- Top of page
- Materials and methods
The capacity of the high-affinity choline transporter (CHT) to import choline into presynaptic terminals is essential for acetylcholine synthesis. Ceramic-based microelectrodes, coated at recording sites with choline oxidase to detect extracellular choline concentration changes, were attached to multibarrel glass micropipettes and implanted into the rat frontoparietal cortex. Pressure ejections of hemicholinium-3 (HC-3), a selective CHT blocker, dose-dependently reduced the uptake rate of exogenous choline as well as that of choline generated in response to terminal depolarization. Following the removal of CHTs, choline signal recordings confirmed that the demonstration of potassium-induced choline signals and HC-3-induced decreases in choline clearance require the presence of cholinergic terminals. The results obtained from lesioned animals also confirmed the selectivity of the effects of HC-3 on choline clearance in intact animals. Residual cortical choline clearance correlated significantly with CHT-immunoreactivity in lesioned and intact animals. Finally, synaptosomal choline uptake assays were conducted under conditions reflecting in vivo basal extracellular choline concentrations. Results from these assays confirmed the capacity of CHTs measured in vivo and indicated that diffusion of substrate away from the electrode did not confound the in vivo findings. Collectively, these results indicate that increases in extracellular choline concentrations, irrespective of source, are rapidly cleared by CHTs.
Using choline-sensitive microelectrodes and an amperometric method for the measurement of extracellular choline concentrations in vivo (Burmeister and Gerhardt 2001; Burmeister et al. 2003; Parikh et al. 2004), the present experiments were designed to determine the contribution of the CHT to the clearance of exogenous choline as well as choline derived from newly released ACh in the rat cortex. The experimental approaches employed to address these issues here correspond with those used by Gerhardt and colleagues for the study of dopamine clearance via dopamine transporters (Cass et al. 1993). Choline clearance was determined in either the presence or the absence of a highly potent and selective inhibitor of CHTs, HC-3 (Guyenet et al. 1973; Simon and Kuhar 1975), in intact animals and following the removal of cholinergic terminals in the recording region. In vitro synaptosomal assays were conducted under conditions that mimicked extracellular choline concentrations determined in vivo, in order to verify the contribution of CHTs to choline clearance determined in vivo. The results provide quantitative insights into the contribution of CHTs in vivo to the clearance of choline hydrolyzed from local increases in ACh release as well as that of exogenous choline.
- Top of page
- Materials and methods
These experiments utilized in vivo and in vitro methods in order to determine changes in extracellular choline concentration and high-affinity CHT function in the cortex in vivo. We report six main findings and will discuss their implications below.
In vivo, cortical choline uptake via HC-3-sensitive mechanisms represents a main mechanism for the rapid clearance of local increases in extracellular concentrations of choline.
CHTs contribute mostly to the clearance of choline during the latter of the two components of the clearance phase (TC2). The CHT-mediated clearance during this latter component did not fundamentally depend on the source of choline (depolarization-induced increases of endogenous choline concentrations versus exogenous choline).
Following 192-SAP-induced cholinergic deafferentation of the recording region, depolarization-induced choline signal amplitudes were almost completely attenuated, confirming that the demonstration of KCl-induced choline signals requires acetylcholine (ACh) release and hydrolysis.
In animals with a loss of cholinergic projections to the recording region, the decrease in clearance of exogenous choline corresponded with the effects of HC-3 in intact animals. Furthermore, the density of CHTs in the recording region of lesioned animals correlated significantly with the choline uptake rate in the later clearance phase. In intact animals, CHT-immunoreactivty in the recording region also was highly correlated with choline uptake rate (TC2); these data indicate that there is considerable interindividual variability in the density of CHTs in intact animals and that this variability is of functional significance.
Basal extracellular concentration of choline in vivo was determined to be 4.85 µm. Loss of cortical cholinergic inputs to the recording region did not affect basal choline concentration.
CHT-mediated choline uptake was also determined in vitro using a synaptosomal uptake assay and conditions that mirror extracellular choline concentrations following choline pressure-ejections in vivo. As diffusion of the substrate away from the electrode surface in vivo does not represent a variable in synaptosomal assays, collectively these findings support the conclusion that approximately 40% of choline clearance is a result of choline uptake by CHTs.
The attribution of the effects of HC-3 to the inhibition of uptake via CHTs requires comment. HC-3 is a highly selective, highly potent, competitive inhibitor of CHTs (Ki = 1–100 nm; Lockman and Allen 2002). In contrast, the concentrations of HC-3 required to affect low-affinity choline transport (Ki ∼ 100 µm) were not reached in the present in vivo experiments. Based on the ratio between the concentration of pressure-ejected choline and the concentration of choline detected by the electrode, and ignoring the different physico-chemical properties of HC-3, we can cautiously estimate that the highest concentration of pressure-ejected HC-3 (10 µm) may have yielded <100 nm concentrations in the recording field. A more convincing argument in support of the selectivity of the effects of HC-3 is derived from data obtained from animals with a loss of cholinergic projections to the recording field. Loss of cholinergic terminals, which selectively express CHTs (e.g. Misawa et al. 2001; Ferguson et al. 2003; Kus et al. 2003), resulted in a decrease in choline clearance that corresponded well with the effects of HC-3 in intact rats, indicating that the effects of HC-3 specifically reflected the inhibition of CHTs (see also Freeman et al. 1979; Chatterjee et al. 1987; Saltarelli et al. 1987; Ferguson et al. 2003). Furthermore, in lesioned animals, HC-3 was no longer able to generate further inhibition of clearance. This finding further excludes the possibility that the effects of HC-3 were caused by mechanisms unrelated to the inhibition of CHTs located on cholinergic terminals.
A second methodological issue that deserves comment concerns the effects of either HC-3 pressure ejections or loss of cholinergic terminals on choline signal amplitude. Reduced clearance would be expected to be associated with increases in signal amplitude, similar to the evidence described in Cass et al. (1993). As described in the Materials and methods section, volumes of pressure ejections were adjusted to yield identical amplitudes in order to ensure that the proportion of choline cleared by the substrate-regulated low-affinity choline transporter remained roughly similar across HC-3 concentrations, and to minimize amplitude-based biases in the calculation of clearance rates. As would be expected, the volume of pressure-ejected exogenous choline needed to be decreased when co-injected with HC-3, to achieve amplitudes similar to those produced by choline alone (see the increase in amplitude/volume ratios in Table 2).
Unexpectedly, such volume adjustment was not required in experiments assessing the clearance of endogenous choline following KCl pressure ejections (Table 3). As the efficacy of HC-3 to attenuate choline clearance did not differ between KCl-induced increases in choline concentrations and increases caused by choline pressure ejections, the basis for a lack of effects of HC-3 on KCl-induced signal amplitudes remains unclear.
Diffusion of substrate away from the recording electrode represents a variable that is difficult to control in in vivo experiments using substrate-sensitive microelectrodes. Although quantitative diffusion models (e.g. Nicholson 1985) assisted greatly in substantiating the conclusion that dopamine transporters are largely responsible for dopamine clearance (Cass et al. 1993), application of such models to the present data is limited by the unknown proportion of choline clearance associated with the low-affinity choline transporter. We are not aware of an experimental approach available for determining the contribution of the low-affinity transporter to choline clearance. Therefore, the results from our in vitro synaptosomal assays are relevant to the interpretation of the in vivo data. Because diffusion (of the substrate away from the electrode) does not represent a variable in measurements of choline uptake using synaptosomal assays, and although the proportion of low-affinity choline uptake transporters in a crude synaptosomal fraction is unlikely to reflect the density of low-affinity choline transporters in vivo, the observation that the HC-3-sensitive component of choline uptake in vitro agrees quantitatively with the HC-3-sensitive component of choline clearance measured in vivo (40–43% of overall clearance) appears informative. Moreover, this estimate appears to be in general agreement with earlier conclusions concerning the proportion of choline recycled for ACh synthesis (Collier and Katz 1974).
The finding that relatively high exogenous choline concentrations were cleared as rapidly and as extensively as KCl-induced increases in extracellular choline, via CHTs, suggests that the capacity of CHTs does not exclusively depend on cholinergic neuronal activity. For this discussion, it is important to first exclude the possibility that increases in choline concentrations depolarized cholinergic terminals. Choline has also been suggested to act as an agonist at α7 nicotinic receptors (Alkondon et al. 1997). As nicotinic receptor stimulation elicits choline signals (Parikh et al. unpublished observations), choline-induced depolarization of cholinergic neurons, either directly or involving for example the release of excitatory amino acids (Rousseau et al. 2005), could have contributed to the rapid CHT-mediated clearance of exogenous choline. However, given the low micromolar concentrations of basal extracellular choline concentrations, and the micromolar choline signal peak amplitudes observed following both KCl and choline pressure ejections, it seems highly unlikely that the millimolar concentrations of choline required to stimulate nicotinic receptors (Alkondon et al. 1997) were reached in these experiments. Indeed, as it is difficult to envision local extracellular, endogenous choline concentrations higher than those seen following KCl-induced depolarization, reflecting increases in extracellular ACh by several hundreds of percent (Herzog et al. 2003), it is not clear whether concentrations required to stimulate nicotinic receptors can occur in vivo (Ulus et al. 1988). This view is also supported by the observation that choline signals evoked by even higher concentrations of choline (20 mm) were not affected by high concentrations of neostigmine (100 mm), indicating that choline from endogenous sources does not contribute to the signal recorded following such choline pressure ejections (Parikh et al. 2004).
The present data indicate that the rate of clearance of exogenous choline (peak amplitudes, 20–25 µm) via CHTs does not differ fundamentally from the clearance of endogenous choline, generated as a result of terminal depolarization. The suggestion that a low-affinity state of the CHT (KD = 22.8 nm; Chatterjee et al. 1987) represents the functionally active form of the high-affinity precursor uptake system (Chatterjee et al. 1987) corresponds with the ability of the CHT to clear high extracellular concentrations of exogenous choline, specifically if it is also speculated that increased extracellular choline concentrations trigger a conformational change to the low-affinity state. However, our findings on exogenous choline clearance do not suggest that a tight coupling between high-affinity precursor uptake and ACh synthesis (e.g. Atweh et al. 1975) represents an exclusive mode of CHT capacity regulation. Furthermore, our results on the effects of KCl suggest that endogenous choline concentrations, even under conditions of extreme activation of cholinergic neurons, may rarely exceed 10–15 µm, and thus appear to stay well within estimates of the limits of the capacity of CHTs (Ferguson and Blakely 2004). These data also indicate that the conditions which necessitate increased translocation of the CHT to plasma membranes (Ferguson and Blakely 2004; Apparsundaram et al. 2005) deserve more research.
Concerns about the degree to which high-affinity choline uptake is coupled to ACh synthesis and release have been raised previously (e.g. Kessler and Marchbanks 1979; Collier and Ilson 1977). Although information about the regulation of choline acetyltransferase (ChAT) and the vesicular transporter remains incomplete, it is generally believed that these steps do not significantly contribute to the rate-limiting mechanisms of ACh synthesis (Yamamura and Snyder 1972). Therefore, it may be assumed that CHT-mediated transport of choline, including the transport of choline of exogenous origin, serves as a precursor for ACh synthesis. Results from prior microdialysis experiments also indicated that increased choline concentrations, including those from dietary sources, are taken up a HC-3-sensitive mechanisms and yield increases in ACh release (Cohen and Wurtman 1976; Growdon et al. 1977; Ulus et al. 1989; Koshimura et al. 1990; Marshall and Wurtman 1993; Ikarashi et al. 1997; Koppen et al. 1997; Vinson and Justice 1997). The present results correspond with this evidence, collectively indicating that increases in extracellular choline from exogenous sources, and not necessarily involving cholinergic neuronal activity, will be cleared by CHTs.
The notion that the CHT is regulated in part by its substrate would suggest that the functional properties of the CHT are comparable with those of other neurotransmitter transporters, such as the dopamine, norepinephrine or serotonin transporters (Cass et al. 1993; Zahniser and Doolen 2001; Zahniser and Sorkin 2004). If this assumption is correct, it would be important to determine whether substrate-induced trafficking, which was demonstrated for the dopamine transporter (Chi and Reith 2003), also applies to the CHT. However, in contrast to evidence demonstrating that the dopamine transporter fully accounts for dopamine clearance (Cass et al. 1993; Giros et al. 1996; Benoit-Marand et al. 2000), the contribution of the low-affinity choline transporter to choline clearance in vivo remains unsettled.
Finally, the significant correlation between CHT-IR and choline clearance observed in intact animals suggests that the density of CHTs in the cortex varies considerably between animals, and that this variability is of functional significance. In the context of previous results indicating that cognitive performance correlated with CHT-mediated choline uptake and the density of CHTs in plasma membrane in the cortex (Apparsundaram et al. 2005), the present correlational results further support the possibility that CHT-mediated choline clearance serves as a marker predicting individual cognitive performance.