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Methods of Investigating the Brain

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Kevin Silber

on 6 March 2013

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Transcript of Methods of Investigating the Brain


Amino acid autoradiography is where radioactive amino acids are injected into the brain prior to death.

The cells take up these labelled amino acids, make neurotransmitters with them and these are then transported to terminal boutons.

This method can be used to see where various brain regions project to.

Horse-radish Peroxidase (HRP) is taken up by boutons and transported via retrograde axoplasmic flow to cell bodies. Hence this can show where fibres have come from. HISTOLOGICAL TECHNIQUES Post mortem stains can be used to mark a variety of elements.

Cell body stains are taken up by the Nissl substance (DNA, RNA and proteins) in cells. Neutral Red and Cresyl Violet Acetate are examples of such stains.

Myelin stains are taken up by axons. Luxol Fast Blue is the most common of these stains.

Membrane stains such as Golgi-Cox only stain some cells and so can be used to observe axonal and dendritic branching. HISTOLOGICAL TECHNIQUES Immunological Analysis

This method uses slices of living brain tissue. Antibodies attached to special dyes are introduced and these then glow under ultra-violet light.

By detecting where the antigens are located one can detect the sites of viral infections. CHEMICAL AND IMMUNOLOGICAL METHODS Chemical

A glass pipette is used to introduce chemicals into the brain.

This tends to stimulate only the cell bodies and not the axons surrounding and passing through the region.

This contrasts with electrical stimulation.

Microiontophoresis can be used to identify receptors to particular neurotransmitters. BRAIN STIMULATION Electrical

Current is passed through micro- or macro- electrodes but at physiological levels and not lesion levels.

This method can be used to investigate what a region does (e.g. hypothalamic stimulation will cause eating or drinking behaviour).

During surgery, brain stimulation can be used in conjunction with the EEG to identify and treat focal seizures.

This requires an awake patient so a local anaesthetic is used. BRAIN STIMULATION Multiple Unit Activity (MUA)

This involves using large (macro) electrodes to record from hundreds to thousands of cells.

The recording is extracellular.

The electrodes are typically 100-150 microns in diameter. The electrodes are usually made of stainless steel or platinum and are cemented to the skull using dental plastic.

This ensures that the electrodes remain in place during the recording period.

This method is used to record from small structures. RECORDING TECHNIQUES LOBOTOMIES Ablations
Either a knife is used to cut away a region of the brain or a vacuum pump is attached to a pipette and this is used to literally suck away brain tissue. The latter is more frequently used for removing surface structures. ABLATIONS, LESIONS AND LOBOTOMIES This is the name used for some forms of surgery carried out on the brain. It is called this because one can pinpoint the location of the electrode using a stereotaxic atlas.
A stereotaxic atlas is a precise mapping of where brain structures lie relative to markings which can be located on the skull. STEREOTAXIC SURGERY This session will cover:
Stereotaxic Surgery
Ablations and Lesions
Recording Techniques
Brain Stimulation
Chemical and Immunological Techniques
PART 1: INVASIVE METHODS A number of chemicals can be used to show up particular elements of neuronal tissue after post-mortem examination.

Some of these histological chemicals can be introduced before the organism is sacrificed and the resulting distribution of them can indicate a number of processes that have taken place.

Usually, to see the results of histological analysis very thin sections of brain tissue are viewed under powerful microscopes. HISTOLOGICAL TECHNIQUES Chemical Analysis

The method here is to extract a small amount of brain extracellular fluid (ECF) by a method called microdialysis. This is done by pumping in a CSF-like solution which then passes to a collecting tube picking up ECF molecules as it goes.

Using this method one can detect neurotransmitters released by terminal boutons which have escaped from the region of the synaptic cleft. CHEMICAL AND IMMUNOLOGICAL METHODS Single Unit Activity (SUA)

This requires very fine-tipped microelectrodes.

The recording is intracellular.
The electrodes are typically around 5 microns in diameter and is usually made of stainless steel or tungsten.

The electrode is etched to sharpen it.

An alternative to metal electrodes is to use a glass pipette electrode filled with KCl to facilitate conduction.

This method is used to record from inside axons or synapses.

The time for recording is limited. RECORDING TECHNIQUES
An electrode is used to pass DC current. This initiates chemical reactions whose products destroy the tissue around the electrode tip. One drawback is that metal ions are left in the damaged area. LESIONS Summary The lobotomy was first performed by Walter Freeman and was an operation designed to sever the frontal lobes from the rest of the brain. It was used for people suffering from a range of 'mental disorders' but most commonly these were depression or a personality disorder. Latterly they were performed on patients with rare conditions of obsessive compulsive disorder Figure 1. A single optrode as an in vivo electrophysiological recording tool. (a) Optical images of an optrode being slowly driven into the cerebellar cortex of an anesthetized mouse. The inset shows the detailed structure of the optrode. Light could be locally delivered through the aperture of the tapered optical fiber, while simultaneously the neural activities of nearby cells are recorded. (b) SEM image showing that the diameter of the optical aperture is about 10 µm in this optrode. The metal coating around the tapering tip, except the aperture, is indicated. (c) Examples of in vivo recording from an optrode. The left trace is the band pass filtered (300 Hz–10 kHz) recording of multiple unit activity from the cerebellar cortex of an anesthetized mouse as shown in (a). The Vpp noise amplitude was about 30 µV. The right panel shows the waveform samples from two isolated single units recorded by the optrode. From Jing Wang, Fabien Wagner, David A Borton, Jiayi Zhang, Ilker Ozden, Rebecca D Burwell, Arto V Nurmikko, Rick van Wagenen, Ilka Diester and Karl Deisseroth (2012) Integrated device for combined optical neuromodulation and electrical recording for chronic in vivo applications. Journal of Neural Engineering Volume 9 Number 1 Ethanol stimulates DA neuron firing. (A) Example traces of single-unit recording of DA neuron firing before (top) and 5 min after i.v. injection of ethanol (bottom) in an awake rat. Adapted from Mereu et al. (1984). (B) Representative traces of DA neuron firing recorded with a perforated-patch configuration in a brain slice. Note the effect of ethanol on the membrane potential trajectory during the interspike interval. Taken from: Hitoshi Morikawa and Richard A. Morrisett (2010) Ethanol action on dopaminergic neurons in the ventral tegmental area: Interaction with intrinsic ion channels and neurotransmitter inputs. International Review of Neurobiology Volume 91, Pages 235–288 Recording From The Brain Removing parts Of The Brain Stimulating The Brain Electrical Brain Stimulation Deep brain stimulation is being considered in the treatment of depression, epilepsy, obsessive-compulsive disorder, headaches, chronic pain and stroke where patients have not responded well to other treatments.

This link is to an article from The Observer in 2012.
http://www.guardian.co.uk/science/2012/jun/03/electrical-brain-stimulation-treatments Brain stimulation has been carried out in rats for a long time as a way of trying to discover what various parts of the brain do.

In 1957, Olds and Milner discovered that rats will press a lever in a Skinner Box to obtain an electrical buzz to their septum (part of the limbic system). It seemed they had discovered a reward system in the brain.

Rats will press at phenomenal rates (up to 2000 presses per minute) to obtain such a reward. Fig. 6. Single unit recording from the nucleus accumbens core combined with microiontophoresis using a multi-barrel glass microelectrode. The hatched area medial to and ventral to the nucleus accumbens core indicates the nucleus accumbens shell. The micropipettes contained NaCl for recording and for automatic current balance, dye to mark the recording site, glutamate (GLU) to drive the quiescent cells, MDMA, and some combination of 5HT, DA, agonists and antagonists for specific subtypes of 5HT and DA receptors or a specific 5HT uptake blocker. Taken from: N.R White, T Obradovic, K.M Imel and M.J Wheaton (1996) The effects of methylenedioxymethamphetamine (MDMA, “ecstacy”) on monoaminergic neurotransmission in the central nervous system. Progress in Neurobiology Volume 49, Issue 5, Pages 455–479 Chemical Analysis Of The Brain Histological Techniques Histofluorescence Horseradish peroxidase Hippocampal sections from rat (A), guinea pig (B), cat (C), European hedgehog (D), dog (E) and man (F), illustrating the distribution of intensely stained, black Mossy Fiber terminals in the different species Invasive methods provide an alternative perspective on brain functioning than non-invasive methods

They are really the only way to examine the micro detail of brain functioning

They are also proving to have potential in the treatment of brain disorders that are non-responsive to alternative methodologies As the skull is made up of a number of different bones which are fused together, the points where these bones meet can be used as precise markers on any size of skull. Lesions
There are three different kinds of lesions;
Radio Frequency
Neurotoxic Lobotomies
These are where the connections between one lobe and the rest of the brain are severed. Radio Frequency
A high frequency AC current is used to destroy cells by heat. Hence no metal ions are left behind. Neurotoxic
These lesions are more selective as they do not destroy all the tissue in the vicinity of the lesion.
Kainic Acid or Ibotenic Acid are typically used and are delivered via a cannula. These acids destroy the cell bodies but leave the axons intact.
Even more specific is 6-hydroxydopamine (6-HD) which specifically kills dopamine or noradrenaline neurons by being taken up by them.
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