Indirectly gated Cl(-)-dependent Cl(-) channels sense physiological changes of extracellular chloride in the leech.

The maintenance of ion homeostasis requires adequate ion sensors. In leeches, 34 nephridial nerve cells (NNCs) monitor the Cl(-) concentration of the blood. After a blood meal, the Cl(-) concentration of leech blood triples and is gradually restored to its normal value within 48 h after feeding. As previously shown in voltage-clamp experiments, the Cl(-) sensitivity of the NNCs relies on a persistent depolarizing Cl(-) current that is turned off by an increase of the extracellular Cl(-) concentration. The activation of this Cl(-)-dependent Cl(-) current is independent of voltage and of extra- and intracellular Ca(2+). The transduction mechanism is now characterized on the single-channel level. The NNC's sensitivity to Cl(-) is mediated by a slowly gating Cl(-)-dependent Cl(-) channel with a mean conductance of 50 pS in the cell-attached configuration. Gating of the Cl(-) channel is independent of voltage, and channel activity is independent of extra- and intracellular Ca(2+). Channel activity and the macroscopic current are reversibly blocked by bumetanide. In outside-out patches, changes of the extracellular Cl(-) concentration do not affect channel activity, indicating that channel gating is not via direct interaction of extracellular Cl(-) with the channel. As shown by recordings in the cell-attached configuration, the activity of the channels under the patch is instead governed by the Cl(-) concentration sensed by the rest of the cell. We postulate a membrane-bound Cl(-)-sensing receptor, which-on the increase of the extracellular Cl(-) concentration-closes the Cl(-) channel via a yet unidentified signaling pathway.

A model of a segmental oscillator in the leech heartbeat neuronal network.

We modeled a segmental oscillator of the timing network that paces the heartbeat of the leech. This model represents a network of six heart interneurons that comprise the basic rhythm-generating network within a single ganglion. This model builds on a previous two cell model (Nadim et al., 1995) by incorporating modifications of intrinsic and synaptic currents based on the results of a realistic waveform voltage-clamp study (Olsen and Calabrese, 1996). Due to these modifications, the new model behaves more similarly to the biological system than the previous model. For example, the slow-wave oscillation of membrane potential that underlies bursting is similar in form and amplitude to that of the biological system. Furthermore, the new model with its expanded architecture demonstrates how coordinating interneurons contribute to the oscillations within a single ganglion, in addition to their role of intersegmental coordination.

Modeling alternation to synchrony with inhibitory coupling: a neuromorphic VLSI approach.

We developed an analog very large-scale integrated system of two mutually inhibitory silicon neurons that display several different stable oscillations. For example, oscillations can be synchronous with weak inhibitory coupling and alternating with relatively strong inhibitory coupling. All oscillations observed experimentally were predicted by bifurcation analysis of a corresponding mathematical model. The synchronous oscillations do not require special synaptic properties and are apparently robust enough to survive the variability and constraints inherent in this physical system. In biological experiments with oscillatory neuronal networks, blockade of inhibitory synaptic coupling can sometimes lead to synchronous oscillations. An example of this phenomenon is the transition from alternating to synchronous bursting in the swimming central pattern generator of lamprey when synaptic inhibition is blocked by strychnine. Our results suggest a simple explanation for the observed oscillatory transitions in the lamprey central pattern generator network: that inhibitory connectivity alone is sufficient to produce the observed transition.

Intracellular Ca2+ dynamics during spontaneous and evoked activity of leech heart interneurons: low-threshold Ca currents and graded synaptic transmission.

In oscillatory neuronal networks that pace rhythmic behavior, Ca(2+) entry through voltage-gated Ca channels often supports bursting activity and mediates graded transmitter release. We monitored simultaneously membrane potential and/or ionic currents and changes of Ca fluorescence (using the fluorescence indicator Ca Orange) in spontaneously active and experimentally manipulated oscillator heart interneurons in the leech. We show that changes in Ca fluorescence in these interneurons during spontaneous bursting and evoked activity reflect the slow wave of that activity and that these changes in Ca fluorescence are mediated by Ca(2+) entry primarily through low-threshold Ca channels. Spatial and temporal maps of changes in Ca fluorescence indicate that these channels are widely distributed over the neuritic tree of these neurons. We establish a correlation between the amount of transmitter released, as estimated by the integral of the postsynaptic current, and the change in Ca fluorescence. In experiments in which we were able to record presynaptic low-threshold Ca currents, associated IPSCs, and presynaptic changes in Ca fluorescence from fine neuritic branches of heart interneurons near their region of synaptic contact with their contralateral partner, there was a close association between the rise in Ca fluorescence and the rise of the postsynaptic conductance. The changes in Ca fluorescence that we record at the end of fine neuritic branches appear to reflect changes in [Ca(2+)](i) that mediate graded synaptic release in leech heart interneurons. These results indicate that widely distributed low-threshold Ca currents play an important role in generating rhythmic activity and in mediating graded transmitter release.

Taking the lead from a model.

It is rare these days that theory leads experiment in the biological sciences, but it still happens. A recent study has experimentally confirmed the predictions of a model aimed at explaining how neural networks interact to produce the coordinated patterns of motor activity necessary for effective behavior.

Motor pattern switching in the heartbeat pattern generator of the medicinal leech: membrane properties and lack of synaptic interaction in switch interneurons.

The motor program for heartbeat in the medicinal leech is produced by a central pattern generator that regularly switches between two alternative coordination states. A pair of switch heart interneurons reciprocally alternate between rhythmically active and inactive states to effect these switches. During spontaneous switches in the activity state of switch interneurons, there was no correlation between the duration of a particular activity state and beat period, indicating that the timing networks for the switch cycle and the beat cycle are relatively independent. Simultaneous recordings from two switch heart interneurons showed that a perturbation in the electrical activity of one does not influence switching of the other and that there is no synaptic interaction between them. Using voltage clamp, we characterized an L-like Ca2+ current (measured as Ba2+ currents), inactivating and non-inactivating K+ currents, a persistent Na+ current, and a hyperpolarization-activated inward current in switch interneurons. Dynamic clamp experiments show that "subtraction" of an artificial switch leak conductance (described previously by Gramoll et al. 1994) from a switch interneuron when it is in the inactive state causes it to display activity associated with the active state. We discuss how the switch leak conductance may interact with the intrinsic currents of switch interneurons to control their activity state.

Cellular, synaptic, network, and modulatory mechanisms involved in rhythm generation.

The membrane properties and the synaptic interactions of individual neurons, as well as the interactions between neuronal networks, all contribute to the formation of the complex patterns of activity that underlie rhythmic motor patterns and slow-wave sleep rhythms. These properties and interactions are potential points of modulation for further refining network output. Recent work illustrates the range of these properties and interactions and suggests how they may be modulated.

A slow outward current activated by FMRFamide in heart interneurons of the medicinal leech.

The endogenous neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2) can accelerate the oscillation of reciprocally inhibitory pairs of interneurons that pace heartbeat in the medicinal leech. A model based on all available biophysical data of a two-cell heart interneuron oscillator provides a theoretical basis for understanding this modulation. Previously observed modulation of K+ currents by FMRFamide cannot account for this acceleratory effect in the model. This observation prompted the present reexamination of K+ currents in heart interneurons. We devised better methods for separation of the various components of K+ current and more accurately measured their activation and deactivation kinetics. Moreover, we demonstrated that FMRFamide activates a previously undetected K+ current (IKF), which has very slow activation and deactivation kinetics. Addition of physiologically measured amounts of IKF to the model two-cell oscillator can account for the acceleratory effect of FMRFamide.

Functional role of Ca2+ currents in graded and spike-mediated synaptic transmission between leech heart interneurons.

We used intracellular recording and single electrode voltage-clamp techniques to explore Ca2+ currents and their relation to graded and spike-mediated synaptic transmissions in leech heart interneurons. Low-threshold Ca2+ currents (activation begins below -50 mV) consist of a rapidly inactivating component (I(CaF)) and a slowly inactivating component (I(CaS)). The apparent inactivation kinetics of I(CaF) appears to be influenced by Ca2+; both the substitution of Ca2+ (5 mM) with Ba2+ (5 mM) in the saline and the intracellular injection of the rapid Ca2+ chelator, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), from the recording microelectrode, significantly increase its apparent inactivation time constant. The use of saline with a high concentration of Ba2+ (37.5 mM) permitted exploration of divalent ion currents over a broader activation range, by acting as an effective charge carrier and significantly blocking outward currents. Ramp and pulse voltage-clamp protocols both reveal a rapidly activating and inactivating Ba2+ current (I(BaF)) and a less rapidly activating and slowly inactivating Ba2+ current with a broad activation range (I(BaS)). Low concentrations of Cd2+ (100-150 microM) selectively block I(BaS), without significantly diminishing I(BaF). The current that remains in Cd2+ lacks the characteristic delayed activation peak of I(BaS) and inactivates with two distinct time constants. I(BaF) appears to correspond to a combination of I(CaF) and I(CaS), i.e., to low-threshold Ca2+ currents, that can be described as T-like. I(BaS) appears to correspond to a Ca2+ current with a broad activation range, which can be described as L-like. Cd2+ (100 microM) selectively blocks spike-mediated synaptic transmission between heart interneurons without significantly interfering with low-threshold Ca2+ currents and plateau formation in or graded synaptic transmission between heart interneurons. Blockade of spike-mediated synaptic transmission between reciprocally inhibitory heart interneurons with Cd2+ (150 microM), in otherwise normal saline, prevents the expression of normal oscillations (during which activity in the two neurons consists of alternating bursts), so that the neurons fire tonically. We conclude that graded and spike-mediated synaptic transmission may be relatively independent processes in heart interneurons that are controlled predominantly by different Ca2+ currents. Moreover, spike-mediated synaptic inhibition appears to be required for normal oscillation in these neurons.

Activation of intrinsic and synaptic currents in leech heart interneurons by realistic waveforms.

Leech heart interneurons were voltage-clamped with realistic waveforms to investigate the currents underlying the oscillation in the cells. By estimating the leak current parameters in regions in which there was little contamination by voltage-gated currents, it was possible to measure the Ca2+ current, the persistent Na+ current, Ip, and the hyperpolarization-activated inward current, Ih. The experiments verified a prediction of a computer model of HN cells that the shape of the typical waveform was such that the low-threshold Ca2+ currents were partially inactivated during a slow up-ramp to a plateau potential. A step within the same range of the membrane potential as the realistic waveform produced > 4 times as much Ca2+ current. In two-cell voltage-clamp experiments, the step produced 20 times more graded inhibition than the normal presynaptic waveform. When the presynaptic heart interneuron oscillated with spikes, the graded inhibition was larger. The difference may arise from integration of a slowly decaying component of the spike-mediated inhibition. The persistent Na+ current had a very low threshold. During the most hyperpolarized phase of the waveform, Ip deactivated to 50% of its maximum conductance. A substantial part of Ip, therefore, was effectively contributing to the leak current in the HN cells. The h-current increased for waveforms that had longer periods, whereas increasing the h-current in the model reduced the period. The h-current thus provides negative feedback to perturbations that alter the period of the oscillation.

Principles of rhythmic motor pattern generation.

Rhythmic movements are produced by central pattern-generating networks whose output is shaped by sensory and neuromodulatory inputs to allow the animal to adapt its movements to changing needs. This review discusses cellular, circuit, and computational analyses of the mechanisms underlying the generation of rhythmic movements in both invertebrate and vertebrate nervous systems. Attention is paid to exploring the mechanisms by which synaptic and cellular processes interact to play specific roles in shaping motor patterns and, consequently, movement.

Oscillation in motor pattern-generating networks.

Oscillation in motor pattern generators is driven either by pacemaker neurons with inherent bursting properties or through network interactions. In a few examples in invertebrates and lower vertebrates, the mechanisms by which reciprocal inhibition combines with inherent membrane properties to produce network oscillation are beginning to emerge. A recently developed theoretical framework provides a context for understanding and comparing these findings.

Outward currents in heart motor neurons of the medicinal leech.

1. Outward currents were studied in isolation in heart motor neurons in the medicinal leech, using the single-electrode voltage-clamp technique. The currents were divided into four distinct types on the basis of their time and voltage characteristics and sensitivity to external Ca2+ concentration. 2. The four types were a fast transient current, IKA; a slow transient current. IK1; a noninactivating current, IK2, all measured in a bathing solution in which Co2+ was substituted for Ca2+; and a calcium-sensitive current. IK1Cal which was revealed in a bathing solution containing normal levels of Ca2+. 3. The outward currents in heart motor neurons studied in different ganglia possessed differences of quality. For example, heart motor neurons from ganglia 3 or 4 had significantly less IK2 and IK1 than neurons recorded from more posterior ganglia. Heart motor neurons from ganglion 3 often had little or no IK1. Soma input resistance, electrotonic length, and soma capacitance measured in heart motor neurons from both anterior and posterior ganglia exhibited no significant differences. 4. IKA started to activate near -45 mV with half-maximal activation at about -20 mV and was fully inactivated by 0 mV: IK1 started to activate near -45 mV with half-maximal activation at about -10 mV and was not fully inactivated by 0 mV; IK2 started to activate near -50 mV; IK1Cal started to activate near -35 mV. The time constant of removal of inactivation for IKA was 25 ms, measured at -80 mV, and that for IK1 was 380 ms, measured at -40 mV. 5. Tetraethylammonium acetate (TEA) allowed to diffuse from the inside of the recording microelectrode effectively blocked IKA, IK1, and IK2. Bath-applied TEA (25 mM) acted similarly but was less effective, particularly at blocking Ik2. Bath-applied 4-aminopyridine effectively blocked the transient currents IKA and IK1. A reversal potential of -65 mV was found for the outward currents, corresponding to a mix of IK1 and IK2.

Segment-specific effects of FMRFamide on membrane properties of heart interneurons in the leech.

1. The effects of Phe-Met-Arg-Phe (FMRF)amide (10(-6) M) on membrane properties of heart interneurons in the third, fourth, and fifth segmental ganglia [HN(3), HN(4), and HN(5) cells, respectively] of the leech were studied using discontinuous current-clamp and single-electrode voltage-clamp techniques. FMRFamide was focally applied onto the soma of the cell under investigation. 2. Application of FMRFamide depolarized HN(3) and HN(4) cells by evoking an inward current. These responses were subject to pronounced desensitization. The inward currents evoked by application of FMRFamide were associated with an increase in membrane conductance and appeared to be voltage dependent. Currents were enhanced at more depolarized potentials. 3. The responsiveness of the HN(3) and HN(4) cells was not affected when the Ca2+ concentration in the bath saline was reduced from normal (1.8 mM) to 0.1 mM. The depolarizing response on application of FMRFamide was blocked when Co2+ was substituted for Ca2+. 4. HN(3) and HN(4) cells did not respond to FMRFamide application in Na(+)-free solution. Inward currents were largely reduced when bath saline with 30% of the normal Na+ concentration was used. When Li+ was substituted for Na+ in the saline, application of FMRFamide still evoked depolarizing responses in HN(3) and HN(4) cells. 5. We conclude that focal application of FMRFamide onto the somata of HN(3) and HN(4) cells evokes a voltage-dependent inward current, carried largely by Na+. 6. Focal application of FMRFamide onto somata of HN(5) cells hyperpolarized these cells by activating a voltage-dependent outward current. 7. HN(5) cells were loaded with Cl- until inhibitory postsynaptic potentials carried by Cl- reversed. Cl(-)-loaded cells still responded with a hyperpolarization when FMRFamide was applied onto their somata. Therefore the outward current evoked by FMRFamide appears to be mediated by a K+ conductance increase. 8. Application of FMRFamide onto the somata of HN(5) cells enhanced outward currents that were evoked by depolarizing voltage steps from a holding potential of -45 mV. 9. We conclude that the hyperpolarizing response of HN(5) cells to focal application of FMRFamide onto their somata is the result of an up-regulation of a voltage-dependent K+ current.

Modeling the leech heartbeat elemental oscillator. I. Interactions of intrinsic and synaptic currents.

We have developed a biophysical model of a pair of reciprocally inhibitory interneurons comprising an elemental heartbeat oscillator of the leech. We incorporate various intrinsic and synaptic ionic currents based on voltage-clamp data. Synaptic transmission between the interneurons consists of both a graded and a spike-mediated component. By using maximal conductances as parameters, we have constructed a canonical model whose activity appears close to the real neurons. Oscillations in the model arise from interactions between synaptic and intrinsic currents. The inhibitory synaptic currents hyperpolarize the cell, resulting in activation of a hyperpolarization-activated inward current Ih and the removal of inactivation from regenerative inward currents. These inward currents depolarize the cell to produce spiking and inhibit the opposite cell. Spike-mediated IPSPs in the inhibited neuron cause inactivation of low-threshold Ca++ currents that are responsible for generating the graded synaptic inhibition in the opposite cell. Thus, although the model cells can potentially generate large graded IPSPs, synaptic inhibition during canonical oscillations is dominated by the spike-mediated component.

Modeling the leech heartbeat elemental oscillator. II. Exploring the parameter space.

In the previous paper, we described a model of the elemental heartbeat oscillator in the leech. Here, the parameters of our model are explored around the baseline canonical model. The maximal conductances of the currents and the reversal potential of the leak current are varied to reveal the effects of individual currents and the interaction between synaptic and intrinsic currents in the model. The model produces two distinct modes of oscillation as the parameters are varied, S-mode and G-mode. These two modes are defined, their origin is identified, and the parameter space is mapped into S-mode and G-mode oscillation and no oscillation. Finally, we will make predictions for how the period can be modulated in heart interneurons.

Heartbeat control in the medicinal leech: a model system for understanding the origin, coordination, and modulation of rhythmic motor patterns.

We have analyzed in detail the neuronal network that generates heartbeat in the leech. Reciprocally inhibitory pairs of heart interneurons form oscillators that pace the heartbeat rhythm. Other heart interneurons coordinate these oscillators. These coordinating interneurons, along with the oscillators interneurons, form an eight-cell timing oscillator network for heartbeat. Still other interneurons, along with the oscillator interneurons, inhibit heart motor neurons, sculpting their activity into rhythmic bursts. Critical switch interneurons interface between the oscillator interneurons and the other premotor interneurons to produce two alternating coordination states of the motor neurons. The periods of the oscillator interneurons are modulated by endogenous RFamide neuropeptides. We have explored the ionic currents and graded and spike-mediated synaptic transmission that promote oscillation in the oscillator interneurons and have incorporated these data into a conductance-based computer model. This model has been of considerable predictive value and has led to new insights into how reciprocally inhibitory neurons produce oscillation. We are now in a strong position to expand this model upward, to encompass the entire heartbeat network, horizontally, to elucidate the mechanisms of FMRFamide modulation, and downward, to incorporate cellular morphology. By studying the mechanisms of motor pattern formation in the leech, using modeling studies in conjunction with parallel physiological experiments, we can contribute to a deeper understanding of how rhythmic motor acts are generated, coordinated, modulated, and reconfigured at the level of networks, cells, ionic currents, and synapses.

An endogenous peptide modulates the activity of a sensory neurone in the leech Hirudo medicinalis.

Sensory and neurosecretory innervation of each leech excretory complex, a nephridium and its bladder, is accomplished by a single neurone, the nephridial nerve cell (NNC). The NNC monitors the extracellular Cl- concentration, which ranges between 20 and 100 mmol l-1 depending on the physiological state. The NNC contains FMRFamide in its soma and sensory terminals in the nephridium. Bath or focal application of FMRFamide leads to hyperpolarization and decreases the rate of firing of the NNC, suggesting autoregulation of peptide release. Experiments under single-electrode current-clamp and voltage-clamp show that FMRFamide turns off the receptor-specific Cl- current of the NNC, indicating that FMRFamide also modulates the receptor gain.

A persistent sodium current contributes to oscillatory activity in heart interneurons of the medicinal leech.

1. Normal activity in bilateral pairs of heart interneurons, from ganglia 3 or 4, in the medicinal leech (Hirudo medicinalis) is antiphasic due to their reciprocally inhibitory connections. However, Ca(++)-free Co(++)-containing salines lead to synchronous oscillations in these neurons. 2. Internal TEA+ allows expression of full plateaus during Co++ induced oscillations in heart interneurons; these plateaus are not blocked by Cs+. Similar plateaus are also observed with internal TEA+ alone, but under these conditions activity in heart interneurons from ganglia 3 or 4 is antiphasic. 3. Plateaus in heart interneurons induced by Co++ and internal TEA+ involve a conductance increase. 4. A voltage-dependent inward current, IP, showing little inactivation, was isolated using single-electrode voltage-clamp in heart interneurons. This current is carried at least in part by Na+; the current is reduced when external Na+ is reduced and is carried by Li++ when substituted for Na+. 5. Calcium channel blockers such as La3+ and Co++ block neither the TEA+ induced plateaus nor IP, suggesting that Na+ is not using Ca++ channels. Moreover, IP is enhanced by Ca(++)-free CO(++)-containing salines. Thus, IP is correlated with the TEA(+)- and Co(++)-induced plateau behavior.

Modulation of high-threshold transmission between heart interneurons of the medicinal leech by FMRF-NH2.

1. We examined high-threshold synaptic transmission between oscillatory pairs of leech heart interneurons. Inhibitory postsynaptic currents (IPSCs) could be reliably evoked by depolarizing the presynaptic neuron in voltage clamp from a holding potential of -35 mV. At this presynaptic potential, the Ca2+ currents underlying graded transmission are completely inactivated, and we conclude that a high-threshold Ca2+ current is extant in heart interneurons. Further evidence for this was that inhibitory postsynaptic currents were blocked when Co2+ replaced Ca2+ in the saline and thus high-threshold transmission was dependent on the presence of external Ca2+. 2. When IPSCs were evoked by a 200-ms duration voltage step from a holding potential of -35 mV in the presynaptic neuron, the time course of turn-on of the IPSC consisted of a fast (time-to-peak = 17.5 +/- 1.93 (SE) ms [n = 7]) and a slow (time-to-peak = 250 +/- 28.5 ms [n = 8]) component. FMRF-NH2 reduced the amplitude of the fast component but did not affect the slow component. When the presynaptic voltage step was ended the IPSC turned off with a single exponential time course. FMRF-NH2 slowed the time course of turn-off of the IPSC. 3. When IPSCs were evoked by a 1500-ms duration voltage step from a holding potential of -35 mV in the presynaptic neuron, these IPSCs peaked around 300 ms. Following the peak, the IPSC decayed with a single exponential time course. FMRF-NH2 accelerated the time course of this decay. At potentials of 0 mV and +5 mV, FMRF-NH2 produced a significant decrease in the peak current and at potentials of -5 mV and 0 mV, produced a significant decrease in the current integral. 4. High-threshold IPSCs could also be evoked by a spike in the presynaptic neuron. Bath application of 1 microM FMRF-NH2 decreased the amplitude of the spike-evoked IPSC and slowed the time course of its falling phase. 5. We examined the effect of FMRF-NH2 on the quantal synaptic transmission. Bath-application of FMRF-NH2 increased binomial p, the probability of release, and decreased binomial n, the number of units available for release. FMRF-NH2 had no effect on q, the unit size, when calculated from the distributions of PSPs, and increased the coefficient of variation (CV). 6. The lack of a change in q and the increase in CV suggested that FMRF-NH2 acted at a presynaptic location.(ABSTRACT TRUNCATED AT 400 WORDS)