Neuroscience - WEEK 7 Examination Notes PDF

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WEEK 6: NEUROSCIENCE METHODS Neural Tracing – Structural Connectivity 

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Neural tracing is the methodology that allows us to unravel the complexity of neural pathways o Connectivity between neurons gives rise to the functional diversity/capabilities of the nervous system o Complexity of neural pathways has increased throughout vertebrate evolution - increased number of functional connections Neural pathways are composed of many neural connections working in concert o Complex – terminology complex, diversity of connections is complex o Convoluted – things rarely travel in a straight line, large numbers of connections/diversions o Homogeneous – white/grey matter, neural pathways cannot be detected through macroscopic/microscopic detection Degeneration Studies  Earliest studies conducted on animals to study neural pathways and circuits  Involved applying a scalpel to a region of the nervous system to create a lesion o Lesions cause axon degeneration in the desired neuronal pathway  The soma contains the metabolic machinery of a neuron and is separated from the axon through lesioning o Lack of nutrient supply to axon/axon terminal induces morphological changes in the NS through axonal degeneration  Axonal degeneration allows us to identify a neuronal pathway and its site of termination o Morphological changes within the NS 

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Example: Studying the thalamocortical projection (green cells) o Lesion the thalamus of the NS o Gradual axonal degeneration throughout neural pathway Limitations: o Visualisation of degenerating axons is difficult microscopically o Axon degeneration is time dependant - occurs at a different rate depending on the site of induced lesion o Lesioning is imprecise – inadvertently damage other axons passing through lesion area producing false positive results

Neuronal Tracers  Substances that are taken up and transported along neuron length  Two major types depending on direction of travel: o RETROGRADE (backwards)  Travel towards the cell body from axon terminal  Allows us to determine the origin of neural pathway o ANTEROGRADE (forwards)  Travel away from the cell body to axon terminal  Allows us to determine the termination of neural pathways  Neural tracers become more specific throughout evolution o Tracers don’t travel past axon terminal – not transsynaptic o Non-specific  Horseradish Peroxidase

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Enzyme causes damage to nervous system after injection Neurons uptake enzyme allowing us to visualise neural projections Any neuron that is damaged via injection can uptake the enzyme (NONSPECIFIC)  Lipophillic Tracers  Dissolve/diffuse along the lipid bilayer of axons/neurons  Any neuron that is damaged during administration can uptake the tracer (NON - SPECIFIC)  Slow – diffusion occurs slowly  Image: Tracer applied to a ganglion in PNS, fluorescent red visualisation of neuronal pathway Site- specific  Only taken up by intact neurons at injection site  Plant extracts (proteins)  Molecules that have high binding affinity to carbohydrates expressed on neuron plasma membrane  Binding triggers endocytosis of tracer via vesicles  Wheat – germ agglutinin (WGA)  Phaseolus vulgaris leucoagglutinin (PHA-L)

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Population-specific  Tracers are taken up by functional subclasses of neurons  Plant extracts (proteins)  Molecules that have high binding affinity to carbohydrates expressed on neuron plasma membrane  Binding triggers endocytosis of tracer via vesicles  Bandeiraea simplicifolia isolectin B4 (IB4)  Black = primary sensory neurons in ganglion  Only some neurons uptake the tracer due to carbohydrate specificity

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Transsynaptic  Allows us to highlight an entire neuronal pathway  Rabies virus – continually replicates and travels retrogradely

Electrophysiology – Functional  Study of electrical activity in excitable tissues (nervous system): cover entire range of resolutions o Whole Brain o Single Cells o Single Channels o Potential Difference  Equipment: experiments performed in Faraday’s cage to reduce electrical noise o Recording the potential difference between the electrode and ground (0 mV) o Amplifier required as signals recorded are small o Filters allow us to ignore background noise (parts of signals we interested in studying)

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Oscilloscope plots potential difference against time Stimulator provides electrical stimulation in the form of a square pulse to depolarise neurons adjacent to leads

Electroencephalogram (EEG) 

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Spontaneous electrical activity of brain o Recorded by large numbers of electrodes placed on the scalp o Each trace shows the potential difference across two electrodes Different pairs of electrodes give different viewpoint of electrical activity in brain Poor spatial resolution o The brain is electrically far away from electrodes o Difficult to pinpoint the region of brain associated with each trace Good temporal resolution o Electrical activity is changing in real time Applications o Diagnosis of epilepsy– irregular high electrical activity in regions of the brain o Diagnosis of sleep disorders o Criteria for diagnosing brain death

Field Potentials  

Increased resolution Evoked electrical activity in central nervous system: o Recorded by a single electron on/in brain or spinal cord o Produced by synchronous activity of 1000’s of neurons o Often very small so requires signal averaging:  Signal averaging is used to increase the resolution of trace  Noise is random – electrical noise isn’t related to experiment  Signal is time-locked to stimulus

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Signal Averager:  Enhances signal  Reduces noise

Applications: o Identifying functional areas:  Establish connections (low resolution)  Identify regions to lesion

Compound Action Potentials 

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Evoked electrical activity in the peripheral nervous system: o Stimulus is required o Recorded by a pair of electrodes on/close to peripheral nerves (e.g sciatic nerve, crual nerve)  Stimulator produces AP at one end of the peripheral nerve  AP travel along nerve  Potential difference is recorded at opposing end o Produced by synchronous activity of 1000’s axons Applications: o Clinical diagnosis

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Peripheral neuropathies: Traumatic nerve injury (e.g burning pain to lateral surface of the hand) Diabetic neuropathy

Experiment: o Provide electrical stimulation at the elbow/wrist o Record the potential difference at the motor or sensory recording sites o Action potentials travel to the motor/sensory sites after stimulation o Allows us to determine if there is damage to the median nerve (e.g during its course though carpal tunnel)

Extracellular Single Unit Recording  Responses in single neurons: o Microelectrode with conductor is placed close to single cell  Microelectrode = glass pipette with strong NaCl solution for electrical continuity  NaCl is the conductor o Stepping-drive motor is used to drive microelectrode into brain/spinal cord  Electrode is moved as close as possible to the neuron o Electrical activity of a single cell isolated  Applications: o Establishing basic functions of individual neurons:  Psychophysical studies  Try to correlate activity in a single cell with what the subject may report  E.g polymodal nociceptors are responsible burning pain o Experimentally, record the action potential frequency in a single cell via burning the skin o Ask the subject to rate their degree of burning pain o Determine a linear relationship between degree of pain and action potential frequency  Clinical lesions o Surgeon lesions a portion of the brain after using microelectrodes to perform mapping o Mapping allows them to pinpoint the neural circuit responsible for epileptic seizures etc.  Pharmacology of nervous system  Record the electrical activity in a neuron with and without a drug  Allows us to determine the effects of drug (e.g does it increase/decrease action potential frequency)  Advantages: o Studying single cells – high resolution o Fairly easy to perform  Disadvantages: o Only see action potentials  No EPSP or IPSP  No insight into integrative processes – sub threshold excitation/inhibition not visible o No idea what cells you are recording from.  Cannot determine the physical attributes of neuron or the mechanism of action (e.g type of neurotransmitter use) Intracellular Recording 

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Recording membrane potential of a single cell o Microelectrode is place within the cell to record potential difference  Fine micropipette filled with KCl (potassium – chloride) solution  Potassium as the intracellular concentration is innately higher  Stepping-drive motor used to drive microelectrode into the cell  Classical picture of an AP is visualised Applications:

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o Establishing the ionic basis of the membrane potentials (AP) o Studying synaptic inputs (e.g EPSP or IPSP can be visualised) Advantages: o Higher resolution image  study EPSP or IPSP o Can fluorescently stain neurons for subsequent analysis  Identify the neuron that you are recording  Identify the structure, type of neurotransmitter used and cell surface carbohydrates that are expressed  Electrical, structural and biochemical properties can be determined Disadvantages: o Technically demanding (small diameter of neurons)

Single Channel Recording 

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Recording electrical activity of a single ion channel: o Developed by Erwin Neher (physiologist) and Bert Sakmann (engineer)  Nobel Prize Physiology – 1991 Recording from a small patch of membrane o Isolated cells maintained in vitro (culture) o Enzymatically cleaned  Remove lipid bilayer, proteins, carbohydrates that are expressed extracellularly o Pipette applied to the cell surface o Suction applied – the portion of the membrane associated with pipette sticks o Small patch of membrane removed o Containing single channel Applications: o Confirmed theories regarding the ionic basis of action potentials o Mechanism of action of ligand – gated ion channels o Molecular structure of ion channels  Determining the portion of the channel responsible for gating Advantages: o Understand the molecular basis of membrane potentials o Exceptionally high resolution Disadvantages o No information on what cell does in nervous system

Functional Brain Imaging 

Insight into higher cognitive functions: o Visual processing o Memory o Language Functional Magnetic Resonance Imaging (fMRI)

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MRI:    fMRI  

Functional development for MRI High resolution images of brain structure Based upon differences in density throughout the brain

lower resolution images of brain function Based on changes of blood oxygenation level dependant signal  Blood oxygenation levels are dependant on an increase/decrease in activity of the brain  Increase in neuronal (electrical) activity  Increase in rCBF [Increased redirection of cerebral blood flow, reflex change]  Change in magnetic field [Dependent on the structure of haemoglobin] Procedure:  Subject lies in MRI scanner  High resolution structural scan  Control functional scan  Without task applied to subject  Task functional scan  Subject applies task  Change in rCBF = task functional scan – control functional scan  Images overlayed Applications:  Mapping brain functions – reinforce theories  Understanding pathophysiology – determine what occurs in disease states  Clinical diagnostic Advantages:  Non-invasive  Good correlation with structure – structural components can be easily compared to functional components  Non ionising radiation involved Disadvantages:  Poor temporal resolution – cannot visualise rapid changes in regional blood flow  Susceptible to movement artefacts  Stick your head in a big magnet – claustrophobic...