Leech Hivudo medicinal has for many years gained popularity in the neuroscience; this is mainly due to their medicinal benefit. They serve as an ideal preparation for neurobiology experiment, and helps scholars to understand certain cells in the human being nervous system. The same cells are also present in leech's nervous system. The field of neuroscience involves the measurement of the electrical activity of neuron. This is done precisely through an intercellular recording technique, a study that explores the electrical properties of biological cells and tissue (electrophysiology).This study also includes the measurement of the action potential properties.
Leech segmental ganglion holds the evidence describing the interaction between homogenous neurons in the nervous system .Similar single neurons visible in the leech ganglion are also be present and can be identified in other different animals. This is particularly due do their similarity in sizes and shape characteristics. These same neurons have a similar way of responding to certain stimulants.
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To understand physiological and morphological result of certain neurons, intercellular recording technique to identify neurons by their position and membrane properties is applied. Also the observation of neuronal morphology by application of fluorescent dyes, and a determination of receptive fields of sensory neurons is good reference frame in trying to answer some of the neuroscience questions pertaining nervous system. Neurons are proposed to normally progress in two definite stages during their early development. In one of the stages, these neurons are insensitive to interaction with their homologous neurons and a second in which they are sensitive to the homologous neurons. Different morphological and physiological measures can be conveniently serve to identify different neurons.
For quite great deal of years now, certain single neurons have been easy to identify due to their distinct characteristic that are easy to identify. These characteristics include the shape and size of the same neurons in different animals. Retzius cells serves a good example, it is in the centre of the vertical surface of leech segmental ganglion.Retzius cells have a distinguished characteristic of size, they are exceptionally large in size as compared to other cells on the leech segmental ganglion and thus they easily visible.
Mammalians Retzius cells produces substantially higher amount of the reelin. Reelin serves as a signal to dissociation for migrating neurons, and also a neuronal positioning in the developing brain. Reelin continues developing in adult and is also found in the spinal cord and blood and other organs and tissues in human body.
Leech neurobiology was started in the 1960's, by john Nicholls seconded by others like Bullock who proposed the concept of equivalent sets of adressable neurons in higher animals. The outcome of Bullock's concept was that; neurons exhibit a wide spectrum of morphological and physiological properties which are unique and defines the neuron. The doctrine of neuron by the Swedish anatomist Gustav Retzius, proposed that that, nervous system contain variety of discrete cells.
Bullock's concept of neurons in higher animals, laid a staring ground that enabled the understanding neural function and the control of behaviors in terms of the properties and interaction of single neurons. With the help of the intracellular recording, approximately 200pairs of bilaterally symmetrical neurons as well as several unpaired neurons are found to be contained in the leech's ganglion. 8 or 9possibly 9 of the 200 are readily accessible due to their large sizes. This are the Retzius cells, the touch sensitive neurons are equal to 3 pairs, the pressure sensitive neurons are 2 pairs, the nociceptive neurons 2 pairs, the anterior pagoda 1 pair and possibly one pair of the annulus erectors.
These neurons respond to the mechanical stimulation of the body wall in a way suggested in their names. Leech ganglion is highly ordered and is divided into 6 packet characterized by; 2 anteriolateral, 2 postero-lateral, 1 central anterior and central posterior. A given set of this neuron is always in the same packet in a characterized position and every neuron in packets has a matching homologue on the other side.
The morphology of the neuron is possible. By examining the projection of axon and the dendrites structure, this is revealed by the intracellular injection of dye, fluorescent dye Lucifer yellow CH, introduced by Walter Stewart in 1978 is used in neuronal basis of behavior study. This is because, it is easy to inject and one can always know which neuron provides the physiological data. Since each neuron ha as characteristic membrane properties, individual neuron can be distinguished. This is done with the aid of their characteristic branching patterns in the roots; the absence of branches in the roots is usually indicative of interneuron.
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Leech was dissected and individual ganglia in a dish. The ganglionswere pinned on the plastic resin contained in the Petri dish so that the ventral surface is uppermost. A working sketch of the ganglion was made, drawing the margins as they appeared in the preparation and the shape of the largest neurons.
The cells were numbered to help keep track of them as their physiology was being determined. The electronic instruments and the computer were set ready. One of the Retzius cell was penetrated followed by a pressure (P) and nociceptive (N) then touch sensory cells were recorded. Anterior pagoda (AP) and finally annulus erector (AE) were tested for, each time penetrating a neuron and noting the number in the diagram. To make sure that no cell was lost, the experiment was done much quickly collecting data only without the analysis.
In the second part of the experiment, a drawing o the ganglion was made, and all the possible cells identified using their difference in physiological characteristics. Upon identification the all the cells were numbered to assist in keeping track of the cells that were penetrated.
A Lucifer yellow electrode filled with 2M LiCl was mounted on the electrode holder. As the cells were penetrated, some physiological characteristics of the cell were established.
When the penetration appeared to be stable, hyperbolize pulses with the stimulator were continuously delivered, traces of action potential were captured. The pulse was carefully turned off and the electrode withdrawn.
The instructor mounted the ganglion on a slide in glycerol with a cover slip. The preparation was then taken to the fluorescence microscope and the neuron photographed. All cell body position was noted and the major neuritis branching pattern. This was done quickly to avoid gradual bleaching of the fluorescence by the intense light
In the last part of the experiment, a ganglion attached to its piece of segmental skin was provided. The dissection consisted of a slitting opening at the back of the leech, it was pinned out and a small hole cut in the ventral skin to expose the ganglion. The ganglion was observed through the hole made in the ventral surface. A drawing of each dissection of the receptive fields was made to help indicate where exactly the stimulation of the cell was.
Since ganglia are usually much healthier, and always last much longer when attached to the skin as compared to the isolated ganglia, this was handled more carefully since only a limited supply of these intricately dissected preparations were available. As soon as the bath was entered, the electrodes were checked for any breakage and also all the electrode that had leaked into the dish replaced with the ringer's.
Once a reasonable electrode of about 20-50microohms, they were placed away to avoid hitting of the electrode. The following order of cells was used in the first attempts, as it is obvious that P, T, N and then AE. The P cells are large enough within some time are easily recognized for the first attempt of the day although the ganglion were under tension and could be distorted. Once the P cell was penetrated, action potential was extracted by the current injection. Once an action potential was obtained by touching, its shape was recorded and compared to the spike elicited by current injection, any difference in the height and the rise in impulse was noted and recorded
The receptive fields were carefully mapped paying attention to the anatomic landmarks, defining whether the cell was sensitive to dorsal or ventral skin stimulation. The boundaries of the receptive cells were observed and carefully noted in the drawing. Once done with the mapping of the receptive field for one of the P cells and a good data obtained the same procedure was used on T cell. Done with the P and T cells, N cell was tried. When all of the above possibilities have been attempted, and the ganglions were still alive, recording from the AE cells was tried.
In this four weeks experiment, the first two weeks the neurons are identified by their position and membrane properties, using the intercellular recoding techniques. The third week involves the observation of the neuronal morphologies by intercellular application, while the fourth week, the receptive fields of sensory neurons of recording from ganglia attacked to segmental skin.
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Identification by physiology of sensory, neurons are easy due to certain neurons are present in different animals. To help in support of this doctrine, leech neurons were used as example to support the concept that nervous system contains variety of discrete cells.
Neuroscience includes measurement of voltage or currents across a membrane of a cell. This is attained by the intercellular recording techniques. Axograph or oxcoscope assists in the interpretation of the electrical activities of neurons. By penetrating a neuron it can be classified according to the physiological properties. Each neuron has a particular intensity of impulse which can be used to identify certain neuron.
The resting potential differs from the time immediately the neuron is penetrated, to time later after penetration .For a healthy leech neuron,the resting membrane potential is supposed to be between -40 to -50 mN. Also, most healthy neurons shows smooth amplitude in their impulse and does not overshot, this is always the same at the start of penetration and also later after penetration.
During analysis, V, the deviation from the resting potential is plotted as a function of me the injected current. The slope to the curve v-I is supposed to be a straight line where the slope giving resistance R. whether the curve s linear or there is a rectification the results are only as good as the electrode compensation.
For higher current intensities, the data may alternate even after being done carefully. To avoid this, its advisable to use two electrodes in this experiment so that, one electrode can be used for passing current and the other one for recording the change in voltage.
From the data obtain, the different factors were calculated, to calculate the input resistance, it can be obtained from the graph of v-I as it is the slope. Also R=V/I is a better formulae to calculate the input resistance. When there is a potential change across the membrane, the new steady state is slowly attained. This is because the membrane has the ability to hold charge and therefore is a capacitor. When a current is applied to a capacitor the voltage changes exponentially to reach a steady state. This exponential change by the time constant Tau (τ) which can be calculated with the following formula: τ = RxC. From the earlier calculation of input resistance, r can be used in calculations of the time constant here another way of calculating the time constant, is by the graphical method .plotting of all logarithms of voltage for each time point on a charging curve can be done Since Vt=V0 e -t/Rc .where T=time Vt= voltage at time t, Vo=voltage before the decay.
Membrane capacitance can also be calculated using the values obtained from the input resistance and the time constant. In this method, C = τ/ R, a surface area of the cell can be estimated from this formulae. By assuming that the cell is spherical, diameter (D) can be estimated also. This is supported by the mathematical fact that a sphere = (S/π). This estimated diameter can be compared with the measured diameter of soma visualized following Lucifer Yellow injection.
Another important calculation done in this experiment is the Sodium current density. This calculation is done to estimate the sodium current density for each cell. For an isopotential neuron, the flow of ionic current into the cell is exactly enough to charge the membrane capacitance to a new steady state membrane potential. Since there is no net flow of current, this current is equal but opposite to the capacitive current. The maximal rate of depolarization, also the maximum slope in the upswing is due to activation of sodium current. Sodium current can be given as a function of the capacitance and voltage change, thus giving the following formulae; Sodium current= -C x dV/dt.
Alternatively, sodium current from different neurons can be stabilized by dividing the value sodium current for each cell by the estimated cell's surface area. This stabilization allows for the direct comparison of the properties of different neurons.
The identification of other neurons can be done with the aid of actions such as the magnitude.
To identify T cell, P cell and N cells, morphology of the neurons records is put into consideration, the three cells are indeed unique and this can only be verified by the help of the projections of axons and the dendrites structure revealed by the intracellular injection dye. Fluorescent dye Lucifer Yellow CH best fits the task due to its ability to easily inject and identify which neuron provided the physiological data. It can as well delete the individual labeled neurons from a circuit by irradiating them with ultraviolet light. Fluorescence microscopy works by illuminating the preparation with a short ultraviolet light wavelength light.
The low wavelength light is deflected onto the specimen by what is called a diachronic mirror and is seen through oculars.
Using the lithium salt Lucifer Yellow CH is injected into the cell with the passage of hyperpolarizing current, this is because; the dye is both bivalent and negatively charged.