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Choline chloride is often, and N-methyl-d-glucamine (NMDG) sometimes, used to replace sodium chloride in studies of sodium-activated potassium channels. Given the high concentrations used in sodium replacement protocols, it is essential to test that it is not the replacement substances themselves, as opposed to the lack of sodium, that cause any observed effects. We therefore compared, in lobster stomatogastric neurons and leech Retzius cells, the effects of applying salines in which choline chloride replaced sodium chloride, and in which choline hydroxide or sucrose was added to normal saline. We also tested, in stomatogastric neurons, the effect of adding NMDG to normal saline. These protocols allowed us to measure the direct effects (i.e., effects not due to changes in sodium concentration or saline osmolarity or ionic strength) of choline on stomatogastric and leech currents, and of NMDG on stomatogastric currents. Choline directly reduced transient and sustained depolarization-activated outward currents in both species, and NMDG directly reduced transient depolarization-activated outward currents in stomatogastric neurons. Experiments with lower choline concentrations showed that adding as little as 150 mM (stomatogastric) or 5 mM (leech) choline reduced at least some depolarization-activated outward currents. Reductions in outward current with choline chloride or NMDG replacement alone are thus not evidence of sodium-activated potassium currents. NEWNOTEWORTHY We show that choline or N-methyl-d-glucamine (NMDG) directly (i.e., not due to changes in extracellular sodium) decrease outward currents. Prior work studying sodium-activated potassium channels in which sodium was replaced with choline or NMDG without an addition control may therefore be artifactual.

Measuring neuron capacitance is important for morphological description, conductance characterization, and neuron modeling. One method to estimate capacitance is to inject current pulses into a neuron and fit the resulting changes in membrane potential with multiple exponentials; if the neuron is purely passive, the amplitude and time constant of the slowest exponential give neuron capacitance (Major G, Evans JD, Jack JJ. Biophys J 65: 423–449, 1993). Golowasch et al. (Golowasch J, Thomas G, Taylor AL, Patel A, Pineda A, Khalil C, Nadim F. J Neurophysiol 102: 2161–2175, 2009) have shown that this is the best method for measuring the capacitance of nonisopotential (i.e., most) neurons. However, prior work has not tested for, or examined how much error would be introduced by, slow voltage-dependent phenomena possibly present at the membrane potentials typically used in such work. We investigated this issue in lobster ( Panulirus interruptus) stomatogastric neurons by performing current clamp-based capacitance measurements at multiple membrane potentials. A slow, voltage-dependent phenomenon consistent with residual voltage-dependent conductances was present at all tested membrane potentials (−95 to −35 mV). This phenomenon was the slowest component of the neuron's voltage response, and failure to recognize and exclude it would lead to capacitance overestimates of several hundredfold. Most methods of estimating capacitance depend on the absence of voltage-dependent phenomena. Our demonstration that such phenomena make nonnegligible contributions to neuron responses even at well-hyperpolarized membrane potentials highlights the critical importance of checking for such phenomena in all work measuring neuron capacitance. We show here how to identify such phenomena and minimize their contaminating influence.

Synapses show short-term activity-dependent dynamics that alter the strength of neuronal interactions. This synaptic plasticity can be tuned by neuromodulation as a form of metaplasticity. We examined neuromodulator-induced metaplasticity at a graded chemical synapse in a model central pattern generator (CPG), the pyloric network of the spiny lobster stomatogastric ganglion. Dopamine, serotonin, and octopamine each produce a unique motor pattern from the pyloric network, partially through their modulation of synaptic strength in the network. We characterized synaptic depression and its amine modulation at the graded synapse from the pyloric dilator neuron to the lateral pyloric neuron (PD→LP synapse), driving the PD neuron with both long square pulses and trains of realistic waveforms over a range of presynaptic voltages. We found that the three amines can differentially affect the amplitude of graded synaptic transmission independently of the synaptic dynamics. Low concentrations of dopamine had weak and variable effects on the strength of the graded inhibitory postsynaptic potentials (gIPSPs) but reliably accelerated the onset of synaptic depression and recovery from depression independently of gIPSP amplitude. Octopamine enhanced gIPSP amplitude but decreased the amount of synaptic depression; it slowed the onset of depression and accelerated its recovery during square pulse stimulation. Serotonin reduced gIPSP amplitude but increased the amount of synaptic depression and accelerated the onset of depression. These results suggest that amine-induced metaplasticity at graded chemical synapses can alter the parameters of synaptic dynamics in multiple and independent ways.

Dopamine (DA) modifies the motor pattern generated by the pyloric network in the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus , by directly acting on each of the circuit neurons. The 14 pyloric neurons fall into six cell types, and DA actions are cell type specific. The transient potassium current mediated by shal channels ( IA) is a common target of DA modulation in most cell types. DA shifts the voltage dependence of IAin opposing directions in pyloric dilator (PD) versus lateral pyloric (LP) neurons. The mechanism(s) underpinning cell-type specific DA modulation of IAis unknown. DA receptors (DARs) can be classified as type 1 (D1R) or type 2 (D2R). D1Rs and D2Rs are known to increase and decrease intracellular cAMP concentrations, respectively. We hypothesized that the opposing DA effects on PD and LP IAwere due to differences in DAR expression patterns. In the present study, we found that LP expressed somatodendritic D1Rs that were concentrated near synapses but did not express D2Rs. Consistently, DA modulation of LP IAwas mediated by a Gs-adenylyl cyclase-cAMP-protein kinase A pathway. Additionally, we defined antagonists for lobster D1Rs (flupenthixol) and D2Rs (metoclopramide) in a heterologous expression system and showed that DA modulation of LP IAwas blocked by flupenthixol but not by metoclopramide. We previously showed that PD neurons express D2Rs, but not D1Rs, thus supporting the idea that cell specific effects of DA on IAare due to differences in receptor expression.

Phase-locked activity is thought to underlie many high-level functions of the nervous system, the simplest of which are produced by central pattern generators (CPGs). It is not known whether we can define a theoretical framework that is sufficiently general to predict phase-locking in actual biological CPGs, nor is it known why the CPGs that have been characterized are dominated by inhibition. Previously, we applied a method based on phase response curves measured using inputs of biologically realistic amplitude and duration to predict the existence and stability of 1:1 phase-locked modes in hybrid networks of one biological and one model bursting neuron reciprocally connected with artificial inhibitory synapses. Here we extend this analysis to excitatory coupling. Using the pyloric dilator neuron from the stomatogastric ganglion of the American lobster as our biological cell, we experimentally prepared 86 networks using five biological neurons, four model neurons, and heterogeneous synapse strengths between 1 and 10,000 nS. In 77% of networks, our method was robust to biological noise and accurately predicted the phasic relationships. In 3%, our method was inaccurate. The remaining 20% were not amenable to analysis because our theoretical assumptions were violated. The high failure rate for excitation compared with inhibition was due to differential effects of noise and feedback on excitatory versus inhibitory coupling and suggests that CPGs dominated by excitatory synapses would require precise tuning to function, which may explain why CPGs rely primarily on inhibitory synapses.

The mechanism(s) of olfactory transduction in invertebrates remains to be fully understood. In lobster olfactory receptor neurons (ORNs), a nonselective sodium-gated cation (SGC) channel, a presumptive transient receptor potential (TRP)C channel homolog, plays a crucial role in olfactory transduction, at least in part by amplifying the primary transduction current. To better determine the functional role of the channel, it is important to selectively block the channel independently of other elements of the transduction cascade, causing us to search for specific pharmacological blockers of the SGC channel. Given evidence that the Na+/Ca2+exchange inhibitor, KB-R7943, blocks mammalian TRPC channels, we studied this probe as a potential blocker of the lobster SGC channel. KB-R7943 reversibly blocked the SGC current in both inside- and outside-out patch recordings in a dose- and voltage-dependent manner. KB-R7943 decreased the channel open probability without changing single channel amplitude. KB-R7943 also reversibly and in a dose-dependent manner inhibited both the odorant-evoked discharge of lobster ORNs and the odorant-evoked whole cell current. Our findings strongly imply that KB-R7943 potently blocks the lobster SGC channel and likely does so directly and not through its ability to block the Na+/Ca2+exchanger.

A fundamental question in systems biology addresses the issue of how flexibility is built into modulatory networks such that they can produce context-dependent responses. Here we examine flexibility in the serotonin (5-HT) response system that modulates the cycle frequency (cf) of a rhythmic motor output. We found that depending on the preparation, the same 5-min bath application of 5-HT to the pyloric network of the California spiny lobster, Panulirus interruptus, could produce a significant increase, decrease, or no change in steady-state cf relative to baseline. Interestingly, the mean circuit output was not significantly different among preparations prior to 5-HT application. We developed pharmacological tools to examine the preparation-to-preparation variability in the components of the 5-HT response system. We found that the 5-HT response system consisted of at least three separable components: a 5-HT2βPan-like component mediated a rapid decrease followed by a sustained increase in cf; a 5-HT1αPan-like component produced a small and usually gradual increase in cf; at least one other component associated with an unknown receptor mediated a sustained decrease in cf. The magnitude of the change in cf produced by each component was highly variable, so that when summed they could produce either a net increase, decrease, or no change in cf depending on the preparation. Overall, our research demonstrates that the balance of opposing components of the 5-HT response system determines the direction and magnitude of 5-HT–induced change in steady-state cf relative to baseline.

Modulation of synaptic transmission is a major mechanism for the functional reconfiguration of neuronal circuits. Neurotransmitter release and, consequently, synaptic strength are regulated by intracellular Ca2+ levels in presynaptic terminals. In identified neurons of the lobster pyloric network, we studied localized, voltage-induced Ca2+ accumulation and its modulation in varicosities on distal neuritic arborizations, which have previously been shown to be sites of synaptic contacts. We previously demonstrated that dopamine (DA) weakens synaptic output from the pyloric dilator (PD) neuron and strengthens synaptic output from the lateral pyloric (LP) and pyloric constrictor (PY) neurons. Here we show that DA modifies voltage-activated Ca2+ accumulation in many varicosities in ways that are consistent with DA's effects on synaptic transmission: DA elevates Ca2+ accumulation in LP and PY varicosities and reduces Ca2+ accumulation in PD varicosities. However, in all three neuron types, we also found varicosities that were unaffected by DA. In the PY neurons, we found that DA can simultaneously increase and decrease voltage-evoked Ca2+ accumulation at different varicosities, even within the same neuron. These results suggest that regulation of Ca2+ entry is a common mechanism to regulate synaptic strength in the pyloric network. However, voltage-evoked local Ca2+ accumulation can be differentially modulated to control Ca2+-dependent processes in functionally separate varicosities of a single neuron.

We cloned 10 full-length variants of PIIH, the gene for Ih from the spiny lobster, Panulirus interruptus, using reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). This gene shows a significant amount of alternative splicing in the S3–S4 and S4–S5 linkers, in the P-loop and the entire S6 transmembrane domain, in the cyclic nucleotide binding domain (CNBD), and near the 3′ end of the gene. Functional expression of seven splice variants in Xenopus oocytes generated slowly activating hyperpolarization-activated inward currents, which were blocked by the Ih channel blockers CsCl and ZD7288. The different splice variants had markedly varying activation kinetics and voltage dependence of activation. Bath application of 8-Br-cAMP shifted the V1/2 to more positive potentials and accelerated the activation kinetics in an isoform-specific manner. Two variants containing a segment with an ER-retention motif in the S4-S5 loop did not produce currents in oocytes. Overexpression of one splice variant, PIIH ABS -I, in pyloric dilator (PD) neurons in the lobster stomatogastric ganglion produced an average threefold increase in Ih without evoking a compensatory increase in IA. The voltage for half-maximal activation of Ih in PIIH ABS-I-expressing PDs was shifted in the depolarizing direction by 9 mV, whereas the slope factor decreased by 3.8 mV. Moreover, its activation kinetics were significantly faster than in control PDs. PIIH ABS-I overexpression enhanced PD neuron rhythmic firing in an amplitude-dependent manner above a minimal threshold two- to threefold increase in amplitude.

Rhythmically bursting neurons are fundamental to neuronal network function but typically are not considered in the context of primary sensory signaling. We now report intrinsically bursting lobster primary olfactory receptor neurons that respond to odors with a phase-dependent burst of action potentials. Rhythmic odor input as might be generated by sniffing entrains the intrinsic bursting rhythm in a concentration-dependent manner and presumably synchronizes the ensemble of bursting cells. We suggest such intrinsically bursting olfactory receptor cells provide a novel way for encoding odor information.

We studied the functional role and modulation of the hyperpolarization-activated inward current ( Ih) in the pyloric network of the lobster stomatogastric ganglion. In isolated neurons, Ih is a small current with a hyperpolarized voltage of half-activation ( VAct) and a slow time constant of activation (τAct). Bath application of dopamine (DA), octopamine (OCT), or serotonin (5HT) modified Ih in selected synaptically isolated pyloric neurons. DA significantly enhanced Ih in the anterior burster (AB) neuron by depolarizing its VAct, accelerating its τAct, and enhancing its maximal conductance ( gmax). DA more weakly enhanced Ih in the pyloric constrictor (PY) and ventricular dilator (VD) neurons. OCT weakly depolarized VAct and accelerated τAct in the VD and inferior cardiac (IC) neurons. 5HT depolarized VAct in the IC neuron. Under control conditions with intact modulatory inputs from other ganglia, the pyloric rhythm cycles strongly at about 1–2 Hz. Bath application of the Ih blocker cesium (Cs+) caused a mean increase in the period of 8%, although this effect was highly variable. When Cs+ was applied to an isolated ganglion where the pyloric rhythm had been activated only by DA, the cycle period was consistently increased by 13.5%, with no other strong changes in rhythm parameters. These results suggest that Ih regulates the pyloric rhythm by accelerating AB pacemaker frequency, but that this effect can vary with the modulatory conditions.

In addition to ionotropic effects, glutamate and acetylcholine have metabotropic modulatory effects on many neurons. Here we show that in the stomatogastric ganglion of the lobster, glutamate, one of the main ionotropic neurotransmitters, modulates the excitability of gastric mill neurons. The neurons in this well-studied system produce rhythmic output to a subset of lobster foregut muscles. Recently, metabotropic glutamate receptor (mGluR) agonists were suggested as modulators of the rhythmic output, in addition to the previously described muscarinic modulation by acetylcholine. However, the cellular mechanisms responsible for these effects on the pattern are not known. Using intracellular recording methods and calcium imaging, we show that glutamate has an excitatory effect on specific neurons in the stomatogastric ganglion, which is mediated by mGluRs. Responses to the application of mGluR type I agonists are transient oscillations in the system, probably arising from network interactions. We show that the excitatory effect is sensitive to phospholipase-C and IP3and is G-protein dependent. The G-protein dependency was demonstrated by GDPβS and GTPγS injection into identified neurons. The depolarizations and oscillations were accompanied by an increase of intracellular Ca2+levels and correlated Ca2+oscillations. By using cyclopiazonic acid, an endoreticular Ca2+uptake inhibitor, we show that some internal calcium release may augment the response, but is not crucial for its production. Interestingly, although Ca2+concentration increase is typically associated with the phosphoinositide pathway, in the lobster, the Ca2+concentration increase—either voltage dependent or independent—cannot account for the observed depolarization.

The fast transient potassium or A current ( IA) plays an important role in determining the activity of central pattern generator neurons. We have previously shown that the shal K+ channel gene encodes IA in neurons of the pyloric network in the spiny lobster. To further study how IA shapes pyloric neuron and network activity, we microinjected RNA for a shal-GFP fusion protein into four identified pyloric neuron types. Neurons expressing shal-GFP had a constant increase in IA amplitude, regardless of cell type. This increase in IA was paralleled by a concomitant increase in the hyperpolarization-activated cation current Ih in all pyloric neurons. Despite significant increases in these currents, only modest changes in cell firing properties were observed. We used models to test two hypotheses to explain this failure to change firing properties. First, this may reflect the mislocalization of the expressed shal protein solely to the somata and initial neurites of injected neurons, rendering it electrically remote from the integrating region in the neuropil. To test this hypothesis, we generated a multicompartment model where increases in IA could be localized to the soma, initial neurite, or neuropil/axon compartments. Although spike activity was somewhat more sensitive to increases in neuropil/axon versus somatic/primary neurite IA, increases in IA limited to the soma and primary neurite still evoked much more dramatic changes than were seen in the shal-GFP–injected neurons. Second, the effect of the increased IA could be compensated by the endogenous increase in Ih. To test this, we modeled the compensatory increases of IA and Ih with a cycling two-cell model. We found that the increase in Ih was sufficient to compensate the effects of increased IA, provided that they increase in a constant ratio, as we observed experimentally in both shal-injected and noninjected neurons. Thus an activity-independent homeostatic mechanism maintains constant neuronal activity in the face of dramatic increases in IA.

Delayed rectifier potassium currents [ IK(V)] generate sustained, noninactivating outward currents with characteristic fast rates of activation and deactivation and play important roles in shaping spike frequency. The pyloric motor network in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus, is made up of one interneuron and 13 motor neurons of five different classes. Dopamine (DA) increases the firing frequencies of the anterior burster (AB), pyloric (PY), lateral pyloric (LP), and inferior cardiac (IC) neurons and decreases the firing frequencies of the pyloric dilator (PD) and ventricular dilator (VD) neurons. In all six types of pyloric neurons, IK(V)is small with respect to other K+currents. It is made up of at least two TEA-sensitive components that show differential sensitivity to 4-aminopyridine and quinidine, and have differing thresholds of activation. One saturable component is activated at potentials above −25 mV, whereas the second component appears at more depolarized voltages and does not saturate at voltage steps up to +45 mV. The magnitude of the components varies among cell types but also shows considerable variation within a single type. A subset of PY neurons shows a marked enhancement in spike frequency with DA; DA evokes a pronounced reversible increase in IK(V)conductance of ≤30% in the PY neurons studied, and on average significantly increases both components of IK(V). The AB neuron also shows a reversible 20% increase in the steady state IK(V). DA had no effect on IK(V)in PD, LP, VD, and IC neurons. The physiological roles of these currents and their modulation by DA are discussed.

Recent studies have found electrical coupling to be more ubiquitous than previously thought, and coupling through gap junctions is known to play a crucial role in neuronal function and network output. In particular, current spread through gap junctions may affect the activation of voltage-dependent conductances as well as chemical synaptic release. Using voltage-clamp recordings of two strongly electrically coupled neurons of the lobster stomatogastric ganglion and conductance-based models of these neurons, we identified effects of electrical coupling on the measurement of leak and voltage-gated outward currents, as well as synaptic potentials. Experimental measurements showed that both leak and voltage-gated outward currents are recruited by gap junctions from neurons coupled to the clamped cell. Nevertheless, in spite of the strong coupling between these neurons, the errors made in estimating voltage-gated conductance parameters were relatively minor (

We report that a Na+-activated nonselective cation channel described previously in lobster olfactory neurons, in which phosphoinositide signaling mediates olfactory transduction, can also be activated by Ca2+. Ca2+activates the channel in the presence of Na+, increasing the open probability of the channel with a K1/2of 490 nM and a Hill coefficient of 1.3. Ca2+also increases the sensitivity of the channel to Na+. In some cells, the same channel is Ca2+insensitive in a cell-specific manner. The nonspecific activator of protein phosphatases, protamine, applied to the intracellular face of patches containing the channel irreversibly eliminates the sensitivity to Ca2+. This effect can be blocked by okadaic acid, a nonspecific blocker of protein phosphatases, and restored by the catalytic subunit of protein kinase A in the presence of MgATP. The Ca2+-sensitive form of the channel is predominantly expressed in the transduction zone of the cells in situ. These findings imply that the Ca2+sensitivity of the channel, and possibly its regulation by phosphorylation, play a role in olfactory transduction and help tie activation of the channel to the canonical phosphoinositide turnover pathway.

We examined the dopamine (DA) modulation of calcium currents (ICa) that could contribute to the plasticity of the pyloric network in the lobster stomatogastric ganglion. Pyloric somata were voltage-clamped under conditions designed to block voltage-gated Na+, K+, and H currents. Depolarizing steps from –60 mV generated voltage-dependent, inward currents that appeared to originate in electrotonically distal, imperfectly clamped regions of the cell. These currents were blocked by Cd2+ and enhanced by Ba2+ but unaffected by Ni2+. Dopamine enhanced the peak ICa in the pyloric constrictor (PY), lateral pyloric (LP), and inferior cardiac (IC) neurons and reduced peak ICa in the ventricular dilator (VD), pyloric dilator (PD), and anterior burster (AB) neurons. All of these effects, except for the AB, are consistent with DA's excitation or inhibition of firing in the pyloric neurons. Enhancement of ICa in PY and LP neurons and reduction of ICa in VD and PD neurons are also consistent with DA-induced synaptic strength changes via modulation of presynaptic ICa. However, the reduction of ICa in AB suggests that DA's enhancement of AB transmitter release is not directly mediated through presynaptic ICa. ICa in PY and PD neurons was more sensitive to nifedipine block than in AB neurons. In addition, nifedipine blocked DA's effects on ICa in the PY and PD neurons but not in the AB neuron. Thus the contribution of specific calcium channel subtypes carrying the total ICa may vary between pyloric neuron classes, and DA may act on different calcium channel subtypes in the different pyloric neurons.

Cardiac sac network activity (cycle period tens of seconds to minutes) has long been known to alter pyloric network activity (cycle period approximately 1 s), but these effects have not been quantified. Some pyloric muscles extract cardiac sac timed variations in pyloric motor neuron firing, and consequently produce cardiac sac timed movements even though no cardiac sac neurons innervate them. Determining pyloric behavior therefore requires detailed description of cardiac sac effects on pyloric neural output. Pyloric muscle activity correlates well with motor neuron overall spike frequency (OSF, number of spikes per burst divided by cycle period). We therefore quantified the effects of cardiac sac activity on the OSF of all pyloric neurons in the lobster, Panulirus interruptus. The ventricular dilator (VD) neuron had a biphasic response, with its OSF first increasing and then decreasing during cardiac sac bursts. Lateral pyloric (LP) neuron OSF decreased during cardiac sac activity. The pyloric (PY) neurons had two responses, with OSF either decreasing or increasing just after the beginning of cardiac sac activity. The pyloric dilator (PD) neurons had a triphasic response, with OSF increasing slightly at the beginning of cardiac sac activity, decreasing during the cardiac sac burst, and strongly increasing after cardiac sac activity ended. The inferior cardiac (IC) neuron had a biphasic response, with OSF decreasing at the beginning of cardiac sac activity and strongly increasing when cardiac sac activity ceased. These data provide the quantitative description of cardiac sac effects on pyloric activity necessary to predict pyloric movement from pyloric neural output.

Rhythm generation by the pyloric motor network in the stomatogastric ganglion (STG) of the spiny lobster requires permissive neuromodulatory inputs from other central ganglia. When these inputs to the STG are suppressed by cutting the single, mainly afferent stomatogastric nerve (stn), pyloric neurons cease to burst and the network falls silent. However, as shown previously, if such a decentralized quiescent ganglion is maintained in organ culture, pyloric network rhythmicity returns after 3–4 days and, although slower, is similar to the motor pattern expressed when the stn is intact. Here we use current- and voltage-clamp, primarily of identified pyloric dilator (PD) neurons, to investigate changes in synaptic and cellular properties that underlie this transition in network behavior. Although the efficacy of chemical synapses between pyloric neurons decreases significantly (by ≤50%) after STG decentralization, the fundamental change leading to rhythm recovery occurs in the voltage-dependent properties of the neurons themselves. Whereas pyloric neurons, including the PD, lateral pyloric, and pyloric cell types, are unable to generate burst-producing membrane potential oscillations in the short-term absence of extrinsic modulatory inputs, in long-term decentralized ganglia, the same cells are able to oscillate spontaneously, even after experimental isolation in situ from all other elements in the pyloric network. In PD neurons this reacquisition of rhythmicity is associated with a net reduction in outward tetraethylammonium-sensitive ionic currents that include a delayed-rectifier type potassium current ( I Kd) and a calcium-dependent K+ current, I KCa. By contrast, long-term STG decentralization caused enhancement of a hyperpolarization-activated inward current that resembles I h. These results are consistent with the hypothesis that modulatory inputs sustain the modulation-dependent rhythmogenic character of the pyloric network by continuously regulating the balance of membrane conductances that underlie neuronal oscillation.

Subthreshold ionic currents, which activate below the firing threshold and shape the cell's firing properties, play important roles in shaping neural network activity. We examined the distribution and synaptic roles of the hyperpolarization-activated inward current ( Ih) in the pyloric network of the lobster stomatogastric ganglion (STG). Ih channels are expressed throughout the STG in a patchy distribution and are highly expressed in the fine neuropil, an area that is rich in synaptic contacts. We performed double labeling for Ih protein and for the presynaptic marker synaptotagmin. The large majority of labeling in the fine neuropil was adjacent but nonoverlapping, suggesting that Ih is localized in close proximity to synapses but not in the presynaptic terminals. We compared the pattern of Ih localization with Shal transient potassium channels, whose expression is coregulated with Ih in many STG neurons. Unlike Ih, we found significant levels of Shal protein in the soma membrane and the primary neurite. Both proteins were found in the synaptic fine neuropil, but with little evidence of colocalization in individual neurites. We performed electrophysiological experiments to study a potential role for Ih in regulating synaptic transmission. At a synapse between two identified pyloric neurons, the amplitude of inhibitory postsynaptic potentials (IPSPs) decreased with increasing postsynaptic activation of Ih. Pharmacological block of Ih restored IPSP amplitudes to levels seen when Ih was not activated. These experiments suggest that modulation of postsynaptic Ih might play an important role in the control of synaptic strength in this rhythmogenic neural network.

Many rhythmically active networks involve heterogeneous populations of pacemaker neurons with potentially distinct synaptic outputs that can be differentially targeted by extrinsic inputs or neuromodulators, thereby increasing possible network output patterns. To understand the roles of heterogeneous pacemaker neurons, we characterized differences in synaptic output from the anterior burster (AB) and pyloric dilator (PD) neurons in the lobster pyloric network. These intrinsically distinct neurons are strongly electrically coupled, coactive, and constitute the pyloric pacemaker ensemble. During pyloric oscillations, the pacemaker neurons produce compound inhibitory synaptic connections to the follower lateral pyloric (LP) and pyloric constrictor (PY) neurons, which fire out of phase with AB/PD and with different delay times. Using pharmacological blockers, we separated the synapses originating from the AB and PD neurons and investigated their temporal dynamics. These synapses exhibited distinct short-term dynamics, depending on the presynaptic neuron type, and had different relative contributions to the total synaptic output depending on waveform shape and cycle frequency. However, paired comparisons revealed that the amplitude or dynamics of synapses from either the AB or PD neuron did not depend on the postsynaptic neuron type, LP or PY. To address the functional implications of these findings, we examined the correlation between synaptic inputs from the pacemakers and the burst onset phase of the LP and PY neurons in the ongoing pyloric rhythm. These comparisons showed that the activity of the LP and PY neurons is influenced by the peak phase and amplitude of the synaptic inputs from the pacemaker neurons.

The phasing of neuronal activity in a rhythmic motor network is determined by a neuron's intrinsic firing properties and synaptic inputs; these could vary in their relative importance under different modulatory conditions. In the lobster pyloric network, the firing of eight follower pyloric (PY) neurons is shaped by their intrinsic rebound after pacemaker inhibition and by synaptic input from the lateral pyloric (LP) neuron, which inhibits all PY neurons and is electrically coupled to a subset of them. Under control conditions, LP inhibition is weak and has little influence on PY firing. We examined modulation that could theoretically enhance the LP's synaptic contribution to PY firing. We measured the effects of dopamine (DA) on LP→PY synapses, driving the LP neuron with trains of realistic waveforms constructed from prerecorded control and DA LP oscillations, which differed in shape and duration. Under control conditions, chemical inhibition underwent severe depression and disappeared; in the mixed synapses, electrical coupling dominated. Switching between control and DA LP waveforms (with or without DA present) caused only subtle changes in synaptic transmission. DA markedly enhanced synaptic inhibition, reduced synaptic depression and weakened electrical coupling, reversing the sign of the mixed synapses. Despite this, removal of the LP from the intact network still had only weak effects on PY firing. DA also enhances PY intrinsic rebound properties, which still control the onset of PY firing. Thus in a rhythmic network, the functional importance of synaptic modulation can only be understood in the context of parallel modulation of intrinsic properties.

Short-term dynamics such as facilitation and depression are present in most synapses and are often target-specific even for synapses from the same type of neuron. We examine the dynamics and possible functions of two synapses from the same presynaptic neuron in the rhythmically active pyloric network of the spiny lobster. Using simultaneous recordings, we show that the synapses from the lateral pyloric (LP) neuron to the pyloric dilator (PD; a member of the pyloric pacemaker ensemble) and the pyloric constrictor (PY) neurons both show short-term depression. However, the postsynaptic potentials produced by the LP-to-PD synapse are larger in amplitude, depress less, and recover faster than those produced by the LP-to-PY synapse. The main function of the LP-to-PD synapse is to slow down the pyloric rhythm. However, in some cases, it slows down the rhythm only when it is fast and has no effect or to speeds up when it is slow. In contrast, the LP-to-PY synapse functions to delay the activity of the PY neuron; this delay increases as the cycle period becomes longer. Using a computational model, we show that the short-term dynamics of synaptic depression observed for each of these synapses are tailored to their individual functions and that replacing the dynamics of either synapse with the other would disrupt these functions. Together, the experimental and modeling results suggest that the target-specific features of short-term synaptic depression are functionally important for synapses efferent from the same presynaptic neuron.

Irregular intrinsic behavior of neurons seems ubiquitous in the nervous system. Even in circuits specialized to provide periodic and reliable patterns to control the repetitive activity of muscles, such as the pyloric central pattern generator (CPG) of the crustacean stomatogastric ganglion (STG), many bursting motor neurons present irregular activity when deprived from synaptic inputs. Moreover, many authors attribute to these irregularities the role of providing flexibility and adaptation capabilities to oscillatory neural networks such as CPGs. These irregular behaviors, related to nonlinear and chaotic properties of the cells, pose serious challenges to developing deterministic Hodgkin-Huxley-type (HH-type) conductance models. Only a few deterministic HH-type models based on experimental conductance values were able to show such nonlinear properties, but most of these models are based on slow oscillatory dynamics of the cytosolic calcium concentration that were never found experimentally in STG neurons. Based on an up-to-date single-compartment deterministic HH-type model of a STG neuron, we developed a stochastic HH-type model based on the microscopic Markovian states that an ion channel can achieve. We used tools from nonlinear analysis to show that the stochastic model is able to express the same kind of irregularities, sensitivity to initial conditions, and low dimensional dynamics found in the neurons isolated from the STG. Without including any nonrealistic dynamics in our whole cell stochastic model, we show that the nontrivial dynamics of the membrane potential naturally emerge from the interplay between the microscopic probabilistic character of the ion channels and the nonlinear interactions among these elements. Moreover, the experimental irregular behavior is reproduced by the stochastic model for the same parameters for which the membrane potential of the original deterministic model exhibits periodic oscillations.

Odors activate lobster olfactory receptor neurons (ORNs) through phosphoinositide signaling that appears to target a Na+-gated nonselective cation channel. The Na+-gated channel is a potential member of the growing family of transient receptor potential (TRP) channels. Here, we test the effect of potential antagonists on the channel in cell-free patches from cultured lobster ORNs. We show that the channel is antagonized by H+ and the TRP channel blockers 2-aminoethoxydiphenyl borate, SKF96365 , ruthenium red, Al3+, Gd3+, and La3+. We then use this enhanced antagonist profile together with the agonists Na+ and Ca2+ to implicate the channel in signal amplification in the cells.

The lobster pyloric network has a densely interconnected synaptic connectivity pattern, and the role individual synapses play in generating network activity is consequently difficult to discern. We examined this issue by quantifying the effect on pyloric network phasing and spiking activity of removing the Lateral Pyloric (LP) and Ventricular Dilator (VD) neurons, which synapse onto almost all pyloric neurons. A confounding factor in this work is that LP and VD neuron removal alters pyloric cycle period. To determine the effects of LP and VD neuron removal on pyloric activity independent of these period alterations, we altered network period by current injection into a pyloric pacemaker neuron, hyperpolarized the LP or VD neuron to functionally remove each from the network, and plotted various measures of pyloric neuron activity against period with and without the LP or VD neuron. In normal physiological saline, in many (or most) cases removing either neuron had surprisingly little effect on the activity of its postsynaptic partners, which suggests that under these conditions these neurons play a relatively small role in determining pyloric activity. In the cases in which removal did alter postsynaptic activity, the effects were inconsistent across preparations, which suggests that either despite producing very similar neural outputs, pyloric networks from different animals have different cellular and synaptic properties, or some synapses contribute to network activity only under certain modulatory conditions, and the “baseline” level of modulatory influence the network receives from higher centers varies from animal to animal.

The pyloric network of the lobster stomatogastric nervous system is one of the best described assemblies of oscillatory neurons producing bursts of action potentials. While the temporal patterns of bursts have been investigated in detail, those of spikes have received less attention. Here we analyze the intraburst firing patterns of pyloric neurons and the synaptic interactions shaping their dynamics in millisecond time scales not performed before. We find that different pyloric neurons express characteristic, cell-specific firing patterns in their bursts. Nonlinear analysis of the interspike intervals (ISIs) reveals distinctive temporal structures (‘interspike interval signatures’), which are found to depend on the synaptic connectivity of the network. We compare ISI patterns of the pyloric dilator (PD), lateral pyloric (LP), and ventricular dilator (VD) neurons in 1) normal conditions, 2) after blocking glutamatergic synaptic connections, and 3) in various functional configurations of the three neurons. Manipulation of the synaptic connectivity results in characteristic changes in the ISI signatures of the postsynaptic neurons. The intraburst firing pattern of the PD neuron is regularized by the inhibitory synaptic connection from the LP neuron as revealed in current-clamp experiments and also as reconstructed with a dynamic clamp. On the other hand, mutual inhibition between the LP and VD neurons tend to produce more irregular bursts with increased spike jitter. The results show that synaptic interactions fine-tune the output of pyloric neurons. The present data also suggest a way of processing of synaptic information: bursting neurons are capable of encoding incoming signals by altering the fine structure of their intraburst spike patterns.

The transient potassium current ( I A) plays an important role in shaping the firing properties of pyloric neurons in the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus. The shal gene encodes I A in pyloric neurons. However, when we over-expressed the lobster Shal protein by shal RNA injection into the pyloric dilator (PD) neuron, the increased I A had somewhat different properties from the endogenous I A. The recently cloned K-channel interacting proteins (KChIPs) can modify vertebrate Kv4 channels in cloned cell lines. When we co-expressed hKChIP1 with lobster shal in Xenopusoocytes or lobster PD neurons, they produced A-currents resembling the endogenous I A in PD neurons; compared with currents evoked by shal alone, their voltage for half inactivation was depolarized, their kinetics of inactivation were slowed, and their recovery from inactivation was accelerated. We also co-expressed shal in PD neurons with lobster frequenin, which encodes a protein belonging to the same EF-hand family of Ca2+ sensing proteins as hKChIP. Frequenin also restored most of properties of the shal-evoked currents to those of the endogenous A-currents, but the time course of recovery from inactivation was not corrected. These results suggest that lobster shal proteins normally interact with proteins in the KChIP/frequenin family to produce the transient potassium current in pyloric neurons.