Analysis of autoradiograms demonstrated that the injection tracts terminated in the thalamic relay nuclei (Figures 2.4. and 2.6 ). The distribution of GABAg site binding varied between the five brain areas studied which included the CAl region of the hippocampus, the VL and VPL thalamic relay nuclei, the NRT and the CPu. The relative values of GABAg site binding under control conditions in these brain regions fi'om highest to lowest is VL>VPL>CAl>CPu>NRT (Figures 2.5. and 2.7.). Analysis of the autoradiograms following the 40 min treatment with PTx, denatured-PTx and vehicle (Figure 2.4.) demonstrated that GABAg site binding on the right and left hand sides of the brain in all of the brain regions investigated was not altered by PTx treatment (Figure
Chapter 2 : Intrathalamic pertussis toxin treated GAERS
Total SWD duration (s per 40 min recording period)
Vehicle Denatured- PTx (n=3) PTx (n=3) (n=6) Control 528 ± 95 538 ± 1 8 0 594 ± 63 Injection Day 1 467 ± 38 473 ± 164 224 ± 73* Day 2 447 ± 22 447 ± 64 118±58*(axa) Day 4 333 ± 68* 437 ± 87 43 ± 1 8 * (b )(e ) Day 6 500 ± 83 533 ± 128 22 ± 12*(c)(f)
Table 2.2. Total SWD duration in GAERS following an in vivo 6 day treatment
with intrathalamic pertussis toxin.
On days seven to nine following implantation of cortical electrodes, the total cumulative SWD duration (s) per 40 min recording period was determined for three groups o f GAERS designated for future treatment with vdiicle (i^3), denatured-PTx (n=3) and PTx (n=6). The mean SWD duraticm value for these three days was designated as the control value for each animal. Following injecticxi o f the designated GAERS with either PTx, denatured-PTx or vdiicle the EEG was recorded far 40 min paiods and the total SWD duraticm (s) determined on days 1 ,2 ,4 and 6 after injection. Data represent mean db s.e.mean. There was no significant difference between the ccmtrol values for the three groups (uiq)aired Student t-test). PTx treatment was found to significantly deoease the total SWD duration cm days 1 ,2 ,4 and 6 when compared with the control value (* p < 0.01, rq)eated measures ANOVA followed by Dunnett's test). The total SWD duration on day 4 following vehicle treatmmt was also found to be significantly reduced when compared with the control value (* p < 0 .0 1 , rqxated measures ANOVA followed by Dunnett's test). The total SWD duration o f GAERS following PTx- treatment was significantly lower than that recorded in vehicle treated GAERS on days 2, 4 and 6 ( (a) p < 0.01, (b)p <0.001 and (c)p <0.0001, uiç>aired Student t-test) and in denatured-PTx treated GAERS cm days 2 ,4 and 6 ((d) p < 0 .0 1 , (e )p < 0.0005 and (f) p < 0.001, unpaired Student t-test).
(a) PTx (b) Denatured-PTx
(d) Non-specific (c) Vehicle
Figure 2.4. Autoradiogram s o f pH ]-G ABA binding to GABAg sites in G A ER S following an in vivo 40 min treatm ent with intrathalam ic pertussis toxin.
Example autoradiograms of total [^H]-GABA binding to GABAg sites in coronal brain sections of GAERS following intrathalamic injection and subsequent 40 min treatment with (a) PTx, (b) denatuied-PTx and (c) vehicle, plus (d) an example of non-specific binding. The binding scale (fmol/mg of tissue) shades different regions different colours depending on the level of binding. The areas analysed on both the right and left hand sides of the brain (see (c)) were the CAl region of the hippocampus (CAl), ventrolateral thalamic nucleus (VL), ventral posterolateral thalamic nucleus (VPL), nucleus reticularis thalami (NRT) and caudate putamen (CPu). Experiments were performed using 50nM [^H]-GABA as described in experimental procedures and the autoradiograms represented aie typical.
Chapter 2 : Intrathalamic pertussis toxin treated GAERS (a) 100 1 o# £ o E 00
i
m < O VPL NRT CPu (b) 100 n o> 80 -1
sz. 60 - s o 40 - m 1 < m 20 - < <3Figure 2.5. [^H]-GABA binding to GABAg sites in GAERS following an in vivo 40 min
treatment with intrathalamic pertussis toxin.
pH]-GABA binding to GABAg sites in specific regions of the (a) right and (b) left hand sides o f the brain o f GAERS following intrathalamic injection and subsequent 40 min treatment with vehicle (open bars, n=3), denatured-PTx (hatched bars, n=4) and PTx (shaded bars, n=3). As described in Figure 2.4. the brain areas analysed were the C A l, VL, VPL, NRT and CPu. Experiments were performed using 50nM pH]-GABA as described in experimental procedures. Data represent specific binding o f [^H]-GABA (mean ± s.e.mean, finol/mg o f tissue). N o significant difference between vehicle, denatured-PTx and PTx treatments, except in the NRT o f the right hand side, where binding was significantly greater in PTx treated when compared with vehicle treated (* p < 0.05, unpaired Studait t-test).
(a) PTx (b) Denatured-PTx
(d) Non-specific (c) Vehicle
Figure 2.6. Autoradiogram s of pH ]-G ABA binding to GABAg sites in G A E R S follow ing an in vivo 6 day treatm ent with intrathalam ic pertussis toxin.
Example autoradiograms of total [^H]-GABA binding to GABAg sites in coronal brain sections of GAERS following intrathalamic injection and subsequent 6 day treatment with (a) PTx, (b) denatured-PTx and (c) vehicle, plus (d) an example of non-specific binding. The binding scale (fmol/mg of tissue) shades different regions different colours depending on the level of binding. The areas analysed were as descnbed m Figure 2.4.. Experiments were performed using 50nM pH]-GABA as described in experimental procedures and the autoradiograms represented are typical.
Chapter 2 : Intrathalamic pertussis toxin treated GAERS (a) 100 CA l VL VPL NRT CPU (b) 100 1 a t E o E 0 jO 00 1 < CO < o
Figure 2.7. [^H]-GABA binding to GABAg sites in GAERS following an in vivo 6 day
treatment with intrathalamic pertussis toxin.
pH]-GABA binding to GABAg sites in specific regions o f the (a) right and (b) left hand sides o f the brain o f GAERS following intrathalamic injection and subsequent 6 day treatment with vehicle (open bars, n=3), denatured-PTx (hatched bars, n=3) and PTx (shaded bars, n=6). As described in Figure 2.4. the brain areas analysed were the C A l, VL, VPL, NRT and CPu. Experiments were performed using 50nM pH]-GABA as described in experimental procedures. Data represent specific binding o f pH]-GABA (mean ± s.e.mean, finol/mg o f tissue). PTx significantly decreased binding in the VL and VPL in both sides o f the brain when con^ared with vehicle ( (a) p < 0.(X)05, unpaired Student t-test) and denatured-PTx ( (b) p < 0.005, (c) p < 0.001, (d) p < 0.0005, unpaired Student t-test).
2.5.). An exception was the NRT of the right hand side of the brain of PTx treated GAERS, where GABAg site binding was significantly greater than equivalent binding in vehicle treated GAERS, but not that of denatured-PTx treated GAERS (Figure 2.5.). The recorded level of GABAg site binding in the NRT is below the level of the lowest standard, suggesting that the relevance of this difference may be questionable. Furthermore this difference was not recorded in the 6 day data, thus indicating that it probably represents an experimental artifact. After the 6 day treatment with PTx, analysis of the autoradiograms (Figure 2.6.) dononstrated that GABAg site binding in the VL and VPL was significantly decreased by between 70 and 80% on both the right and left hand sides of the brain when compared with denatured-PTx and vehicle treated GAERS (Figure 2.7 ). In the same thalamic nuclei there was no significant difference between denatured- PTx and vehicle. In the CAl, NRT and CPu there was no significant difference between PTx, denatured-PTx and vehicle.
2.4.4. The effect of intrathalamic pertussis toxin on GABA^ site binding