The intracellular recording and stimulation techniques described here provide a foundation for pharmacological assays and research in neuroethology and development. Understanding the synaptic connections within the animal as well as recreating the same type of connections in a culture dish are powerful tools for investigating synaptogenesis 16-18 as well as pharmacological profiling of synapses 19. Some connections are bidirectional while others are unidirectional chemically but bidirectional electrically 7. The ability of some leech neurons to form both electrical and chemical communication is intriguing. The connections that are used which underlie a swim pattern 13,14 and rhythmicity of the heart 15 have been well characterized in the leech. In principle one can hope that the study of less complex systems such as the leech could help to define basic mechanisms used in higher animals. In mammalian brains it is a daunting task to explain behavior in terms of the properties and connections of individual identified neurons. The leech CNS has also served as a valuable preparation for investigating mechanisms involved with neuronal repair and regeneration in the whole animal and in culture 4-6,9-12. Studies in the leech ganglia have provided insight into the development of functional circuits that can be measured with electrophysiology as well as with whole animal behavior studies. From investigations in the pattern of innervation and branching of individual neurons within the CNS, the synaptic connections have been reproduced in culture with identified neurons 2,8. Ionic currents that give rise to the various shapes of the characteristic action potentials in various neurons have been identified 4-7. Due to the relatively large somata of the neurons within a ganglion they are visible with a dissecting microscope, and the somata can be selectively impaled with microelectrodes for recording their electrical properties.ĭyes and large molecules can be injected into individual cells and channel properties studied by patch clamp. Knowledge of the identity and function of a neuron is advantageous for investigating neural circuits and for making experiments with a given cell type. The unique location and morphology of neurons in leech ganglia were noted by Retzius as early as 1891 3. Moreover neurons can be removed from the animal and cultured in a suitable medium in which the cells maintain their electrical characteristics for as long as 45 days 1,2.Ī distinct advantage of the leech ganglion for learning how to make electrophysiological recordings is that cells are characteristically and reliably arranged in the ganglion in a pattern that allows identification of distinct cell types from ganglion to ganglion and from animal to animal. The useful property of leech neurons is that the shape of their action potentials and biophysical properties are characteristic for a given cell type ( i.e. The leech preparation is useful for investigating the function of identifiable (sensory, motor and inter-neurons within the animal's central nervous system (CNS). They are also an appropriate training module for neuroscience or physiology teaching laboratories. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology.
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