Ronald L Calabrese

Animal-to-animal variability of connection strength in the leech heartbeat central pattern generator

The heartbeat central pattern generator (CPG) in medicinal leeches controls blood flow within a closed circulatory by programming the constrictions of two parallel heart tubes. This circuit reliably produces a stereotyped fictive pattern of activity and has been extensively characterized. Here we determined, as quantitatively as possible, the strength of each inhibitory synapse and electrical junction within the core circuit of the heartbeat CPG. We also examined the animal-to-animal variability in strengths of these connections and, for some, determined the correlations between connections to the same postsynaptic target. The core CPG is composed of seven bilateral pairs of heart interneurons connected via both inhibitory chemical synapses and electrical junctions. Fifteen different connections within the core CPG were measured for strength using extracellular presynaptic recordings and postsynaptic voltage-clamp recordings across a minimum of seven individuals each, and the animal-to-animal variability was characterized. Connection strengths within the core network varied three to more than sevenfold among individuals (depending on the specific connection). The balance between two inputs onto various postsynaptic targets was explored by within-individual comparisons and correlation across individuals. Of the seven comparisons made within the core CPG, three showed a clear correlation of connection strengths, while the other four did not. We conclude that the leech heartbeat CPG can withstand wide variability in connection strengths and still produce stereotyped output. The network appears to preserve the relative strengths of some pairs of inputs, despite the animal-to-animal variability.

Constancy and Variability in the Output of a Central Pattern Generator

Experimental and corresponding modeling studies have demonstrated a twofold to fivefold variation of intrinsic and synaptic parameters across animals, whereas functional output is maintained. These studies have led to the hypothesis that correlated, compensatory changes in particular parameters can at least partially explain the biological variability in parameters. Using the leech heartbeat central pattern generator (CPG), we selected three different segmental motor neurons that fire in a functional phase progression but receive input from the same four premotor interneurons. Previous work suggested that the phase progression arises because the pattern of relative strength of the four inputs varies systematically across the segmental motor neurons. Nevertheless, there was considerable animal-to-animal variation in the absolute strengths of these connections. We tested the hypothesis that functional output is maintained in the face of variation in the absolute strength of connections because relative strengths onto particular motor neurons are maintained. We found that relative strength is not strictly maintained across animals even as functional output is maintained, and animal-to-animal variations in relative strength of particular inputs do not correlate strongly with output phase. In parallel with this variation in synaptic strength, the firing phase of the premotor inputs to these motor neurons varies considerably across individuals. We conclude that the number (four) of inputs to each motor neuron, which each vary in strength, and the phase diversity of the temporal pattern of input from the CPG diminish the influence of individual inputs. We hypothesize that each animal arrives at a unique solution for how the network produces functional output.

Coping with Variability in Small Neuronal Networks

Experimental and corresponding modeling studies indicate that there is a 2- to 5-fold variation of intrinsic and synaptic parameters across animals while functional output is maintained. Here, we review experiments, using the heartbeat central pattern generator (CPG) in medicinal leeches, which explore the consequences of animal-to-animal variation in synaptic strength for coordinated motor output. We focus on a set of segmental heart motor neurons that all receive inhibitory synaptic input from the same four premotor interneurons. These four premotor inputs fire in a phase progression and the motor neurons also fire in a phase progression because of differences in synaptic strength profiles of the four inputs among segments. Our work tested the hypothesis that functional output is maintained in the face of animal-to-animal variation in the absolute strength of connections because relative strengths of the four inputs onto particular motor neurons is maintained across animals. Our experiments showed that relative strength is not strictly maintained across animals even as functional output is maintained, and animal-to-animal variations in strength of particular inputs do not correlate strongly with output phase. Further experiments measured the precise temporal pattern of the premotor inputs, the segmental synaptic strength profiles of their connections onto motor neurons, and the temporal pattern (phase progression) of those motor neurons all in the same animal for a series of 12 animals. The analysis of input and output in this sample of 12 individuals suggests that the number (four) of inputs to each motor neuron and the variability of the temporal pattern of input from the CPG across individuals weaken the influence of the strength of individual inputs. Moreover, the temporal pattern of the output varies as much across individuals as that of the input. Essentially, each animal arrives at a unique solution for how the network produces functional output.

Contribution of motoneuron intrinsic properties to fictive motor pattern generation

Previously, we reported a canonical ensemble model of the heart motoneurons that underlie heartbeat in the medicinal leech. The model motoneurons contained a minimal set of electrical intrinsic properties and received a synaptic input pattern based on measurements performed in the living system. Although the model captured the synchronous and peristaltic motor patterns observed in the living system, it did not match quantitatively the motor output observed. Because the model motoneurons had minimal intrinsic electrical properties, the mismatch between model and living system suggests a role for additional intrinsic properties in generating the motor pattern. We used the dynamic clamp to test this hypothesis. We introduced the same segmental input pattern used in the model to motoneurons isolated pharmacologically from their endogenous input in the living system. We show that, although the segmental input pattern determines the segmental phasing differences observed in motoneurons, the intrinsic properties of the motoneurons play an important role in determining their phasing, particularly when receiving the synchronous input pattern. We then used trapezoidal input waveforms to show that the intrinsic properties present in the living system promote phase advances compared with our model motoneurons. Electrical coupling between heart motoneurons also plays a role in shaping motoneuron output by synchronizing the activity of the motoneurons within a segment. These experiments provide a direct assessment of how motoneuron intrinsic properties interact with their premotor pattern of synaptic drive to produce rhythmic output.

Using a Model to Assess the Role of the Spatiotemporal Pattern of Inhibitory Input and Intrasegmental Electrical Coupling in the Intersegmental and Side-to-Side Coordination of Motor Neurons by the Leech Heartbeat Central Pattern Generator

Previously we presented a quantitative description of the spatiotemporal pattern of inhibitory synaptic input from the heartbeat central pattern generator (CPG) to segmental motor neurons that drive heartbeat in the medicinal leech and the resultant coordination of CPG interneurons and motor neurons. To begin elucidating the mechanisms of coordination, we explore intersegmental and side-to-side coordination in an ensemble model of all heart motor neurons and their known synaptic inputs and electrical coupling. Model motor neuron intrinsic properties were kept simple, enabling us to determine the extent to which input and electrical coupling acting together can account for observed coordination in the living system in the absence of a substantive contribution from the motor neurons themselves. The living system produces an asymmetric motor pattern: motor neurons on one side fire nearly in synchrony (synchronous), whereas on the other they fire in a rear-to-front progression (peristaltic). The model reproduces the general trends of intersegmental and side-to-side phase relations among motor neurons, but the match with the living system is not quantitatively accurate. Thus realistic (experimentally determined) inputs do not produce similarly realistic output in our model, suggesting that motor neuron intrinsic properties may contribute to their coordination. By varying parameters that determine electrical coupling, conduction delays, intraburst synaptic plasticity, and motor neuron excitability, we show that the most important determinant of intersegmental and side-to-side phase relations in the model was the spatiotemporal pattern of synaptic inputs, although phasing was influenced significantly by electrical coupling.

Neural circuits controlling behavior and autonomic functions in medicinal leeches.

In the study of the neural circuits underlying behavior and autonomic functions, the stereotyped and accessible nervous system of medicinal leeches, Hirudo sp., has been particularly informative. These leeches express well-defined behaviors and autonomic movements which are amenable to investigation at the circuit and neuronal levels. In this review, we discuss some of the best understood of these movements and the circuits which underlie them, focusing on swimming, crawling and heartbeat. We also discuss the rudiments of decision-making: the selection between generally mutually exclusive behaviors at the neuronal level.

Identifying Crucial Parameter Correlations Maintaining Bursting Activity

Recent experimental and computational studies suggest that linearly correlated sets of parameters (intrinsic and synaptic properties of neurons) allow central pattern-generating networks to produce and maintain their rhythmic activity regardless of changing internal and external conditions. To determine the role of correlated conductances in the robust maintenance of functional bursting activity, we used our existing database of half-center oscillator (HCO) model instances of the leech heartbeat CPG. From the database, we identified functional activity groups of burster (isolated neuron) and half-center oscillator model instances and realistic subgroups of each that showed burst characteristics (principally period and spike frequency) similar to the animal. To find linear correlations among the conductance parameters maintaining functional leech bursting activity, we applied Principal Component Analysis (PCA) to each of these four groups. PCA identified a set of three maximal conductances (leak current, Leak; a persistent K current, K2; and of a persistent Na+ current, P) that correlate linearly for the two groups of burster instances but not for the HCO groups. Visualizations of HCO instances in a reduced space suggested that there might be non-linear relationships between these parameters for these instances. Experimental studies have shown that period is a key attribute influenced by modulatory inputs and temperature variations in heart interneurons. Thus, we explored the sensitivity of period to changes in maximal conductances of Leak, K2, and P, and we found that for our realistic bursters the effect of these parameters on period could not be assessed because when varied individually bursting activity was not maintained.

Na + /K + pump interacts with the h -current to control bursting activity in central pattern generator neurons of leeches

The dynamics of different ionic currents shape the bursting activity of neurons and networks that control motor output. Despite being ubiquitous in all animal cells, the contribution of the Na+/K+ pump current to such bursting activity has not been well studied. We used monensin, a Na+/H+ antiporter, to examine the role of the pump on the bursting activity of oscillator heart interneurons in leeches. When we stimulated the pump with monensin, the period of these neurons decreased significantly, an effect that was prevented or reversed when the h-current was blocked by Cs+. The decreased period could also occur if the pump was inhibited with strophanthidin or K+-free saline. Our monensin results were reproduced in model, which explains the pump’s contributions to bursting activity based on Na+ dynamics. Our results indicate that a dynamically oscillating pump current that interacts with the h-current can regulate the bursting activity of neurons and networks. DOI: http://dx.doi.org/10.7554/eLife.19322.001

Bringing up the rear: new premotor interneurons add regional complexity to a segmentally distributed motor pattern

Central pattern generators (CPGs) pace and pattern many rhythmic activities. We have uncovered a new module in the heartbeat CPG of leeches that creates a regional difference in this segmentally distributed motor pattern. The core CPG consists of seven identified pairs and one unidentified pair of heart interneurons of which 5 pairs are premotor and inhibit 16 pairs of heart motor neurons. The heartbeat CPG produces a side-to-side asymmetric pattern of activity of the premotor heart interneurons corresponding to an asymmetric fictive motor pattern and an asymmetric constriction pattern of the hearts with regular switches between the two sides. The premotor pattern progresses from rear to front on one side and nearly synchronously on the other; the motor pattern shows corresponding intersegmental coordination, but only from segment 15 forward. In the rearmost segments the fictive motor pattern and the constriction pattern progress from front to rear on both sides and converge in phase. Modeling studies suggested that the known inhibitory inputs to the rearmost heart motor neurons were insufficient to account for this activity. We therefore reexamined the constriction pattern of intact leeches. We also identified electrophysiologically two additional pairs of heart interneurons in the rear. These new heart interneurons make inhibitory connections with the rear heart motor neurons, are coordinated with the core heartbeat CPG, and are dye-coupled to their contralateral homologs. Their strong inhibitory connections with the rearmost heart motor neurons and the small side-to-side phase difference of their bursting contribute to the different motor and beating pattern observed in the animals rear.

Variation in motor output and motor performance in a centrally generated motor pattern

Central pattern generators (CPGs) produce motor patterns that ultimately drive motor outputs. We studied how functional motor performance is achieved, specifically, whether the variation seen in motor patterns is reflected in motor performance and whether fictive motor patterns differ from those in vivo. We used the leech heartbeat system in which a bilaterally symmetrical CPG coordinates segmental heart motor neurons and two segmented heart tubes into two mutually exclusive coordination modes: rear-to-front peristaltic on one side and nearly synchronous on the other, with regular side-to-side switches. We assessed individual variability of the motor pattern and the beat pattern in vivo. To quantify the beat pattern we imaged intact adults. To quantify the phase relations between motor neurons and heart constrictions we recorded extracellularly from two heart motor neurons and movement from the corresponding heart segments in minimally dissected leeches. Variation in the motor pattern was reflected in motor performance only in the peristaltic mode, where larger intersegmental phase differences in the motor neurons resulted in larger phase differences between heart constrictions. Fictive motor patterns differed from those in vivo only in the synchronous mode, where intersegmental phase differences in vivo had a larger front-to-rear bias and were more constrained. Additionally, load-influenced constriction timing might explain the amplification of the phase differences between heart segments in the peristaltic mode and the higher variability in motor output due to body shape assumed in this soft-bodied animal. The motor pattern determines the beat pattern, peristaltic or synchronous, but heart mechanics influence the phase relations achieved.