italy W. Winlow Department of Veterinary Preclinical Science, The University of Liverpool, Liverpool L69 7JZ, UK W. Winlow (&) NPC Newton, 32 Hill Crescent, Newton, Preston PR4 3TR, UK e-mail:
[email protected]
Introduction The pond snail Lymnaea stagnalis is a highly tractable model for investigating the cellular mechanisms of learning and memory by exhibiting a variety of rhythmic behaviors that are controlled by relatively simple neural circuits (Benjamin et al. 2000; Lukowiak et al. 2003, 2006). These networks have been termed central pattern generators (CPG) due to their ability to generate repetitive and periodic motor outputs in the absence of peripheral sensory inputs (Marder and Bucher 2001; Marder et al. 2005). The respiratory CPG (rCPG), which generates its aerial respiratory behavior under hypoxic conditions (Jones 1961), is one of the best understood neuronal circuit in L. stagnalis. Lymnaea utilises cutaneous respiration in normoxic water, but a reduction in oxygen content drives the animal to the water surface to perform aerial respiration via the opening of its primitive lung, the pneumostome (Jones 1961). The rCPG, whose sufficiency and necessity for the generation of respiratory motor output has been demonstrated, encompasses three interneurons: (1) right pedal dorsal 1 (RPeD1), which initiates the respiratory rhythm; (2) input 3 interneuron (IP3), which causes pneumostome opening (expiration); and (3) visceral dorsal 4 (VD4), which controls pneumostome closing (inspiration) (Lukowiak and Syed 1999; Syed et al. 1990, 1992; Winlow and Syed 1992). The generation of the respiratory rhythm is the result of emerging network properties: the three cells in isolation are not capable of producing a rhythmic output (Syed et al. 1990; Taylor and Lukowiak 2000). As expected from a CPG (Marder and Bucher 2001; Marder et al. 2005), such a neuronal circuit is still able to produce a fictive respiratory rhythm when the central nervous system (CNS) is isolated from the snail and placed in a recording chamber containing the physiological saline (Inoue et al.
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1996; Lukowiak and Syed 1999). The motor output generated by RPeD1 in the isolated ganglion appears as periodical bursts of action potentials which are driven by ligand-dependent ion channels operated by synapticallyreleased neurotransmitter(s) (Syed et al. 1990; Taylor and Lukowiak 2000). RPeD1 may be considered as a multifunctional CPG interneuron (Lukowiak et al. 2006): it not only establishes synaptic connections within the circuit to drive aerial respiration rhythmogenesis, but makes synaptic contacts with peripheral sensory and motor elements in the pneumostome area to adjust the breathing output according to the behavioral needs of the snail (Bell et al. 2007; Haque et al. 2006; Lukowiak et al. 2003, 2006). Accordingly, RPeD1 does not initiate the fictive respiratory output (i.e., is quiescent) in the so-called ‘‘semi-intact preparation’’, where the CNS retains the innervation to (and perhaps from) the pneumostome area (Inoue et al. 1996, 2001). Nevertheless, a drop in oxygen levels in the perfusate causes the activation of osphradial cells, which are located in the pneumostome area and serve as oxygen-sensing neurons (Bell et al. 2007). The latter, in turn, convey an excitatory output to RPeD1 via a direct synaptic connection, which triggers the patterned rCPG activity and consequently, aerial respiration (Bell et al. 2007; Inoue et al. 2001). Furthermore, pneumostome opening at the saline (water)/air interface relays an additional excitatory outflow to RPeD1 (Haque et al. 2006). Both in the isolated CNS and in ‘‘semi-intact preparations’’, the rhythm is maintained by IP3, which is the only other known central activator of RPeD1 and re-excites RPeD1 sufficiently to initiate further cycles of rCPG activity (Syed et al. 1990; Lukowiak and Syed 1999). In addition to serving as a model to study the cellular control of breathing, L. stagnalis constitutes an ideal model to investigate how operant-conditioned changes in respiratory behavior occur (learning) and are retained by the animal (memory) (Lukowiak et al. 2003, 2006). For instance, the snails can be trained to reduce aerial respiration in a hypoxic environment by applying an aversive tactile stimulus to the pneumostome as they attempt to open it (Lukowiak et al. 2006; McComb et al. 2005; Spencer et al. 2002). Consistent with its multifaceted role, RPeD1 provides the necessary site for memory formation and accordingly, its electrophysiological properties may be altered in conditioned animals: for instance, the bursting activity is reduced and it is less prone to initiate the respiratory discharge upon depolarization (Lukowiak et al. 2006; McComb et al. 2005; Spencer et al. 2002). Despite the wealth of information on the role served by RPeD1 in the control of aerial respiratory and in behavioral phenomena of memory formation and consolidation, the blend of ion channels that regulates its rhythmic electrical
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activity is still unclear. Such an issue is not trivial, as the mode of operation of rCPG is shaped by the interplay of the neurotransmitter-activated ionotropic receptors expressed on the plasma membrane of the interneuron that drives the circuit (Grillner 1999, 2003), which lacks autorhythmic activity (Lukowiak et al. 2006). In addition, these channels are likely to trigger the intracellular signaling pathways mediating the plasticity of RPeD1 in operantly-conditioned snails. Glutamate (Glu) is a putative candidate as the neurotransmitter initiating the regular bursting activity which drives aerial respiration. This hypothesis stems from the observation that Glu depolarizes RPeD1 (Nesic et al. 1996) and that ionotropic glutamate receptor (GluR) polypeptides with high homology sequence to a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)sensitive GluR subunits (GluR1-4) and kainate-selective GluR subunits (GluR5-7) have been cloned from the Lymnaea nervous system (Hutton et al. 1991; Stu¨hmer et al. 1996). NMDA-like receptors were first described in the CNS of Lymnaea by Moroz et al. (1993) and effects on RPeD1 were first described by Mishmast-Nehi and Winlow (1995). More recently, two molluscan ionotropic NMDAtype receptors have been cloned both from Lymnaea nervous system (Ha et al. 2006). In the mammalian CNS, glycine (Gly) and c-aminobutyric acid (GABA) are the main inhibitory neurotransmitters and have been shown to shape the respiratory motor output from the pre-Bo¨tzinger complex, a limited region of ventrolateral medulla which contains mammal rCPG (Rekling and Feldman 1998). For instance, GABAA receptor-mediated Cl- current has been shown to switch from depolarizing (excitatory) to hyperpolarizing (inhibitory) within the first postnatal week (Ritter and Zhang 2000). However, neither Gly- nor GABA-dependent inhibition of the behaviorally relevant discharges generated by RPeD1 has been reported in freshwater snails (Cheung et al. 2006; Rubakhin et al. 1996). The aim of the current study was to shed light on the blend of ion channels that determine the rhythmical activity of RPeD1, which drives the fictive respiratory output of the rCPG, in isolated CNS. In particular, we were interested in addressing the role of GluRs in neuron excitation by exploiting the electrical bursting exhibited by the neuron under such conditions. Here we provide evidence that GABA, but not Gly, hyperpolarizes RPeD1 by activating picrotoxin-sensitive GABAA receptors. Furthermore, we demonstrate that the electrical discharges of RPeD1 disappear upon desensitization of GluRs with AMPA, whereas kainate and NMDA are less efficacious and rather serve a modulatory role. Finally, we attempt to integrate these findings into the large body of knowledge on the plastic control of breathing by rPeD1.
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Materials and methods Animals Specimens of the freshwater pond snail, L. stagnalis, were obtained from animal suppliers (Blades Biological, Cowden, Kent, UK) or from the Department of Cellular and Molecular Neurobiology Vrije Universiteit of Amsterdam. The specimens were reared in a dedicated laboratory environment in the animal care facility of the Department of Functional and Structural Biology. The animals were maintained in well aerated artificial pond water at room temperature (21°C) and fed lettuce. Snails with a shell length of approximately 20–25 mm (2–3 months old) were used for all experiments. Isolated brain preparations Brains were removed and prepared according to the method of Holden et al. (1982). Briefly, snails were anesthetized by placing them in a solution of 25% Listerine for 10 min. Once anesthetized, the animals were de-shelled with forceps and pinned dorsal surface up to a dissecting dish containing standard Lymnaea saline (40.0 mM NaCl, 1.7 mM KCl, 4.1 mM CaCl2, 1.5 mM MgCl2, 5.0 HEPES) buffered to pH 7.9. A mid-dorsal incision was made from the base of the mantle to the head and the central ring ganglion (CRG) was dissected out with a pair of fine scissors. The CRG was rapidly transferred to a siliconcovered recording dish containing Lymnaea saline and pinned out. The connective tissue sheath surrounding the ganglion was removed with fine forceps and scissors and the inner sheath was softened by applying non-specific solid protease (Sigma Chemicals, St Louis, MO, USA; Sigma type IX) for 20 min. Thereafter, the ganglion was allowed to stabilize by perfusing Lymnaea saline for at least 30 min before recording. Such a preparation provides a valuable model system to elucidate the nature of ionotropic ion channels that shape the bio-electrical behavior of RPeD1. It has been shown that, in the isolated CNS, RPeD1 frequently exhibits a spontaneously occurring respiratory discharge, similar to that recorded in ‘‘semiintact preparations’’ following direct current injection or mechano-chemical stimulation of the neuron (Bell et al. 2007; Haque et al. 2006; Holden et al. 1982; Inoue et al. 1996, 2001; McComb et al. 2003). Such spontaneous activity depends on neurotransmitter release from yet to identify presynaptic terminals. It is conceivable that pharmacological manipulation of the bursting might gain insights on the mechanisms leading to RPeD1 excitation. Conversely, in a minor percentage of isolated CNS, the latter neuron is quiescent and displays a significantly more negative resting membrane potential (Vm) (Holden et al.
1982; McComb et al. 2003). Therefore, analysis of quiescent neurons should help in unraveling the chemical nature of the peripheral afferent that suppresses the periodical discharge of RPeD1 in situ (Inoue et al. 1996, 2001; McComb et al. 2003). Electrophysiology Conventional intracellular recordings were performed by impaling RPeD1 in the left pedal dorsal ganglion with sharp microelectrodes (filament type, 1.5 mm o.d.; Harvard Apparatus, UK), which were filled with a saturated solution of K2SO4 and had a resistance of 80–90 MOhm. Such a relatively high resistance might explain the membrane potential drift seen in some experiments. Electrodes were pulled by using a vertical pipette puller (Palmer Bioscience, Sheerness, UK). Signals were amplified by a headstage connected to a Neurolog NL103 pre-amplifier and an associated NL125 filter system. The changes in Vm were digitized with a Minidigi 1A (Molecular Devices Corp., Union City, CA, USA) driven by Axoscope software (Molecular Devices Corp., Union City, CA, USA). The ganglion was perfused with standard Lymnaea saline at a flow rate of 2–4 mL/min. The saline was maintained normoxic by directly bubbling air into the beaker containing the solution. In experiments aiming at inhibition of polysynaptic transmission, a high divalent cation saline (69 Ca2?/69 Mg2? in mM: 35.0 NaCl, 1.7 KCl, 24.0 CaCl2, and 9.0 MgCl2) was superfused onto the ganglion. All experiments were performed at room temperature (21–22°C). Statistics Pooled data were given as mean ± standard error (SE) and n values in the text refer to the number of tested cells. The significance of differences between the averages was evaluated by Student’s t test for paired or unpaired observations and P \ 0.05 was considered significant. The fictive respiratory rhythm was evaluated by counting the number of electrical discharges per 10 min. Unless otherwise stated, action potential frequency was determined by counting the number of action potentials within the last bursts in the presence of the drug. This value was compared with that obtained during the last burst recorded before drug addition (control). Chemicals NMDA, AMPA, kainate, and CNQX were purchased from Tocris Cookson (Bristol, UK). All other chemicals were of analytical grade and obtained from Sigma (Sigma Chemicals, St Louis, MO, USA).
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Results The resting Vm sets the spontaneous, rhythmical electrical activation of RPeD1 The bio-electrical behavior of RPeD1 was monitored intracellularly in isolated CRG exposed to normoxic saline. As reported elsewhere (Holden et al. 1982; Inoue et al. 2001), soon after impalement, RPeD1 exhibited either regular bursting activity or an irregular firing pattern in most preparations (active neurons, 29 out of 34; Fig. 1a). The resting Vm, measured during the inter-burst intervals, and the mean frequency of regular bursts were -39.7 ± 1.48 mV (n = 29) and 11.5 ± 1.8 discharges per 10 min (n = 13), respectively. In the remaining five cells, no spontaneously occurring patterned respiratory activity could be recorded (silent neurons; Fig. 1b) and the resting Vm averaged -65.9 ± 1.7 mV (n = 4), which is significantly more negative (P \ 0.0001) than the value measured in spiking neurons. However, in nine bursting neurons, the resting potential hyperpolarized by 21.0 ± 2.5 mV within 10–15 min of the onset of recording (n = 9) (Fig. 1c), a feature which led to the inhibition of neuronal activity. These cells could, thus, be classified as silent. Pooling together the values measured in both groups of quiescent neurons (i.e., those which were electrically
Fig. 1 Electrophysiological behavior of the central pattern generating neuron, RPeD1. a Intracellular recording performed soon after impalement reveals that the central pattern generator neuron, RPeD1, may either display rhythmical bursts of action potentials (n = 13) or be quiescent (b) (n = 5). Note that the resting Vm of bursting cells is more positive than in silent neurons. c Trace depicting an initially
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silent from the beginning and those which underwent the negative shift in Vm), the mean resting Vm was -63.3 ± 1.8 mV (n = 13), which is in the same range as that reported for RPeD1 in semi-intact preparations (-59 ± 1 mV; Spencer et al. 2002). Notably, Holden et al. (1982) had previously reported that RPeD1 in isolated CRG may be either silent, with a resting Vm as negative as -70, or exhibit a background discharge, with an interspike Vm of about -40 mV. A closer inspection of the bioelectrical signal during the quiescent period revealed a complex waveform, consisting of repetitive tonic excitatory postsynaptic potentials (EPSPs) overlapping the inhibitory inputs (Fig. 1d). Overall, these data suggest that the isolated CRG may retain an inhibitory input that hyperpolarizes RPeD1 (Inoue et al. 1996, 2001), so that Vm is held far below the threshold for neuronal action potentials. GABA hyperpolarizes RPeD1 by activating GABAA receptors In order to elucidate which neurotransmitter(s) are involved in the tonic inhibition of RPeD1, we assessed the role of Gly and GABA, which contribute to respiratory rhythmogenesis in the mammalian CNS (Rekling and Feldman 1998). Superfusion of CRG with either Gly (50 lM) or strychnine (50 lM), a potent inhibitor of Gly receptors in vertebrates
active neuron which undergoes a negative shift in Vm resulting in the failure of spontaneously occurring respiratory discharge. This trace is representative of the behavior manifested by nine neurons. d Expansion of the section of the silent trace boxed in a shows that continuous EPSPs may occur in quiescent cells without reaching the threshold for action potentials generation
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Fig. 2 Picrotoxin initiates firing in silent cells. a Superfusion with 100 lM picrotoxin, a GABAA receptors inhibitor, depolarizes silent RPeD1 neurons and results in action potential discharges (n = 7). b Picrotoxin (100 lM) addition to active cells causes a moderate, although not significant, increase in their rhythmical activation (n = 3; see text for further explanations). In both traces, Vm underwent a positive shift due to the high value of electrode resistance (see ‘‘Materials and methods’’)
(Lynch 2004), did not produce any significant effect on the burst frequency in RPeD1 (not shown). Notably, bath application of strychnine neither elicited action potentials in silent neurons nor affected their resting Vm (not shown). In contrast, exposing silent RPeD1 neurons to picrotoxin (100 lM), a well known blocker of GABAA receptors in Lymnaea CNS (Rubakhin et al. 1996), caused a membrane depolarization which led to the onset of rhythmical bursts of spikes in seven out of ten neurons (Fig. 2a). The appearance of the first action potential occurred within 8.1 ± 7.0 min (n = 7) (Fig. 2a) of drug application and ceased at washout (not shown). When applied onto a bursting neuron, picrotoxin was associated with a slight, but not significant
Fig. 3 GABA is responsible for RPeD1 hyperpolarization by binding to GABAA receptors. a GABA (100 lM) inhibits neuronal bursting by inducing a negative shift in resting Vm with a latency of about 2 min. On agonist removal from the bath, Vm recovers to its control level and action potentials resume. This trace is representative of the findings obtained in seven neurons. The dashed line indicates the resting Vm in this and the following figures. b Picrotoxin (100 lM) causes bursting to resume in the presence of GABA (n = 5). c GABA-elicited hyperpolarization did occur in high divalent cation saline
(P = 0.393; n = 3), increase in the intraburst frequency without a significantly relevant change in Vm (Fig. 2b). Consistent with these findings, superfusion of CRG with GABA (100 lM) inhibited spontaneously occurring RPeD1 discharge in 8 out of 12 RPeD1 cells within 4.6 ± 1.2 min (n = 8) upon drug addition (Fig. 3a). As depicted in Fig. 3a, suppression of firing occurred subsequent to GABA-induced hyperpolarization from -45.5 ± 2.5 to 53.4 ± 3.6 mV (n = 7; P = 0.002). GABA-dependent
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changes in Vm and action potential frequency were reversed either by agonist removal from the bath (Fig. 3a) or by picrotoxin (100 lM) (Fig. 3b). As displayed in Fig. 3b, the hyperpolarizing action of GABA on RPeD1 persisted in the presence of high divalent cation saline (-10 ± 3.9 mV, n = 4, cells held at -60 mV by current injection), a finding that rules out the possibility that the GABA effect was due to the activation of other neurons in the brain (Nesic et al. 1996). Taken together, these results indicate that GABA may suppress RPeD1 activity by causing a negative shift in Vm upon binding to GABAA receptors. Such a mechanism is likely to explain the more hyperpolarized level of Vm recorded in silent RPeD1 neurons. Glutamate is the neurotransmitter mediating the rhythmical activation of RPeD1 The next step was to shed light on the neurochemistry of RPeD1 excitation. Therefore, we considered Glu, which has long been known as an activator of the neuron (Nesic et al. 1996) and might, thus, deliver a stimulatory signal to the snail rCPG. Vertebrate GluR antagonists, such as CNQX, DNQX, and AP-5, are known to lack efficacy on Lymnaea neurons (Ha et al. 2006; Moroz et al. 1993; Nesic et al. 1996; preliminary observations from our group), prompting us to impair glutamatergic transmission by adding desensitizing doses of Glu (1 mM) to the perfusate (Nesic et al. 1996). As shown in Fig. 4a, bath application of 1 mM Glu abolished the bursting activity of RPeD1 within 1 min without any evident change in Vm in 13 out of 15 cells. On average, the firing ceased with a delay of 4.6 ± 2.9 min (n = 13) after agonist addition and resumed following Glu removal. In a small (n = 2) number of cells, Glu-induced desensitization was accompanied by a negative shift in Vm, perhaps due to the unmasking of GABA-dependent hyperpolarization (data not shown). A lower (100 lM) concentration of Glu, which is close to the EC50 value (80 lM) calculated with a focal application system (Nesic et al. 1996), reversibly desensitized the rhythmical discharge only in one out of seven cells, whereas it did not significantly affect the firing rate in the remaining six neurons (Fig. 4b) (P = 0.268). The failure of an EC50 dose of Glu to affect the firing might be due to a marked diffusion barrier across the isolated ganglion and/or to the rapid neurotransmitter removal from the extracellular space (De Santis and Messenger 1989). This result suggests that Glu elicits the bursting activity of RPeD1 under our experimental conditions. The putative role of AMPA receptors in the rhythmical activation of RPeD1 Once we had established the excitatory role of Glu in modulating RPeD1 discharges, and presumably snail
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Fig. 4 Glutamate may mediate rhythmical excitation of RPeD1. a A high concentration of Glu (1 mM) reversibly blocks the spontaneously occurring respiratory discharge (n = 13). b Lower doses of Glu (100 lM) do not significantly affect the electrical activity (n = 6)
respiratory rhythmogenesis, we aimed at elucidating the contribution of NMDA and non-NMDA receptors in RPeD1 activation. Again, we took advantage of GluR desensitization following bath application of Glu agonists, NMDA, AMPA, and kainate (see Nesic et al. 1996). In these circumstances, bath application of 100 lM NMDA has been shown to cross-desensitize the glutamatergic synaptic transmission from VD4 to RPeD2/3 synapse in Lymnaea CNS (Nesic et al. 1996), although the situation may be different using pressure ejection techniques when applying drugs direct to the RPeD1 cell body (MishmastNehi and Winlow 1995). Figure 5a shows that the effect of NMDA was different as compared with Glu. Accordingly, superfusion of NMDA (100 lM) was ineffective in 60% of
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firing cells (not shown), while it increased the action potential frequency by about 95% in the remaining fraction of neurons (Fig. 5a). In the latter case, NMDA often induced a partial depolarization block with reduced action potential amplitude and was rapidly washed off (not shown). When the NMDA action was monitored in silent RPeD1 cells, it induced irregular action potentials in 50% of the neurons (Fig. 5b), while sub-threshold activity occurred in the remainder (Fig. 5c, d). The latter was not due to the well known Mg2?-dependent block of NMDA receptors at resting potential, since NMDA-induced currents are unaffected by Mg2? in Lymnaea (Moroz et al. 1993). The findings described above indicate that NMDA receptors are unlikely to initiate Glu-dependent synaptic activation of RPeD1 under our experimental conditions, because 100 lM NMDA does not desensitize RPeD1, although they could increase the rhythm frequency and thus, modulate the motor output. We therefore hypothesized that non-NMDA receptors were involved in the initiation of the respiratory rhythm. The two ionotropic non-NMDA GluRs that have been cloned in L. stagnalis display a selective sensitivity to the glutamatergic agonists, AMPA and kainate, respectively (Hutton et al. 1991; Stu¨hmer et al. 1996). Therefore, the electrophysiological effects of such agonists on the whole respiratory circuit
were investigated and compared with those exerted by Glu. We reasoned that, if any of AMPA- and kainate-sensitive receptors mediated the respiratory discharge, addition of selective agonists to the perfusate could lead to inhibition of firing by receptor desensitization (Adams and Gillespie 1988; De Santis and Messenger 1989; Nesic et al. 1996). On the other hand, if the receptors were expressed on the plasma membrane, but not primarily responsible for the rhythmical acrivation of the neuron, they could by activated by the agonists and cause a change either in Vm or in action potential frequency. For instance, synaptic transmission at the squid giant synapse may be blocked by bath application of the GluR agonists, Glu, AMPA, and kainate, but not NMDA (Adams and Gillespie 1988; De Santis and Messenger 1989). These findings, corroborated by the cloning of a putative GluR subunit whose primary structure was homologous to mammalian AMPA subunits GluR1-4 (Battaglia et al. 2003), led to the notion that Glu is the neurotransmitter operating at this synapse (for a review, see Di Cosmo et al. 2006). AMPA (100 lM) induced bursts of spikes in five out of five quiescent RPeD1 cells with a latency of 3.9 ± 1.7 min (n = 5) (Fig. 6a). Consistent with this finding, when AMPA (100 lM) was added onto spontaneously firing neurons, the electrical action potentials ceased within 8.2 ± 1.6 min (n = 5) with no
Fig. 5 NMDA does not desensitize the spontaneous activation of RPeD1. a NMDA (100 lM) may increase the firing frequency of an active cell without desensitizing its rhythmical activation (n = 4). When applied onto silent cells, NMDA (100 lM) may either produce an excitatory effect (b) (n = 4) or elicit a subthreshold activity (c) (n = 4). d Expansion of the box on the trace in c depicts at higher magnification the sub-threshold potentials which may be evoked by NMDA in quiescent cells
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Fig. 6 AMPA receptors are responsible for the periodical excitation of RPeD1. a AMPA (100 lM) stimulates silent RPeD1 neurons to produce action potential discharges (n = 5). b When added to the saline perfusing an active cell, AMPA (100 lM) rapidly causes a failure in action potential generation without any detectable change in resting Vm (n = 5)
detectable change in Vm (Fig. 6b). Notably, the bursting resumed upon washout of the agonist (Fig. 6b). The cessation of bursting activity upon desensitization of AMPAsensitive GluRs suggests that the latter underpin the rhythmical excitation of RPeD1 by Glu (De Santis and Messenger 1989; Nesic et al. 1996). Accordingly, preincubation with 1 mM Glu prevented the electrophysiological effects of AMPA in three out of three cells (Fig. 7a). A similar protocol showed that kainate was ineffective when applied onto cells exposed to 1 mM Glu (Fig. 7b). Nevertheless, under control conditions, bath application of kainate (100 lM) exerted a complex triphasic change in resting potentials of three silent RPeD1 cells. In all three cells, kainate first induced a transient membrane hyperpolarization by 13.7 ± 2.0 mV (n = 3),
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Fig. 7 Desensitization of GluRs prevents the effect of both AMPA and kainate on RPeD1. Fifteen minutes pre-treatment with a desensitizing dose of Glu (1 mM) prevents both AMPA- and kainate-elicited changes in Vm. Both Glu agonists were applied at 100 lM and n was equal to 3 for both agonists
followed by a mid-phase repolarization slightly above Vm (?4.1 ± 0.4 mV, n = 3), and finally a short lasting hyperpolarization (Fig. 8a). The latency of kainate-induced changes in resting potential averaged 2.0 ± 0.1 min (n = 3). In seven out of seven firing neurons, kainate (100 lM) caused a negative shift in Vm by 15.8 ± 3.0 mV (n = 6) with a mean delay of 3.3 ± 0.8 min (n = 7) (Fig. 8b). Kainate-elicited hyperpolarization reversibly inhibited the electrical activity of RPeD1 in three out of seven cells (Fig. 8b). In the remaining four neurons, action potentials resumed at a significantly higher frequency than under control conditions and kainate had to be rapidly washed out to prevent the partial depolarization block (Fig. 8c). The kainate effect is dramatically different as compared with AMPA and Glu, and is not consistent with the full desensitization of ongoing discharge (see below). The triphasic change in Vm indicates an interference with presynaptic activity. Indeed, to determine whether kainate
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Discussion
Fig. 8 Complex effect of kainate on the resting Vm of RPeD1. a Kainate (100 lM) may elicit a complex triphasic change in resting Vm of silent RPeD1 cells, which consists of an early hyperpolarization, a repolarization above resting Vm and a late hyperpolarization (n = 3). In this trace, kainate-induced hyperpolarization occurs with a delay of about 1 min. b When added onto active cells, kainateinduced hyperpolarization reversibly inhibits action potentials firing in 43% of neurons (n = 3). c In the remaining 57% (n = 4), action potentials may resume at higher frequency than in control conditions. The trace in the inset in b shows that picrotoxin (100 lM) did not prevent kainate-induced hypepolarization
inhibited RPeD1 via a polysynaptic pathway, the CRG was bathed in high divalent cation saline. Under these conditions, the drop in Vm disappeared (n = 3; not shown), a result which is in accordance with previous findings (Nesic et al. 1996) and shows the indirect nature of kainateinduced hyperpolarization. The latter did not involve GABAergic transmission, as picrotoxin (100 lM) did not rescue kainate-induced inhibition of rCPG (n = 4; data not shown). Overall, these findings indicate that kainate GluRs may modulate the rhythmical excitation of RPeD1 by AMPA, but are unlikely to contribute to its initiation.
The present investigation demonstrated for the first time that GABAA receptors and AMPA-selective GluRs play a pivotal role in determining whether RPeD1, the neuron which initiates breathing rhythmogenesis in the model system, L. stagnalis, is excited or not. The onset of patterned respiratory activity is controlled by both excitatory and inhibitory inputs mainly conveyed to RPeD1 from the pneumostome area (Bell et al. 2007; Haque et al. 2006; Inoue et al. 2001; Lukowiak et al. 2006; McComb et al. 2003). Strikingly, this behavior can undergo a dramatic operant conditioning and the resulting changes (i.e., associative learning) be consolidated into both intermediateand long-term memory (Lukowiak et al. 2006; McComb et al. 2005; Spencer et al. 2002). In particular, it has been shown that RPeD1 provides a necessary site for memory formation and storage following conditioning-induced alteration in gene expression and new protein synthesis (see Lukowiak et al. 2006 for a comprehensive review). It is conceivable that highlighting the receptors on the plasma membrane of RPeD1 which are involved in the shaping of respiratory discharge will: (1) aid in unraveling the intracellular signaling cascade which underlie behaviorally relevant performances; (2) help to reveal novel targets to investigate the molecular basis of learning. Inoue et al. (2001); see also Spencer et al. (2002) recently demonstrated that RPeD1 receives a regulatory suppressive input from the periphery that prevents the initiation of the respiratory rhythm. The neuronal pathway for inhibition is still unclear, although it is likely to originate adjacent to the pneumostome area (Inoue et al. 2001; see below). We took advantage of the minor fraction of quiescent RPeD1 neurons in isolated CNS to investigate the mechanism whereby these cells may be silenced. Three pieces of evidence indicate that the classical inhibitory neurotransmitter, GABA, suppresses RPeD1 firing by binding to GABAA receptors: (1) picrotoxin, a blocker of GABAA receptors, reversibly induces action potentials in quiescent RPeD1 cells; (2) GABA induces a reversible and picrotoxin-sensitive block of respiratory discharge in active RPeD1 neurons; and (3) the amino acid Gly, another well known inhibitory neurotransmitter in vertebrates, does not affect rCPG activity, and strychnine, an antagonist of Gly receptors, did not restart firing in silent RPeD1 cells. Consistent with the involvement of GABAA receptors in determining the onset of the respiratory rhythm, a mature polypeptide with an approximately 35–50% identity to vertebrate GABAA receptor has been found in L. stagnalis (Barnard et al. 1989; Harvey et al. 1991). Since RPeD1 is the neuron driving aerial respiration rhythmogenesis in Lymnaea, it is conceivable that GABA-induced interruption of its electrical bursting will suppress any behaviorally
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relevant output from the rCPG (Inoue et al. 1996, 2001; McComb et al. 2003). The notion that GABA is a suitable molecule to deliver an inhibitory signal to the rCPG in freshwater snails is supported by a large body of work conducted in the vertebrates. Although not essential for rhythm generation in the neonatal rodent, GABA may affect the frequency of respiratory motor output from the pre-Bo¨tzinger complex, a limited region of ventrolateral medulla which is essential for rhythmic breathing in mammals (Rekling and Feldman 1998; see ‘‘Introduction’’). Indeed, GABA induces a hyperpolarization of Vm in neonatal rat respiratory medullary neurons and a suppression of respiratory frequency (Ren and Greer 1996). The lamprey provides an additional model to understand the role of GABA in patterning the respiratory motor output. Consistently, the frequency of respiratory discharge produced by isolated brainstem preparations is significantly increased by picrotoxin and suppressed by the GABAA receptor agonist, muscimol (Bongianni et al. 2006). Two main differences in the GABAergic control of breathing between the gastropod mollusc, L. stagnalis, and vertebrates should be appreciated: (1) GABA is accompanied by Gly in the inhibitory control of respiratory burst in rodents (Rekling and Feldman 1998; Ren and Greer 1996) and lamprey (Bongianni et al. 2006), while Gly is ineffective in snails (present study); and (2) GABA modulates the frequency, but not the onset, of respiratory motor output in vertebrates (Bongianni et al. 2006; Rekling and Feldman 1998; Ren and Greer 1996). In contrast, GABA signaling does prevent the excitation of Lymnaea RPeD1 by causing a negative shift in Vm, which may be relieved by the block of GABAA receptors. This hypothesis is supported by the finding that spontaneous EPSPs may occur during RPeD1 inhibition, but do not result in spiking as GABA maintains resting Vm far below the threshold for action potentials generation. Once the firing has been initiated, the frequency of respiratory discharges is determined by the emerging properties of the CPG where RPeD1 is embedded (Lukowiak and Syed 1999; Syed et al. 1990, 1992; Winlow and Syed 1992). This feature would explain why the frequency of respiratory discharges is unaffected by picrotoxin. In other words, the delivery of GABAergic inputs decides whether RPeD1 is activated or not by Glu (see below), but does not set the rhythm of the motor output. The physiological source of GABA might reside in the osphradium of L. stagnalis, which has been shown to contain GABA-like immunoreactive nerve cells (Nezlin and Voronezhskaya 1997). These nerve cells project into the CNS and might, thus, deliver the hyperpolarizing signal to RPeD1. Nevertheless, we cannot rule out the involvement of GABA-like immunoreactive neurons within the CNS of Lymnaea in GABAergic inhibition of RPeD1 (Hatakeyama et al. 2007). Notably, the osphradial origin of
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GABA signaling concurs with the notion that RPeD1 receives a peripheral suppressive drive from the pneumostome area (Inoue et al. 1996, 2001). The data presented here are in contrast with those obtained by Rubakhin et al. (1996) and Cheung et al. (2006). These authors reported a depolarizing action of GABA when superfused onto isolated CRG under normoxic conditions. Cheung et al. (2006), however, observed that GABA is responsible for RPeD1 hyperpolarization when the saline is made hypoxic. Such a switch in GABA effect on Vm might be due to a change in intracellular Clconcentration ([Cl-]i) following a hypoxia-induced decrease in the activity of Na?/K?/Cl- (NKCCl) cotransporter. Importantly, these recordings were performed on non-identified neurons of neuronal cluster F on the dorsal surface of right pedal ganglia. In our experiments, we used the same solutions and reproduced the same conditions as those employed in the above mentioned studies (see ‘‘Materials and methods’’): (1) the perfusion saline was maintained normoxic by bubbling air directly into the solution; (2) recordings were carried out at room temperature. The reason(s) for such a discrepancy, therefore, is likely to be ascribed to the different intracellular Clhomeostasis between RPeD1 (this study) and the neurons of neuronal cluster F. The following pieces of evidence indicate a central role for Glu in the rhythmical excitation of RPeD1 following activation of AMPA-selective GluRs: (1) superfusion of isolated CRG with a desensitizing concentration of Glu rapidly inhibits the spontaneously occurring, patterned respiratory activity; (2) AMPA is the only agonist to completely desensitize the rhythmical firing, with lower (if none) efficacy displayed by kainite and NMDA-selective GluRs; (3) AMPA induces electrical firing in silent RPeD1 neurons; and (4) a mature polypeptide, with a 37–46% amino acid identity to the rat AMPA subunits GluR1-6, has been cloned from Lymnaea CNS (Hutton et al. 1991), albeit its pharmacological profile is different to the Glu response on RPeD1 (Nesic et al. 1996). In this regard, it is noticeable that the Lymnaea GluR subunit is unable to produce detectable ion currents when expressed in Xenopus oocytes. It has been proposed that it requires at least one additional subunit to form a fully functional receptor complex in vivo (Hutton et al. 1991). The central role played by AMPA-sensitive GluRs in the onset of aerial breathing in L. stagnalis concurs with the results obtained in vertebrates (Rekling and Feldman 1998). AMPA receptor activation is essential in rhythm generation in spontaneously bursting neurons within the preBo¨tzinger complex of newborn rats (Ge and Feldman 1998). Although these neurons are sensitive to both AMPA and kainate, respiratory output is abolished only following selective inhibition of AMPA-sensitive GluRs (Ge and
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Feldman 1998). In addition, transgenic mice lacking the NMDAR1 gene produce a respiratory rhythm virtually identical to control in a Mg2?-containing medium (Funk et al. 1997). Similar results have been reported in the lamprey, whose respiratory activity relies on the activation of ionotropic AMPA, but not NMDA, receptors (Bongianni et al. 1999; Martel et al. 2007). These data strongly support the role of Glu as neurotransmitter in rhythm generator networks in Lymnaea. Consistently, it has recently been shown that Glu mediates the responses generated by the N2v rasp phase neurons on postsynaptic cells of the Lymnaea feeding network (Brierley et al. 1997). Interestingly, Glu-induced effects on Vm are mimicked by AMPA, rather than by kainate, also in feeding neurons (Brierley et al. 1997). However, though desensitization experiments hint at the pivotal role played by AMPA-sensitive GluRs in the excitation of RPeD1, the function of kainate and NMDA receptors must be discussed. In this regard, the complex triphasic change (initial hyperpolarization, mid-phase repolarization, and a final short lasting depolarization) in Vm exerted by bath application of kainate depends on the activation of a polysynaptic circuit. In agreement with this hypothesis, when polysynaptic inputs were blocked in the presence of a high divalent cation saline, kainate depolarized RPeD1, as reported in the pioneering paper by Nesic et al. (1996). Notably, kainate-elicited hyperpolarization does not depend on the activation of a GABAergic neuron, as it is not prevented by picrotoxin. It is conceivable that kainate-dependent modulation of RPeD1 firing is, at least partially, mediated by a polypeptide with a 44–48% identity to the mammalian kainate-selective GluR5–7 subunits that has been cloned from buccal ganglion and is present within all Lymnaea CNS (Stu¨hmer et al. 1996). We speculate that kainate, as well as NMDA, GluRs are not the main target of rhythmically-liberated Glu, but are located to distinct synaptic sites. Based on the observations reported at the squid giant synapse (Adams and Gillespie 1988; De Santis and Messenger 1989), this feature would explain why bath addition of such agonists does not efficaciously desensitise the firing, but rather induces a change either in Vm or in action potential frequency. We, therefore, suggest that the rhythmical excitation of RPeD1 is mainly driven by AMPA receptors and modulated by kainate- and NMDA-selective GluRs. The different compartmentalization of GluRs would also account for the difference between our results and those reported by Nesic et al. (1996), who found kainate to be more effective than AMPA in exciting the neuron by using a focal perfusion system. As for GABA, the source for glutamatergic transmission to RPeD1 is unclear, however, Glu-like immunoreactive sensory neurons have recently been found in the head–foot complex of Lymnaea, the region where the pneumostome is located (Hatakeyama et al. 2007).
Nevertheless, the contribution of Glu-like immunoreactive neurons to AMPA-mediated activation of RPeD1 cannot be ruled out by the present data. A major caveat in the interpretation of the findings reported in this study concerns the evidence that Lymnaea rCPG may behave differently when it is isolated from the periphery (Inoue et al. 1996, 2001; McComb et al. 2003). As aforementioned, RPeD1 is quiescent in ‘‘semi-intact preparations’’ bathed in normoxic physiological saline and is activated by a peripheral excitatory drive encompassing two components: a mechanosensory input from the pneumostome (Haque et al. 2006) and a chemosensory input from the osphradium (Bell et al. 2007). Nevertheless, this feature does not impair the conclusion that the interplay between GABAA- and AMPA-receptors is central in shaping the rhythmical activation of RPeD1, although it makes it likely that other neurotransmitters participate in the modulation of the circuit in vivo. For instance, it has recently been shown that acetylcholine mediates the excitatory synaptic input from the osphradial cells to RPeD1 (Bell et al. 2007). It is tempting to try to integrate our data into the substantial literature regarding the control of RPeD1, the rCPG interneuron that determines the rhythm of aerial respiration in Lymnaea. Accordingly RPeD1 exhibits plasticity and undergoes the biophysical alterations essential to enable the snail to change its breathing behavior (‘‘learn’’) and memorize the changes. This leads us to the conclusions below. Evidence has been presented that GABA inhibits the rhythmical bursting of RPeD1 by holding its Vm at a hyperpolarized level following the activation of GABAA receptors. Therefore, we hypothesize that GABAergic transmission participates in silencing RPeD1 (and consequently, the rCPG) when the snail is submerged in normoxic water and does not need to switch into the aerial breathing mode. When this occurs, due to a decrease in O2 content (hypoxic stimulation), and the animal surfaces to expose its pneumostome to air (mechanical stimulation), Glu aids Ach in delivering the excitatory signal to RPeD1 by activating AMPA-sensitive receptors. The novel insights into the neuromodulation of CPG brought about by these data might help to unravel the molecular mechanisms of learning and memory formation in Lymnaea. As mentioned above, RPeD1 provides a necessary site for memory formation and consolidation in operant-conditioned animals (Lukowiak et al. 2006). Studies conducted on semi-intact preparations from snails trained to perform cutaneous respiration in a hypoxic environment have shown that RPeD1 undergoes dramatic electrophysiological changes after application of an aversive mechanical stimulus to the pneumostome as the animal attempts to open it. More specifically, its respiratory activity decreases, while a larger current must be injected to induce pneumostome opening (McComb et al. 2005; Spencer et al. 2002). The reduced excitability of RPeD1 in
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conditioned animals might be due to an increase in GABAergic transmission, which would lead to the gating of additional chloride channels on the plasma membrane (or to an increase in the open state probability of already open channels) and to the reduction in input resistance. On the other hand, the reduction in action potential frequency might result from training-dependent posttranslational modifications of AMPA receptors. In support of this hypothesis, an increasing body of evidence indicates that, at many excitatory synapses in vertebrates, changes in phosphorylation and cellular distribution of AMPA GluRs underlie the synaptic alterations observed during long-term potentiation and depression, respectively (Santos et al. 2008). Acknowledgments The work described in the present study has been supported by the Minister of Instruction, University and Research—Project FIRB n RBAU017KSA.
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