Parallel Processing in the Leech Central Nervous System

The aim of this thesis is to shed light on the parallel processing in the leech nervous system. Two main problems have been addressed: 1) how a mechanical stimulus is coded by mechanosensory neurons at the first stage of the information processing in the central nervous system (CNS). 2) which statistical properties of the electrical activity of neurons in the segmental ganglion are relevant for infom1ation processing and in particular for motor reactions. A semi-intact preparation consisting of a single segment innervated by its own ganglion and the adjacent ganglia was used. Sensory stimulation was delivered by touching the skin with an appropriate device. Previous observations indicate that each point of the skin surface is innervated by several mechanosensory neurons (Nicholls and Baylor, 1968; Yau, 1976; Lewis and Kristan, 1998-c). In my thesis I analyzed how sensory information is coded in the electrical activity of a population of mechanosensory neurons. I developed and used an experimental set-up allowing the recording of the simultaneous activation of all mechanosensory neurons belonging to three consecutive segmental ganglia. The experiments were performed in high Mg2+ Ringer fluid in order to block all chemical synapses so as to eliminate synaptic responses and muscle contraction leading to a motion of the skin resulting in an additional sensory feedback. In these conditions it is possible isolate the responses of sensory neurons directly activated by the stimulus. The extent of the activation of mechanosensory neurons in three adjacent ganglia after a stimulation was analyzed. The results of this mapping show that all mechanosensory neurons are consistently activated also in their accessory receptive fields. The reproducibility of their response is the same as that observed in mechanosensory neurons responding in major receptive fields. T cells activation presents the same characteristics for stimulation \ub7of the major or minor receptive fields, indeed they respond to the transient phase of the stimulus. P cells show a different dynamics of activation if stimulated in their major or minor receptive field. In presence of a prolonged stimulation, P cells of the central ganglion fire action potentials continuously while P cells of accessory ganglia adapt. N cells are always activated in major and minor receptive fields showing approximately the same dynamics. When the skin is touched with a moderate tactile stimulus, i.e. exerting a force ofless than 20 mN, many different neurons (T and P cells) of the three ganglia fire action potentials. Thus sensory coding is initially redundant. If the stimulation is prolonged, many of these sensory neurons rapidly adapt and only P cells of the central ganglion respond. Thus sensory coding is dynamical and becomes very sharply tuned. In the second part of my PhD work, a different stage of the information flow has been investigated. The aim of the research was to study which statistical properties of the electrical activity of neurons in the leech ganglion are important for neural processing. In particular I analyzed the statistics of the evoked activity in response to a sensory input. The ganglion was considered as a black box with inputs and outputs; inputs are represented by sensory neurons and outputs by motor neurons or other neurons innervating roots and com1ectives. The responses of mechanosensory neurons to touch stimuli show trial-to-trial reproducibility, i.e. spikes in sensory neurons occur at very precise times. This does not happen for motor neurons. The experiment consisted in stimulating repeatedly a particular mechanosensory neuron by evoking action potentials with an intracellular electrode. The evoked activity in response to this input was recorded by suction electrodes. The extracellular recordings contain the superimposed activity of many neurons which can be separated by spike sorting procedure. The first and second order statistics were studied. Two main results were obtained by this analysis: 1) the response is distributed on many neurons; many neurons respond to the same stimulus and the same neurons are recruited for different stimuli. 2) the response is characterized by spatio-temporal variability, i.e. different neurons respond in different trials and neurons respond with jitter in time occurrences of spikes. The comparison between reproducibility of mechanosensory neurons evoked by touch stimulus and motor neurons evoked by mechanosensory neurons. activation shows that sensory cells are very reproducible, therefore the origin of motor neuron variability has to be found in synaptic transmission. The analysis of the second order statistics was used to check correlation among coactivated neurons. Joint entropies and joint probabilities for each pair of neurons were computed and compared with the sum of individual entropies and the product of individual probabilities, respectively. In all cases these quantities are consistently equal. This means that coactivated neurons are statistically independent. This property of the system can be functional for neural proeessmg. If the response of the network is distributed, then the pooling of the electrical activity of all individual responses is important and may be the key feature of the network. In this perspective, a distributed process consisting in a large number of statistically independent unreliable elements leads to a reproducible response, when the response is pooled over all elements. There is theoretical justification for this statement, indeed it is demonstrated that the pooling of a large number of statistical independent stochastic processes affected by high variability lead to a stochastic process with low variability. In conclusion, increasing the number of elements pooled, the response is more and more reliable

Optical delivery of liposome encapsulated chemical stimuli to neuronal cells

Spatially confined and precise time delivery of neuroactive molecules is an important issue in neurophysiology. In this work we developed a technique for delivering chemical stimuli to cultured neurons consisting in encapsulating the molecules of interest in liposomes. These vectors were then loaded in reservoirs consisting of glass capillaries. The reservoirs were placed in the recording chamber and single liposomes were trapped and transported out by optical tweezers to the site of stimulation on cultured neurons. Finally, the release of liposome content was induced by application of UV-pulses, breaking the liposome membrane. The efficiency of encapsulation and release were first evaluated by loading the liposomes with fluorescein. In order to test the effect of the UV-induced release, liposomes with diameter ranging from 1 to 10 μm (fL to pL volumes), were filled with KCl and tested on neuronal cells. Neuronal cultures, loaded with Ca(2+) dye, were monitored by imaging intracellular Ca(2+). An efficient release from the liposomes was demonstrated by detectable calcium signals, indicating stimulated depolarization of the neuronal cells by KCl. The present technique represents an alternative method for focal chemical stimulation of cultured cells that circumvents some of the limitations of microejection and photorelease of caged compounds

Use of optical tweezers technology for long-term, focal stimulation of specific subcellular neuronal compartments

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Improved neuron culture using scaffolds made of three-dimensional PDMS micro-lattices

Tissue engineering strives to create functional components of organs with different cell types in vitro. One of the challenges is to fabricate scaffolds for three-dimensional (3D) cell culture under physiological conditions. Of particular interesting is to investigate the morphology and function of the central nervous system (CNS) cultured using such scaffolds. Here, we used an elastomer, polydimethylsiloxane (PDMS), to produce lattice-type scaffolds from a photolithography defined template. The photomask with antidot arrays was spin-coated by a thick layer of resist and downward mounted on a rotating stage at angle of 45\ub0. After exposure for three or more times keeping the same exposure plan but rotated by the same angle, the photoresist was developed to produce a 3D porous template. Afterward, a pre-polymer mixture of PDMS was poured in and cured, followed by a resist etch, resulting in lattice-type PDMS features. Before cell culture, the PDMS lattices were surface functionalized. Culture test has been done using NIH-3T3 cells and primary hippocampal cells from rats, showing homogenously cell infiltration and 3D attachment. As expected, a much higher cell number was found in 3D PDMS lattices than in 2D culture. We also found a higher neuron to astrocyte ratio and a higher degree of cell ramification in 3D culture compared to 2D culture, due to the change of scaffold topography and the elastic properties of the PDMS micro-lattices. Our results demonstrate that the 3D PDMS micro-lattices improve the survival and growth of cells as well as the network formation of neurons. We believe that such an enabling technology is useful for research and clinical applications including disease modeling, regenerative medicine, and drug discovery/drug cytotoxicity studies

Dynamics and reproducibility of a moderately complex sensory-motor response in the medicinal leech

Local bending, a motor response caused by mechanical stimulation of the leech skin, has been shown to be remarkably reproducible, in its initial phase, despite the highly variable firing of motoneurons sustaining it. In this work, the reproducibility of local bending was further analyzed by monitoring it over a longer period of time and by using more intact preparations, in which muscle activation in an entire body segment was studied. Our experiments showed that local bending is a moderately complex motor response, composed of a sequence of four different phases, which were consistently identified in all leeches. During each phase, longitudinal and circular muscles in specific areas of the body segment acted synergistically, being co-activated or co-inhibited depending on their position relative to the stimulation site. Onset and duration of the first phase were reproducible across different trials and different animals as a result of the massive co-activation of excitatory motoneurons sustaining it. The other phases were produced by the inhibition of excitatory and activation of inhibitory motoneurons, and also by the intrinsic relaxation dynamics of leech muscles. As a consequence, their duration and relative timing was variable across different preparations, whereas their order of appearance was conserved. These results suggest that, during local bending, the leech neuromuscular system 1) operates a reduction of its available degrees of freedom, by simultaneously recruiting groups of otherwise antagonistic muscles and large populations of motoneurons; and 2) ensures reliability and effectiveness of this escape reflex, by guaranteeing the reproducibility of its crucial initial phase