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Preincubation o f M D L 72222 (10 fxM) attenuated the depolarizing effects o f 5- HT on DVMs to 85.1 ± 3.8 % of control responses (i.e. 4.7 ± 1 . 7 mV to 4 ± 1 . 5 mV, n = 9 , figure 4.6a, figure 4.8). The responses to 5-HT were never completely abolished and in two neurones MDL 72222 had no effect.

ICS-205-930 (1 fiM) also reduced partially the depolarizing effects o f 5-HT in all neurones tested to 86.3 ± 3.1 % of control responses (i.e. 6.4 ± 1 . 9 mV to 5.2 ± 1.3 mV, n = 3 , figure 4.6b, figure 4.8). ICS-205-930 at a higher concentration (10 fiM) increased the partial attenuations to 70.8 ± 5.8 % of control responses (i.e. 5.6 ± 1 .7 mV to 4 ± 0.4 mV, n = 4 , figure 4.6c, figure 4.8). The attenuations by MDL 72222 or ICS-205-930 at both concentrations did not attenuate significantly (p > 0 .0 5 ) the excitatory responses of 5-HT on DVMs. The preincubation of 5-HT3 receptor antagonists did not have any effects on membrane potential, the levels of baseline noise or input resistances.

2-Methyl-5-HT (100-300 /xM, 2 min, figure 4.3c) excited 5 out o f 9 DVMs tested. The responses consisted of depolarizations (3.8 ± 0.6 mV, 100 /xM, n = 3 ) combined with decreases in apparent input resistance (17.4 ± 2.2 %, n = 3 ) and increases in baseline noise which indicated increases in PSPs.

Figure 4.6

The effects of 5-HT3 receptor antagonists on three different DVMs.

a) Superfiision of M DL 72222 (10 fiM) attenuated the number of action potentials elicited by 5-HT (20 fxM, 2 min) after a 15 min preincubation period.

b) Superfiision o f ICS-205-930 (1 jixM) also produced a small reduction in the depolarizing effect of 5-HT (20 ^M , 2 min) after a 20 m in preincubation period.

c) At a higher concentration, superfiision of ICS-205-930 (10 /xM) reduced the number of action potentials evoked by an application of 5-HT (20 juM, 2 min) after a preincubation period of 25 min.

In all three recordings, the neurones were lost before recovery could be obtained.

a) MDL 72222 10 ^lM 5-HT 20 5-HT 20 i^M b) IC S-205-930 1 5-HT 20 5-HT 20 i^M c) ÏC S-205-930 10 5-HT 20 laM 5-HT 20 laM 20 m V

Figure 4.7

The effects o f a selective 5-HT4 receptor antagonist GR 113808A on the depolarizing responses of 5-HT and 5-MEOT on a DVM.

a) The excitatory effect of 5-HT (20 /iM , 2 min) on a DVM was not attenuated by GR 113808A (1 /iM) after a 40 m in preincubation period.

b) In the same neurone, 5-MEOT (50 /iM , 2 min) still mimicked the depolarizing effect of 5-HT (20 /iM, 2 min) after GR 113808A (1 /iM) had been continually superfused for over 1 hr.

a)

5-HT 20 nM 5-i i r 20 nM 5-M i'O r 50 [iM

léÉÊIIIIiiliÊlItlÉm

20 mV

4 .3 d Effects o f G R 113808A an d 5-M EO T

GR 113808A (1 jLiM) had no effect on the depolarizing responses of 5-HT (96.4 3.6 % of control responses, 5.3 ± 0.6 mV to 5.2 ± 0.7 mV, n = 3 , figure 4.7a, figure 4.8) even after preincubation periods of over 1 hour.

Applications of 5-MEOT (50 and 100 jitM, 2 min) mimicked the effects of 5- HT in 17 out o f 19 DVMs tested, evoking slow depolarizations (6.6 ± 0.8 m V, 50 jiiM, 2 min, n = 13, figure 4.9) that were maintained in synaptic block medium (figure 4.9a). In the majority of neurones these depolarizations were accompanied by increases in apparent input resistance (25.6 ± 3.6 %, n = 14). O f the remaining three neurones, one evoked a 11 % decrease in apparent input resistance whilst the apparent input resistances of the other two DVMs were not changed. In all cells tested, the responses of 5-MEOT (50 (jM ) were attenuated significantly by ketanserin (1 /xM) to 10.1 ± 1.2 % of control responses (i.e. 6.3 ± 0.9 mV to 0.7 ± 0.2 mV, n = 3 , figure 4.9b). In one neurone, ketanserin abolished completely the slow depolarization elicited by 5-MEOT and after 40 minutes of wash out a partial recovery was obtained (62 % recovery, 4 mV to 2.5 mV, figure 4.9b). In another DVM, a prolonged application of GR 113808A for over 1 hour did not prevent 5-MEOT from mimicking the response of 5-HT (figure 4.7b).

4.3e Effects of other 5-H T recep to r antagonists an d agonists

Pindobind. 5-HT^^^ (5 /xM) had no effect on the excitatory responses of 5-HT on DVMs (97.5 ± 3.4 % of control responses, i.e. 8 ± 0.6 mV to 7.8 to 0.7 mV, n = 5 , figure 4.10a, figure 4.8). Applications of (+ )-8 -0 H -D P A T (1-50 /xM, 2 min) also had no effect on the membrane potentials, spontaneous PSPs or input resistances of any DVMs tested (n = 4 , figure 4.10b).

Figure 4.8

A bar chart showing the % reduction of control depolarizing responses to 5-HT (20 ^M , 2 min) by the various 5-HT receptor antagonists used in this study.

Ketanserin, spiperone and LY 53,857 all significantly attenuated the control responses. * = p < 0 .0 5 and * * = p < 0 .0 1 . ICS-205-930, M DL 72222, GR 113808A and pindobind.5-HTi^ did not significantly inhibit the responses to 5- HT.

5-HT response (%) O O CJ)

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Figure 4.9 The effects o f 5-M EOT on two different DVMs.

a) Application o f 5-M EOT (50 fxM, 2 min) elicited a slow depolarization with a peak amplitude of 6 mV which was large enough to reach the threshold for action potential generation. The excitatory effect was maintained in a medium which blocked synaptic transmission. The presence of TTX blocked the generation o f action potentials.

b) In another neurone, an application of 5-MEOT (50 juM, 2 min) elicited a slow depolarization which was blocked by ketanserin (1 juM) after a preincubation period o f 15 min. Partial recovery (62 %) was observed after 40 mins wash out.

a)

5-MEOT

ITX 1

[iM, CNQX 10 nM, AP-5 50 |aM,

b i c u c u l l i n e 10 p M