Leech Neurons Research Articles

In Memoriam: Lawrence B. Cohen (1939–2023)

In Memoriam: Lawrence B. Cohen (1939–2023)

Order in chaos: Structure of chaotic invariant sets of square-wave neuron models.

Bursting phenomena and, in particular, square-wave or fold/hom bursting, are found in a wide variety of mathematical neuron models. These systems have different behavior regimes depending on the parameters, whether spiking, bursting, or chaotic. We study the topological structure of chaotic invariant sets present in square-wave bursting neuron models, first detailed using the Hindmarsh-Rose neuron model and later exemplary in the more realistic model of a leech heart neuron. We show that the unstable periodic orbits that form the skeleton of the chaotic invariant sets are deeply related to the spike-adding phenomena, typical from these models, and how there are specific symbolic sequences and a symbolic grammar that organize how and where the periodic orbits appear. Linking this information with the topological template analysis permits us to understand how the internal structure of the chaotic invariants is modified and how more symbolic sequences are allowed. Furthermore, the results allow us to conjecture that, for these systems, the limit template when the small parameter ε, which controls the slow gating variable, tends to zero is the complete Smale topological template.

Transcriptional profiling of identified neurons in leech

BackgroundWhile leeches in the genus Hirudo have long been models for neurobiology, the molecular underpinnings of nervous system structure and function in this group remain largely unknown. To begin to bridge this gap, we performed RNASeq on pools of identified neurons of the central nervous system (CNS): sensory T (touch), P (pressure) and N (nociception) neurons; neurosecretory Retzius cells; and ganglia from which these four cell types had been removed.ResultsBioinformatic analyses identified 3565 putative genes whose expression differed significantly among the samples. These genes clustered into 9 groups which could be associated with one or more of the identified cell types. We verified predicted expression patterns through in situ hybridization on whole CNS ganglia, and found that orthologous genes were for the most part similarly expressed in a divergent leech genus, suggesting evolutionarily conserved roles for these genes. Transcriptional profiling allowed us to identify candidate phenotype-defining genes from expanded gene families. Thus, we identified one of eight hyperpolarization-activated cyclic-nucleotide gated (HCN) channels as a candidate for mediating the prominent sag current in P neurons, and found that one of five inositol triphosphate receptors (IP3Rs), representing a sub-family of IP3Rs absent from vertebrate genomes, is expressed with high specificity in T cells. We also identified one of two piezo genes, two of ~ 65 deg/enac genes, and one of at least 16 transient receptor potential (trp) genes as prime candidates for involvement in sensory transduction in the three distinct classes of leech mechanosensory neurons.ConclusionsOur study defines distinct transcriptional profiles for four different neuronal types within the leech CNS, in addition to providing a second ganglionic transcriptome for the species. From these data we identified five gene families that may facilitate the sensory capabilities of these neurons, thus laying the basis for future work leveraging the strengths of the leech system to investigate the molecular processes underlying and linking mechanosensation, cell type specification, and behavior.

Effects of calcium-activated potassium channel modulators on afterhyperpolarizing potentials in identified motor and mechanosensory neurons of the medicinal leech.

Calcium-activated potassium (KCa) channels contribute to multiple neuronal properties including spike frequency and afterhyperpolarizing potentials (AHPs). KCa channels are classified as KCa1.1, KCa2, or KCa3.1 based on single-channel conductance and pharmacology. Ca2+-dependent AHPs in vertebrates are categorized as fast, medium, or slow. Fast and medium AHPs are generated by KCa1.1 and KCa2 channels, respectively. The KCa subtype responsible for slow AHPs is unclear. Prolonged, Ca2+-dependent AHPs have been described in several leech neurons. Unfortunately, apamin and other KCa blockers often prove ineffective in the leech. An alternative approach is to utilize KCa modulators, which alter channel sensitivity to Ca2+. Vertebrate KCa2 channels are targeted selectively by the positive modulator CyPPA and the negative modulator NS8593. Here we show that AHPs in identified motor and mechanosensory leech neurons are enhanced by CyPPA and suppressed by NS8593. Our results indicate that KCa2 channels underlie prolonged AHPs in these neurons and suggest that KCa2 modulators may serve as effective tools to explore the role of KCa channels in leech physiology.

Ultrasound Stimulations Induce Prolonged Depolarization and Fast Action Potentials in Leech Neurons.

Objective: Ultrasound (US) stimulation carries the promise of a selective, reversible, and non-invasive modulation of neural activity without the need for genetic manipulation of neural structures. However, the mechanisms of US-induced generation of action potentials (APs) are still unclear. Methods: Here we address this issue by analyzing intracellularly recorded responses of leech nociceptive neurons to controlled delivery of US. Results: US induced a depolarization linearly accumulating in time and outlasting the duration of the stimulation. Spiking activity was reliably induced for an optimal US intensity range. Moreover, we found that APs induced by US differ in smaller amplitude and faster repolarization from those induced by electrical stimulation in the same cell but display the same repolarization rate. Conclusions: These results shed light on the mechanism by which spikes are induced by US and pave the way for designing more efficient US stimulation patterns.

Non-synaptic Plasticity in Leech Touch Cells.

The role of Na+/K+-pumps in activity-dependent synaptic plasticity has been described in both vertebrates and invertebrates. Here, we provide evidence that the Na+/K+-pump is also involved in activity-dependent non-synaptic cellular plasticity in leech sensory neurons. We show that the resting membrane potential (RMP) of T cells hyperpolarizes in response to repeated somatic current injection, while at the same time their spike count (SC) and the input resistance (IR) increase. Our Hodgkin–Huxley-type neuron model, adjusted to physiological T cell properties, suggests that repetitive action potential discharges lead to increased Na+/K+-pump activity, which then hyperpolarizes the RMP. In consequence, a slow, non-inactivating current decreases, which is presumably mediated by voltage-dependent, low-threshold potassium channels. Closing of these putative M-type channels due to hyperpolarization of the resting potential increases the IR of the cell, leading to a larger number of spikes. By this mechanism, the response behavior switches from rapidly to slowly adapting spiking. These changes in spiking behavior also effect other T cells on the same side of the ganglion, which are connected via a combination of electrical and chemical synapses. An increased SC in the presynaptic T cell results in larger postsynaptic responses (PRs) in the other T cells. However, when the number of elicited presynaptic spikes is kept constant, the PR does not change. These results suggest that T cells change their responses in an activity-dependent manner through non-synaptic rather than synaptic plasticity. These changes might act as a gain-control mechanism. Depending on the previous activity, this gain could scale the relative impacts of synaptic inputs from other mechanoreceptors, versus the spike responses to tactile skin stimulation. This multi-tasking ability, and its flexible adaptation to previous activity, might make the T cell a key player in a preparatory network, enabling the leech to perform fast behavioral reactions to skin stimulation.

Transitions from chimeras to coherence: An analytical approach by means of the coherent stability function.

Chimera states have been a vibrant subject of research in the recent past, but the analytical treatment of transitions from chimeras to coherent states remains a challenge. Here we analytically derive the necessary conditions for this transition by means of the coherent stability function approach, which is akin to the master stability function approach that is traditionally used to study the stability of synchronization in coupled oscillators. In chimera states, there is typically at least one group of oscillators that evolves in a drifting, random manner, while other groups of oscillators follow a smoother, more coherent profile. In the coherent state, there thus exists a smooth functional relationship between the oscillators. This lays the foundation for the coherent stability function approach, where we determine the stability of the coherent state. We subsequently test the analytical prediction numerically by calculating the strength of incoherence during the transition point. We use leech neurons, which exhibit a coexistence of chaotic and periodic tonic spiking depending on initial conditions, coupled via nonlocal electrical synapses, to demonstrate our approach. We systematically explore various dynamical states with the focus on the transitions between chimeras and coherence, fully confirming the validity of the coherent stability function. We also observe complete synchronization for higher values of the coupling strength, which we verify by the master stability function approach.

Ultrasound Stimulations Induce Prolonged Depolarization and Fast Action Potentials in Leech Neurons

Ultrasound stimulation (US) carries the promise of a selective, reversible, and non-invasive modulation of neural activity without the need for genetic manipulation of neural structures. However, the mechanisms of US-induced generation of action potentials (APs) are still unclear. Here we address this issue by analyzing intracellularly recorded responses to controlled delivery of US of leech nociceptive neurons. US induced a depolarization linearly accumulating in time and outlasting the duration of the stimulation. Spiking activity was reliably induced for an optimal US intensity range. Moreover, we found that APs induced by US differ in smaller amplitude and faster repolarization from those induced by electrical (EL) stimulation in the same cell, but keep the same repolarization rate. These results shed light on the mechanism by which spikes are induced by US and pave the way for designing more efficient US patterns.

Extrasynaptic Neurotransmission Mediated by Exocytosis and Diffusive Release of Transmitter Substances.

This review article deals with the mechanisms of extrasynaptic release of transmitter substances, namely the release from the soma, axon and dendrites in the absence of postsynaptic counterparts. Extrasynaptic release occurs by exocytosis or diffusion. Spillover from the synaptic cleft also contributes to extrasynaptic neurotransmission. Here, we first describe two well-known examples of exocytosis from the neuronal soma, which may release copious amounts of transmitter for up to hundreds of seconds after electrical stimulation. The mechanisms for somatic exocytosis of the low molecular weight transmitter serotonin, and the peptides oxytocin and vasopressin have been studied in detail. Serotonin release from leech neurons and oxytocin and vasopressin from rodent neurons have a common multi-step mechanism, which is completely different from that for exocytosis from presynaptic endings. Most transmitters and peptides released extrasynaptically seem to follow this same mechanism. Extrasynaptic exocytosis may occur onto glial cells, which act as intermediaries for long-term and long-distance transmission. The second part of this review article focuses on the release upon synthesis of the representative diffusible molecules nitric oxide (NO) and endocannabinoids. Diffusible molecules are synthesized “on demand” from postsynaptic terminals in response to electrical activity and intracellular calcium elevations. Their effects include the retrograde modulation of presynaptic electrical activity and transmitter release. Extrasynaptic neurotransmission is well exemplified in the retina. Light-evoked extrasynaptic communication sets the gain for visual responses and integrates the activity of neurons, glia and blood vessels. Understanding how extrasynaptic communication changes the function of hard-wired circuits has become fundamental to understand the function of the nervous system.

9-Phenanthrol modulates postinhibitory rebound and afterhyperpolarizing potentials in an excitatory motor neuron of the medicinal leech.

Postinhibitory rebound (PIR) responses in leech dorsal excitatory motor neurons (cell DE-3) are eliminated by Ca2+ channel blockers but also exhibit a strong dependence on extracellular Na+. These features could be explained by a voltage-gated Ca2+ current acting in concert with a Ca2+-activated nonspecific current (ICAN). In vertebrates, ICAN is associated with TRPM4 channels which are blocked selectively by 9-phenanthrol. Here, we show that 9-phenanthrol selectively inhibits a late phase of PIR and simultaneously enhances afterhyperpolarizing potentials (AHPs). Bath application of NNC 55-0396 or Cd2+ combined with ion substitution experiments indicate that a low-voltage-activated Ca2+ current plays a key role in generating PIR and that Ca2+ influx through low- or high-voltage-activated Ca2+ channels can trigger AHPs via activation of a Ca2+-dependent K+ current. We also demonstrate modulation of rebound responses by other ICAN blockers such as gadolinium and flufenamic acid, as well as the calmodulin antagonist W-7. We discuss how these results provide additional insights into the specific types of ionic currents underlying rebound responses of motor neuron DE-3 in the medicinal leech.

Scalable Semisupervised Functional Neurocartography Reveals Canonical Neurons in Behavioral Networks.

Large-scale data collection efforts to map the brain are underway at multiple spatial and temporal scales, but all face fundamental problems posed by high-dimensional data and intersubject variability. Even seemingly simple problems, such as identifying a neuron/brain region across animals/subjects, become exponentially more difficult in high dimensions, such as recognizing dozens of neurons/brain regions simultaneously. We present a framework and tools for functional neurocartography-the large-scale mapping of neural activity during behavioral states. Using a voltage-sensitive dye (VSD), we imaged the multifunctional responses of hundreds of leech neurons during several behaviors to identify and functionally map homologous neurons. We extracted simple features from each of these behaviors and combined them with anatomical features to create a rich medium-dimensional feature space. This enabled us to use machine learning techniques and visualizations to characterize and account for intersubject variability, piece together a canonical atlas of neural activity, and identify two behavioral networks. We identified 39 neurons (18 pairs, 3 unpaired) as part of a canonical swim network and 17 neurons (8 pairs, 1 unpaired) involved in a partially overlapping preparatory network. All neurons in the preparatory network rapidly depolarized at the onsets of each behavior, suggesting that it is part of a dedicated rapid-response network. This network is likely mediated by the S cell, and we referenced VSD recordings to an activity atlas to identify multiple cells of interest simultaneously in real time for further experiments. We targeted and electrophysiologically verified several neurons in the swim network and further showed that the S cell is presynaptic to multiple neurons in the preparatory network. This study illustrates the basic framework to map neural activity in high dimensions with large-scale recordings and how to extract the rich information necessary to perform analyses in light of intersubject variability.

Multiplexed Population Coding of Stimulus Properties by Leech Mechanosensory Cells.

Multiplexing, the simultaneous encoding of different stimulus properties by distinct neuronal response features, has recently been suggested as a mechanism used in several sensory systems, including primate somatosensation. While a rigorous experimental verification of the multiplexing hypothesis is difficult to accomplish in a complex vertebrate system, it is feasible for a small population of individually characterized leech neurons. Monitoring the responses of all four mechanoreceptors innervating a patch of skin revealed striking similarities between touch encoding in the primate and the leech: summed spike counts represent stimulus intensity, whereas relative timing of first spikes encodes stimulus location. These findings suggest that multiplexed population coding is a general mechanism of touch encoding common to species as different as man and worm.

The dependence of synchronization transition processes of coupled neurons with coexisting spiking and bursting on the control parameter, initial value, and attraction domain

As the value of a control parameter decreases, monostable bursting changes to monostable spiking via the coexistence of spiking and bursting in a Leech neuron model, and the attraction domain of the bursting decreases, while that of the coexisting spiking increases for coexisting behaviors. As the coupling strength increases, the two coupled Leech neurons with the coexisting spiking and bursting manifest transitions from non-synchronization to complete synchronization (CS) through a complex process, which is dependent of the values of the control parameter, cases of configurations (one neuron spiking and the other bursting, both bursting, and both spiking), and the initial values of the two neurons. The transition processes involve various spiking and bursting or chaos synchronization states for the first two cases given here, but contain only spiking patterns for the last case. Corresponding with the attraction domain, the probability of initial values that can lead to bursting patterns of CS decreases, but the probability of initial values that can induce spiking of CS increases for the former two cases, as the control parameter is decreased. This phenomenon can also be interpreted by the structure and volume of the attraction domain with which all initial values can only induce CS of spiking for case 3. The results may improve understanding of the synchronization dynamics of the coupled neurons with coexisting behaviors.

Cell dialysis by sharp electrodes can cause nonphysiological changes in neuron properties.

We recorded from lobster and leech neurons with two sharp electrodes filled with solutions often used with these preparations (lobster: 0.6 M K2SO4 or 2.5 M KAc; leech: 4 M KAc), with solutions approximately matching neuron cytoplasm ion concentrations, and with 6.5 M KAc (lobster, leech) and 0.6 M KAc (lobster). We measured membrane potential, input resistance, and transient and sustained depolarization-activated outward current amplitudes in leech and these neuron properties and hyperpolarization-activated current time constant in lobster, every 10 min for 60 min after electrode penetration. Neuron properties varied with electrode fill. For fills with molarities ≥2.5 M, neuron properties also varied strongly with time after electrode penetration. Depending on the property being examined, these variations could be large. In leech, cell size also increased with noncytoplasmic fills. The changes in neuron properties could be due to the ions being injected from the electrodes during current injection. We tested this possibility in lobster with the 2.5 M KAc electrode fill by making measurements only 10 and 60 min after penetration. Neuron properties still changed, although the changes were less extreme. Making measurements every 2 min showed that the time-dependent variations in neuron properties occurred in concert with each other. Neuron property changes with high molarity electrode-fill solutions were great enough to decrease neuron firing strongly. An experiment with (14)C-glucose electrode fill confirmed earlier work showing substantial leak from sharp electrodes. Sharp electrode work should thus be performed with cytoplasm-matched electrode fills.

Control of neuronal morphology and connectivity: Emerging developmental roles for gap junctional proteins

Recent evidence indicates that gap junction (GJ) proteins can play a critical role in controlling neuronal connectivity as well as cell morphology in the developing nervous system. GJ proteins may function analogously to cell adhesion molecules, mediating cellular recognition and selective neurite adhesion. Moreover, during synaptogenesis electrical synapses often herald the later establishment of chemical synapses, and thus may help facilitate activity-dependent sculpting of synaptic terminals. Recent findings suggest that the morphology and connectivity of embryonic leech neurons are fundamentally organized by the type and perhaps location of the GJ proteins they express. For example, ectopic expression in embryonic leech neurons of certain innexins that define small GJ-linked networks of cells leads to the novel coupling of the expressing cell into that network. Moreover, gap junctions appear to mediate interactions among homologous neurons that modulate process outgrowth and stability. We propose that the selective formation of GJs between developing neurons and perhaps glial cells in the CNS helps orchestrate not only cellular synaptic connectivity but also can have a pronounced effect on the arborization and morphology of those cells involved.

Interaction of leech neurons with topographical gratings: comparison with rodent and human neuronal lines and primary cells

Controlling and improving neuronal cell migration and neurite outgrowth are critical elements of tissue engineering applications and development of artificial neuronal interfaces. To this end, a promising approach exploits nano/microstructured surfaces, which have been demonstrated to be capable of tuning neuronal differentiation, polarity, migration and neurite orientation. Here, we investigate the neurite contact guidance of leech neurons on plastic gratings (GRs; anisotropic topographies composed of alternating lines of grooves and ridges). By high-resolution microscopy, we quantitatively evaluate the changes in tubulin cytoskeleton organization and cell morphology and in the neurite and growth cone development. The topography-reading process of leech neurons on GRs is mediated by filopodia and is more responsive to 4-µm-period GRs than to smaller period GRs. Leech neuron behaviour on GRs is finally compared and validated with several other neuronal cells, from murine differentiated embryonic stem cells and primary hippocampal neurons to differentiated human neuroblastoma cells.

The spontaneous electrical activity of neurons in leech ganglia

Using the newly developed voltage-sensitive dye VF2.1.Cl, we monitored simultaneously the spontaneous electrical activity of ∼80 neurons in a leech ganglion, representing around 20% of the entire neuronal population. Neurons imaged on the ventral surface of the ganglion either fired spikes regularly at a rate of 1–5 Hz or fired sparse spikes irregularly. In contrast, neurons imaged on the dorsal surface, fired spikes in bursts involving several neurons. The overall degree of correlated electrical activity among leech neurons was limited in control conditions but increased in the presence of the neuromodulator serotonin. The spontaneous electrical activity in a leech ganglion is segregated in three main groups: neurons comprising Retzius cells, Anterior Pagoda, and Annulus Erector motoneurons firing almost periodically, a group of neurons firing sparsely and randomly, and a group of neurons firing bursts of spikes of varying durations. These three groups interact and influence each other only weakly.

Once more unto the leech: production of functional motor patterns in leech heart motor neurons

Leech heart motor neurons are the output component of the leech heartbeat pattern generator. Although the inhibitory synaptic input they receive from the central pattern generator (CPG) is the primary determinant of the rhythmic output they produce, the neurons themselves can contribute in a meaningful way to the patterns they produce [1-3]. In this study, we seek to understand the interaction of the inhibitory synaptic inputs, electrical coupling, and active membrane currents, as well as the interaction and balance between membrane currents. Towards this goal, we developed a 7 compartment GEneral NEural SImulation System (GENESIS 2.3) model which incorporated the currents known to be present in leech heart motor neurons. This model was parameterized by the maximal conductance for each of these currents, and an initial instance of this model was hand tuned to produce acceptable activity. We then used a multi-objective evolutionary algorithm (MOEA) to generate model instances which correctly produced a target output pattern within an acceptable error bound given the corresponding input pattern. The MOEA we used was previously applied to models of crustacean stomatogastric ganglion neurons [4]. We adapted it to run on a ~1000 node high-performance computing cluster (ELLIPSE, Emory IT) and to interact with our 7 compartment leech heart motor neuron model. This evolutionary algorithm independently selects the parents for each generation on each fitness metric separately, obviating the need to create a unified fitness function. Thus, a model instance that is excellent on only one metric can contribute to the subsequent generation even if it performs poorly on all other metrics. We executed 5 runs of this evolutionary algorithm with different, randomly selected initial populations and our post-hoc analyses examined the millions of resulting models from all runs of the MOEA. The fitness metrics we used to evaluate model instances are composed of two sets: output attributes that directly affect the muscles which the motor neurons innervate and attributes which capture other key identifying attributes of leech heart motor neurons. The former are the relative phase, as measured against the peristaltic interneuron in ganglion 4, and the intra-burst spike frequency for each neuron simulated. The latter are the duty cycle, spike height, and the slow wave height. The targets for each of these fitness metrics are drawn from our unique database of complete, individual-animal sets of spike time pattern, synaptic weight profile, and output pattern for leech heart motor neurons in ganglia 8 and 12. We have 431 model instances which met our targets on all fitness metrics and thousands that achieved the targets for only ganglia 8 or 12, but not both. We examined the contribution and balance of currents in these models and have found relationships between sets of currents and a strong differential effect of electrical coupling between pairs of leech heart motor neurons in different ganglia.

Parameter-dependent synchronization transition of coupled neurons with co-existing spiking and bursting

A firing pattern transition is simulated in the Leech neuron model, firstly from bursting to co-existence of spiking and bursting and then to spiking. The attraction domain of spiking and bursting for three different parameter values are calculated. Synchronization transition processes of two coupled Leech neurons, one is bursting and the other the co-existing spiking, are simulated for the three parameters. The three synchronization processes appear similar as the coupling strength increases, beginning from non-synchronization to complete synchronization through a complex dynamical procedure, but their detailed processes are different depending on the parameter values. The transition procedure is complex and the complete synchronization is in bursting for larger parameter values, while the process is simple with complete synchronization of spiking for smaller values. The potential relationship between complete synchronization and the attraction domain is also discussed. The results are instructive to understanding the synchronization behaviors of the coupled neuronal system with co-existing attractors.

Identification of leech embryonic neurons that express a Hox gene required for the differentiation of a paired, segment-specific motor neuron

This study investigated the embryonic expression and function of the Hox gene Lox1 in the simple, well-characterized central nervous system (CNS) of the medicinal leech Hirudo medicinalis. Lox1 was expressed in an anterior–posterior domain, extending from the posterior aspect of the fourth segment (rostral neuromere 4, R4) to the seventeenth segment (midbody ganglion 13, M13). Lox1 expression was also found in both sex organ primordia (male and female). Lox1 expression was not detected in every cell of the ganglia included in its domain. It was detected in a specific subset that included several segmentally iterated neurons and segment-specific neurons. Several central neurons (neurons located in the central nervous system – CNS) that coexpressed both Lox1 and FMRFamide-like peptides were identified using antibody staining of leech embryos and epifluorescence and confocal microscopy. RNA interference was used to block the expression of Lox1. The expression pattern and the effect of RNA interference indicate that Lox1 is required for the differentiation of a segment-specific pair of motor neurons, the RPE (rostral penile evertor) neurons, which appear only in midbody ganglion 6 (M6) and innervate the male sex organ.