Toolbox/Lecture 1

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WIKIPEDIA BIO154/254: Molecular and Cellular Neurobiology

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Contents


Lecture 1 Model Systems

What are the advantages of each?

Mammalian visual system

The Mammalian visual system, of which the human visual system is an important member, is among the most complex in the animal world. The mammalian visual system is composed of several components, listed in the order which they act: the eye itself (consisting most importantly of the lens and the retina), the optic nerve, the optic chiasm, the left and right optic tracts, the lateral geninculate nucleus, the optic radiation and lastly the visual cortex itself. The light focused by the lens falls on photoreceptors in the retina, which, in humans, are bound to two different proteins called opsins: rodopsins (in rods) and cone opsins (in cones). The human, and some primates', visual system is different from most other mammals in that we have one extra type of cone opsin that allows us to distinguish colour. Most mammals are colourblind; colour evolved fairly recently in a sub-branch of the primate evolutionary tree. Despite this, the mammalian visual system (i.e. that of mammals other than the higher primates) is a powerful model for study as most of its components are analogous to those in humans. For further information on the specific components of the visual system, see lecture 8 below.

Mammalian olfactory system

Mammalian sense of smell depends on chemoreceptors. The olfactory sensors are sensory neurons embedded in a layer of epithelial tissue at the top of the nasal cavity. These neurons directly project their axons to the olfactory bulb of the brain (i.e. they are not relayed through the thalamus like other senses' axons). Their dendrites end in olfactory hairs on the surface of the nasal epithelium. Each functional olfactory receptor protein that is expressed is found in a limited number of sensory cells in the olfactory epithelium. All of the cells that express the same receptor protein project to the same regions in the olfactory bulb. A given odorant molecule may bind to one or to more than one receptor protein. Therefore each odorant molecule can excite a unique combination of cells in the olfactory bulb, so an olfactory system with even hundred of different receptor proteins can discriminate a large number of cells. Interestingly, the more odorant molecule that bind to receptors, the more action potentials are generated, and the greater the intensity of the perceived smell.

Spinal cord motor neurons

The spinal cord may be divided up into sections based on the nerve roots that extend from it. At each segment, rootlets come out of both the dorsal and ventral halves of the spinal cord. In fact, nerves go in through the dorsal side and then exit through the ventral face. The dorsal horns respond to sensory perception by receiving axons from the periphery via the dorsal root. The ventral horns contain motor neurons with axons that leave the cord via the ventral roots and travel to stimulate the muscles. The dorsal root ganglion contains a collection of cell bodies that have all the receptor neurons sending processes to the peripheral muscles. These are the motor neurons located in the spinal cord. Lower motor neurons are either alpha or gamma cells. Alpha cells comprise the principle motor neurons of the spinal cord and are a part of the main portion of the common reflex pathway. These neurons conduct rapid motor impulses; each alpha cell innervates about 200 muscle fibers. Gamma neurons, also part of the common pathway, are only half as numerous as alpha cells. They conduct slower motor impulses and their major function is to stretch muscle spindles.

Human brain

As the most anterior part of the central nervous system, the human brain acts as the primary control center for the peripheral nervous system. Autonomic functions of the brain include controlling heartbeat, digestion, respiration, sensation, and movement. Higher order functions of the human brain include conscious activities like thinking and reasoning. The human brain is unique from other mammalian and vertebrate brains because it contains millions of billions of synaptic connectins, resulting in a complex and dense neural circuit. Neuroscience is the study of the brain and its functions; psychology is the study of the mind and behavior; and neurophysiology is the study of normal healthy brain activity. Advantages of using the human brain system for study is that results obtained will be the closest model system for determining the causes and pathways of mental illness in humans. Neuroimaging allow scientists to study the brain not only in detail, but in real time (functional neuroimaging). The human brain is most often compared with a computer: individual neurons are analogous to microchips, and specific areas of the brain are said to resemble graphic cards. However, no clear sequential (one-to-one command) set of instructions are consistently observed in the brain, and the study of the human brain directly cannot be replaced with computer models with great accuracy.

Lecture 1 Techniques

What can these be used for?

Golgi staining

Golgi staining labels a subset of cells within the brain in black and brown, while leaving others completely unstained. The stains are performed by injecting potassiumdichromate and silver nitrate; the resulting brown-black color stems from the microcrystallization of silver chromate. In the affected subset of neurons, this technique stains the cell soma, as well as the axons and dendrites along their entire length. The exact mechanisms behind the Golgi stain are not well understood: it is not clear why it labels a small percentage of neurons in their entirety, while leaving the rest unstained. Nevertheless, this property of the stain makes it very useful for neuroanatomists trying to visualize, track, and map neural circuits. Indeed, if the Golgi stain affected all neurons and cellular processes, it would be hard to discern the structure of individual neurons.

Tissue culture

Tissue cultures allow researchers to grow tissues and/or cells outside of the organism under investigation. Primary cell cultures usually have a finite life span in culture compared to abnormal cell lines or transformed cell lines. The availability of tissue cultures enable the study of cells in a controlled environment without the external influences found in the organisms' physiological environment. The advantages of performing a tissue culture (as opposed to working in vivo) include the ability to study specific cellular mechanisms without the influence of surrounding tissues, and the opportunity to manipulate cell lines to better understand developmental abnormalities. In Lecture 1, we discussed an example of tissue culture with Ross Harrison's Tissue Culture Experiments that sought to resolve the conflict between Golgi (reticulum) and Ramon y Cajal (neuron doctrine).

Electron microscopy

Through the use of electrons to create an image of the object, electron microscopy provides higher magnification and superior resolving power than a light microscope by almost a magnitude of two million. Various electron microscopy techniques exist for exploring morphology and mechanisms: scanning electron microscopes give a 3D image of the sample; transmission electrion microscopes produce 2D images at impressive magnifications (up to 500 million times); and scanning tunneling microscopes determine the height of the sample surface. Electron microscopy provided a much more detailed look at the finer structures of the nervous system. This allowed for clear identification of the presynaptic terminal with vesicles, postsynaptic cell, as well as the synaptic cleft.

Biolistic transfection (gene gun)

This technique injects cells with a heavy metal coated with plasmid DNA, and is capable of transforming almost all types of cells including their genetic information and cellular organelles. Gene guns are also effective in delivering DNA vaccines to mammals for therapy.

Genetic labeling

A recent method developed for detecting transposition and may genetically label conjugative plasmids that do not result in an apparently identifiable phenotype. Genetic labeling results in the transformation of the host organism with a plasmid containing a heterologous DNA fragment. This DNA labels genetic areas of interest which may then be visualized. The method is also beginning to be studied in vivo, where mice can be orally inoculated with genetically-labelled probiotic bacteria plasmids to study products of the digestive tract.

Patch clamp

The patch clamp method allowed detailed understanding of the action potential after it was invented by Kenneth Cole in the 1940s. This method enables us to measure the membrane potential, or voltage, at any level desired by the experimenter through use of a microelectrode placed inside the cell. There are severable variations of the technique. An “inside out” patch is created when a portion of the cell membrane is ripped off, leaving the intracellular surface of the membrane oriented towards the media. This enables the study of intracellular components’ (e.g. ligands) that influence ion channels. An “outside-out” patch, on the other hand, allows the researcher to study the properties of an ion channel when it is exposed to a novel extracellular environment while having the same intracellular components. The voltage clamp technique reveals how membrane potential influences ionic current flow across the membrane, and was instrumental in providing Hodgkin and Huxley with information leading to membrane ion gradients and the action potential.

Electrical stimulation

Coupled with the patch clamp technique, electrical stimulation is a form of manual current that may be delivered to the neuron under investigation in order to study its electrophysiological properties in a controlled manner. Usually only a few milliAmps are applied to the neuron to evoke passive responses. Greater current is introduced to evoke action potentials. Through this manner, the firing threshold for various types of neurons may be quantitatively determined with great accuracy. Furthermore, recent experiments have used this method to purposefully stimulate neocortical neurons to study the effects of prolonged activity (or "stimulation") on axonal growth.

fMRI

Functional magnetic resonance imaging is a technique used to visualize not only the neural anatomical images created by traditional MRI scans, but also overlaid images of event-related hemodynamic responses in the brain. The hemodynamic activation levels refer to the amount of blood oxygenation ocurring at a particular "voxel" of the image, which is a kind of three-dimensional pixel. This hemodynamic response is often referred to as BOLD (blood-oxygen level dependent) contrast. High BOLD contrast reflects a decreased amount of deoxygenated hemoglobin present in the brain. General changes in BOLD signal are highly correlated with changes in blood flow to different regions of the brain.

Images of both anatomical and functional (BOLD) data are recorded every few seconds. Data can be analyzed in such a way as to contrast the activations associated with two separate paradigms, effectively subtracting the activation of one dataset from another and presenting the difference visually. This technique is generally applied to psychophysical ventures, quantifying the results of a multitude of psychological questions.

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