Encoding of Tactile Stimuli by Mechanoreceptors and Interneurons of the Medicinal Leech

For many animals processing of tactile information is a crucial task in behavioral contexts like exploration, foraging, and stimulus avoidance. The leech, having infrequent access to food, developed an energy efficient reaction to tactile stimuli, avoiding unnecessary muscle movements: The local bend behavior moves only a small part of the body wall away from an object touching the skin, while the rest of the animal remains stationary. Amazingly, the precision of this localized behavioral response is similar to the spatial discrimination threshold of the human fingertip, although the leech skin is innervated by an order of magnitude fewer mechanoreceptors and each midbody ganglion contains only 400 individually identified neurons in total. Prior studies suggested that this behavior is controlled by a three-layered feed-forward network, consisting of four mechanoreceptors (P cells), approximately 20 interneurons and 10 individually characterized motor neurons, all of which encode tactile stimulus location by overlapping, symmetrical tuning curves. Additionally, encoding of mechanical force was attributed to three types of mechanoreceptors reacting to distinct intensity ranges: T cells for touch, P cells for pressure, and N cells for strong, noxious skin stimulation. In this study, we provide evidences that tactile stimulus encoding in the leech is more complex than previously thought. Combined electrophysiological, anatomical, and voltage sensitive dye approaches indicate that P and T cells both play a major role in tactile information processing resulting in local bending. Our results indicate that tactile encoding neither relies on distinct force intensity ranges of different cell types, nor location encoding is restricted to spike count tuning. Instead, we propose that P and T cells form a mixed type population, which simultaneously employs temporal response features and spike counts for multiplexed encoding of touch location and force intensity. This hypothesis is supported by our finding that previously identified local bend interneurons receive input from both P and T cells. Some of these interneurons seem to integrate mechanoreceptor inputs, while others appear to use temporal response cues, presumably acting as coincidence detectors. Further voltage sensitive dye studies can test these hypotheses how a tiny nervous system performs highly precise stimulus processing

Spontaneous and evoked electrical activity of neurons in leech Hirudo medicinalis studied by a new generation of voltage sensitive dyes

By using the newly developed voltage sensitive dye VF2.1.Cl invented by Miller and colleagues (Miller et al. 2012), I monitored simultaneously the spontaneous electrical activity of approximately 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 , Leydig 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. I was able to obtain long optical recordings for several minutes. I studied, also, the evoked response of nervous system by stimulating mechanosensory neurons. This work paves the way for further studies of multicellular networks using the new voltage sensitive dye

A double-sided microscope to realize whole-ganglion imaging of membrane potential in the medicinal leech

Studies of neuronal network emergence during sensory processing and motor control are greatly facilitated by technologies that allow us to simultaneously record the membrane potential dynamics of a large population of neurons in single cell resolution. To achieve whole-brain recording with the ability to detect both small synaptic potentials and action potentials, we developed a voltage-sensitive dye (VSD) imaging technique based on a double-sided microscope that can image two sides of a nervous system simultaneously. We applied this system to the segmental ganglia of the medicinal leech. Double-sided VSD imaging enabled simultaneous recording of membrane potential events from almost all of the identifiable neurons. Using data obtained from double-sided VSD imaging, we analyzed neuronal dynamics in both sensory processing and generation of behavior and constructed functional maps for identification of neurons contributing to these processes

From neuronal networks to behavior: dynamics of spontaneous activity and onset of movement in the leech

Animal behavior was once seen as a chain of reactions to stimuli from the environment. From chemotaxis in bacteria to mammals withdrawing from painful stimuli, most of the actions taken by animals are clearly driven by external inputs. Reflexes were among the first phenomena to be studied to have an insight on the dynamics of the nervous system. Later, a step forward was the discovery of central pattern generators: once a behavior is started by a stimulus, some neuronal networks are able to maintain it without further inputs from the environment. The nervous system of all animals, however, is so complex that is displaying a rich dynamics even in the absence of external inputs or, in a more realistic situation, when no single input is able to drive a clear-cut reaction. In the same way, at the motor output level, animals keep moving in the absence of evident stimuli. These spontaneous behaviors are still far from being understood. Difficult problems are often easier to solve in simple systems. The leech has a relatively simple nervous system, composed of ~103 neurons disposed in a regular structure, but at the same time displays a variety of different behaviors. It seems then a good preparation to approach the spontaneous dynamics problem. The aim of my PhD research is to describe the spontaneous behavior of the leech and the spontaneous activity of its nervous system. A first, necessary step for this study was to develop a method of automatic classification and analysis of the leech movements. Thanks to this method we described accurately the properties of the different behaviors: we focused particularly on the largely unknown irregular exploratory behavior, which is found to display a broad range of oscillation frequencies and displacement speeds, but with some recurrent movement patterns. Finding the complete list of the leech spontaneous behaviors, and the probability of the transitions between them, it was possible to demonstrate that decision making in the leech is a Markovian process. The spontaneous activity in the isolated leech ganglion was found to be characterized by long-term correlations and a large variability in bursts size and duration. The same dynamics was observed in dissociated culture of rat hippocampal neurons, despite the difference in the structure between the two networks. We studied the effects of pharmacological modulations of inhibitory and excitatory processes on the spontaneous activity, and the role of single identified motor neurons in spontaneous bursts. Finally we proposed a simple statistical model accounting for experimental results. We studied then the spontaneous activity of the leech ganglion when it was connected to the other ganglia and in the semi-intact moving animal. Inputs received from the head and tail brain caused a drastic change in the activity of the ganglion, increasing synchronization among neurons and leading to a regime dominated by very large bursts. By recording at the same the movements of the leech and its nervous activity it was possible to have a better understanding of the relationship between the motor neuron bursts and the onset of movements

Timescale-invariant representation of acoustic communication signals by a bursting neuron

Acoustic communication often involves complex sound motifs in which the relative durations of individual elements, but not their absolute durations, convey meaning. Decoding such signals requires an explicit or implicit calculation of the ratios between time intervals. Using grasshopper communication as a model, we demonstrate how this seemingly difficult computation can be solved in real time by a small set of auditory neurons. One of these cells, an ascending interneuron, generates bursts of action potentials in response to the rhythmic syllable-pause structure of grasshopper calls. Our data show that these bursts are preferentially triggered at syllable onset; the number of spikes within the burst is linearly correlated with the duration of the preceding pause. Integrating the number of spikes over a fixed time window therefore leads to a total spike count that reflects the characteristic syllable-to-pause ratio of the species while being invariant to playing back the call faster or slower. Such a timescale-invariant recognition is essential under natural conditions, because grasshoppers do not thermoregulate; the call of a sender sitting in the shade will be slower than that of a grasshopper in the sun. Our results show that timescale-invariant stimulus recognition can be implemented at the single-cell level without directly calculating the ratio between pulse and interpulse durations

Identification and Characterization of electrical patterns underlying stereotyped behaviours in the semi-intact leech

Neuroscience aims at understanding the mechanisms underlying perception, learning, memory, consciousness and acts. The present Ph.D. thesis aims to elucidate some principles controlling actions, which in a more scientific and technical language is referred to as motor control. This concept has been studied in a variety of preparations in vertebrate and invertebrate species. In this PhD thesis, the leech has been the subject of choice, because it is a well known preparation, highly suitable for relating functional and behavioural properties to the underlying neuronal networks. The semi-intact leech preparation (Kristan et al., 1974) has been the main methodological strategy performed in the experiments. Its importance lies in the fact that it gives the possibility to access the information from the leech\u2019s central nervous system (CNS) and compare simultaneously some stereotyped behaviours. Thus, entering in this work it is necessary to make a brief summary of the steps followed before arriving to the conclusions written ahead. The main objective followed in this work has been the analysis, identification and characterization of electrical patterns underlying different behaviours in Hirudo medicinalis. This main objective has been reached focusing the project on three particular objectives, which have been pursued during the author\u2019s Philosophical Doctorate course

On the Dynamics of the Spontaneous Activity in Neuronal Networks

Most neuronal networks, even in the absence of external stimuli, produce spontaneous bursts of spikes separated by periods of reduced activity. The origin and functional role of these neuronal events are still unclear. The present work shows that the spontaneous activity of two very different networks, intact leech ganglia and dissociated cultures of rat hippocampal neurons, share several features. Indeed, in both networks: i) the inter-spike intervals distribution of the spontaneous firing of single neurons is either regular or periodic or bursting, with the fraction of bursting neurons depending on the network activity; ii) bursts of spontaneous spikes have the same broad distributions of size and duration; iii) the degree of correlated activity increases with the bin width, and the power spectrum of the network firing rate has a 1/f behavior at low frequencies, indicating the existence of long-range temporal correlations; iv) the activity of excitatory synaptic pathways mediated by NMDA receptors is necessary for the onset of the long-range correlations and for the presence of large bursts; v) blockage of inhibitory synaptic pathways mediated by GABA(A) receptors causes instead an increase in the correlation among neurons and leads to a burst distribution composed only of very small and very large bursts. These results suggest that the spontaneous electrical activity in neuronal networks with different architectures and functions can have very similar properties and common dynamics