Chapter 11

Question 0

What does the Nissl stain do?

Answer 0

The Nissl stain attaches itself to RNA and gives it a different colour based on what dye it uses. RNA is most concentrated in the ribosomes of the rough endoplasmic reticulum of the neurons and glia and therefore the cell body is highlighted. Some of the dyes Nissl stain can use are thionin, cresyl violet, and fluorescent compounds.

Question 1

How is the brain sliced into thin sections?

Answer 1

The tissues has to chemical preserved with chemicals such as formalin.
Fresh tissue can be sectioned using a microtome with a vibrating blade.
Frozen tissue can also be sectioned.

The tissue which is going to be prepared is dehydrated in alcohol and then embedded in paraffin wax or celloidin.

Question 2

How can the sections be stained for myelinated axons?

Answer 2

Some preparation methods deposits silver haematoxylin in the protein scaffold of the membranes. Other myelin stains uses dyes such as Luxol fast blue or osmium salts, which stains the the fatty content of the myelin sheath. The Weigert stain method is also commonly used.

Question 3

What is something funny about the Golgi stain?

Answer 3

The Golgi stain deposits a silver dye, which then crystallises in the membranes of the individual cells. The cell is now highlighted in black. This method only works because only 1% of the neurons are stained in this manner. If all the neurons were stained, the whole thing will be just black. The reason why only 1% of the neurons are stained is unknown.
Only the cell body and dendrites are stained, myelin sheath is not stained.

Question 4

What are histochemical stains?

Answer 4

Stains that are used to mark the particular parts of the cells in the brain based on their chemical and physical properties. They render the cellular components into particular chemical states where the dye is either attracted or reacts with it to leave coloured products on the structures.

For staining enzymes usually works best on lightly or unfixed sections, whereas some of the chemically aggressive reactions require robustly preserved tissues.

Question 5

What histochemical stain is used to mark enzymes that synthesise nitric oxide?

Answer 5

NADPH-d(nicotine adenine dinucleotide phosphate-diaphorase) is used to identify cells with enzymes that synthesise nitric oxide. This stain is the strongest in the caudate, putamen and in some parts of the amygdala.

Question 6

What does cytochrome oxidase histochemistry indicate?

Answer 6

CO combines with a chromagen(DAB) with a substrate molecule to indicate where this mitochondrial enzyme is present. Since the majority of cytochrome oxidase is found in inhibitory neurons, the stain indicates long term inhibition in the particular areas.

Question 7

What does acetylcholine esterase biochemistry indicate?

Answer 7

Acetylcholine esterase breaks down acetylcholine in the synapses. Areas with this enzyme highlight high cholinergic activity. This biochemistry uses s-acetylcholine iodide and ethopropazine to leave a brown deposit in the presence of the breakdown enzyme.

Question 8

What dyes can be used to visualise DNA?

Answer 8

4’,6’-diamidino-2-phenylenidole dihydrochloride(DAPI) or Hoechst 33258, which forms fluorescent complexes with DNA. These dyes indicate both the nucleus of the neurons and glia alike.

Question 9

What is used to visualise apoptosis?

Answer 9

The dye TUNEL(deoxynucleotidlytransferase-mediated dUTP nick-end labelling) is used to indicate cell death or damaged DNA.

Question 10

What can be used to study the movement of calcium ions?

Answer 10

Calcium dyes can be tracked by using fluorescent dyes such as fura-2 and indo-1 in live subjects or cell culture enabling the regulation and traffic of this important ion to be tracked in real time.

Question 11

What is used to indicate zinc? What does zinc mean?

Answer 11

Zinc and other heavy metals are stained using silver stains like Timm-Danscher or fluorescent dyes. Zinc is used in DNA regulation and synaptic plasticity as well as other cellular signalling. So zinc stains show long term patterns of activity and change.

Question 12

What are immunohistochemical stains?

Answer 12

Immunohistochemical stains can detect the presence of particular proteins in neurons. The most common way is based on deriving specific immunoglobulin G(IgG) antibodies from common lab animals. There are two types of antibodies polyclonal and monoclonal. Polyclonal Ig bodies are produced by animals when in response to inoculation of its body with foreign proteins.
Monoclonal is derived from a mass of antibody producing cell. The antibody is then attached to a fluorescent dye or catalytic molecule which can be stained.

Question 13

What is the most common catalytic molecule used?

Answer 13

Peroxidase that can deposit a chromagen such as DAB(3,3’-diaminobenzidine) for permanent labelling.

Question 14

Why is it sometimes better to use a fluorescent dye in immunohistochemical stains?

Answer 14

To label multiple different proteins at once. It can also be used in controlled laser techniques such as confocal and two photon microscopy for 3D localisation.

Question 15

What are some of the challenges of immunhistochemistry?

Answer 15

Ensuring the antibody only attaches to needed protein. This is tackled by using the antibodies in a control.

Techniques used to fix and stabilise tissue can drastically alter the structure of the protein, which is the basis of immunohistochemistry. For example, aldehyde fixation strongly cross links proteins, which interfere with antibody binding to the proteins.

Question 16

Name immunohistochemical markers for calcium binding proteins.

Answer 16

Calcium binding proteins, such as calbindin, calretinin, and parvalbumin, as they are found in only certain cell groups.

Question 17

How is NeuN use to count neurons?

Answer 17

Suzana Herculano-Houzel developed a clever technique to count neurons in the part or the whole of the brain. The tissue is homogenised with detergent in a blender in order to free the nuclei from the cells. A small sample is withdrawn and is stained with NeuN so that the neurons can be accurately counted.

Question 18

What are lectin stains?

Answer 18

Lectins are plant proteins that bind extremely strongly to specific patterns of sugars in a carbohydrate molecule. Since neurons also exhibit glycoprotein structures on their cell membranes, biotin conjugated lectins can be applied to tissue sections to label them. Areas are classified by the protein distribution.

Question 19

What is cell culture, and how it is used?

Answer 19

Cell culture is the study of a cell or a group of cell in a medium that is similar to its original extra cellular fluid. If interaction with gases or gases are bubbled into the liquid, the cell(s) can be kept alive for
weeks.

A culture consisting of single neuron is called monoculture, and the advantage of this is the behaviour of this neuron can generalised to similar neuron. However, the neuron is deprived of connections and therefore they not necessarily behave in the same manner.

Co-cultures use multiple cell types grown on the same structure or close to each other to better reproduce the interactions between the cells. When the type of the cells matches a part in the brain, it is called organotypic culture.

Slice culture is when a 1 mm slice of the living brain is placed in a medium. It is used to study quite complex interconnection between parts.

Question 20

What is hodology?

Answer 20

Hodology is the study of connections in the nervous system.

Question 21

What are some of the tracers used in living subjects?

Answer 21

Tracers can be either solid inserted by pressure injection or by iontophoresis(in which a polar compound is infused by passing an electric current through a pipette electrode containing the tracer), or via infectious harmless virus like pseudorabies or adenovirus.

Some anterograde(cell body to axon) tracers are cholera toxin b, and dextranbiotin. Some retrograde(axon to cell body) tracers are wheat germ, flurogold and fast blue. Dil is a tracer that is bidirectional.

Question 22

Where can tracers be placed?

Answer 22

In regions identifiable during surgery and areas next to it. It should be within the stereotaxic atlas. The stereotaxic frame is a precise 3 dimensional positioning apparatus that allows us to accurately insert the tracer inside the cells.

Question 23

How can tracers be detected?

Answer 23

Using fluorescent techniques, or with histochemical staining methods.

Question 24

What is in-situ hybridization?

Answer 24

In situ hybridisation is where a radioactive tagged replicated DNA attaches to a RNA which is trying to make that particular protein. In concventional ISH, the radioactive tags make dark dots on photographic emulsion to show regions of this protein being formed.

Question 25

What is gene targeting?

Answer 25

Gene targeting is the ability to add new genes or remove genes in a developing cell. This was first discovered by Cappechi, Evans and, Smithies in 2007. This allowed to study the role of particular genes in our system and what is the effect of gene mutations. Inducible knock ins is the ability to insert a gene that can be activated at any particular point through an external stimuli. The most common induction system is to trigger an estrogen receptor with the drug Tamoxifen.

Question 26

What are reporter molecules?

Answer 26

Reporter molecules are molecules that are formed during the expression of an introduced gene that can be viewed by histochemical visualisation methods, or fluorescence microscopy.

Question 27

What are some non invasive imaging techniques?

Answer 27

MR

DTI

Question 28

Why did the MR replace the x-Ray?

Answer 28

The x-Ray could not reveal much information about the brain, sine it is all soft tissue. However, it could be used if a contrast media was injected into the blood stream, or if air was introduced in the ventricular system. Unfortunately, these techniques were technically difficult and occasionally dangerous. In the 1980s, there was a huge improvement to the X-Ray, which allowed the computers to construct a 3d version of the brain based on mathematics and several x-Rays from different angles. This is now known as CR scan and its use is limoted because it shows the difference in density which only changes in tumour or stroke. It gives a basic outline of the brain parts.

However, when the MR was released in the 1990, it took over because it could give detailed delineation between structures and the resolution can be adjusted with the force of the magnetic field. The MR works by exciting specific molecules such as the hydrogen in water and then uses this to create a systematic 3D representation of the brain.

Question 29

How has the MR resolution increased?

Answer 29

The MR in those times used 1-3 tesla units of field strength which yielded 1-3 millimetre resolution. Today’s small volume MR scanner, produces fields of strength 4-16 tesla causing a resolution of 20-50 micrometers,

Question 30

What is DTI?

Answer 30

Diffusion tensor imaging uses MR scanner in rapid pulses to obtain information of how freely the molecules are moving by diffusion along axons. Every point in the brain is assigned an intensity value and a tensor. The image gives an indication on how axons move in the brain,

Question 31

What is fMRI and how does it work?

Answer 31

Functional magnetic resonance imagining uses the same principle of MRI to measure the blood oxygen level at every pint of the brain while resting and is compared to the measurements when the participant is doing a designed task. The comparison could say which parts of the brain became more active. The problem with fMRI is the greatest changes occuring in the cortex could only be measured and even though a different part of the brain might have responded, it is undetected. Another drawback is we don’t know is the cortical activation is to excite other areas, suppress other areas or are just peripheral processing to the task.

Question 32

What is PET and SPECT? What is the major drawback?

Answer 32

Positron emission tomography and single photon emission computed tomography both uses radioactive markers that are absorbed by arrive neurons and are resistant to breakdown and emits gamma rays for a brief time. This is then detected by gamma sensors and are computed for an image. The problem with this is it cannot be frequently used on children.

Question 33

What are some electrophysiology tests?

Answer 33

Using electrodes to monitor, digitise, time, average and classify AP. These electrodes use amplifiers to amplify the change in voltage and records them. The results can vary depending on the construction of electrodes. High impedance electrodes can just focus on one neuron or low impedance neurons can measure from 4 to 100 neurons activity.

Question 34

What is patch clamping?

Answer 34

Patch clamp is enclosing a part of the neuronal membrane in glass electrode tip which becomes a part of an electrical circuit. Our current electronic technology allows us to measure the entering and exiting of ions in great detail. The glass pipette amplifier is so sensitive that opening of a single ion channel can be recorded in nanosecond scale.

Question 35

Name the immunohistochemical stain that stains neurofilaments.

Answer 35

Structural proteins that make neurofilaments which have transport-related functions are marked by SMI-32 labelling the neurofilaments triplet proteins when in dephosphorylated states. Due to the varying expression of these proteins, SMI-32 is a popular label.

Question 36

Name some immunohistochemicals that stain the different glial cells.

Answer 36

GFAP(glial fibrillary acidic protein) identifies astrocytes in their reactive state. Micro glia are marked by protein ferritin. Oligodendrocytes are marked by a range of markets such as myelin basic protein and CNP(2’3’- cyclic nucleotide-3’-phosphohydrolase). Polydendrocytes are labelled with NG2, proteoglycan molecule in the cell surface.

Question 37

What is NeuN?

Answer 37

NeuN, an antibody derived from mice when inoculated with purified neuronal nuclei attaches to this unknown protein synthesised by all neurons except mitral cells of the olfactory bulb and the purkinje cell of the cerebellum. No glial cells are marked.

Question 38

What helps us to visualise DNA replication and repair?

Answer 38

Bromodeoxyuridine/BrDU/thymidine analog is injected with a cell body. This molecule is only taken up be cells that are exhibiting DNA replication or repair. Coupled with other markers, areas of neurogenesis can be identified. A newer technique is the use of ethynyldeoxyuridine(EdU) which directly incorporates a fluorescent nucleotide substitute into newly synthesised DNA.