APLICACIÓN CON GENERADORES EQUIVALENTES

k- MAIN SCREW

CANNULA

COLLAR

SKULL SURFACE

ELECTRODES

Figure 6a. Diagram showing basic features of the "poor lady" microdrive. Most o f the rats in the present study were implanted with this type o f microdrive.

Dental cement is used to connect the cannula to the main screw and frame. After introducing the electrodes into the brain (eg 1.5mm as shown beneath the skull surface), dental cement is used to affix the sleeve and feet of the drive to the skull. The diagram does not show electrical wire connections from electrodes to the plug.

Mechanical features o f the drive are briefly described. If we ignore the cannula on the right o f the diagram in Figure 6a, there are basically two posts. These two posts are joined at the bottom (soldering joint near word “frame” in Figure 6a) and at the top

(with a flange and nut). These two posts comprise the stationary part o f the drive. One o f the posts (right hand side in Figure 6a) bears a piece o f tubing carrying a screw- thread (“main screw”). The dimensions o f these two tubes are chosen such that the second tube can turn freely on the first without wobbling. Two small sections o f heatshrink tubing are fitted onto the microdrive, one onto each post. When the heatshrink tubing is shrunk well, the tubing round the screw thread on the right-hand post beeomes moulded to the thread in the process and forms a “nut”. Dental cement is then applied around these pieces o f tubing. This forms the basis o f the moveable part o f the microdrive, and is cemented to the cannula (shown).

Not shown are the plug and connection wires mounted onto this moveable part o f the microdrive, using dental cement. The standard set up is to mount 8 wires, 4 on each side, onto the moveable part. However, 16 wires, 8 on each side were mounted onto it for rl079. The wires are soldered at the plug end, so that electrical signals in the wires can reach the headstage, when the headstage is connected to the plugs. At the other end, the wires are cut o ff to form post: each wire o f the tetrode is tightly wound round one o f the four posts relating to that tetrode.

When the feet o f the drive are fixed in space, turning the screw (“screw-tumer”) will cause the moveable part o f the drive to go up or down. A 360 degree turn o f the screw moves the cannula and electrodes down by 200 microns. In general, no turn is made

o f less than 45 degrees (ie. 25 microns). The point o f the spring (see top o f right-hand post “spring”) is to confer smoothness and stability on the movements o f the screw.

Broadly similar principles are behind the design o f the large microdrive used for rl0 0 4 and rl020, onto which 8 tetrodes can be loaded. However, because it is difficult to get many tetrodes all in the pyramidal layer simultaneously, the large microdrive was designed (by John O ’Keefe and David Edwards) with 8 separate screws; this allows for independent movement o f each tetrode. The large microdrive contains 8 posts arranged in a circle. The tetrodes connected to each screw are all fed, and move, through a circular array o f individual cannulae. At the bottom o f the drive, the

tetrodes fit into a sleeve, which is like an 8-bullet gun revolver. As with the standard small “poor lady” microdrive, in surgery dental cement fixes the feet o f the drive and the sleeve to the skull.

Tetrodes - further details

Tetrodes were constructed by twisting four individual fine pieces o f wire together. This is done by taking one piece o f wire, sticking the ends together, and creating two loops. The top o f the two loops were hung on a post, and the wires were twisted clockwise (about 15-20 turns per mm). The top o f the loops are cut to form free ends. These free ends are tied around the posts o f the drive. The other end is cut to form the tetrode tips which will be implanted into the brain.

For experiments 1 and 2, tetrodes used in the standard microdrive, which does not allow for independent movement o f each tetrode, were sometimes made and cut so that the tips o f each tetrode were located at similar dorsoventral levels (eg. rl079,

rl062, ri 029). In other words, the aim was to achieve a state o f recording, at some stage in the experiment, with two or more tetrodes recording from the same pyramidal layer simultaneously. This was simply to increase the potential yield from any one microdrive.

Surgery

Rats always underwent surgical implantation o f movable microelectrodes before the given experiment. Rats were anaesthetized using isoflurane and nitrous oxide, and given an i.m. injection o f buprenorphine (45pg) for intra- and postoperative analgesia, and an s.c. injection o f enrofloxacin (2.5mg) as a prophylactic antibiotic.

Rats were first anaesthetized using the gas preparation as follows: Oxygen at 3 litres per minute, with isoflurane at 3% o f the gas volume. Before the earbars are placed in the animal, nitrous oxide is introduced at 3 litres per minute, and the oxygen flow is reduced to 1.5 litres per minute. Around this point, the i.m. injection o f buprenorphine and s.c. injection o f enrofloxacin are given. The percentage o f isoflurane is gradually decreased throughout surgery, which usually ends at levels o f 0.5 to 1.2 %.

The target coordinates for electrodes in animals implanted with a single microdrive were centred, in either hemisphere, as follows: AP -3 .8 to -4.0 mm; ML 2.4 to 2.7 mm, (both AP and ML relative to bregma); DV 1.5 to 1.7 below pial surface. For animals implanted with two microdrives, the two targets were a) more anterior and medial (AP -3 .0 to 3.3 mm; ML 1.3 to 2.0mm) and b) more posterior and lateral (AP —4.0 to -4.8 mm; ML 2.5 to 3.0 mm) with the same DV distance. A 2mm diameter hole was drilled into the skull above the target coordinates.

The tetrode-microdrive assembly was fixed to the skull by a) screwing in jew eller’s screws into the skull, and b) applying dental acrylic around the feet o f the assembly and on the skull and screws. One screw soldered to a gold Amphenol pin served as the ground attachment. A plastic screw, screw end down, is cemented to the skull,

providing the attachment for the LED-holder part o f the headstage. Other plastic screws are also cemented where necessary to protect the drive from any grooming movements by the animal.

Unit recording

Each rat was connected to the recording equipment via a headstage (or two

headstages) which fitted onto the plug(s) o f the mierodrive(s). The headstages used were unity-gain buffer amplifiers; the implanted electrodes were AC-coupled to these amplifiers, which served to isolate the electrodes from the wires carrying their signals to the recording system. One or two small, infrared light-emitting diodes (LEDs) were attached to the rat (anchored via a plastic screw cemented to the rats'

skull during surgery) for the purpose o f tracking the rats' movement with a video camera and position-detection hardware. Lightweight hearing-aid wires 2 to 3 metres in length connected the headstage(s) to a preamplifier (gain 1000). The outputs o f the latter passed through a switching matrix, and then to the filters and amplifiers o f the recording system. The switching matrix is an array o f analogue switches to which all o f the electrode signal inputs and all the amplifier boards are connected. Under software control, the switching matrix allows the user to control which signals get recorded differentially with respect to which other signals.

Signals were amplified (c. 15-50 thousand times) and bandpass filtered (500 Hz-7 kHz). Each o f the four channels o f a given tetrode was recorded differentially with respect to a channel on another tetrode. Differential recording is now a standard technique, used to subtract noise and artifact from the signal. If two channels pick up the same artifact signal, and a cellular signal, differential recording will pick up only the cellular signal. Occasionally (rarely), it was necessary to record from two “active” tetrodes where each was referenced to a channel on the other. Fortuitously, in all such cases, no obviously overlapping place fields were recorded, and in any event, the data in this thesis will not be used for correlation purposes. Each channel was continuously monitored at 20-|us intervals, and potentials were captured with 50 sampling points per channel (1ms, with 200 ps pre-threshold and 800 ps post-threshold) whenever the signal from any o f the pre-specifled recording channels exceeded a given threshold set by the experimenter (ie when a presumptive neuronal spike occurred). Each spike event was spatiotemporally stamped with: a) the time since the start o f the recording, and b) the x, y location o f the LED, or LEDs, as determined by the position-detecting hardware (position sampling rate 50Hz). All such data were stored on a hard drive and transferred to a SUN workstation, for later analysis. Most o f the animals were

recorded with two LEDs, and thus head-direction information, as well as x,y location could be determined.

All the microdrives, headstages, preamplifiers, tracking and recording systems, were purpose built (John O ’Keefe Laboratory members; Gignomai Ltd; Axona Ltd).

Long-term recording

One aim in experiments lb and 2 was to be able to follow cells over several days, and even weeks if possible. This was o f course to try to observe experience-dependent or learning-based changes at the level o f the individual cell. Accordingly, once amplifier gains and thresholds were set for a tetrode, they were changed as little as possible. This helped to identify a cell recorded over a long period. In order to achieve a high level o f stability, recording did not usually begin until several weeks after the surgical implant. In order to prevent accidental movement o f the electrodes, blue tack was sometimes affixed to the screw-tumer on the microdrive(s). Efforts to achieve long­ term stability were particularly important in experiment 2.

Screening for activitv and ensuring stabilitv

Rats were screened for cell activity on the holding platform, and the experimenter checked for stable place cells on the holding platform, before recording. Screening generally did not begin until at least a week after surgery. As decribed in Chapter 3, on the physiology o f the hippocampus, various BEG and cell firing characteristics can be used to determine the whereabouts o f the electrodes in the hippocampus. Particular attention was paid to the high-frequency “ripples” state, seen when the animal is still and at low arousal levels (including sleep). The electrodes were moved down towards CA l in multiples o f 50 or 100 micron steps until signs o f low amplitude “ripples” were seen. When ripples were seen, the electrodes were moved down in 25 micron steps. The basic idea was to leave the electrodes above the layer or simply left at that dorsoventral level to gradually drift into the C A l layer. At some point, place cell activity would be seen on the holding platform. This was almost never recorded, but

the experimenter would make rough diagrams o f place fields based on this activity. Once stable fields were seen over days, recording would begin.

General Laboratory layout -