48 A
drug infusion 2 ml h means that drug concentrations at In addition, no statistical comparisons were made between
sites distant from the target nuclei will be low compared to Bis I and V at the 200 mM dose, because of the small
the injection site [36,44]. The infusion rate used in this sample sizes involved n52 per group.
study is comparable to that of some osmotic minipumps. Second, the diameter of the injection needle and inner
2.8. Histology cannula were identical in order to minimise drug backflow
up the sides of the injection needle. Cannula tip positions were verified for each animal in
these experiments. At the conclusion of the experiment, 2.5. Unilateral vestibular deafferentation UVD
animals were given an overdose of pentobarbital followed by a 0.5–1.0 ml cannula injection of dye Alcian blue
One week following cannula surgery, all animals were 8GX, 5 w v to mark the infusion site. Animals were
given a right global surgical labyrinthectomy UVD, as then transcardially perfused with 0.9 saline followed by
described in detail elsewhere [34]. Briefly, guinea pigs a phosphate-buffered formaldehyde 10 solution. The
were anesthetised and prepared for surgery in an identical brainstem and cerebellum were removed and stored in a
manner to that described for the cannula surgery above. A formaldehyde solution see above until sectioned. The
horse-shoe-shaped incision was made over the right ear fixed tissue was mounted on a microtome stage, frozen
and the temporal bone exposed following blunt dissection with CO and 80–90 mm coronal sections cut serially with
2
of the temporalis muscle and its subsequent removal with a Leitz microtome. The coronal sections were mounted on
surgical scissors. The bony labyrinth was exposed with a gelatin-coated slides and air dried for 24 h before staining
high speed drill, the horizontal and anterior semicircular with thionin blue and coverslipping. At least one slide
canal ampuliae and otoliths were subsequently destroyed from each animal was photographed Kodak Ektachrome
under microscopic control using a fine burr, and their 64T film.
contents aspirated. Our previous studies have shown that this UVD procedure results in complete destruction of the
vestibular receptors [5,11,23,33–35,39].
3. Results
2.6. Behavioural measurements 3.1. Histological verification of cannula tips
Spontaneous nystagmus SN, roll head tilt RHT and Alcian blue dye spots were used to mark the injection
yaw head tilt YHT were measured for each animal at 6, site and hence the position of the cannula tip, but were not
8,10, 12, 20, 25, 30, and 50 h post-UVD. Measurements intended to measure drug spread from the injection site.
could not be carried out earlier than 6 h post-UVD due to Cannula tips of all animals were located within the ipsi-
the possibility of lingering anesthetic affects. All behav- VNC or on its border Fig. 1A–B or in the IVth ventricle
ioural measurements for experiments using PKC inhibitors in the case of the additional control animals described
were carried out double-blind. SN, YHT and RHT were above data not shown.
measured using video techniques and the procedures we have described in detail previously e.g., [5,11]. Mean SN,
3.2. Spontaneous nystagmus compensation YHT and RHT were calculated for each measurement time
within each group of animals. Animals treated with the PKC inhibitor Bis I 5 or 50
mM, had significantly higher levels of SN compared to 2.7. Statistical procedures
controls Bis V or vehicle groups Fl, 1556.70; P,0.05; Fig. 2A–B. Analysis of simple main effects showed that
All data were analysed using a three factor analysis of the Bis I-induced increase in SN was significantly different
variance ANOVA with repeated measures using the from controls only at 6 and 8 h post-UVD F1, 1559.77;
statistical software package SPSS v6.1.1. Drug and dose P,0.05. These times correspond to 5 and 7 h following
represented between-subjects factors while time repre- Bis I infusion. Following this, SN frequencies began to
sented the within-subjects factor i.e. the repeated mea- decrease and became similar to those of controls by
sure. A significant drug or dose interaction with time approximately 20 h post-UVD, and remained this way
represented evidence of a drug or dose effect on the rate of throughout the course of compensation Fig. 3A–B. The
compensation. Analysis of between-group simple main 50 mM dose of Bis I produced the greatest increase in SN
effects was carried out using a standard two factor at 8 h post-UVD; 87 and 88 higher than Bis V and
ANOVA drug and dose at the first two time points vehicle groups, respectively Fig. 3B. Although 50 mM
alpha50.05 [17]. Only data from the 5 and 50 mM doses Bis I resulted in higher mean SN across all time points
of Bis I and V or vehicle were subjected to statistical compared to the 5 mM dose Fig. 3A c.f. 3B, this
analysis since the 200 mM Bis solutions contained a higher difference was not statistically significant. There were no
concentration of DMSO i.e., 10 cf. 5 for lower doses. significant drug or dose interactions with time, suggesting
A .J. Sansom et al. Brain Research 882 2000 45 –54
49
Fig. 1. Schematic illustration of cannula placements in the ipsi-VNC. Animals were infused with either A myr-AIP black circles, AlP grey circles or vehicle light grey circles; B Bis I black circles, Bis V grey circles or vehicle light grey circles. Schematic adapted from Ref. [14]. Dvn, descending
vestibular nucleus; Mvn, medial vestibular nucleus; PH, prepositus hypoglossi; TS; tractus solitarius; TSpV, spinal tract of the trigeminal nerve; SpV, spinal nucleus of the trigeminal nerve; iCP, inferior cerebellar peduncle; x, cell group x; dCN, dorsal cochlea nucleus; vCN, ventral cochlea nucleus; Sa, stria
acoustica; Flm, medial longitudinal fasciculus.
that neither Bis I or Bis V, at 5 or 50 mM, altered the rate tion process. However, since the effect was present in only
of SN compensation. Importantly, there was no significant a subset of animals from both the drug and control groups,
difference in SN compensation between Bis V 5 or 50 mM it seems unlikely that an underestimation of SN frequency
doses and vehicle groups, indicating that the less potent in these animals could account for differences in SN
PKC inhibitor Bis V, had no effect on the compensation between groups. The possible reduction in quick phase
process. These results contrast with the obvious lack of amplitude was not due to an action of DMSO per se, since:
difference in SN between 200 mM Bis I and Bis V at any 1 not all animals were affected; 2 the magnitude of the
time point Fig. 2C, although the data were not subjected effect did not change with increasing DMSO concentration
to statistical analysis see Methods. the vehicle for the 200 mM dose condition contained 10
Detection of the quick phase of SN was difficult in some DMSO c.f. 5 in other groups; 3 there were no
animals at early measurement times i.e. 8 24 animals at significant differences in compensation of SN between
6–12 h in the Bis I, V and Bis vehicle groups and was animals infused with a vehicle containing 5 DMSO in
probably due to a reduction in the amplitude of the quick mACSF and mACSF alone data not shown. VOR op-
phase. Fewer animals 4 24 showed this effect up to 30 h tokinetic reflex eye movements could be evoked in all of
post-op. It is therefore possible that SN frequency was these animals by rotation in light. One possible explanation
underestimated on these occasions early in the compensa- is that the effect was due to a combination of DMSO and
50 A
Fig. 3. The change in spontaneous nystagmus SN frequency expressed as of vehicle 100 produced by either 5 mM A or 50 mM B Bis I
or Bis V over time post-UVD. Symbols represent the ratio of two means. When 5 and 50 mM dose groups were combined, the difference between
Bis I and Bis V at 6 and 8 h post-UVD was statistically significant P,0.05.
Fig. 2. Effect of 5 mM A, 50 mM B or 200 mM C of either the selective PKC inhibitor Bis I, the less selective PKC inhibitor Bis V,
or vehicle on the compensation of spontaneous nystagmus SN. Symbols
in the compensation patterns of RHT or YHT between
represent means61 S.E. When 5 and 50 mM dose groups were combined,
animals treated with the PKC inhibitor, Bis I 5 or 50 mM
the difference between Bis I and Bis V at 6 and 8 h post-UVD was
and controls data not shown. As has been reported in
statistically significant P,0.05. Bar represents the drug infusion period.
numerous other studies, the compensation of RHT and YHT
within groups
was extremely
variable precise cannula placement within the VNC. It is conceiv-
[9,33,34,37,39]. able that differences in the effects of the anesthesia were
A number of animals in both the mAIP 2 11 animals responsible; however, we have not observed such effects in
and AIP 5 10 groups, and one animal 1 3 in the previous studies e.g. [5].
vehicle group, could not be measured for either RHT or Comparison of 5 and 50 mM Bis I infused into the right
YHT at early time points because they could not stand VNC versus the IVth ventricle indicated no significant
unassisted. Since a greater number of animals in the AIP difference in the effects on SN P.0.05, suggesting that
group were affected, statistical comparisons were made Bis I injected into the right VNC might be diffusing to
between mAIP and vehicle mACSF data only, beginning some extent into other areas of the brainstem and cere-
at 10-h post-UVD. There were no statistically significant bellum data not shown; see Discussion.
