What makes a neuron a neuron? Using pond snails to explore intracellular properties of neurons. Stephen Hauptman 1, Carol Ann Paul 2, Patsy Dickinson 1, and Bruce Johnson 3 1 Department of Biology, Bowdoin College, Brunswick, ME 04011, 2 Department of Biological Sciences, Wellesley College, Wellesley, MA Department of Neurobiology and Behavior, Cornell University, Ithaca, NY Support Contributed by: NSF Due and DEB Active Properties Cells in the buccal ganglia are part of the snail’s feeding pattern and show regular spontaneous postsynaptic potentials. Cells showing spontaneous psps can be hyperpolarized to find the reversal potential. Psp amplitude can be plotted as a function of membrane potential. The x-intercept of the resulting line is the reversal potential. Synaptic Properties Introduction To demonstrate the functional properties of neurons to students, we use the criteria enumerated by Henriette van Praag et al. 1 to determine whether newborn neurons behaved as functional neurons, as defined by their physiological properties: Through these exercises, students can start to answer the question: what makes a neuron a neuron? We use pond snails because they are easy to maintain, moderately easy to dissect, cells are amazingly easy to visualize and cells are extremely active. Many of the cells have also been identified and mapped. The cells in pond snail preps show a range of spontaneous activity patterns, from cells that are quiet at rest, to beating cells, to cells showing regular bursts. These exercises may take several weeks in the lab. Students who do these exercises end up not only with an excellent understanding of physiological properties of a neuron, but also with a thorough grounding in the use of electrophysiological equipment. 1 van Praag H, Schinder A, Christie B, Toni N, Palmer T & Gage F. 2002, Functional neurogenesis in the adult hippocampus Nature 415: Input resistance can be determined over a range of membrane potentials by creating a V-I curve for both hyperpolarizing and depolarizing current pulses, and determining the slope. Notice in this example that input resistance decreases once threshold is reached (as indicated by the decreased slope), indicating the opening of voltage-gated channels. Action potential frequency can be plotted as a function of current pulse amplitude. Students can consider why the frequency levels off and the action potentials diminish in amplitude. The action potential threshold can be determined by finding the membrane potential at which any further depolarization triggers an action potential. In this example, the threshold is -52 mV. Many cells show post-inhibitory rebound. This can be demonstrated by hyperpolarizing the cell and then releasing the inhibition. Membrane conductivity can be compared during psps and between psps by injecting current pulses long enough to come to a new steady-state voltage. The amplitudes are compared. Smaller amplitude indicates reduced resistance and increased conductance. In this way, students can determine whether channels are opening or closing during the psps. Time constants can also be compared as another measure of changed conductance. During pspBetween psps epsps In this student lab, we use and expand on these criteria in pond snail species such as Lymnaea or Helisoma. We have developed a set of exercises based on these snails, which can also be done with any intracellular prep. The Pond Snail Preparation The pond snail brain is located just posterior to the buccal mass. There are large, easily penetrable cells in the parietal, visceral and buccal ganglia. To get microelectrodes through the sheath that surrounds the ganglia, the ganglia must first be treated with 0.5% protease (45 seconds for buccal ganglia, one minute for ring ganglia). Input resistance can be calculated at a given membrane potential by calculating V/I. The overall capacitance of the cell can be determined by calculating Tau/R. Passive Properties Resting membrane potential can be established upon successful impalement. The time constant can be determined by injecting a current pulse and calculating the time it takes to reach 63% of the voltage change. epsps Acknowledgements We would like to thank the students in Bowdoin’s Bio 253, Neurophysiology, for the use of data collected in that class. Students can easily master the dissection with the assistance of the instructional videos available at l/theneuronconnection.html. ipsp s Conclusion These exercises clearly reinforce the idea that neurons are special types of cells. This study of snail passive and active membrane properties is useful in helping students define what properties make neurons special and different from other cell types. It answers the question: what makes a neuron a neuron?