Chapter 5 research Experimental Ablation Definition The removal - - PDF document

Chapter 5

Methods and strategies of research

Experimental Ablation

• Definition

– The removal or destruction of a portion of the brain of a laboratory animal; presumably, the functions that can no longer be performed are the ones the region previously controlled.

• Distinction between brain function and

behavior

– Destroy a nucleus in a mice, trying to prove it involves visual information processing, how to test?

Producing brain lesions

• Tissue removal/ aspiration

– Outer part of the brain, cortex

• Radio frequency lesions (RF)

– Destroy both neural cell bodies and axons bypassing

• Excitotoxic lesions

– Destroy neural cell bodies only

• Neurochemical lesions (6-hydroxydopamine, 6-

HD)

– Selectively destroy noradrenergic or dopaminergic neurons

• The Importance of sham lesions, (placebo)

Stereotaxic surgery

Histological Methods

• Histological methods are used to verify the placement of

a lesion

– Perfuse (to remove blood) – Fix in formalin (to solidify tissue and prevent autolysis) – Slice into sections – Stains (to highlight selective neural elements)

• Golgi stain—stains full cell (membrane)
• Nissl stain—stains cell bodies (RNA)
• Weil stain—Stains axon (Myelin)
• Electron microscopy

– Transmission electron microscope – Scanning electron microscope

• Confocal laser scanning microscope

Tracking neural connections

• Neurons send outputs
• Efferent (exiting) axons

– Anterograde labeling (cell bodies to axons to terminal buttons) – Eg.PHA-L – Use immunocytochemistry with antibodies to proteins

• r peptides
• Neurons receive inputs
• Afferent (arriving) axons

– Retrograde labeling (from axons to cell bodies) – Eg. Flourgold

Visualizing a living human brain

• To see anatomy & functioning
• Detect tumors or lesions or abnormalities

– Computerized tomography (CT) uses an X-ray beam to scan the brain from all angles, there scans are then summarized in an image of the skull and brain – Magnetic Resonance Imaging (MRI) uses a magnetic field and radio waves to excite hydrogen molecules, the resulting information is combined to form an image of tissue

• Which one has higher resolution?
• Other brain imaging technique: EEG, PET

Alzheimer's brain

Human Brain Imaging

• The PET scan measures metabolic activity in

brain

– Human subject is injected with radioactive substance, which is taken up by brain cells – As the redioactive molecules decay they emit positrons that can be detected by a scanner – A PET scan indicates the relative activity of different brain regions during mental states

• Functional MRI (fMRI) scans detect the level of
• xygen in brain blood vessles

– Current fMRI scanners have a higher resolution than do PET scanners

Recording neural activity

• Microelectrodes record single neuron

activation, acute or chronic

• Macroelectrodes record summated

electrical activity of large regions of brain

• Surface electrodes placed on human scalp

are used to record brain activity

• Electroencephalogram: EEG
• Events related potential: ERP (stimulus

locked EEG)

Recording metabolic & synaptic activity

• The 2-deoxyglucose (2-DG) methods measures

relative glucose utilization

– 2-DG cannot be metabolized, is trapped in cells and accumulates – Radioactive 2-DG is then quantitaed using autoradiography

• FOS (nuclear protein) is expressed when a

neuron is activated

– Neuronal activation is associated with activation of genes in the neuron nuleus; can localize Fos within the nucleus, indicate relative degree of activation

Secretions & Stimulation of the Brain

• The secretion of neurotransmitter within a

discrete brain region can be measured using microdialysis, then analyze its contents

• Neurons in a region can be artificially activated

to assess the role of a region in behavior

– Electrical stimulation involves passing electrical current through a wire inserted into brain – Chemical stimulation can involve infusion of small amount of an excitatory amino acid such as glutamate into a region, microiontophoresis

Behavioral effects of electrical brain stimulation

• Transcanial magnetic

stimulation (TMS)

– Stimulation of the cerebral cortex by means

• f magnetic fields

produced by passing pulses of electricity through a coil of wire placed next to the skull; interferes with the functions of the brain region that is stimulated. – Eg, primary somatosensory cortex and pain localization

Localization of neurotransmitters

• Three approaches to the localization of a

neurochemicals in the brain:

– Localize the chemical

• Peptides or proteins localized directly via

immunocytochemistry

– Localize the enzyme that produces the chemical or neurotransmitter

• If it is not a peptide (or protein) can still use stain with

antibody to the enzyme

– Localize the messenger RNA involved in chemical synthesis, in situ hybridization

• Peptides & protein with tracers, anterograde or retrograde

(chemicals + connections in double labeling)

Localizing particular receptors

• Receptors can be localized in brain tissue using
• Autoradiography

– Sections of brain are exposed to solutions containing a radioactive ligand (chemical that binds), washed, and placed on film – The resulting file images show spots at which radioactivity exposed to the film

• Immunocytochemistry

– Antibodies are developed for the receptor protein, are tagged with a fluorescent dye – The tissue is exposed to the antibody/dye – The section is then examined under a microscope for the presence of dye in specific regions

Genetic Methods

• Genetic research methods seek demostration

the linkage between genes and behavior

– Twin studies examine the impact of varying degress

• f genetic similarity on behavioral similarity

– Adoption studies examine a trait similarity of an adopted person compared to their adopted parents and their biological parents – Targeted mutations involve the insertion of a defective (knockout) genes into the chromosome of mice – Antisense oligonucleotide uses modified strand of RNA or DNA that binds with a specific molecule of mRNA and prevents it from producing its protein