En la opinión del entrevistado se aduce que, no siempre; hay fallas ya que siempre se regatea, para lograr un acuerdo lo que conlleva a no catar lo

dopamine in the nucleus accumbens. Therefore, ‘recovery’ might be useful as an index of dopamine uptake in vivo.

In more recent studies, Justice and colleagues applied the same method to investigate the effects of various neuronal processes on the in vivo ‘recovery’ of 5-HT and noradrenaline (Cosford et al. 1996). As with dopamine, only changes in the rate of clearance changed the ‘recovery’ of the two monoamines. As a consequence, ‘recovery’ could be taken as an index of rate of reuptake of these neurotransmitters.

In this chapter, the No Net Flux method was used to determine the ‘recovery’ of noradrenaline. By carrying out measurements simultaneously in the frontal cortex and the hypothalamus of freely-moving rats, it was possible to investigate whether there were any differences, between these two regions, in active processes governing the clearance of the neurotransmitter. Since the present work focused on the determination of ‘recovery’, high concentrations of noradrenaline (much greater than those usually encountered in in vivo

microdialysis) were infused through the probes. This was to minimise any confounding influence of endogenous noradrenaline release on the measurements of noradrenaline reuptake. Under these conditions, differences in release, and its regulation by (%2-adrenoceptors, would make a negligible contribution to any difference in ‘recovery’

3.2/ Methods

3.2.1/ Protocols

All procedures complied with the Animals (Scientific Procedures) Act 1986. Experiments were carried out on male Sprague-Dawley rats (n = 6, 240-280g) obtained from the colony at University College London. Probes were implanted as described in section 2.3.2.1. On the day after surgery, the microdialysis experiment started at 08:00 a.m. (for more details refer to section 2.3.2.3). Ringer’s solutions, containing different concentrations of noradrenaline, were infused successively (150, 250, 400, 600 and 900 fmol/40/il) into both brain areas of every rat. For each perfusate, 2 dialysis samples were collected at 20-min intervals after a 40-min equilibration period. The perfusate was then switched to the next, higher, concentration until all the solutions were perfused.

The exact concentration of noradrenaline in the perfusate was determined by HPLC-ECD. The mean of three consecutive samples was considered the best approximation of the concentration of the solution. This measure was done for every perfusate and every experiment. The mean concentration of the 2 diaiysates obtained after the period of equilibration was used for determination of noradrenaline ‘recovery’. All the concentrations are expressed in fmol/ 40/xl.

3.2.2/ Data analysis

The loss of noradrenaline from the perfusate versus the concentration infused was plotted for each rat individually. A linear regression indicated the ‘recovery’ (the slope of the regression line) for each individual. A Student’s paired r-test was used to compare any difference between the mean sample ‘recovery’ of the two brain areas.

Chapter 3: Differences in noradrenaline reuptake between the frontal cortex and the hypothalamus

3.3/ R esu lts

The mean ‘recovery’ for noradrenaline in the frontal cortex was 71.9% ±2.9% and in the hypothalamus 53.6% ± 5.3% (Table 3.1). The ‘recovery’ in the frontal cortex was significantly greater than that in the hypothalamus (paired r-test, P = 0.015). This is illustrated in Figure 3.1 in which is plotted the mean loss of noradrenaline versus the mean of the concentrations infused, according to the No Net Flux method.

T able 3.1 Individual ‘recoveries’ in the frontal cortex and the hypothalamus.

Frontal Cortex Hypothalamus

Recovery Reg. Coeff. Recovery Reg. Coeff.

Rat 1 0.828 0.994 0.501 0.985 Rat 2 0.766 0.997 0.451 0.951 Rat 3 0.659 0.876 0.378 0.742 Rat 4 0.639 0.966 0.518 0.978 Rat 5 0.688 0.995 0.737 0.993 R a te 0.736 0.996 0.634 0.999 Mean 0.719 ±0.029 0.536 ± 0.053

Values indicate mean ± standard error of the mean.

FCx y = 0.7178x - 36.767F f = 0.9968 y = 0.5386X + 2.3617 R2 = 0.9979 700 1 _Hyp 600 - ZJ O 500 - 400 - S 300 - Ü 2 0 0 - 100 - 200 0 400 600 800 1000 Cin (fmol/40ul)

Figure 3.1 Determination o f noradrenaline ‘recovery’ by the No Net Flux method.

‘Cin’ is the concentration in the perfusate at the inlet of the probe and ‘Gout’ is the concentration in the sample collected at the outlet of the probe. The slope of the lines is the recovery of the

3.4 / D iscussion

As seen in section 1.5, the ‘recovery’ of noradrenaline depends on many factors: characteristics of the microdialysis probe (type of membrane, surface area); parameters of the experiment (perfusion flow rate, composition of the perfusate); characteristics of the substance measured (chemical structure leading to interactions with the membrane and the solution/tissue surrounding the probe); and finally characteristics of the solution/tissue. These are subdivided into two categories: the passive processes, which include the diffusion properties (dependent on the extracellular volume space fraction) and active processes: cellular uptake, metabolism and exchange mechanisms. The active processes usually account for the difference between the in vivo and the in vitro

‘recovery’.

The first observation to emerge from the present results is that, for all but one animal, the regression coefficient of the slope determining the ‘recovery’ was close to 1. The linearity of the regression lines implies that the ‘recovery’ was constant over the range of concentrations studied.

Secondly, the values for the in vivo ‘recoveries’ (in the frontal cortex: 71.9% ± 2.9% and in the hypothalamus: 53.6% ± 5.3%) were both greater than in vitro {c.f. 36%; see 2.3.1.3), under the same experimental conditions (probe, flow rate, etc...). The only differences between the in vivo and in vitro experiments were the concentration of noradrenaline in the perfusate and the characteristics of the environment into which the probe was inserted. It has already been shown that noradrenaline ‘recovery’ is a function of the concentration gradient across the dialysis membrane (Lonnroth 1987). Since a much greater concentration of noradrenaline was used for the in vivo experiment than for the in vitro study, it is not surprising that a greater ‘recovery’ was obtained in vivo.

However, this would not account for the difference in ‘recovery’ between the two brain areas, which has to be explained by regional differences in the characteristics of the brain tissue surrounding the probe. The profile of the concentration gradient around the probe, which is directly related to ‘recovery’, is a function of the capacities of the tissue to clear the substance of interest (Stable 1991). The greater ‘recovery’ (slope; refer to

Chapter 3: Differences in noradrenaline reuptake between the frontai cortex and the