High affinity choline transport and acetylCoA production in brain and their roles in the regulation of acetylcholine synthesis

This review describes recent advances made in the understanding of the regulation of acetylcholine synthesis in brain with regard to the availability of its two precursors, choline and acetylCoA. Choline availability appears to be regulated by the high affinity choline transport system. Investigations of the localization and inhibition of this system are reviewed. Procedures for measuring high affinity choline transport and their shortcomings are described. The kinetics and effects of previous in vivo and in vitro treatments on high affinity choline transport are reviewed. Kinetic and direct coupling of the transport and acetylation of choline are discussed.Recent investigations of the source of acetylCoA used for the synthesis of acetylcholine are reviewed. Three sources of acetylCoA have recently received support: citrate conversion catalyzed by citrate lyase, direct release of acetylCoA from mitochondria following its synthesis from pyruvate catalyzed by pyruvate dehydrogenase, and production of acetylCoA by cytoplasmic pyruvate dehydrogenase. Investigations indicating that acetylCoA availability may limit acetylcholine synthesis are reviewed. A model for the regulation of acetylcholine synthesis which incorporates most of the reviewed material is presented.     

Interaction between nerve growth factor and GM1 monosialoganglioside in preventing cortical choline acetyltransferase and high affinity choline uptake decrease after lesion of the nucleus basalis

Monosialoganglioside GM₁ and nerve growth factor (NGF) were administered alone or concomitantly to adult male rats with a unilateral ibotenic acid lesion of the nucleus basalis magnocellularis (NBM). High-affinity choline uptake (HACU) rate and choline acetyltransferase (ChAT) activity were measured, 4 and 21 days after surgery, respectively, in the frontal and parietal cortices of both hemispheres. A 33–34% decrease in HACU rate and a 43-39% decrease in ChAT activity was found in the ipsilateral cortices 4 and 21 days, respectively, after the lesion. If the lesioned rats received NGF (10 μg i.c.v.) twice a week or daily administrations of GM₁ (30 mg/kg, i.p.), beginning immediately after surgery the decrease in HACU rate and ChAT activity was smaller. If NGF and GM, were given concomitantly no decrease in HACU rate and ChAT activity was detected in the lesioned hemisphere and a slight increase occurred in the contralateral hemisphere. However, after the concurrent administration of NGF (10 μg i.c.v.) and the inactive dose of GM₁ 10 mg/kg i.p. no decrease in HACU and ChAT activity was also found in the lesioned rats. The latter finding indicates a potentiation by GM₁ of NGF effects on the cholinergic neurons of the IBM. The two drugs may either antagonize the neurotoxic effects of ibotenic acid or stimulate a compensatory activity in the remaining neurons.     

MKC-231, a choline uptake enhancer: (3) mode of action of MKC-231 in the enhancement of high-affinity choline uptake

MKC-231, a putative cholinergic activity, is reported to improve learning and memory impaired in AF64A-treated animals. MKC-231 enhances high-affinity choline uptake (HACU) known as the rate-limiting step of acetylcholine (ACh) synthesis. We investigated the mode of action (MOA) of HACU enhancement by MKC-231. Intracerebroventricular (i.c.v.) injections of AF64A (3 nmol/brain) resulted in significant HACU reduction in hippocampal synaptosomes. Treatment with MKC-231 increased Vmax of HACU and Bmax of [3H]-HC-3 binding 1.6 and 1.7-fold, respectively. In studies of [3H]-MKC-231 binding and Biacore analysis, MKC-231 showed noticeable affinity for cloned high-affinity choline transporters (CHT1). The present study suggests that MKC-231 directly affects trafficking of CHT1 and increases the numbers of transporter, working for HACU, at the synaptic membrane.     

Interaction between nerve growth factor and GM1 monosialoganglioside in preventing cortical choline acetyltransferase and high affinity choline uptake decrease after lesion of the nucleus basalis

Monosialoganglioside GM₁ and nerve growth factor (NGF) were administered alone or concomitantly to adult male rats with a unilateral ibotenic acid lesion of the nucleus basalis magnocellularis (NBM). High-affinity choline uptake (HACU) rate and choline acetyltransferase (ChAT) activity were measured, 4 and 21 days after surgery, respectively, in the frontal and parietal cortices of both hemispheres. A 33–34% decrease in HACU rate and a 43-39% decrease in ChAT activity was found in the ipsilateral cortices 4 and 21 days, respectively, after the lesion. If the lesioned rats received NGF (10 μg i.c.v.) twice a week or daily administrations of GM₁ (30 mg/kg, i.p.), beginning immediately after surgery the decrease in HACU rate and ChAT activity was smaller. If NGF and GM, were given concomitantly no decrease in HACU rate and ChAT activity was detected in the lesioned hemisphere and a slight increase occurred in the contralateral hemisphere. However, after the concurrent administration of NGF (10 μg i.c.v.) and the inactive dose of GM₁ 10 mg/kg i.p. no decrease in HACU and ChAT activity was also found in the lesioned rats. The latter finding indicates a potentiation by GM₁ of NGF effects on the cholinergic neurons of the IBM. The two drugs may either antagonize the neurotoxic effects of ibotenic acid or stimulate a compensatory activity in the remaining neurons.     

Ultrastructural localization of high‐affinity choline transporter in the rat anteroventral thalamus and ventral tegmental area: Differences in axon morphology and transporter distribution

The high‐affinity choline transporter (CHT) is a protein integral to the function of cholinergic neurons in the central nervous system (CNS). We examined the ultrastructural distribution of CHT in axonal arborizations of the mesopontine tegmental cholinergic neurons, a cell group in which CHT expression has yet to be characterized at the electron microscopic level. By using silver‐enhanced immunogold detection, we compared the morphological characteristics of CHT‐immunoreactive axon varicosities specifically within the anteroventral thalamus (AVN) and the ventral tegmental area (VTA). We found that CHT‐immunoreactive axon varicosities in the AVN displayed a smaller cross‐sectional area and a lower frequency of synapse formation and dense‐cored vesicle content than CHT‐labeled profiles in the VTA. We further examined the subcellular distribution of CHT and observed that immunoreactivity for this protein was predominantly localized to synaptic vesicles and minimally to the plasma membrane of axons in both regions. This pattern is consistent with the subcellular distribution of CHT displayed in other cholinergic systems. Axons in the AVN showed significantly higher levels of CHT immunoreactivity than those in the VTA and correspondingly displayed a higher level of membrane CHT labeling. These novel findings have important implications for elucidating regional differences in cholinergic signaling within the thalamic and brainstem targets of the mesopontine cholinergic system. J. Comp. Neurol. 518:1908–1924, 2010. © 2010 Wiley‐Liss, Inc.     

Immunocytochemical localization of choline acetyltransferase in rat cerebral cortex: a study of cholinergic neurons and synapses

Choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme and a definitive marker for cholinergic neurons, was localized immunocytochemically in the motor and somatic sensory regions of rat cerebral cortex with monoclonal antibodies. ChAT-positive (ChAT+) varicose fibers and terminal-like structures were distributed in a loose network throughout the cortex. Some immunoreactive cortical fibers were continuous with those in the white matter underlying the cortex, and many of these fibers presumably originated from subcortical cholinergic neurons. ChAT+ fibers appeared to be rather evenly distributed throughout all layers of the motor cortex, but a subtle laminar pattern was evident in the somatic sensory cortex, where lower concentrations of fibers in layer IV contrasted with higher concentrations in layer V. Electron microscopy demonstrated that immunoreaction product was concentrated in synaptic vesicle-filled profiles and that many of these structures formed synaptic contacts. ChAT+ synapses were present in all cortical layers, and the majority were of the symmetric type, although a few asymmetric ones were also observed. The most common postsynaptic elements were small to medium-sized dendritic shafts of unidentified origin. In addition, ChAT+ terminals formed synaptic contacts with apical and, probably, basilar dendrites of pyramidal neurons, as well as with the somata of ChAT-negative nonpyramidal neurons. ChAT+ cell bodies were present throughout cortical layers II-VI, but were most concentrated in layers II-III. The somata were small in size, and the majority of ChAT+ neurons were bipolar in form, displaying vertically oriented dendrites that often extended across several cortical layers. Electron microscopy confirmed the presence of immunoreaction product within the cytoplasm of small neurons and revealed that they received both symmetric and asymmetric synapses on their somata and proximal dendrites. These observations support an identification of ChAT+ cells as nonpyramidal intrinsic neurons and thus indicate that there is an intrinsic source of cholinergic innervation of the rat cerebral cortex, as well as the previously described extrinsic sources.     

Morphological and physiological properties of enhanced green fluorescent protein (EGFP)‐expressing wide‐field amacrine cells in the ChAT‐EGFP mouse line

Mammalian retinas comprise a variety of interneurons, among which amacrine cells represent the largest group, with more than 30 different cell types each exhibiting a rather distinctive morphology and carrying out a unique function in retinal processing. However, many amacrine types have not been studied systematically because, in particular, amacrine cells with large dendritic fields, i.e. wide‐field amacrine cells, have a low abundance and are therefore difficult to target. Here, we used a transgenic mouse line expressing the coding sequence of enhanced green fluorescent protein under the promoter for choline acetyltransferase (ChAT‐EGFP mouse) and characterized a single wide‐field amacrine cell population monostratifying in layer 2/3 of the inner plexiform layer (WA‐S2/3 cell). Somata of WA‐S2/3 cells are located either in the inner nuclear layer or are displaced to the ganglion cell layer and exhibit a low cell density. Using immunohistochemistry, we show that WA‐S2/3 cells are presumably GABAergic but may also release acetylcholine as their somata are weakly positive for ChAT. Two‐photon‐guided patch‐clamp recordings from intact retinas revealed WA‐S2/3 cells to be ON‐OFF cells with a homogenous receptive field even larger than the dendritic field. The large spatial extent of the receptive field is most likely due to the extensive homologous and heterologous coupling among WA‐S2/3 cells and to other amacrine cells, respectively, as indicated by tracer injections. In summary, we have characterized a novel type of GABAergic ON‐OFF wide‐field amacrine cell which is ideally suited to providing long‐range inhibition to ganglion cells due to its strong coupling.     

PET radiotracer development for imaging high‐affinity state of dopamine D2 and D3 receptors: Binding studies of fluorine‐18 labeled aminotetralins in rodents

Imaging the high‐affinity, functional state (HA) of dopamine D2 and D3 receptors has been pursued in PET imaging studies of various brain functions. We report further evaluation of ¹⁸F‐5‐OH‐FPPAT, and the newer ¹⁸F‐5‐OH‐FHXPAT and ¹⁸F‐7‐OH‐FHXPAT. Syntheses of ¹⁸F‐5‐OH‐FHXPAT and ¹⁸F‐7‐OH‐FHXPAT were improved by modifications of our previously reported procedures. Brain slices and brain homogenates from male Sprague‐Dawley rats were used with the 3 radiotracers (74‐111 kBq/cc). Competition with dopamine (1–100 nM) and Gpp(NH)p (10–50 µM) were carried out to demonstrate binding to dopamine D2 and D3 HA‐states and binding kinetics of ¹⁸F‐5‐OH‐FPPAT measured. Ex vivo brain slice autoradiography was carried out on rats administered with ¹⁸F‐5‐OH‐FHXPAT to ascertain HA‐state binding. PET/CT imaging in rats and wild type (WT) and D2 knock‐out mice were carried out using ¹⁸F‐7‐OH‐FHXPAT (2‐37 MBq). Striatum was clearly visualized by the three radiotracers in brain slices and dopamine displaced more than 80% of binding, with dissociation rate in homogenates of 2.2 × 10^?2 min^?1 for ¹⁸F‐5‐OH‐FPPAT. Treatment with Gpp(NH)p significantly reduced 50–80% striatal binding with faster dissociation rates (5.0 × 10^?2 min^?1), suggesting HA‐state binding of ¹⁸F‐5‐OH‐FPPAT and ¹⁸F‐5‐OH‐FHXPAT. Striatal binding of ¹⁸F‐5‐OH‐FHXPAT in ex vivo brain slices were sensitive to Gpp(NH)p, suggesting HA‐state binding in vivo. PET binding ratios of ¹⁸F‐7‐OH‐FHXPAT in rat brain were ventral striatum/cerebellum = 2.09 and dorsal striatum/cerebellum = 1.65; similar binding ratios were found in the D2 WT mice. These results suggest that in vivo PET measures of agonists in the brain at least in part reflect binding to the membrane‐bound HA‐state of the dopamine receptor.     

Ex vivo [11C]‐(+)‐PHNO binding is unchanged in animal models displaying increased high‐affinity states of the D2 receptor in vitro

Dopamine (DA) D₂ receptor supersensitivity has been linked to an increase in the density of the D₂ high‐affinity state as measured in vitro. The two‐ affinity‐state model of the D₂ receptor predicts that the ex vivo specific binding of [¹¹C]‐(+)‐PHNO, an agonist radiotracer thought to bind selectively to the high‐affinity state in vivo, should be increased in animal models that display in vitro increases in the proportion of receptors in the D₂ high‐affinity state. Here, we test this hypotheses by comparing the ex vivo SBR of [¹¹C]‐(+)‐PHNO with that of the antagonist radiotracer [³H]‐raclopride in three dopaminergically supersensitive rat models—AMPH‐sensitized rats, rats withdrawn from chronic ethanol, and unilaterally 6‐OHDA‐lesioned rats—using ex vivo dual‐radiotracer biodistribution studies. We find that in AMPH‐sensitized rats and rats withdrawn from chronic ethanol treatment, models that exhibited ～4‐fold increases in the D₂ high‐affinity state in vitro, the SBRs of [¹¹C]‐(+)‐PHNO and [³H]‐raclopride are unchanged relative to control rats. In unilaterally 6‐OHDA‐lesioned rats, we find that the increase in [¹¹C]‐(+)‐PHNO SBR is no different than that observed for the antagonist radiotracer [³H]‐raclopride (54% ± 16% and 52% ± 14%, respectively). In addition, the effect of acute AMPH pretreatment (4 mg/kg, i.v.) on the SBRs of [¹¹C]‐(+)‐PHNO and [³H]‐raclopride is equivalent in AMPH‐sensitized (?38% ± 12% and ?36% ± 8%, respectively) and in control rats (?40% ± 11% and ?38% ± 7%). These data emphasize a significant discrepancy between in vitro and in vivo measures of D₂ agonist binding, indicating that the two‐affinity‐state model of the D₂ receptor may not apply veridically to living systems. The potential implications of this discrepancy are discussed. Synapse 63:998–1009, 2009. © 2009 Wiley‐Liss, Inc.     

Dopamine β‐hydroxylase‐deficient mice have normal densities of D2 dopamine receptors in the high‐affinity state based on in vivo PET imaging and in vitro radioligand binding

In vitro, D₂ dopamine receptors (DAR) can exist in low‐ and high‐affinity states for agonists and increases of D₂ receptors in high‐affinity state have been proposed to underlie DA receptor supersensitivity in vivo. Deletion of the gene for dopamine β‐hydroxylase (DBH) causes mice to become hypersensitive to the effects of psychostimulants, and in vitro radioligand binding results suggest an increased percentage of D₂ receptors in a high‐affinity state. To determine whether DBH knockout mice display an increase of high‐affinity state D₂ receptors in vivo, we scanned DBH knockout and control mice with the agonist PET radioligand [¹¹C]MNPA, which is thought to bind preferentially to the high‐affinity state of the D₂ receptor. In addition, we performed in vitro binding experiments on striatal homogenates with [³H]methylspiperone to measure B_max values and the percentages of high‐ and low‐affinity states of the D₂ receptor. We found that the in vivo striatal binding of [¹¹C]MNPA was similar in DBH knockout mice and heterozygous controls and the in vitro B_max values and percentages of D₂ receptors in the high‐affinity state, were not significantly different between these two groups. In summary, our results suggest that DBH knockout mice have normal levels of D₂ receptors in the high‐affinity state and that additional mechanisms contribute to their behavioral sensitivity to psychostimulants. Synapse 64:699–703, 2010. © 2010 Wiley‐Liss, Inc.     

Intricate paths of cells and networks becoming "Cholinergic" in the embryonic chicken retina

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are the decisive enzymatic activities regulating the availability of acetylcholine (ACh) at a given synaptic or nonsynaptic locus. The only cholinergic cells of the mature inner retina are the so‐called starburst amacrine cells (SACs). A type‐I SAC, found at the outer border of the inner plexiform layer (IPL), forms a synaptic subband “a” within the IPL, while a type‐II SAC located at the inner IPL border projects into subband “d.” Applying immunohistochemistry for ChAT and AChE on sections of the chicken retina, we here have revealed intricate relationships of how retinal networks became dominated by AChE or by ChAT reactivities. AChE⁺ cells were first detectable in an embryonic day (E)4 retina, while ChAT appeared 1 day later in the very same cells; at this stage all are Brn3a⁺, a marker for ganglion cells (GCs). On either side of a faint AChE⁺ band, indicating the future IPL, pairs of ChAT⁺/AChE^?/Brn3a^? cells appeared between E7/8. Type‐I cells had increased ChAT and lost AChE; type‐II cells presented less ChAT, but some AChE on their surfaces. Direct neighbors of SACs tended to express much AChE. Along with maturation, subband “a” presented more ChAT but less AChE; in subband “d” this pattern was reversed. In conclusion, the two retinal cholinergic networks segregate out from one cell pool, become locally opposed to each other, and become dominated by either synthesis or degradation of ACh. These “cholinergic developmental divergences” may also have significant physiologic consequences. J. Comp. Neurol., 520:3181–3193, 2012. © 2012 Wiley Periodicals, Inc.     

The synthesis and preclinical evaluation in rhesus monkey of [18F]MK‐6577 and [11C]CMPyPB glycine transporter 1 positron emission tomography radiotracers

Two positron emission tomography radiotracers for the glycine transporter 1 (GlyT1) are reported here. Each radiotracer is a propylsulfonamide‐containing benzamide and was labeled with either carbon‐11 or fluorine‐18. [¹¹C]CMPyPB was synthesized by the alkylation of a 3‐hydroxypyridine precursor using [¹¹C]MeI, and [¹⁸F]MK‐6577 was synthesized by a nucleophilic aromatic substitution reaction using a 2‐chloropyridine precursor. Each tracer shows good uptake into rhesus monkey brain with the expected distribution of highest uptake in the pons, thalamus, and cerebellum and lower uptake in the striatum and gray matter of the frontal cortex. In vivo blockade and chase studies of [¹⁸F]MK‐6577 showed a large specific signal and reversible binding. In vitro autoradiographic studies with [¹⁸F]MK‐6577 showed a large specific signal in both rhesus monkey and human brain slices and a distribution consistent with the in vivo results and those reported in the literature. In vivo metabolism studies in rhesus monkeys demonstrated that only more‐polar metabolites are formed for each tracer. Of these two tracers, [¹⁸F]MK‐6577 was more extensively characterized and is a promising clinical positron emission tomography tracer for imaging GlyT1 and for measuring GlyT1 occupancy of therapeutic compounds. Synapse, 2011. © 2010 Wiley‐Liss, Inc.     

Mice overexpressing wild‐type human alpha‐synuclein display alterations in colonic myenteric ganglia and defecation

Background Prevalent non‐motor symptoms of Parkinson’s disease (PD) include gastrointestinal motor impairments and advanced stage PD displays pathological aggregates of α‐synuclein in colonic enteric neurons. We previously showed that 12 months old mice overexpressing human wild type (WT) α‐synuclein under the Thy1 promoter (Thy1‐aSyn) displayed colonic motor dysfunction. We investigated functional gut alterations at earlier ages and histological correlates. Methods Defecation, gastric emptying (GE), and immunostaining for α‐synuclein, peripheral choline acetyltransferase (pChAT), tyrosine hydroxylase (TH), neuronal nitric oxide synthase (nNOS), and vasoactive intestinal peptide (VIP) in distal colon myenteric plexuses were assessed in male Thy1‐aSyn compared to littermate WT mice. Key Results Thy1‐aSyn mice aged 2.5–3 or 7–8 months old had 81% and 55% reduction in fecal pellet output, respectively, in the first 15 min of exposure to a novel environment. The reduction remained significant in the older group for 2‐h, and subsequent refeeding resulted also in a 60% and 69% reduction of defecation in the first hour, respectively. Thy1‐aSyn mice (8–10 months) displayed increased α‐synuclein in the myenteric plexuses with abundant varicose terminals surrounding pChAT‐immunoreactive (ir) neurons, and only a few, nNOS‐ir neurons. There were no conspicuous changes in pChAT‐ and nNOS‐ir neurons, or TH‐ and VIP‐ir nerve fibers. Thy1‐aSyn mice aged 4–18 months had normal GE. Conclusions & Inferences The occurrence of over‐production of pre‐synaptic α‐synuclein in colonic myenteric ganglia several months before the loss of striatal dopamine may provide an anatomical basis for interference with cholinergic neuronal activation, causing an early impairment in defecation to stimuli.     

Low‐ and high‐frequency transcutaneous electrical acupoint stimulation induces different effects on cerebral μ‐opioid receptor availability in rhesus monkeys

Although systematic studies have demonstrated that acupuncture or electroacupuncture (EA) analgesia is based on their accelerating endogenous opioid release to activate opioid receptors and that EA of different frequencies is mediated by different opioid receptors in specific areas of the central nervous system, there is little direct, real‐time evidence to confirm this in vivo. The present study was designed to investigate the effects of transcutaneous electrical acupoint stimulation (TEAS), an analogue of EA, at low and high frequencies on μ‐opioid receptor (MOR) availability in the brain of rhesus monkeys. Monkeys underwent 95‐min positron emission tomography (PET) with ¹¹C‐carfentanil three times randomly while receiving 0, 2, or 100 Hz TEAS, respectively. Each TEAS was administered in the middle 30 min during the 95‐min PET scan, and each session of PET and TEAS was separated by at least 2 weeks. The results revealed that 2 Hz but not 100 Hz TEAS evoked a significant increase in MOR binding potential in the anterior cingulate cortex, the caudate nucleus, the putamen, the temporal lobe, the somatosensory cortex, and the amygdala compared with 0 Hz TEAS. The effect remained after the end of TEAS in the anterior cingulate cortex and the temporal lobe. The selective increase in MOR availability in multiple brain regions related to pain and sensory processes may play a role in mediating low‐frequency TEAS efficacy. © 2014 Wiley Periodicals, Inc.     

Changes in tyrosine hydroxylase, glutamic acid decar☐ylase and choline acetyltransferase after local microinoculation of scrapie agent into the nigrostriatal system of the golden hamster

A microinjection of a homogenate of scrapie agent-infected brain (strain 263 K) into the nigrostriatal system in the golden hamster is followed by the progressive development of the disease which terminates by the death of animals around the 4th month postinoculation. These intracerebral inoculations induce more rapid changes in neuronal activity which can be revealed by the assessment of the specific synthesizing enzymes of neurotransmitter systems. The microinoculation of a homogenate of an infected brain unilaterally into the substantia nigra (SN) provokes a decrease in tyrosine hydroxylase (TH), the synthesizing enzyme for dopamine in the dopaminergic neurones, in the striatum ipsilateral to the injected SN. This biochemical response, specifically induced by the active pathogen, is detectable as soon as the 5th day postinoculation and is detectable towards the 80th day. A return of TH levels to control values is detected after this period. At the end of the incubation period and towards the death of the animals, TH is not different from control TH measured from intact animals. The decrease in TH is concomitant with an increase in striatal glutamic acid decar☐ylase (GAD), the synthesizing enzyme for γ-aminobutyric acid (GABA), measured 20 days postinoculation with no change in choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Studies of the biochemical responses associated locally to the scrapie agent inoculation have been performed at the striatal level. The intrastriatal administration of the infective agent induces 20 days postinoculation an increase in GAD with no change in TH and ChAT. Ninety days postinoculation, a decrease in GAD was detected associated with an increase in TH with no change in ChAT. The decrease in striatal TH 5 days after inoculation of the scrapie agent in the ipsilateral SN represents in the hamster an early marker of the slow progressive pathologic process which can only be revealed about 80 days postinoculation. The influence of the pathogen in the nigrostriatal system could be specific for certain categories of neurones. The cholinergic neurones are apparently not affected. The dopaminergic neurones seem not to be permanently damaged, as seen by the return of TH to control values during the development of the disease. The GABAergic neurones could represent a preferential target for the pathogen as shown by the early GAD activation and tardive decrease activity.