sections taken at lambda and at 1,2,
3 and 4 mm behind lambda. Standard
representations o f these levels are
pictured on the left with overlaid
symbols representing cannula tip
placements for each subject. The
deep nuclei are outlined in pink.
The calibration bar on the right
provides an indication o f the level
of radioactivity, and therefore
muscimol binding in each subject.
Tritiated muscimol provides little
indication of PBS spread.
and is likely to spread further than muscimol. The image is, however, included to demonstrate that the cannulae are cortically placed in these subjects and that the phase 5 performance effect seen in these subjects could be attributed to cortical rather than nuclear spread. This autoradiography was essential in the CON group because, unless HVI location and fully effective performance testing could be established in these subjects, then it would not be valid to make any conclusions about muscimol’s lack of effect on already existent CRs during phase 3.
These images also illustrate the fact that there was no nuclear spread o f muscimol in the CTX subjects and that cortical spread was limited in the DN animals to regions that are not presently known to be important in eyeblink conditioning. All of these subjects had limited damage from the cannulation procedure, because there was little obvious gliosis below or around the cannula, and all were capable o f learning. That the drug was sufficiently close to key components of the eyeblink circuitry is confirmed by the results of the phase 5 performance testing. CR performance was completely abolished for at least one time-point in this test for all subjects included in the final analysis.
The rejected subjects are worth considering in themselves because in some cases they confirm the main result presented above and in no case do they challenge it. Some animals were excluded from the analysis for excessive damage incurred by the cannulation process (see methods). A few of these cases had initially been treated as controls and learned within phase 1. They were therefore unlikely to be more
damaged in key areas than some of those included in the analysis. These animals could not, however, be included post-hoc. It was necessary to adhere to the criteria for rejection set in place prior to the experiment. Histological assessment was carried out blind to the acquisition data otherwise experimental bias would have been introduced.
O f those individuals receiving experimental treatments, it was found that rejected animals tended to uphold the dissociation of muscimol effect between cortex and nuclei. Two particularly interesting rejected cases are depicted in fig 2.09. These cases retain their original experimental codes because they were not included within the final analysis. KG 110 was an animal that had been cannulated to target HVI and
Autoradiography Perform ance Cannulae
a:,
..«am
4 6 à Time (hrs) K0110 2 4 6 8 Time (hrs) K0807 P h a s e 1 Acquisition P h a s e 2 P h a s e 3 100 M 8 0 - 6 0 - (/} q:o
o ' 4 0 - 2 0- ^ CTX K0807 K0110 0 2 4 6 8 10 12 S essionFigure 2.09: Off-target placements - Two example rejected cases. KOI 10 was an intended cortical placement and K0807 an intended deep nuclear placement. The failure in targeting these sites is revealed by autoradiography on the left and phase 5 performance testing in the centre. The graph on the right depicts the learning rate of these two individuals relative to the CTX group. Despite mis-placement both animals are consistent with the main findings. KOllO was not fully affected in performance testing, suggesting that eyeblink circuitry was mis-targeted by muscimol, and is accordingly not severely affected in conditioning. K0807 has significant cortical spread of muscimol
was given muscimol infusions after training across phase 1. This animal was excluded from analysis due to a failure on the phase 5 performance test. Failure on this test indicates that the drug had reached key components o f the eyeblink circuitry some time after the infusion and was not at sufficient concentration to prevent the circuit from functioning fully in the production of CRs. This animal, unlike those included in the CTX group for analysis, developed CRs during phase 1. No other case given this treatment performed any CRs within phase 1. K0807 was a deep nuclear-implanted case in which the muscimol infusion at the beginning of the performance test
completely abolished previously acquired CRs. Analysis of the drug spread, however, revealed a significant amount of muscimol in the area of HVI that had previously been identified as crucial to CR performance (Attwell et al., 1999). This individual was rejected, again blind to the acquisition data, on the grounds of cortical spread. In complete concordance with the results presented above, and consistent with an important role for lobule HVI in the consolidation process, conditioning was found to be heavily retarded in this case.
Study 2
Muscimol does not spread greatly over time
All of the subjects included in this study show complete abolition o f CRs in the performance test. In many cases this abolition is seen almost immediately after the muscimol infusion. In other cases, however, the muscimol does not fully affect the system until one or even two hours later. All cases included showed a complete performance effect. The latest the muscimol can have a complete performance effect and the subject still be considered for inclusion is two hours after the initial infusion (Further discussion of these timing issues can be found in the next chapter). All subjects included were completely affected in their performance o f CRs two hours into the test. With this in mind the subjects were sacrificed two hours after the final ^H-muscimol infusion so that the spread could be assessed at a time-point consistent across all subjects. Figure 2.10 shows the results of a simple study that was conducted to assess the spread of muscimol at these different times. It was essential to know that
% hour 1 hour 2 hours 4 hours