Comodulation of h- and Na+/K+ Pump Currents Expands the Range of Functional Bursting in a Central Pattern Generator by Navigating between Dysfunctional Regimes.

Central pattern generators (CPGs), specialized oscillatory neuronal networks controlling rhythmic motor behaviors such as breathing and locomotion, must adjust their patterns of activity to a variable environment and changing behavioral goals. Neuromodulation adjusts these patterns by orchestrating changes in multiple ionic currents. In the medicinal leech, the endogenous neuromodulator myomodulin speeds up the heartbeat CPG by reducing the electrogenic Na+/K+ pump current and increasing h-current in pairs of mutually inhibitory leech heart interneurons (HNs), which form half-center oscillators (HN HCOs). Here we investigate whether the comodulation of two currents could have advantages over a single current in the control of functional bursting patterns of a CPG. We use a conductance-based biophysical model of an HN HCO to explain the experimental effects of myomodulin. We demonstrate that, in the model, comodulation of the Na+/K+ pump current and h-current expands the range of functional bursting activity by avoiding transitions into nonfunctional regimes, such as asymmetric bursting and plateau-containing seizure-like activity. We validate the model by finding parameters that reproduce temporal bursting characteristics matching experimental recordings from HN HCOs under control, three different myomodulin concentrations, and Cs+ treated conditions. The matching cases are located along the border of an asymmetric regime away from the border with more dangerous seizure-like activity. We found a simple comodulation mechanism with an inverse relation between the pump and h-currents makes a good fit of the matching cases and comprises a general mechanism for the robust and flexible control of oscillatory neuronal networks.SIGNIFICANCE STATEMENT Rhythm-generating neuronal circuits adjust their oscillatory patterns to accommodate a changing environment through neuromodulation. In different species, chemical messengers participating in such processes may target two or more membrane currents. In medicinal leeches, the neuromodulator myomodulin speeds up the heartbeat central pattern generator by reducing Na+/K+ pump current and increasing h-current. In a computational model, we show that this comodulation expands the range of central pattern generator's functional activity by navigating the circuit between dysfunctional regimes resulting in a much wider range of cycle period. This control would not be attainable by modulating only one current, emphasizing the synergy of combined effects. Given the prevalence of h-current and Na+/K+ pump current in neurons, similar comodulation mechanisms may exist across species.

Output variability across animals and levels in a motor system.

Rhythmic behaviors vary across individuals. We investigated the sources of this output variability across a motor system, from the central pattern generator (CPG) to the motor plant. In the bilaterally symmetric leech heartbeat system, the CPG orchestrates two coordinations in the bilateral hearts with different intersegmental phase relations (Δϕ) and periodic side-to-side switches. Population variability is large. We show that the system is precise within a coordination, that differences in repetitions of a coordination contribute little to population output variability, but that differences between bilaterally homologous cells may contribute to some of this variability. Nevertheless, much output variability is likely associated with genetic and life history differences among individuals. Variability of Δϕ were coordination-specific: similar at all levels in one, but significantly lower for the motor pattern than the CPG pattern in the other. Mechanisms that transform CPG output to motor neurons may limit output variability in the motor pattern.

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.

Maturation of peptide-positive synaptic arbors in the medicinal leech requires rhythmic target activity.

The formation and refinement of synaptic connections are dependent on the activity that emerges from nascent synaptic connections. Such activity has the effect of regulating the production and release of specific neurotransmitters. To determine the role of activity in regulating the production of peptide-positive synapses, we used antibodies against Phe-Met-Arg-Phe-NH2 and acetylated α-tubulin as well as intracellular injections of Neurobiotin to examine varicosities belonging to heart excitor (HE) neurons on the heart tubes of medicinal leeches, Hirudo spp. We found that the production of peptide-positive varicosities increased considerably during the last week of embryogenesis, which coincided with the emergence of rhythmic activity of the heart tube. When we compromised central input to HE neurons with bicuculline or by surgical ablation of the central pattern generator during early embryogenesis, we found that activity in the heart tubes and its rhythmicity were greatly diminished. Furthermore, the activity of HE neurons had also lost its rhythmicity and appeared tonic, and production of peptide-positive varicosities was substantially reduced as well. Partial surgical ablations that preserved rhythmic activity in the heart tube while disrupting heart tube innervation by some HE neurons still resulted in peptide-positive varicosity production. Taken together, our results suggest that postsynaptic rhythmic activity of the heart tube is necessary and sufficient for the development and maturation of peptide-positive synapses.

Centrally patterned rhythmic activity integrated by a peripheral circuit linking multiple oscillators.

The central pattern generator for heartbeat in the medicinal leech, Hirudo generates rhythmic activity conveyed by heart excitor motor neurons in segments 3-18 to coordinate the bilateral tubular hearts and side vessels. We focus on behavior and the influence of previously un-described peripheral nerve circuitry. Extracellular recordings from the valve junction (VJ) where afferent vessels join the heart tube were combined with optical recording of contractions. Action potential bursts at VJs occurred in advance of heart tube and afferent vessel contractions. Transections of nerves were performed to reduce the output of the central pattern generator reaching the heart tube. Muscle contractions persisted but with a less regular rhythm despite normal central pattern generator rhythmicity. With no connections between the central pattern generator and heart tube, a much slower rhythm became manifest. Heart excitor neuron recordings showed that peripheral activity might contribute to the disruption of centrally entrained contractions. In the model presented, peripheral activity would normally modify the activity actually reaching the muscle. We also propose that the fundamental efferent unit is not a single heart excitor neuron, but rather is a functionally defined unit of about three adjacent motor neurons and the peripheral assembly of coupled peripheral oscillators.

Targeting a neuropeptide to discrete regions of the motor arborizations of a single neuron.

The heart excitor (HE) motor neuron in the leech Hirudo releases acetylcholine (ACh) and a peptide, FMRFamide, to regulate the contractile activity of the heart tube and associated side vessels. Consistent with Dale's principle, it was assumed that both neurotransmitters were localized to all presynaptic varicosities. However, we found discrete peptide-positive and peptide-negative varicosities associated with particular sites of innervation on the heart tube. We produced dual-labeled HE neurons by pressure injecting Neurobiotin into single HE cell bodies and applied anti-FMRFamide antibodies on the same preparations. Consistent with initial expectations, peptide-labeled varicosities were numerous and widely distributed along the heart tube and at one of the three side vessels, the latero-abdominal vessel. Nevertheless, some Neurobiotin-labeled varicosities along the heart tube lacked peptide label entirely. Moreover, there were dense and distinct peptide-negative innervations at the valve junctions of the latero-dorsal and latero-lateral vessels at each segment. Nevertheless, the peptide label was found in HE axons and varicosities that projected distally along the side vessels. Therefore, the more proximal peptide-negative clusters cannot simply be the result of restricted transport or deficient staining of peptide. Rather, we infer that FMRFamide is transported to (or selectively excluded from) discrete locations and that ACh is present in varicosities that lacked peptide. Such targeting of neurotransmitters could be described using a discrete targeting model of synaptic transmission. Compared with Dale's principle, this model may provide a more complete perspective of chemical communication than previously understood.

Effects of gamma-hydroxybutyrate (GHB) and its metabolic precursors on delayed-matching-to-position performance in rats.

The purpose of the present study was to provide further information about the effects of gamma-hydroxybutyrate (GHB) on memory. Initially, the acute effects of gamma-butyrolactone (GBL, 75-200 mg/kg IP), 1,4-butanediol (1,4-BD, 100-300 mg/kg IP), and ethanol (1.0-3.0 g/kg, oral), as well as GHB (100-300 mg/kg IP), were examined in rats responding under a delayed-matching-to-position (DMTP) procedure with delays from 0 to 32 s. Acute administration of all four drugs reduced the number of trials completed and also reduced accuracy during delay trials, but not during trials without a delay. Some tolerance developed to the disruptive effects of GHB following exposure to 300 mg/kg/day for 29 consecutive days. These data indicate that GHB can disrupt working memory and speed of responding, and that tolerance can develop to these effects. Moreover, the acute effects of GHB under the DMTP procedure resemble those of its metabolic precursors, GBL and 1,4-BD, and of the prototypical CNS depressant drug, ethanol.

Reinforcement schedule effects in rats trained to discriminate 3,4-methylenedioxymethamphetamine (MDMA) or cocaine.

RATIONALE: Relatively few studies have compared the discriminative stimulus effects of 3,4-methylenedioxymethamphetamine (MDMA) and cocaine, and findings from different laboratories are somewhat inconsistent. One possible reason for discrepant results may be the use of different reinforcement schedules during discrimination training. OBJECTIVE: The present study compared fixed ratio (FR) 20 and variable interval (VI) 15-s reinforcement schedules to determine their influence on discrimination acquisition, response rates, frequency of reinforcements, and stimulus generalization in rats trained to discriminate cocaine or MDMA. MATERIALS AND METHODS: Thirty-two male Sprague-Dawley rats were trained to discriminate cocaine (10 mg/kg; n=16) or MDMA (1.5 mg/kg; n=16) from saline under either a FR 20 or a VI 15-s schedule of food reinforcement. Stimulus generalization tests were conducted with a range of doses of cocaine, MDMA, d-amphetamine, and lysergic acid diethylamide in all four training groups. RESULTS: The FR 20 schedule facilitated more rapid discrimination acquisition compared to the VI 15-s schedule and established differential response rates and frequency of reinforcement under drug and vehicle conditions. However, reinforcement schedule had little influence on stimulus generalization between MDMA and cocaine. Cocaine produced partial substitution for MDMA in both training groups (FR 20, 51%; VI 15-s, 58%). Likewise, MDMA produced only partial substitution for cocaine in both training groups (FR 20, 40%; VI 15-s, 72%). CONCLUSIONS: The present findings suggest that the number of sessions required to establish discriminative stimulus control varies with different reinforcement schedules. Nevertheless, training schedules alone do not appear to have significant effects on stimulus generalization between MDMA and cocaine.