Differences in noradrenaline reuptake between the frontai cortex and the hypothalamus

Figure 3.1) in the frontal cortex compared with the hypothalamus suggests that the clearance of noradrenaline is more efficient in the former brain area.

Measuring ion diffusion in the rat cerebellum using iontophoresis, Nicholson and Phillips (1981) found an extracellular fluid space volume fraction of 0.2. Although there is no report of any difference in extracellular fraction between brain areas, specifically the frontal cortex and the hypothalamus, regional differences in noradrenaline diffusion could contribute to regional differences in uptake (clearance).

However, whether or not regional differences in noradrenaline diffusion contribute to regional differences in uptake, active processes could also be responsible for any differences in clearance between these two brain areas. As explained in the Introduction (section 3.1), in the conditions of the present experiment, the amount of released noradrenaline should be negligible when compared with the concentrations infused. Consequently, metabolism and reuptake are the only relevant parameters to discuss. Since uptake is the primary mechanism of clearance for monoamines, it is reasonable to speculate that differences in noradrenaline clearance between the two brain areas could imply differences in reuptake. This could indicate differences in synaptic geometry, with differences in transporter location, differences in transporter density and/or pharmacological properties, or existence of different transporters with different distributions in the frontal cortex and the hypothalamus.

Fritschy and Grzanna (1989) reported differences in the action of DSP-4, a neurotoxin whose primary site of action is the noradrenaline transporter, on noradrenergic neurones projecting from the locus coeruleus and those projecting from the lateral tegmental system (see section 3.1). Although they found that the two groups of neurones differed morphologically, these observations could also suggest that the uptake of DSP-4 was greater in the frontal cortex (which is innervated by neurones projecting from the locus coeruleus) than in the hypothalamus (which is innervated primarily by neurones projecting from the lateral tegmental system).

In 1990, Zaczek et a l confirmed the selectivity of action of DSP-4 on noradrenaline axons in the cerebral cortex over axons in the hypothalamus. Moreover, in

the same study, they measured DSP-4 transport in crude synaptosomal fractions prepared from rat cortex and hypothalamus. They used 10 pM desipramine to determine non­ specific noradrenaline uptake. They found that the inhibition constant o f DSP-4 (.^i) for the noradrenaline transporter (Uptake 1) was lower in the frontal cortex than in the hypothalamus (179 ± 39 nM versus 460 ± 35 nM). These findings indicated that the pharmacology of the noradrenaline transporter differs in the frontal cortex and the hypothalamus and that this could account for the differential effects of DSP-4 in these two brain regions.

Moreover, in the same study, Zaczek et aL measured active noradrenaline transport by the desipramine-sensitive noradrenaline transporter (Uptake 1). Analysing the Eadie-Hofstee plots, they reported different pharmacological properties for the transporter in the two brain regions: the affinity of the transporter for noradrenaline in the frontal cortex was greater than that in the hypothalamus (Km for noradrenaline was 3-fold lower in the cortex [39.5 ± 7.5 nM] than in the hypothalamus [100.3 ± 12.1 nM]). There was also a difference in relative density of transport sites (Vmax 2-fold lower in the cortex than in the hypothalamus). These observations confirmed that the pharmacology of the high-affinity noradrenaline transporter differs in these two brain areas. Moreover, the affinity o f noradrenaline for Uptake 1 is 3-fold greater in the frontal cortex than in the hypothalamus and so total transport of noradrenaline in the former brain area might be greater than that in the hypothalamus. This could contribute to the regional differences in clearance observed in the present study.

The distribution of the Uptake 1 transporter has been studied by immunolocalisation. Using rabbit polyclonal noradrenaline transporter antisera against the -COOH terminus of the mouse noradrenaline transporter, Schroeter et a l (2000) found that the A1 and A2 cell bodies contained little expression of the noradrenaline transporter, compared to other noradrenergic nuclei. As explained in section 1.1.2, the hypothalamus is mainly innervated by neurones projecting from A1 and A2 nuclei. Therefore, poor expression of Uptake 1 in the hypothalamus compared with the frontal cortex could contribute to the difference in uptake mechanisms and noradrenaline clearance in these two brain regions.

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

The Uptake 1 transporter couples to existing ion gradients to achieve reuptake of noradrenaline into terminals; substrate and ions cross the membrane in fixed stoichiometry (see section 1.3.1). However, it has been shown that the Na"^- and Cl -dependent noradrenaline transporter can also exhibit a “channel-type” conductance (Galli et a i 1996). When Uptake 1 works as an ion channel, an arbitrary number of ions depending on the channel open time is carried with the neurotransmitter. This conductance, although occurring much less than the normal “transporter-type”, increases appreciably the rate of noradrenaline uptake. Assuming that switches from the “transporter-mode” to the “channel-mode” occur at a greater frequency in the frontal cortex than in the hypothalamus (which has not been investigated yet), the frontal cortex would be more apt at coping with the high concentrations of noradrenaline infused in this study. This would support the present findings of a more efficient clearance in the frontal cortex than in the hypothalamus.

Finally, Uptake 2 (called OCT3 in the rats [Wu et a i 1998a] and EMT in human [extraneuronal transporter for monoamine transmitters or corticosterone-sensitive extraneuronal transporter, Grundemann 1998]), is the only cationic transporter found in the brain at appreciable concentrations (refer to section 1.3.1.2). It has a broad tissue distribution and its mRNA transcript is found in the central nervous system. Kristufek et a i (2002) observed mRNA for OCT3 in the rat superior cervical ganglion cells, implying a functional role of 0CT3 in nerve cells. Wu et al (1998a) also reported the appreciable expression of OCT3 mRNA transcript in neural cell populations of the rat cerebellum, hippocampus and cerebral cortex. However, it is not clear whether this transporter is located in neurones, glial cells or both. Although these findings do not guarantee protein expression and functional activity of 0CT3 in these brain regions, they suggest that 0CT3 might be a second line of defence in the frontal cortex, inactivating released monoamine transmitters that escape uptake 1 and preventing uncontrolled spreading of the signal. As yet, OCT3 has not been found in the hypothalamus: the absence of expression of OCT3 in this brain region could contribute to the regional differences observed in the present study, therefore.

In this Chapter, the ‘recovery’ for noradrenaline was measured simultaneously in the frontal cortex and the hypothalamus. Under the present conditions, the ‘recovery’ in

the frontal cortex was greater than that in the hypothalamus, suggesting that the processes involved in the clearance of noradrenaline were more efficient in the former brain area. This is supported by previous findings showing that Uptake 1 is poorly expressed in neurones innervating the hypothalamus and that noradrenaline displays a higher affinity for the high-affinity noradrenaline transporter in the frontal cortex than in the hypothalamus. The presence of 0CT3 as a support for Uptake 1 in the frontal cortex but not in the hypothalamus also supports the present observations. Finally, new transporters, such as the newly discovered organic cation transporter, OCTN (Wu et al. 1998b), could have different distributions in the frontal cortex and the hypothalamus; this could further contribute to the regional differences in uptake.

Key findings:

• The rate of noradrenaline clearance is greater in the frontal cortex than in the hypothalamus.

• This could be due to regional differences in noradrenaline reuptake.

Chapter 4: The effects of systemic administration of 6-amphetamine on noradrenaiine effiux in the