Techniques in Neuroscience - Question 1

Discuss why and how Magnetic Resonance Imaging (MRI) scanners are used to measure brain function with fMRI.

Answer: The Functional Magnetic Resonance (or fMRI) is a particular type of magnetic resonance imaging employed in the field of neuroradiological, to detect those brain areas which are activated (how to speak, read, think or move a hand). Its main principle is based on a hydrogen atom nucleus possessing the same features as that of a small sized magnet. The principle functioning of nuclear magnetic response is employed by functional MRI. The main components of a magnetic resonance imaging system are: a magnet; three generators of magnetic fields of variable intensity in space and time (gradients); a radio frequency generator / receiver system; a computerized system capable of controlling all components, calculating and displaying images and MRI data, managing archiving, printing and transfer of studies. MRI employ strong magnets that develop powerful magnetic field which forces the protons in order to align with particular field.

The fMRI is a technical medical imaging non-invasive, which provides a map of the brain regions functionally eloquent. It is used for both clinical and research purposes. It allows you to check whether, in or near a brain injury, important functional activity persists in order to be able to preserve it. The detection thus allows to plan pre-operatively the optimal surgical approach and intra-operatively helps the neurosurgeon to orient himself by providing structural and functional information.

In fMRI, a magnetic field is implemented for alignment of all the hydrogen atoms. The cores absorb energy after implementation of a radio frequency magnetic pulse at an appropriate level of frequency. This leads to the generation of a radio signal that is easily detected by the spirals of radio frequency that exist in MRI setting. Through varying the sequence relevant with the radio frequency various images related to the specific brain area can be selected. The relevant duration existent between implementation of the pulse and acquisition of signal is echo.

In fMRI, two different relaxation times, T1 and T2, are used to characterize a tissue. The longitudinal configuration of the relaxation time equates to the time constant that conducts measurement of the time that is spent by the hydrogen atoms that are excited for further alignment with the external magnetic field. On the other hand, the transverse relaxation time or T2 is the time constant that measures the time spent by the excited hydrogen atoms at reach balance or exits the phase (phase shift). T2-weighted e) TR and TE short scans (of T1-weighted (longest TE and TR) are the most common sequences used in fMRI

Impaired level of neural activity can be easily determined by the variations in the overall flow of blood. Like the tapping of fingers of the hand can lead to an increase in the overall flow of blood. This leads to change in the blood print which is easily detected by the magnetic resonance signal.

In basic fMRI, a qualitative review is provided by bold printed signal. This overview provides the changes that are a resultant of the stimulation of brain in comparison with the resting condition.

The room where the scan takes place is surrounded by a radio frequency screen whose function is to prevent high-power RF pulses from radiating the rest of the hospital and that RF signals from television stations and radio stations can be picked up by the 'device. Usually, the scanning rooms are also surrounded by a magnetic shield which prevents the magnetic field from extending outside a certain limit. In more recent magnets this screen is an integral part of the magnet itself (self-shielded magnets).

Techniques in Neuroscience - Question 2

Describe the principles of the polymerase chain reaction (PCR). Give an example of an application that requires the use of PCR and indicate why this technique is appropriate

Answer: Since DNA duplication was obtained in the laboratory, it has been possible to develop various DNA copies. The polymerase chain reaction or PCR from Polymerase Chain Reaction is a technique that automates this process by copying a stretch of DNA many times in a test tube. This technique was invented by the American biochemist Kary Mullis in 1983 for which he then obtained the Nobel Prize for Chemistry in 1993. The reaction practically reproduces what happens in the nucleus of the cells, that is, the replication of DNA, performed artificially.

The PCR principle is based on the fact that DNA is composed of two complementary strands and that with each synthesis cycle by DNA polymerase, the number of sequence copies available for the subsequent reaction cycle doubles. Consequently, under optimal conditions, the amount of DNA produced by a PCR reaction is 2 n times the initial amount where n is the number of cycles. In other words, after 30 PCR cycles, which correspond to less than 2 hours of reaction under standard conditions, an amount of DNA equal to about 1 billion times the initial quantity was produced. If it is true that in its most basic conditions PCR requires very little technology, at the limit only three thermostatic baths, it is also true that PCR has been a stimulus for technological innovations ranging from the production and purification of thermostable DNA polymerase (with different characteristics processivity and fidelity to the design), to the production of ever faster and more precise cyclers. (Williams, 1990)

PCR produces various copies of a sequence of DNA, be it a gene or part of it. In current practice the main components of PCR are:

  • DNA template, containing fragment for amplification;
  • Two primers (Forward and Reverse), for determination of beginning and end region for amplification;
  • DNA Polymerase, copying region for amplification (DNA is used polymerase of thermophilic bacteria, which are stable at high temperatures; es. Taq Polymerase);
  • Nucleotides, for the synthesis of new DNA
  • Buffer, which provides the ideal chemical environment for DNA polymerase

To set up a PCR reaction it is necessary to have tools such as a thermal cycler, which is a kind of oven where samples are placed, usually placed in a 96-well multiplate. The reaction has the function of amplifying a certain stretch of genetic material by obtaining multiple copies of it. In order to make this process possible it is necessary to prepare a sample containing: (Kadri, 2019)

a) The fragment of DNA to be amplified, present as a double strand

b) Dree triphosphate oligonucleotides, which are needed as raw material to synthesize the new filaments that will be copies of the filament initial

c) Primers (one Forward and one Reverse), which are designed on the basis of certain software (which can be downloaded from the Internet as Free Download), which are needed as initiators of the filament extension process, i.e. as a handbook

An enzyme that provides for the extension of the filaments, called Taq Polymerase, or a heat-resistant DNA polymerase extracted from the microorganism, belonging to the Kingdom of the Archaea, Thermus aquatics. As this polymer has a characteristic of withstanding with temperature of about 100 degrees that is sufficient for denaturing most of proteins.

The Thermus aquatics is a bacterium residing in the ocean near hydrothermal sources to the ocean ridges. Its DNA polymerase is resistant to heat and works within a well-defined temperature range. In addition to these ingredients, a Mg ++ magnesium ion buffer must be added. Once the samples have been prepared, it is necessary to insert them subsequently inside a thermal cycler, in turn it is controlled by a computer directly connected to it, and proceed with the chemical synthesis. This automated synthesis consists of cycles, divided into three phases: the first is called the denaturation phase and takes place bringing the temperature to 94 ° C for 1 minute. It allows denaturing the double strands of DNA that must be amplified, thus obtaining two single strands of which, each of them must serve as Template Strands or Mold Filaments for the synthesis of the complementary strand.

Techniques in Neuroscience - Question 3

How is the membrane potential established in a resting neuron? What happens to the resting potential when: a) current clamp is applied? b) voltage clamp is applied?

The membrane potential is the electrical voltage, measurable in a cell, between the cytoplasmic side, which presents negative charges, and the extracellular side, which presents positive charges.

For transference of information a resting potential should be produced by neurons. Another requirement is the difference of voltage between both the intracellular as well as extracellular environments that further go about the membrane of the cell. This difference in the voltage value in unexcited state of neuron is equivalent to the resting potential which further leads to the generation of an active level of potential.

The communication of nerve cells is based precisely on the membrane and action potential. The electrical signal of neurons is generated when, in response to certain stimuli, the door channels of the membrane open / close. When these channels are activated, the permeability to an ion changes, and therefore there will be a tendency to bring the membrane potential close to the equilibrium potential of that ion for example; when the channels for Na + are opened and this enters, the membrane potential approaches + 55mV.

Voltage Cramp:

It is applied for measuring the ion current across membrane. Under the clamp conditions of voltage, the inn channels open and then close normally. Along with this the apparatus of the voltage clamp provides compensation for the variations in the current of ion for the maintenance of a constant potential of the membrane. The specified membrane holding potential is set by the investigator which is referred to as the holding voltage and command potential. It is different from current clamp. The experimentalist provides the value of the membrane voltage and the relevant value of current is injected by circuit in the amplifier circuit. This provides a counter against any variation in the membrane voltage that would take place without the voltage clamp.

Among the constituents of the intra- and extracellular environment there are the sodium ions (Na +), chloride (Cl -), organic anions (A -) and, above all, potassium (K +). After restoration of resting potential and excitation of neuron, the concentration of the K + ions are greater inside than outside the neuron. Unlike most ions, K + ions can move freely in and out of the cell, through specific ion channels present on the membrane. Guided by the concentration gradient, K + ions they spread outside the neuron, causing a net movement of positive charges. This is followed by development of voltage difference across cell membrane possessing intracellular environment. This is the resting potential, with a value of around -70mV.

Current Cramp:

Under the current clamp voltage is measured across the cellular membrane on injection of constant negative or positive current into the cell. The current apply across the three resistors will be same as they are in series. As per the Ohm’s law, mostly voltage drop will occur at highest resistance. The measurement of the resting potential under the current clamp will be of less than the actual magnitude i.e. -70m V measurement means that the resting potential will be more negative or -70 mV. This underestimation degrees are based on the resistance ratio; more accurate measurement will be in case of large resistance ratio. (Perkins, 2006)

Techniques in Neuroscience - Question 4

Dr Schorge is growing N2A cells (mouse neuroblastoma cells) in vitro for experiments. She defrosted and plated the cells in growth media, but they are not expanding at the expected rate. Which aspects of tissue culture maintenance should Dr Schorge address in order to improve the growth of the N2A cells?

The HPC and OGD models of N2a cells were established, using 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method and lactate dehydrogenase (LDH) ) Leakage rate, in situ end labeling (TUNEL) and Western blot (Western blot) methods were used to detect N2a cell damage, necrosis, apoptosis and autophagy levels. Results OGD 2 and 4 h can significantly reduce the survival rate and mortality of N2a cells (P<0.05, n=6); HPC (20 min) significantly improved the damage of OGD 3 h on N2a cells, while cPKCγ inhibited The agent Go6983 (6 nmol/L) significantly relieved the protective effect of HPC on OGD cells (P<0.05, n=16); OGD 3 h significantly increased the number of apoptosis of N2a cells (P<0.05, n=6), but Neither HPC nor Go6983+HPC can significantly affect the apoptosis level of OGD cells; OGD 3 h can significantly increase the autophagy level of N2a cells. Both Go6983 and HPC+Go6983 can significantly reduce the autophagy level of OGD cells (P<0.05, n=6), while HPC There was no significant effect on the autophagy level of OGD cells; HPC significantly improved the degree of necrosis of N2a cells caused by OGD for 3 h, while Go6983 can relieve the protective effect of HPC (P<0.05, n=16). Conclusion The level of cPKCγ in HPC can improve the OGD damage of N2a cells at the level of isolated cells, and this effect is mainly related to the reduction of the necrosis level of OGD cells. (Agneta Mewes, 2012)

Alzheimer disease (AD) is a primary type of dementia (60%–80%) and the most prevalent neurodegenerative disorder associated with aging. AD is characterized by progressive loss of memory and cognition, brain atrophy, and accumulation of amyloid plaques and neurofibrillary tangle. However, amyloid beta peptide (Aβ) is the main component of amyloid plaques (extracellular deposits found in the brains of patients with AD). In some studies, show that Docosahexaenoic acid (DHA) supplementation caused a significant reduction in Aβ toxic that may have neuroprotection potential. Gold nanoparticles (AuNPs) a novel agent, has good biocompatibility and has been used in biomedical researches. Investigations suggest AuNPs can promote neurite outgrowth of N2A cells and significantly increase the neurite length that can be used as the index of differentiation potential. Recent studies suggested that AuNPs reduced mHtt aggregates in mHtt expressing (Huntington disease, HD) N2A cells.

The result of Transmission Electron Microscopy, we found that the average mitochondrial was shrinkage necrosis in N2A cells from Aβ toxicity. Treatment of AuNPs (chemical and physical) also made average mitochondrial shrinkage necrosis in N2A cells from Aβ toxicity. The DHA with Aβ trial showed that 390 genes were up-regulated and 355 genes were down-regulated compared to the control trial. The AuNPs with Aβ trial showed that 836 genes were up-regulated and 525 genes were down-regulated compared to the control trial. To extending our understanding, we will use QPCR and Western blot to confirm related gene and protein. Our findings will extend our understanding of the central role of DHA and AuNPs in Aβ-related neuronal impairment, which probably increase risks in neurodegenerative diseases.

References for Techniques in Neuroscience

Agneta Mewes, H. F. (2012). Organotypic Brain Slice Cultures of Adult Transgenic P301S Mice—A Model for Tauopathy Studies. 10.1371/journal.pone.0045017.

Kadri, K. (2019). Polymerase Chain Reaction (PCR): Principle and Applications, Synthetic Biology. New Interdisciplinary Science.

Perkins, K. L. (2006). Cell-attached voltage-clamp and current-clamp recording and stimulation techniques in brain slices. J Neurosci Methods.

Williams, J. G. (1990). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic acids research, 6531-6535.

Remember, at the center of any academic work, lies clarity and evidence. Should you need further assistance, do look up to our Neuroscience Assignment Help

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