Transcript Methods 1b
Competition restated fMRI Lesson : You should decide for yourself instead of blindly accepting what some authors claim Which is more difficult? Rocket science Or Brain Surgery Stereotaxic surgery is the first step in many biopsychological experiments The goal is to place an electrode or some other device at a specific target site in the brain. This is a small animal (rat) stereotaxic apparatus Electrode manipulator Electrode Holder Ear bars Incisor bar “U” frame Base plate Stereotaxic Atlas Step 1: Consult the stereotaxic atlas for coordinates of the brain area you are interested in. Step 1a: Find the plate that has the brain region of interest (e.g., the caudate-putamen, CPu). Note that this plate is a coronal section +0.70 mm (anterior) to Bregma. CPu Bregma is a landmark on the skull surface where the coronal suture meets the sagittal suture. Coronal suture Sagittal suture Lambdoid suture Step 1b: Determine the coordinates of the point in the brain where the electrode tip is to be positioned. Suggestion – it is always a good idea to check the literature to see what coordinates others have used. Plane Medial CPu AP 0.7 mm ML 2.0 mm DV 4.4 mm AP (anterior-posterior) ML (media-lateral) DV (dorsal-ventral) CPu Step 2: Use an approved surgical protocol to anesthetize the animal and prepare for surgery. Step 3: Mount the animal in the stereotaxic apparatus by placing incisors over the incisor bar and gently inserting the ear bars so that they enter the auditory canal and meatus. Step 4: Clean and incise the scalp exposing the skull surface. Step 5: use the 3 dials on the stereotaxic manipulator to place the tip of the electrode at the Bregma. DV dial AP dial ML dial Step 6: Read the 3 vernier scales corresponding to the AP, ML and DV dials. DV dial AP dial ML dial How to read a Vernier scale The “0” line on the Vernier is above 3.1 cm (or 31 mm) on the main scale. To determine the 10th of a mm, estimate which line on the Vernier scale lines up best with a line on the main scale. AP = 31.5 mm Vernier Scale Main Scale How to read a Vernier scale Now lets read the ML coordinate. ML = 10.2 mm Vernier Scale Main Scale How to read a Vernier scale And now the DV coordinate. DV = 23.7 mm Vernier Scale Main Scale Step 7: Calculate the new coordinates within the brain relative to the bregma Coordinates just read from the stereotaxic manipulator Plane Plane Bregma AP 31.5 mm ML 10. 2 mm DV 23.7 mm Coordinates from the stereotaxic atlas + +/- NEW Coordinates Medial CPu AP 0.7 mm ML 2.0 mm DV 4.4 mm Plane = = = Medial CPu AP 32.2 mm ML 11.2, 8.2 mm DV 19.3 mm Step 8: Use the new coordinates to reposition the electrode. First, do the AP, then the ML on each side marking each spot. Step 9: Drill holes through the skull at each mark. DV dial AP dial ML dial AP = 32.2 ML = 11.2 ML = 8.2 Step 10: Now lower the electrode into the brain at the new DV coordinate. Step 11: Either pass current or inject chemical to make a lesion. Repeat on other side of brain. DV dial AP dial ML dial Step 12: Suture wound and follow postoperative care procedures. Surgery Complete! With all the rights and privileges hereto forth bestowed This will open the door to many techniques including: 1)Lesion methods 2)Electrical stimulation and recording 3)Central drug infusions Lesion Methods Electrolytic and radiofrequency current Current is passed through an electrode to heat the exposed tip and destroy adjacent tissue. Electrolytic lesions of the CPu A B C Lesion Methods Neurotoxin lesions A chemical is infused through a cannula to selectively destroy cells and spare fibers of passage. Ibotenic acid lesion of hippocampus Lesion Methods Aspiration Lesion Cortical tissue is drawn off by suction through a fine-tipped glass pipette. Aspiration lesion of amygdala and entorhinal cortex (monkey) Lesion Methods Knife cut Cutting a nerve or tract without severely damaging surrounding tissue. Knife cut of the perforant path input to the hippocampus 8 7 6 5 4 3 2 1 Lesion Methods Cryogenic blockade A method of temporary inactivation (reversible lesion). Coolant is pumped through a cryoprobe causing neurons near the tip stop firing. The temperature is maintained above freezing so that there is no structural damage. Electrical stimulation Bipolar electrode Two insulated wires wound tightly together are used to deliver weak pulses of current to increase the firing of neurons near the tip. Electrical stimulation often elicits behavioral responses such as eating, drinking, sleeping, attacking and copulation. The behavioral effects are usually opposite to those produced by a lesion. Electrical recording Intracellular unit recording Used to study the electrophysiological responses of a single neuron. Records graded fluctuations in the neuron’s membrane potential. Usually performed on chemically immobilized animals because it is hard to keep the tip of a microelectrode positioned inside a neuron in a freely moving animal. Extracellular unit recording Records the action potentials of a neuron through a microelectrode whose tip is positioned in the extracellular fluid next to it. It provides no information on the neuron’s membrane potential. Multiple unit recording Large electrodes are used to pick up signals from many neurons, adding the total number of action potentials per unit of time. O’Keefe & Dostrovsky (1971) Fig. 1. Responses of a hippocampal (CA1) unit to a restraining tactile stimulus as a function of the rat's spatial orientation. The arrows and associated letters mark the positions at which the animal was restrained as it was pushed or coaxed in a counterclockwise direction around the test platform. The firing rate of the unit during tiffs procedure is illustrated by the continuous frequency histogram in the middle of the figure. The letters correspond to the positions and the lines indicate the periods when the rat was restrained. In between these periods, the rat sat immobile in the same position for a few seconds and then was moved on to the next position. The bottom two lines show the raw data taken at the onset of the unit response at A (1) and during the absence of a response at D (2). Time calibration for these data is 400 msec. Preliminary evidence A process of elimination… Place units in the hippocampus respond to an animal’s location within the environment, not to a specific sensory stimulus, motor behavior or motivational incentive. A Demonstration of Place Cell Firing Central drug infusions Intracerebroventricular (i.c.v.) Infusion of a drug into the ventricles. This method is used when direct delivery to the brain is desired, limiting systemic effects. Intracerebral (i.c.) Infusion of a drug directly into a brain region of interest. For example, an anesthetic like lidocaine may be infused to produce a temporary lesion or an antagonist can be used to block specific receptors.