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Cholinergic neurons elaborate a hemicholinium-3 (HC-3) sensitive choline transporter (CHT) that mediates presynaptic, high-affinity choline uptake (HACU) in support of acetylcholine (ACh) synthesis and release. Homozygous deletion of CHT (−/−) is lethal shortly after birth (Ferguson et al. 2004), consistent with CHT as an essential component of cholinergic signaling, but precluding functional analyses of CHT contributions in adult animals. In contrast, CHT+/− mice are viable, fertile and display normal levels of synaptosomal HACU, yet demonstrate reduced CHT protein and increased sensitivity to HC-3, suggestive of underlying cholinergic hypofunction. We find that CHT+/− mice are equivalent to CHT+/+ siblings on measures of motor co-ordination (rotarod), general activity (open field), anxiety (elevated plus maze, light/dark paradigms) and spatial learning and memory (Morris water maze). However, CHT+/− mice display impaired performance as a result of physical challenge in the treadmill paradigm, as well as reduced sensitivity to challenge with the muscarinic receptor antagonist scopolamine in the open field paradigm. These behavioral alterations are accompanied by significantly reduced brain ACh levels, elevated choline levels and brain region-specific decreased expression of M1 and M2 muscarinic acetylcholine receptors. Our studies suggest that CHT hemizygosity results in adequate baseline ACh stores, sufficient to sustain many phenotypes, but normal sensitivities to physical and/or pharmacological challenge require full cholinergic signaling capacity.
We identified the murine CHT (Apparsundaram et al. 2001) with the express purpose of establishing novel in vivo models of cholinergic hypofunction derived from genetically determined changes in CHT protein levels. As indicated above, CHT−/− newborn mice are apneic and hypoxic, largely immobile and die within an hour after birth (Ferguson et al. 2004). In contrast, CHT+/− mice survive, reproduce at wild-type rates and are grossly indistinguishable from CHT+/+ mice. Importantly, CHT+/− mice exhibit levels of forebrain synaptosomal high-affinity choline uptake (HACU) equivalent to that of CHT+/+ littermates, despite a 50% reduction in CHT protein levels (Ferguson et al. 2004). These latter findings are consistent with the existence of efficient, post-translational regulation of HACU (Apparsundaram et al. 2005; Ferguson & Blakely 2004; Ferguson et al. 2003). However, CHT+/− mice appear more sensitive to HC-3 (Ferguson et al. 2004), suggesting the existence of adaptive changes engaged to sustain normal behavior that can be exposed by direct manipulations of cholinergic signaling.
In the experiments described below, we sought to elucidate basal behavioral and biochemical phenotypes, and to explore whether altered sensitivity to physical and pharmacological challenges exists in CHT+/− mice. We establish that whereas CHT hemizygosity does not result in overt behavioral phenotypes, reliance on a single CHT gene leads to significantly reduced bulk ACh brain levels and renders animals vulnerable to behavioral and pharmacological challenges of cholinergically mediated behaviors. Moreover, we identify changes in M1 and M2 muscarinic receptor levels that we hypothesize alter the dynamic range of cholinergic signaling. We discuss our findings in relation to mouse genetic models that induce alterations of other components of cholinergic signaling, as well as loss-of-function CHT alleles evident in humans.
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Mouse models featuring genetically established cholinergic deficits have provided new evidence for a dynamic role of CHT as a critical modulator of cholinergic neurotransmission (Bazalakova & Blakely 2006). For example, CHT protein expression is increased in response to decreased catabolism of ACh in AChE knockout mice (Volpicelli-Daley et al. 2003). Conversely, CHT density and function are upregulated in hypocholinergic states, produced by transgenic overexpression of AChE (Beeri et al. 1997). Finally, upregulation of CHT protein and function sustains wild-type levels of AChE activity, as well as tissue ACh content and depolarization-evoked ACh release, in ChAT heterozygote mice (Brandon et al. 2004). In this context, we hypothesized that partial loss of CHT might represent a unique model of cholinergic hypofunction, one where phenotypes would emerge upon behavioral/pharmacological challenges targeting cholinergic signaling. In support of this idea, we previously reported an increase in the sensitivity to the lethal effects of HC-3 in CHT+/− mice compared to wild-type littermates (Ferguson et al. 2004), despite wild-type levels of synaptosomal HACU. Our current studies show that CHT+/− mice achieve normal performance on multiple sensorimotor, anxiety and cognitive tasks. However, physical and/or pharmacological challenges elicit genotype-sensitive phenotypes, consistent with demand-dependent cholinergic hypofunction supported by diminished tissue ACh levels and altered muscarinic receptor expression.
Cholinergic neurotransmission at the NMJ is essential for movement (Brandon et al. 2003; Misgeld et al. 2002) and ACh modulates extrapyramidal motor function (Borison & Diamond 1987; Kaneko et al. 2000). Therefore, we began by characterizing motor function in CHT+/− mice, using the open field chamber paradigm. Whereas overall levels of activity were not different between the genotypes (Fig. 1C), we did observe an interesting pattern of decreased horizontal (Fig. 1A) but increased vertical movement (Fig. 1B) by CHT+/− mice. This phenotype did not appear to be caused by overt musculoskeletal deficits, as CHT+/− mice travel equivalent distances in the Morris water maze and swim at wild-type velocities (data not shown). Additionally, they perform comparably to CHT+/+ mice at slow speeds and short test intervals on the treadmill (Fig. 4) and are not impaired in the rotarod (Fig. 2A), wire hang and inverted screen tests (data not shown). Normal basal performance by CHT+/− mice in the elevated plus maze (Fig. 2B) and light/dark (Fig. 2D) paradigms likely rules out overt anxiety as an alternative reason for the altered locomotor phenotype. The increased rearing, but preserved overall activity levels, possibly represent an altered cognitive response to a novel environment (Sarter & Bruno 1997). Additional studies are needed to explore this possibility and to discern whether modified cholinergic autonomic activity contributes to this phenotype.
The baseline locomotor data described above not only provide controls for studies of stressed motor function but also allowed us to proceed with cognitive studies that rely on normal motor capacities. Similar to motor activity, cognitive performance in CHT+/− mice appeared grossly normal at baseline in multiple behavioral assays, including spontaneous Y maze and rewarded T maze alternation, conditioned freezing, response acquisition and prepulse inhibition (see Supplementary Material). CHT+/− mice were also unimpaired in the Morris water maze—a hippocampus-dependent spatial learning and memory task that investigators have shown can be sensitive to insults on the cholinergic system, including pharmacological challenges with muscarinic antagonists, aging and anatomical lesions of cholinergic pathways (D’Hooge & De Deyn 2001). However, Morris water maze performance is unaffected in other models of cholinergic dysfunction, including M1 knockouts (Miyakawa et al. 2001) and ChAT heterozygous (ChAT+/−) mice (Brandon et al. 2004). Interestingly, the normal water maze performance of ChAT+/− mice has been attributed to wild-type levels of cholinergic neurotransmission capacity, triggered in part by upregulation of CHT (Brandon et al. 2004). In this context, additional tests, such as those developed in the rat to monitor focused attention (Apparsundaram et al. 2005; Sarter & Bruno 1997), may be useful in assessing CHT contributions to cognitive performance.
Having established normal baseline motor and cognitive performance, we asked whether CHT hemizygosity would render CHT+/− mice susceptible to challenges directed at cholinergic neurotransmission. We chose motor performance as our readout, as we could introduce both physical and pharmacological challenges in multiple paradigms. In treadmill tests of physical strain on locomotion, we found that CHT+/− mice remained on the running belt for significantly shorter periods of time (Fig. 4A,C) and were not able to reach and sustain the higher speeds attained by age, gender and weight-matched CHT+/+ littermates (Fig. 4B). Our previous electrophysiological recordings at the NMJ documented normal baseline ACh stores and release in CHT−/− mice, as measured by excitory postsynaptic potential (EPSP) magnitude (Ferguson et al. 2004). However, over time, the ACh release diminishes to a point where EPSPs as well as miniature end plate potentials (mEPPs) are no longer observable in CHT−/− preparations, whereas CHT+/+ NMJs maintain cholinergic neurotransmission. Although we did not measure neuromuscular signaling per se in these studies, previous studies have shown that reduction in mEPP amplitude, reflecting diminished neurotransmitter release, is significantly greater if the prolonged nerve stimulation takes place in the presence of the specific CHT inhibitor HC-3—a pharmacological parallel to the genetic deficit induced in CHT+/− mice (Jones & Kwanbunbumpen 1970). Therefore, a plausible scenario is that CHT+/− treadmill performance is impaired due to inability to sustain ACh turnover at the NMJ under conditions of high demand.
Central cholinergic pathways support the motor program both at brainstem and basal ganglia levels. A paradigm that allowed us to simultaneously investigate central cholinergic drive of locomotion and specific challenge to the muscarinic receptor component of ACh turnover is scopolamine-driven locomotion in the open field paradigm (Sipos et al. 1999). It is well established that scopolamine acts at presynaptic autoinhibitory muscarinic receptors, resulting in elevated ACh release, a corresponding increase in CHT-mediated choline uptake and hyperlocomotion (Douglas et al. 2001; Parikh et al. 2004). Our hypothesis was that, under sustained stimulation, ACh turnover would not be sustainable at CHT+/− synapses, and therefore CHT+/− mice would lack the scopolamine-induced hyperlocomotion evident in their CHT+/+ littermates. Indeed, while a low dose of scopolamine (0.1 mg/kg) failed to elicit a locomotive effect in either genotype, an intermediate dose (0.5 mg/kg) led to significant increases in distance traveled by CHT+/+ mice, but failed to affect CHT+/− mice. This finding was particularly interesting in the context of our previous findings with AChE−/− mice, which display normal basal locomotion in the open field chambers but are hypersensitive to challenge at this same intermediate dose of scopolamine (0.5 mg/kg) (Volpicelli-Daley et al. 2003).
The reverse response to scopolamine can be explained if we view the AChE−/− condition as a potential hypercholinergic state, in contrast with the hypocholinergic status of CHT+/− mice. Consistent with this idea, CHT expression is approximately 50% higher in AChE−/− mice, but 50% lower in CHT+/− mice (Volpicelli-Daley et al. 2003). It is important, however, to recognize that our current experimental design does not allow us to distinguish pre- vs. postsynaptic muscarinic receptor alterations. The latter distinction could, for example, explain the augmented response to a muscarinic agonist (oxotremorine) in the context of a blunted sensitivity to a muscarinic antagonist (scopolamine) in CHT+/− mice. In addition, these differing responses to muscarinic agents, which have also been observed in a mouse line bred for hypersensitivity to anticholinesterase (Overstreet & Russell 1982), may be rooted in the divergent neuroanatomical circuits employed by locomotor- vs. tremor-testing paradigms (Chintoh et al. 2003) used to evaluate the effects of scopolamine and oxotremorine, respectively.
The normal behavioral phenotype of CHT+/− mice in many tasks parallels the wild-type levels of synaptosomal HACU activity we established previously (Ferguson et al. 2004). However, the altered sensitivity to cholinergic pharmacological (HC-3, scopolamine, oxotremorine) challenges suggests that other determinants of ACh signaling are altered in the model. Accordingly, we found CHT+/− mice to have normal ChAT and AChE activities (see Results) but diminished bulk ACh (Fig. 6A) and increased total choline brain levels (Fig. 6B). Whereas total endogenous ACh levels are significantly lower in the CHT+/− brain, preliminary studies show normal evoked [3H]-ACh release, as measured in an in vitro slice-perfusion paradigm (D. Lund and M. Bazalakova, unpublished data), suggesting that a redistribution and altered regulation of ACh release and reserve pools (Neher 1998) may compensate for reduced overall ACh levels.
Decreased bulk ACh and increased total choline brain levels have been observed in several human pathologies and mouse models, including the PDAPP transgenic mouse model of Alzheimer’s disease (Bales et al. 2006). Elevated choline levels in CHT+/− mice may represent a compensatory attempt to enrich the choline supply pool for ACh synthesis. Unlike the impairments seen in AChE transgenic mice (Meshorer et al. 2005), we have little reason to suspect major blood–brain barrier dysfunction leading to increased choline flux in the CHT+/− brain. Therefore, elevated choline levels likely represent either a reduction in consumption of HACU-derived choline for ACh synthesis or increased membrane phosphatidyl turnover, resulting from an attempt to increase choline reserves available as precursors for ACh synthesis (Lee et al. 1993). The decreased bulk ACh levels, but normal exogenous [3H]-choline conversion and release of [3H]-ACh from slices, suggest a possible redistribution of ACh pools (Birks & MacIntosh 1961), where readily releasable pools are maintained at the expense of reserve ACh stores. These observations would predict our behavioral findings of normal baseline performance, but challenge-induced deficits in cholinergically mediated behaviors.
Functional compensation in CHT+/− mice may in part be achieved through a region-specific downregulation of M1 (striatum) and M2 (striatum and cortex) receptor expression (Fig. 7). The downregulation of muscarinic receptor expression is consistent with a hyposensitivity to scopolamine challenge in CHT+/− mice and is reminiscent of compensatory changes in muscarinic expression and function in another model of cholinergic dysfunction—AChE−/− mice (Volpicelli-Daley et al. 2003)—suggesting that loss of presynaptic ACh synthesis, due to a failure to recover choline from released ACh, rather than receptor hyperstimulation, may drive receptor downregulation in vivo. Additionally, M1 and M2 mAChR downregulation in the CHT hemizygous state contrasts with M2 striatal overexpression in AChE transgenics, where CHT density and CHT-mediated choline uptake are elevated at baseline (Beeri et al. 1997; Erb et al. 2001; Svedberg et al. 2003). We were unable to reliably quantify expression of the M3, M4 and M5 receptors, which may also exhibit CHT genotype-dependent modulation. Regardless, our findings reinforce a reliance on presynaptic ACh synthesis and storage to sustain appropriate levels of muscarinic receptors and to maintain normal cholinergic tone.
In conclusion, we have shown that CHT+/− mice perform comparably to wild-type littermates in motor and cognitive tasks, but display impaired performance when cholinergic neurotransmission is subjected to sustained physical or pharmacological challenge. Our findings are particularly relevant in the context of the human CHT single nucleotide polymorphism recently identified by Okuda and colleagues (Okuda et al. 2002). The I89V substitution occurs in approximately 6% of the Ashkenazi Jewish population and at similar or higher frequencies in North American Caucasian populations (B. English and R.D. Blakely, unpublished findings), and results in a 40–50% reduction in choline uptake measured in a transfected cell culture system (Okuda et al. 2002). Our findings predict phenotypes that emerge only upon cholinergic challenge, such as states of high physical activity or sustained focused attention, or which may arise from other alleles/insults, that lessen the activity of additional components of cholinergic signaling pathways. Future studies in CHT+/− mice can provide insights into the possible impact of CHT I89V and other gene variants on human behavior and physiology, and offer a valuable new model for testing theories and therapies related to cholinergic hypofunction.