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10-^M D A I V locust saline 5 X 10-'"M D A (10 ‘*M fluphenazine) V I

(10 M fluphenazine) 0.005% ascorbic acid

Ul l l l . l i l U l H l l i l l

1.8 X IQ-'^M DA on an isolated B A l motoneurone - following bath-application a

membrane depolarization o f approximately Im V in amplitude was evoked. No change in membrane conductance was observed. Upon bath-application of 5 X lO'^M D A the membrane potential was depolarized by approximately 2.5mV. Again no detectable change in membrane conductance was observed (Figure 2.3.9Ü). The effect o f bath-application o f lO'^M D A onto this neurone is shown in Figure 2.3.9iii. In this instance DA evoked approximately 5.5mV membrane depolarization. The small increase in input resistance observed may have been a result o f slight blocking o f the 'voltage' microelectrode, rather than a drug-induced response. The blocking o f the microelectrode results in increased microelectrode resistance, and consequently (from Ohm's Law), larger voltage deflections are associated with the hyperpolarizing current pulses o f constant amplitude. As a control for these DA-induced effects, a test application of locust saline was added to the experimental bath. No significant changes in membrane potential or input resistance were seen following administration (Figure 2.3.9iv).

Attempts to antagonize the DA-induced effects when D A was bath-applied to the isolated motoneurone preparation were unsuccessful under these experimental conditions. The vertebrate D /D2 receptor subtype antagonists

flupenthixol and fluphenazine had previously been described as antagonists of pressure-applied D A responses in the prothoracic inhibitory neurone of the cockroach {Periplaneta americand) (Davis and Pitman, 1991); using a voltage- clamp protocol, they were reported to block DA responses at concentrations o f

lO'^M and lO'^M, respectively. However, in this preparation, when changes in neuronal excitability were studied under current-clamp, they were found to be ineffective antagonists; that is, bath-application o f D A evoked membrane depolarization after 45 to 60 minutes incubation of the isolated neurone with the antagonist (fluphenazine: IG'^^M to 10‘^M, n = 2; flupenthixol; lO’^M, 1 1= 1).

These findings should be compared with results in Chapter 3; data presented in this section showed that fluphenazine blocked the DA-induced response in isolated B A l motoneurones. Figure 2.3.9v shows the response o f an isolated B A l neurone to 5 x 10’^M DA after the preparation had been left in the presence of

fluphenazine for 41 minutes. D A evoked a membrane depolarization of approximately 5mV with no apparent change in membrane conductance, in the presence o f fluphenazine. A similar response was observed following 60 minutes incubation in lO'^M fluphenazine (not shown). One possible explanation for the inability to block the DA response was agonist saturation o f the D A receptor sites. In the presence o f the relatively high concentrations o f DA (as used here to evoke a change in membrane potential) fluphenazine may have been displaced from the binding sites, thus, rendering it ineffective as an antagonist o f the DA-induced response. Preliminary attempts were made to antagonize membrane depolarizations evoked by the pressure-application of DA (lO'^M; lOpsi; up to Is) onto isolated B A l motoneurones. DA applied in this way to the B A l isolated cell body (protocol similar to Pitman and Davis, 1988) evoked 2 to 3mV membrane depolarizations. These small responses to DA were not affected by the bath- application o f 1 0'^M fluphenazine (n = 2).

Another control experiment was performed to demonstrate that the observations were the result o f the bath-application o f DA (Figure 2.3.9vi). Since the stock solution o f D A (lO'^M) was dissolved in 1% ascorbic acid solution to prevent oxidation (which rapidly occurs to D A when it is normally placed in solution), the appropriate concentration o f diluted ascorbic acid (in locust saline) was also used as a control. In some preparations ascorbic acid had no apparent effect on the membrane potential (n = 2 out o f 4), whilst in others it induced small (Im V ) membrane depolarization (n = 2 out of 4). Figure 2.3.9vi shows the bath-application o f 0.005% ascorbic acid, in the presence o f lO'^M fluphenazine.

This concentration o f ascorbic acid was present with the application o f 5 x 10-'^M DA. Ascorbic acid alone induced an approximate Im V membrane depolarization, which was less than that evoked by 5 x 10“^M DA (approximately 2.5mV, see Figure 2.3.9Ü); 0.004% ascorbic acid had a similar effect on the membrane potential in the absence o f fluphenazine in this preparation (not shown). These results infer that the depolarization observed following the application o f DA was a DA-induced response and not the result o f an experimental artefact, associated with the addition o f ascorbic acid.

2.3.5. The effects o f dopamine on the GABA response in isolated B A l motoneurones

The effects o f DA on the response o f isolated B A l motoneurones to the pressure-application o f G ABA were investigated. Pressure-application o f GABA onto isolated B A l motoneurones consistently evoked membrane hyperpolarization (n = 52 out o f 53; see Section 2.3.3). When DA (lO'^^M to lO’^M) alone was bath-applied to isolated B A l motoneurones, it evoked membrane depolarization in 92% o f the preparations (n = 11 out o f 12; see Section 2.3.4); D A alone was never seen to evoke membrane hypeipolarization. However, if D A was applied to the preparation in between the GABA responses, in some neurones it evoked membrane hyperpolarization (n = 5; 2 x lO’^M - lO’^M DA), whilst in others it caused membrane depolarization (n = 6; lO'^M - lO'^M DA). In

one preparation no change in membrane potential was evident. Furthermore, the bath-application o f D A potentiated the amplitude and/or duration o f the response to G ABA in 11 out o f 12 preparations tested.

It was established that the changes in membrane potential and the GABA response observed following D A application had been induced by DA, and not by a time-dependent change in the G ABA response or a decrease in neuronal

viability. In control experiments in which G ABA was pressure-applied to isolated B A l motoneurones for time periods up to 2 hours, the peak amplitude of the G ABA responses was not potentiated (n = 6 out o f 7). In fact, with time, the

responses gradually diminished in amplitude, as the input resistance o f the neurone decreased. When the membrane input resistance fell to approximately IM Q (indicating notable cell deterioration), no response to G ABA was observed (n = 6). Therefore, in isolated B A l motoneurones, the potentiation o f GABA

responses following the bath-application o f D A was a drug-induced effect.

Figures 2.3.10Ai and 2.3.10AÜ show the enhancement o f GABA responses in an isolated B A l motoneurone following the application o f 7.5 X lO'^M D A (resting membrane potential -50mV; continuously perfused with locust saline). Initially, when G ABA was pressure-applied to the neurone (lO'^M; 25s, lOpsi) it evoked 4mV membrane hyperpolarizations. Bath-application o f DA (7.5 X lO'^M) caused a lOmV membrane hyperpolarization which was associated

with a 60% increase in membrane conductance (*,). Following these changes, the GABA response was potentiated and the DA-induced enhancement in membrane conductance o f the neurone gradually returned to that seen before the DA application. The DA-induced potentiation o f G ABA response was long-lasting; that is, the effect did not reverse following washing with locust saline. Figure 2.3.10Aii shows that approximately 75 minutes after washing o ff DA, the peak amplitude and duration o f response had increased further. As long as G ABA was pressure-applied to the neurone, the membrane potential remained hyperpolarized by approximately 5.5mV. However, when GABA was no longer applied the membrane potential returned to -5Im V (*2). It should be noted that the pressure

used to apply G ABA in Figure 2.3.10AÜ had been reduced from lOpsi to 5psi; although the pressure was normally kept constant throughout experiments, it was lowered to (and remained at) 5psi because the GABA-induced response was near

Figure 2.3.10. The effects o f dopamine on the GABA response o f isolated B A l motoneurones.

When D A (10‘^M to lO'^M) is bath-applied to isolated B A l motoneurones in between repeated pressure-applications of GABA, membrane hyperpolarization (n = 5 out o f 12) or depolarization (n = 6 out o f 12) was evoked. Furthermore, DA

potentiated the response evoked by GABA, that is following the bath-application o f DA, the amplitude and/or duration o f the GABA response was increased (see Figures 2.3.lOA and 2.3.1 OB). These effects did not reverse on washing.

Ai. In this preparation (resting membrane potential -50mV; continuously perfused with locust saline), initially the pressure-application o f G ABA (lO'^M; 25ms, lOpsi) was seen to evoke approximately 4m V membrane hyperpolarizations. The bath-application o f D A (7.5 x lO'^M) induced a lOmV membrane hyperpolarization (>K]) which was associated with an increase in membrane conductance. Following this, the G ABA response was potentiated and the change in membrane conductance returned to the control value. The potentiation was seen to be long-lasting, that is, it did not reverse on washing; 75 minutes after washing o ff DA further enhancement o f the GABA response was observed (Aii). The repeated pressure-application o f G ABA appeared to have an effect on the membrane potential during the potentiation o f the GABA-induced membrane hyperpolarization. As long as GABA was applied to the neurone, the membrane potential remained hyperpolarized; on stopping the pressure- application o f GABA, the membrane potential returned to -51mV (4 ^ 2 and as

indicated by the horizontal dotted line).

B. Using a different preparation (resting membrane potential -45mV; continuously perfused with locust saline), initially the pressure-application o f GABA (10‘^M; lOpsi) was seen to evoke approximately 5mV membrane hyperpolarizations. The bath-application o f D A (10‘^M) induced a 14mV membrane depolarization which was associated with an increase in membrane conductance. Following this, the G ABA response was potentiated and the change in membrane conductance returned to the control value (★). The potentiation o f the G ABA response persisted following washing.

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maximal. It was believed that keeping the pressure at lOpsi may have resulted in receptor desensitization, which could have impinged on the DA-induced potentiation o f the G ABA response. Under the conditions used, DA had increased the mean peak amplitude and the mean duration (measured at 1 / 2 peak amplitude)

o f the G ABA response by 344% and 547%, respectively (values obtained by comparing the responses prior to D A application and those shown in Figure 2.3.10AÜ).

W hilst DA could induce membrane hyperpolarization and potentiation o f the G ABA response in some isolated B A l motoneurones (n = 5), in others it induced membrane depolarization which was also associated w ith the potentiation o f G ABA responses (n = 6). This indicated that potentiation o f the GABA

response was not merely a result o f change in membrane potential. Figure 2.3.1 OB shows DA-induced membrane depolarization and potentiation o f the GABA response follow ing bath-application o f lO’^M DA on an isolated B A l motoneurone (resting membrane potential -45mV; continuously perfused w ith locust saline). Prior to DA application the pressure-application o f G ABA evoked 5mV membrane hyperpolarizations o f approximately 1 Is duration at 1/2 peak amplitude which were associated w ith a 34% increase in membrane conductance. The bath-application o f lO'^M DA to this isolated motoneurone evoked a 14mV membrane depolarization w ith a 20% increase in membrane conductance. The membrane potential was prevented from depolarizing further by the subsequent applications o f G ABA, and these responses to GABA were markedly potentiated, both in the presence o f D A and after washing. The DA-induced change in membrane conductance was transient, since the value o f membrane conductance had returned to normal 5.5 minutes after D A application (indicated by ★). Comparing the four G ABA responses obtained follow ing membrane depolarization w ith those obtained before the bath-application o f D A ('control'

responses), it was estimated that the parameters o f the G ABA response had increased as follows: amplitude by 205%, duration at 1/2 peak amplitude by 125% and membrane conductance at peak amplitude by 100%. Despite the slow and gradual decline in neuronal input resistance that developed during this experiment, the G ABA response remained potentiated. As a consequence o f the continued reduction in input resistance o f this preparation, the long-term effects o f DA application (as shown in Figure 2.3.lOA) could not be studied.