Small animal stereotaxy

Transcription

1 Small animal stereotaxy Most experimental manipulation of the brain for example lesions, micro injections, stimulations and recordings etc require the application of stereotaxic procedures. The stereotaxic instrument allows us to precise placement of electrodes and cannulae in to discrete areas of the brain on the basis of the co-ordinates specified in the brain atlases.

2 The stereotaxic instrument has an antero-posterior (AP) scale, a vertical (dorso- ventral)(dv) scale to measure the depth and medio-lateral (ML) to give the position from the midline. The AP is kept at zero and the electrode is fited so that it is in the midline. The earbars are kept at an equal reading on both sides. The precise fixation of the head in a rigid frame is by means of ear bars in the external auditory meatuses and the incisors rest in the incisor bar. Generally during stereotaxic surgery, the coordinates for AP movement are taken from a reference point in the skull. There are mainly two such reference points; a) Bregma- the point of intersection between mid- sagital suture and coronal sutures, b) Lambda- the midpoint of the curve of the best fitalong the lambdoid suture.c) the interaural line the imaginary line passes through the brain at the midpoint of the external auditory meatus. The most commonly followed reference point is the bregma. Stereotaxic implantation of the electrodes for EcoG and LFP recordings. Rats are used for electrode implantation All surgical procedures are performed stereotaxically under asceptic conditions. Rats are anethetized and fixed in astereotaxic apparatus so that the skullis positioned horizontally inorder to keep the bregma and lambda in one plane.

3 The skullsurface is cleaned so that the external landmarks of the surface become visible for the implantation of electrodes using bregma as the referance point. The flat skull stereotaxic co-ordinates are adapted from Paxinos and Watson rat atlas. Holes were drilled in the skull using micro driller ar appropiate watch makers screw driver. Either the screw electrodes or insulated micro electrodes can be used for surface EEG and subcortical EEG. Screw electrodes are used as the reference and ground. All these electrodes are soldered to a 7 pin socketand the connector is fixed to the skull using dental cement. The animals were allowed to recover from the surgery. Recording Procedures. After three to five days of post surgical recovery the implanted electrodes are connected to the Neurologger. Prior one or two days before recordings dummy loggers are connected in order to get habituated to the weight of the Neurologger.

4 Configuring the neurologger. Introduction This manual describes how to use the NeuroLogger. The software package includes two programs. The CommSW program is communicating with the NeuroLogger through a Read Out Station and is used to configure, start and stop the NeuroLogger, and to download and erase recorded data. The HxD program is used to view and edit hex files, the file format in which the NeuroLogger stores data. This package also includes drivers for the CommSW program, and two scripts to import NeuroLogger data into Spike2, a commercially available electrophysiological data analysis software. List of folders Drivers folder with drivers needed to use the Read Out Station Manual this manual s folder Programs folder with the CommSW and HxD programs Spike2scripts folder with two scripts to import hex data into Spike2 Setup Software setup

5 To use the CommSW program to communicate with the NeuroLogger through the Read Out Station (ROS; see the ROS pictures in the NeuroLogger setup section) it needs to be copied to a computer together with the Drivers folder. To install the driver, first connect the ROS to your computer. Wait until the standard Found New Hardware dialog appears. Select Install from a list or specific location (Advanced) and press the Next button. Select Don t search. I will choose the driver to install and press the Next button.

6 Select Show All Devices and press the Next button. Press Have Disk button, select the Driver folder and then the Next button. In order to complete the installation this procedure might need to be repeated twice. It can vary for different systems, but not significantly. NeuroLogger setup

7 To set up the NeuroLogger the CommSW program needs to be installed as described above. Connect the ROS to the computer. It is connected via standard USB cable. To operate the ROS, drivers needs to be installed as described above. Insert the NeuroLogger into the ROS (without batteries) into the hollow (battery holding side down, and connector socket out) and wait until the light blinks on it indicating that the NeuroLogger has been placed correctly into the ROS. Using the CommSW program Run the CommSW program.

8 Press Connect. The program will connect to the NeuroLogger and the different options will become available.

9 The Info section shows how much data is on the logger. The min Page Index is always 64; this data range is used internally. The max Page Index shows how much data is on the NeuroLogger. NumPages= x samples/sec x recordingtime[hrs] Accessing NeuroLogger data is possible in the Download data from device section. After pressing the Download data button, the name for the recording can be given. The data is saved in a hex file format. The download progress is indicated by a fill bar above the Logging section. In the Commands section the settings of the NeuroLogger can be changed by pressing the Configure device button. Pressing the Get config from device button loads the last saved settings. In the DataFormat section there is a possibility to choose between short (8bit) and long (10bit) formats. Delay Mode is used to choose between different ways of starting the NeuroLogger:

10 immediate (no delay) the NeuroLogger will start logging right after the Start Logging button is pressed. timer the NeuroLogger will start logging after the Delay Time interval will pass. event the NeuroLogger will start logging after any event is marked (Marking events is possible through IR light). timer and event the NeuroLogger will start after any event is marked, but not earlier than after the Delay Time interval. timer or event the Neurologger will start after any event is marked, but not later than after the Delay Time interval. Delay Time setting has an effect only if the Delay Mode is set to timer, timer and event or timer or event and means the delay between the time when the device is started by pressing the Start Logging button and the time when data recording starts. In the Time between Samples section the sampling rate of the recording can be set. To get the number that should be entered into that field, needs to be divided by the desired frequency. The result needs to be rounded to the integer. For example if 200 Hz is the required sampling rate, then 164 needs to be entered into the textbox as 32768/200= This is because the NeuroLogger has a standard crystal clock. It is important to keep in mind that the real sampling rate will not be integer. The real sampling rate (199.8 Hz in the example above) is indicated in the header of the hex file and can be viewed with the HxD program. Pressing the Save config to device button, either confirms the previously used settings that appear after pressing the Get config from device button, or saves the newly entered settings. After this, the window needs to be closed in the right top corner of it. The other function in the Commands section is erasing data by pressing the Erase Data button. The NeuroLoggers can be started and stopped in the Logging section, by pressing the relevant button.

11 There is a red light on the top side (battery holding side) of the NeuroLogger (see picture bellow) to indicate if the NeuroLogger functions correctly (short blinks in every few seconds, correct function) or if it has a problem (long blinks almost continuously, malfunction). A usual recording session goes as follows: 1) Insert NeuroLogger into the ROS (without batteries) connect 2) Save/Erase old data from the NeuroLogger disconnect and connect again 3) Set recording parameters (e.g. sampling frequency) 4) Press Start Logging and wait for the correct function blinking 5) Take the NeuroLogger from the ROS 6) Insert batteries and control for the correct function blinking. If that does not occur and/or there is a malfunction blinking, remove the batteries and set up the NeuroLogger again. 7) Mount the NeuroLogger to the connector on the head of the test animal 8) After the desired recording time, remove the NeuroLogger from the animal 9) Take off the batteries 10) Insert the NeuroLogger into the ROS 11) Download data and save it Batteries

12 Their specification: Zinc-Air batteries, p10, 1.4 V. There s a hole on the + side of these batteries through which it gets air. That side should face the battery holding surface on the NeuroLogger. After inserting the batteries, the lamp on the top side of the NeuroLogger s surface should blink in the correct function mode. Otherwise, the batteries need to be removed and inserted again. The top side of the NeuroLogger is shown on the picture above. To the north are the 7 connector sockets, which receive the 7 connector pins implanted into the connector on the head of the test animal. The connections from left to right are as follows: Reference channel 2, EEG channel 4, EEG channel 3, EEG channel 2, EEG channel 1, Reference channel 1, Ground.

13 The NeuroLogger can function in 1 reference channel mode, when the two reference channels are internally connected, or in a 2 reference channel mode, when they are separtated. By default, the NeuroLoggers are in 1 reference channel mode. On request, they can be changed to the 2 reference channel mode. In either case, the connecting pins implanted on the head of the test animal and referring to Ground and reference channel 1 need to be electrically connected. If the NeuroLogger operates in the 1 referenece channel mode, Reference channel 2 is not used, and the corresponding connecting pin should not be connected to any electrodes. Marking events The NeuroLoggers have an infrared (IR) light receiver and are able to distinguish 6 different IR patterns, which are generated by the SynchroBox. The SynchroBox is equipped with two rows of six connectors, 7 push buttons, a red and a green light. 1) The top row of connectors (with a red lamp in the middle) is used to connect IR lamps to the SynchroBox. All 6 connectors produce the same output, so all the lamps connected to the SynchroBox works in a parallel. 2) The red lamp in the middle of the top row of connectors indicates the IR signal. When one button of 6 in the row is pressed, the red lamp blinks for a short amount of time. That means the event was sent to the Neurologger via IR lamps. 3) Green lamp indicates if the device is powered 4) The 6 buttons in the middle row are used to mark 6 different events, each button for one event and producing different IR patterns. The 7 th button(above the button row) is used to reset the device if needed. 5) The connectors in the bottom row are used to generate events and work the same as the corresponding buttons. These can be used by a controlling device instead of pressing buttons manually.

14 To use the event marking functionality the SynchroBox needs to be connected to the power supply, and the IR lamps to the outputs of the SynchroBox. The IR lamps need to be arranged above the space, where the test animas are being recorded, in a manner that the entire space is covered by the IR beam of one or more lamps. Due to the device and program limitations the time interval between each 2 events should be more than half a second (better if it is more than 1 second). Importing NeuroLogger data into Spike2 In order to be able to record 4 EEG channels, a movement channel, and the IR marks up to 48 hours on a device weighing only 3 gr, the NeuroLogger stores recorded data in a highly compressed file format (.hex). One option for visualizing and analyzing the NeuroLogger recordings is the commercially available software, Spike2. This needs two scripts: 1) NeurologgerImp.s2s This script imports.hex files into Spike2 and converts them to.smr files, the Spike2 format. As explained above, the NeuroLogger does not sample integer frequencies, while Spike2 can interpret only integer frequencies. This might lead to time scale problems. A resampling script has been made to counteract these problems.

15 2) Resample.s2s This script display a dialog with a list of the channels present and their respective sample rates, and a box where the required sample rate can be entered. Then it creates a new file with suitable timing parameters, and imports the data into this new file, with an interpolation function resampling the data to the new rate.