Hirudo Medicinalis Local Bending Myomodulin Effects on the P to AP Synapse Masha Day and Eviatar Yemini PHYS 173 BGGN 266 Department of Physics University of California San Diego La Jolla CA 92093 INTRODUCTION Leeches display a hierarchy of reflexive behaviors A moderate side poke elicits local bending in leeches Feeding is high in the hierarchy and local bends are suppressed during feeding The control mechanisms by which this hierarchy is enforced are of great interest within the field In our experiment we investigated the effect of myomodulin on a medicinal leech s local bending circuit Sean Lockery s paper Two forms of sensitization of the local bending reflex of the medicinal leech tested whether the Leydig cell a modulatory neuron that releases myomodulin a known mediator of sensitization decreases the strength of the leech s local bend reflex Originally he found that stimulating the Leydig cell which results in the suppression of local bending released neurotransmitter octopamine However in a later paper he learned that the neurotransmitter released was actually myomodulin This lead us to our experimental question does myomodulin suppress local bending in a leech We investigated this question by looking at paired pulse facilitations to test whether myomodulin decreases the probability of release at P cell synaptic terminals The local bending circuit of the leech begins at the pressure cells P cells continues through local bend interneurons and completes at motor neurons that execute the bend fig 1 Theoretically to test the effect of myomodulin on leech bending it would be best to record from a P cell and an interneuron however the small size of the interneurons involved 10 microns in diameter makes them difficult to impale and record from P cells also synapse onto large anterior pagoda AP cells that are 60 microns in diameter allowing easy impaling and recording If myomodulin has an effect on leech bending then the P synapses may experience a generalized decrease in probability of release and we might see a corresponding paired pulse facilitation onto AP cells SKIN Pv LBI LBI LBI Pd LBI LBI Pv LBI LBI iMN d iMN v iMN v iMN d eMN v eMN d eMN d eMN v LBI LONG ITUDI NAL MUSC LES LBI Pd Fig 1 The Leech Local Bend Circuit THEORY The theory of action potentials on which our experiment is partially based rests on the Hodgkin Huxley model This model uses a set of non linear ordinary differential equation to approximate the electrical characteristics of neurons Originally created to describe the ionic mechanisms of initiation and propagation of action potentials in a squid giant axon it works perfectly to describe the spiking dynamics of other neurons in our case the leech P and AP cells The model accounts for action potentials of a neuron by a triggered rapid inward current of Na ions followed by a slower activated outwards current of K ions The currents result from independent permeability mechanism for Na and K whose conductance changes as a function of time and membrane potential The flow of the ions are independent mechanisms thus voltage gated ion channels and leak currents are physiologically responsible for the action potential The model successfully creates an electrical analog for each component of the excitable cell by statistically determining when channels open and close 1 The formula for the three membrane currents is given below Ik gNa m3h u ENa gK n4 u EK gL u EL The leak channel is described by a voltage independent conductance gL 1 R however the conductance of the other ion channels is voltage and time dependent When the channels are all open currents are transmitted with maximum conductance gNa and gK Their probability of being open is described by variables m n and h M and H controls the Na channels while K are controlled by N Ena Ek and El are reversal potentials whose values are derived from the Nernst equation and based on the ionic concentrations inside and outside the cell 1 The theory is applicable to a wide range of neuronal responses including spiking and EPSP which we observe in the experiment The P and AP cells in the leech from which we recorded are easily accessible for intracellular recording The medicinal leech has a simple well studied nervous system as well as very large distinct nerve signatures that accompany its behaviors swimming feeding etc These make it an ideal animal for physiological research A central nerve cord connects 21 separate ganglia in addition to a head and tail brain fig 2 Each ganglion contains approximately 350 neurons fig 3 of which 4 are pressure P cells and 4 are AP cells 3 Fig 2 Leech Neuroanatomy A central nerve cord connects 21 separate ganglia in addition to a head and tail brain Fig 3 Ganglion Each ganglion contains approximately 350 cell bodies Leeches respond to a moderate poke in the side with a reflexive bend localized in the area of applied pressure However local bending has been to be suppressed in the presence of myomodulin 2 Myomodulin a peptide has been shown to play an important role in the modulation of the properties of leech muscles and neurons that are associated with specific behaviors such as feeding and locomotion Myomodulin is present in neurons that modulate neuromuscular transmission Pressure P and Leydig cells as well as their coupling interneurons are immunoreactive for myomodulin 5 To test the effect of myomodulin on local bending we used paired pulse facilitation PPF to search for a decrease in the probability of release at the synapse from P onto AP To test PPF a P cell is given a train of 2 stimulations fig 4 resulting in spikes and the resulting EPSP responses in the AP cell are recorded A decrease in the probability of release corresponds to less vesicles being released when a spike arrives and results in a smaller recorded EPSP When the second spike arrives with sufficiently small delay residual calcium ions left over from the first spike combine with the new calcium influx to create a larger second EPSP fig 5 We calculate the paired pulse ratio defined as EPSP2 EPSP1 When the ratio has a value greater than 1 it indicates facilitation and correspondingly a decrease in the probability of release of vesicles at the synapse 4 Fig 4 A train of two pulses delivered to the P cell Fig 5 Paired pulse facilitation The second EPSP is larger than the first due to residual calcium ions left over in the presynaptic terminal from the first spike EXPERIMENT SURGERY The leeches that were used for this