Cholinergic forebrain degeneration in the APPswe/PS1DeltaE9 transgenic mouse

The impact of Abeta deposition upon cholinergic intrinsic cortical and striatal, as well as basal forebrain long projection neuronal systems was qualitatively and quantitatively evaluated in young (2-6 months) and middle-aged (10-16 months) APPswe/PS1DeltaE9 transgenic (tg) mice. Cholinergic neuritic swellings occurred as early as 2-3 months of age in the cortex and hippocampus and 5-6 months in the striatum of tg mice. However, cholinergic neuron number or choline acetyltransferase (ChAT) optical density measurements remained unchanged in the forebrain structures with age in APPswe/PS1DeltaE9 tg mice. ChAT enzyme activity decreased significantly in the cortex and hippocampus of middle-aged tg mice. These results suggest that Abeta deposition has age-dependent effects on cortical and hippocampal ChAT fiber networks and enzyme activity, but does not impact the survival of cholinergic intrinsic or long projection forebrain neurons in APPswe/PS1DeltaE9 tg mice.     

Cholinergic neuronal and axonal abnormalities are present early in aging and in Alzheimer disease

A large body of evidence indicates that basal forebrain cholinergic neurons are selectively vulnerable to degeneration early in Alzheimer disease (AD). Recent studies, however, demonstrate reductions in cortical activity of the cholinergic enzyme choline acetyltransferase only in late stages of AD. To address this apparent contradiction, we compared abnormalities in magnocellular basal forebrain cholinergic neurons and their axons in nondemented young (<65 years; n = 6), nondemented old (>65 years; n = 7), pathologically mild (n = 5), and pathologically severe (n = 5) AD cases. Cholinergic axon abnormalities (i.e. thickened fibers and ballooned terminals) were evident in nondemented middle-aged cases, increased in nondemented old cases, and reduced in density in severe AD. This suggests that loss of cortical cholinergic axons in AD occurs preferentially in fibers with these abnormalities. Paired helical filament 1-immunoreactive pretangles and tangles were observed as early as the third decade prior to their appearance in entorhinal/perirhinal cortex; they were increased in mild and severe AD. These results indicate that basal forebrain cholinergic neuron abnormalities are present very early in aging and in the course of AD. Therefore, despite the morphologic alterations, choline acetyltransferase activity, but not necessarily normal neuron functions, may be preserved.     

Inhibitors of serine/threonine phosphatases increase membrane-bound choline acetyltransferase activity and enhance acetylcholine synthesis

The present investigation examines the effects of phosphatase inhibition on short-term regulation of cholinergic function, with particular emphasis on choline acetyltransferase, the enzyme which synthesizes acetylcholine. Rat hippocampal synaptosomes were treated with either okadaic acid (10 nM) or calyculin-A (50 nM) to inhibit protein phosphatases 1 and 2A for 20 min prior to subfractionation of nerve terminals and measurement of choline acetyltransferase activity, or quantification of high-affinity choline transport and acetylcholine synthesis. Inhibition of synaptosomal phosphatases did not alter total or salt-soluble choline acetyltransferase activity, but membrane-bound and water-soluble forms of the enzyme were selectively increased in okadaic acid-treated nerve terminals to 129±11% and 137±10% of control, respectively. High-affinity choline transport was reduced to 77±6% and 76±7% of control in calyculin-A- and okadaic acid-treated nerve terminals, respectively. Acetylcholine synthesis was reduced to 73±6% of control in calyculin-A-treated synaptosomes only; acetylcholine synthesis was at control levels in okadaic acid-treated cultures correlating with enhanced choline acetyltransferase activity in the water-soluble and nonionically membrane-bound fractions. These investigations indicate a role for phosphoprotein phosphatases in the regulation of acetylcholine synthesis in the cholinergic nerve terminal. The observed increases in choline acetyltransferase activity in two subcellular fractions appears to compensate for decreased choline precursor availability, allowing acetylcholine synthesis to be maintained at control levels. The uncoupling of choline transport and acetylcholine synthesis in this situation represents a unique functional role for a subfraction of choline acetyltransferase.     

Abnormal APP, cholinergic and cognitive function in Ts65Dn Down's model mice

We evaluated Ts65Dn Down's syndrome mice and their littermates (LM) at 1-2, 4, and 12 months of age to determine amyloid precursor protein (APP)-related cellular and biochemical changes associated with cognitive deficits. Ts65Dn mice showed cognitive deficits in the Morris water maze compared to LM mice at 4 and 12 months of age. Ts65Dn, but not LM mice, developed a septohippocampal cholinergic neuronal degeneration of choline acetyltransferase (ChAT)-positive neurons at 12 months of age. These cellular changes were compensated by increases in ChAT enzyme activity of remaining cholinergic terminals in the hippocampus. By 12 months of age, Ts65Dn mice had elevations of APP protein levels in the hippocampus compared to their LM. At this age, both Ts65Dn mice and their LM abnormally responded to cholinergic muscarinic M1 agonist treatment in terms of hippocampal APP, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) levels compared to young adult C57BL/6 mice. In summary, the Ts65Dn mice show developmental and progressive age-related behavioral deficits, hippocampal APP, and cholinergic pathology. The relatively better cognitive spatial performance in LM compared to Ts65Dn mice suggests that high APP levels combined with progressive degeneration of the cholinergic system are critical to the pathology and cognitive deficits seen in Ts65Dn mice.     

Stimulation of choline acetyltransferase by C3d, a neural cell adhesion molecule ligand

Septal cholinergic neurons project to the hippocampus and release acetylcholine, a neurotransmitter involved in learning and memory. The enzyme choline acetyltransferase (ChAT) is responsible for synthesizing acetylcholine. Promoting ChAT activity and acetylcholine release can lead to new treatments for neurodegenerative diseases with cholinergic deficits, such as Alzheimer's disease. We present evidence that the synthetic molecule C3d, which is a peptide mimetic of the neural cell adhesion molecule (NCAM), promotes ChAT activity in cultures of rat embryonic septal neurons. Our data demonstrate that ChAT activity triggered by C3d is dependent on the fibroblast growth factor receptor (FGFR) and the mitogen-activated protein kinase (MAPK) pathway. C3d did not affect the number of cholinergic neurons in culture, indicating that NCAM homophilic binding enhances ChAT activity, without affecting cholinergic cell survival. In conclusion, the NCAM mimetic peptide C3d promotes ChAT activity in septal neurons through FGFR and MAPK. These findings are relevant to the design of new strategies aimed at stimulating cholinergic function and improving cognition in disorders such as Alzheimer's disease.     

Pre- and post-synaptic cholinergic dysfunction in aged rodent brain regions: New findings and an interpretative review

Age-related impairment of dynamic aspects of central cholinergic neurotransmission has been indicated by many studies of aged rodents, but the regional distribution of cholinergic deficits and the relative contribution of presynaptic hypofunction and reduced acetylcholine release, loss of synaptic integrity or loss of muscarinic receptors remains unclear. This study therefore compared choline acetyltransferase activity (as a structural marker) and sodium-dependent high affinity choline uptake (which reflects both ongoing cholinergic neuronal activity and structural integrity) in the hippocampus, cortex and striatum of male C57BL mice at 3–4, 10–12 or 28–32 months of age. To evaluate the relationship of changes in muscarinic receptors to presynaptic alterations, binding of the antagonist ³H-quinuclidinyl benzilate was compared in membranes prepared from each of these brain regions. High affinity choline uptake was significantly reduced in all three brain regions by 28–32 months of age. This trend was already evident by 10–12 months of age, especially in hippocampus and cortex. By contrast, choline acetyltransferase activity was unchanged in striatum and actually increased in hippocampus and cortex of aged mice. Muscarinic binding was reduced significantly only in striatum and this effect was significant by 10–12 months of age. This decrease in antagonist binding was accompanied by a small but significant reduction in the relative proportion of high affinity agonist sites as defined by carbachol displacement.

The impairment of high affinity choline uptake in the absence of a parallel reduction of choline acetyltransferase activity suggests a decline of ongoing cholinergic activity rather than loss of terminal integrity as the basis of presynaptic deficits in aging. This functional decline may be exacerbated by reduction of muscarinic receptors in striatum. Despite considerable literature support for the hypothesis that cholinergic mechanisms are impaired with age, several controversies leave important issues unresolved. Therefore, the present results are discussed in the context of a critical review with emphasis on dynamic properties of presynaptic function which require analysis in experimental animal models. The impact of normal aging on brain cholinergic systems is distinguished from the neurodegenerative changes in Alzheimer disease in that presynaptic function is compromised with a relative preservation of the integrity of innervation. Nonetheless, in addition to a role in the behavioral changes associated with normal aging, age-related cholinergic hypoactivity may contribute to the vulnerability of brain cholinergic neurons to degenerative insult and alter the efficacy of drug therapies for this age-dependent disease.     

Ameliorating Effects of SDZ ENA 713 on Age-Associated Decreases in Learning Performance and Brain Choline Acetyltransferase Activity in Rats

In the present study, we have investigated the effects of SDZ ENA 713 on spatial learning deficits in aged rats. Using the same animals, the effect of SDZ ENA 713 on choline acetyltransferase was simultaneously studied to obtain a basis for the behavioral study. In the aged rats, the spatial learning and choline acetyltransferase activity in the frontal cortex were significantly deteriorated compared with young adult rats. SDZ ENA 713 (0.2 mg/kg) significantly shortened the time to reach a hidden platform without affecting swim rates in the water maze task. SDZ ENA 713 (0.1 and 0.2 mg/kg) inhibited aging-induced decreases in choline acetyltransferase activity in the frontal cortex. These results suggest that SDZ ENA 713 ameliorates aging-induced learning deficits and cholinergic dysfunction in rats.     

Impairment of cholinergic neurotransmission in adult and aged transgenic Tg2576 mouse brain expressing the Swedish mutation of human beta-amyloid precursor protein

To address the question of whether beta-amyloid peptides also affect cholinergic neurotransmission in vivo, brain tissue from transgenic Tg2576 mice with Alzheimer plaque pathology at ages ranging from 7 to 24 months were examined by immuno- and histochemical staining for choline acetyltransferase (ChAT) and acetycholinesterase (AChE), by assaying cholinergic enzyme activities and high-affinity choline uptake as well muscarinic and nicotinic cholinergic receptor binding levels by quantitative autoradiography. Cortical and hippocampal activities of AChE and ChAT were not different between transgenic mice and non-transgenic littermates regardless of the postnatal ages examined. However, high-affinity choline uptake was reduced in the hippocampus of 21-month-old transgenic mice. In brains of 8-month-old transgenic mice which do not yet demonstrate cortical beta-amyloids, reduced binding levels of cortical and hippocampal M1-muscarinic cholinergic receptors were observed, which were still reduced in 17-month-old transgenic mouse brains with high plaque load as compared to non-transgenic littermates. M2-muscarinic cholinergic receptor binding was hardly affected in brains from 8-month-old transgenic mice, but in 17-month-old transgenic mice reduced cortical and hippocampal binding levels were observed as compared to non-transgenic controls. Decreased cortical nicotinic cholinergic receptor binding was detected in 17-month-old transgenic mice. The development of changes in cholinergic synaptic markers in transgenic Tg2576 mouse brain before the onset of progressive plaque deposition provides in vivo evidence of a modulatory role of soluble beta-amyloid on cholinergic neurotransmission and may be referred to the deficits in learning and memory also observed in these mice before significant plaque load.     

Relationships between cholinergic phenotype and acetyl-CoA level in hybrid murine neuroblastoma cells of septal origin

High susceptibility of cholinergic neurons to neurotoxic signals may result from their utilization of acetyl-CoA for both energy production and acetylcholine synthesis. SN56 cholinergic cells were transfected stably with cDNA for choline acetyltransferase. Transfected cells (SN56ChAT2) expressed choline acetyltransferase activity and acetylcholine content, 17 times and 2 times higher, respectively, than did nontransfected cells. Transfection did not change pyruvate dehydrogenase but decreased the acetyl-CoA level by 62%. Differentiation by cAMP and retinoic acid caused an increase of choline acetyltransferase activity and decrease of acetyl-CoA levels in both cell lines. Negative correlation was found between choline acetyltransferase activity and acetyl-CoA level in these cells. SN56ChAT2 cells were more susceptible to excess NO than were native SN56 cells, as evidenced by the thiazolyl blue reduction assay. Thus, the sensitivity of cholinergic neurons to pathologic conditions may depend on the cholinergic phenotype-dependent availability of acetyl-CoA.     

Immunohistochemical localization of choline acetyl-transferase in the human cerebellum

Guinea pig antiserum specific to purified bovine choline acetyltransferase was found to cross-react with human enzyme. The peroxidase-antiperoxidase immunohistochemical method was then used to demonstrate the localization of choline acetyltransferase in formalin-fixed and paraffin-embedded human cerebellum from normal as well as from Huntington's disease brains. Choline acetyltransferase was localized exclusively in the mossy fibers and the glomeruli of the cerebellar folia. These immunohistochemical findings reveal the distribution of cholinergic axons and their terminals. The results are not only similar to our previous studies using the same method on the localization of choline acetyltansferase in rabbit cerebellum, but also de onstrate that some mossy fibers are cholinergic as suggested by others.     

Acetylcholine Synthesis in a Primary Culture of Porcine Intermediate Lobe Cells

Previous pharmacological studies with the pituitary gland have suggested that acetylcholine (ACh) might be involved in the regulation of intermediate lobe (IL) function. Whether ACh is endogenous to the IL cells or provided from an extrinsic source is unclear. The present experiments tested the possibility that the endocrine cells of the IL may be a source of ACh by measuring certain cholinergic markers in a primary culture of dissociated porcine cells. The endogenous ACh content was readily measurable in both the freshly dissociated IL cells and in 2- or 4-day primary cultures. Choline acetyltransferase activity was also measurable in the freshly dissociated and cultured IL cells and was reduced by 53% in the presence of a specific inhibitor, napthylvinylpyridine (50 μM). IL cells incubated in the presence of [¹⁴C]choline (1 μM) were able to synthesize [¹⁴C]ACh and the accumulation of the new ACh was inhibited by hemicholinium-3 (30 μM), a competitive inhibitor of high affinity choline uptake at cholinergic nerve terminals. In conclusion, these results demonstrate that the endocrine cells of the IL are capable of synthesizing and storing ACh.     

Thyroid hormone regulation of NGF, NT-3 and BDNF RNA in the adult rat brain.

The effects of peripherally administered thyroid hormone (TH; 500 micrograms/kg; i.p.; q.d.) on the relative abundances of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) RNA were determined by rtPCR in the cortex and hippocampus of young adult rats. Corresponding changes in choline acetyltransferase (ChAT) activity were measured since NGF and BDNF have been shown to enhance the expression of this marker enzyme of central cholinergic pathways. Abundance levels of NGF and NT-3, relative to cyclophilin (cycl), were increased significantly (+50%, P < 0.05) in the hippocampus following TH treatment. Despite enhanced abundance of NGF in the hippocampus, ChAT activity was unchanged, whereas ChAT activity was modestly increased by 28% in the cortex without corresponding changes in NGF, NT-3 or BDNF. These results demonstrate that TH administration is capable of inducing the accumulation of NT-3, in addition to NGF but that the induction levels of RNA cannot be directly correlated with responsivity of the cholinergic system as measured by ChAT activity.     

Degenerative Changes in Forebrain Cholinergic Nuclei Correlate with Cognitive Impairments in Aged Rats

Degenerative changes in the forebrain cholinergic nuclei have been studied morphometrically in behaviourally characterized aged female Sprague-Dawley rats. In all regions analysed (medial septum, diagonal band of Broca, nucleus basalis, and striatum) the acetylcholinesterase-positive neurons were reduced in both size and number in the aged (24-months-old) rats as compared to the young (3-months-old) controls. The overall reduction in cell size amounted to between 20 and 30% and the overall reduction in cell number to between 27 and 45%. Impairment in learning and/or memory performance in the aged rats, as assessed in the Morris' water-maze task, was significantly correlated with both cholinergic cell size and cell number in the medial septum, and with cholinergic cell number in the diagonal band of Broca and in the striatum. In the nucleus basalis there was a trend in the same direction but it did not reach significance. In contrast to these degenerative changes in the cell body regions, no significant differences in cortical or hippocampal choline acetyltransferase activity were detected biochemically between the young and the aged rats, and the enzyme activity levels did not correlate with the degree of behavioural impairment in the aged rats. The present results provide evidence that all major forebrain cholinergic cell groups undergo degenerative changes with age in the rat, and that the most severe changes are found in those rats which display the most profound spatial learning impairments. Despite the severe changes at the cell body level, however, the choline acetyltransferase activity in the cortical projection areas are affected only to a minor degree, perhaps as a result of functional compensatory changes at the terminal level.     

Cholinergic markers are expressed in developing and mature neurons of chick dorsal root ganglia

The presence of acetylcholinesterase has been reported in chick dorsal root ganglia at early developmental stages although acetylcholine is not known to play a role in these ganglia. Recently, we reported that during development the level of acetylcholinesterase increases continuously and the enzyme becomes gradually expressed in all sensory neurons. These observations prompted the study of the developmental pattern of expression of other cholinergic markers, such as choline acetyltransferase (ChAT) and the high affinity transport mechanism for choline. ChAT activity is barely detectable at early developmental stages (E7) and increases markedly thereafter, with an activity profile similar to that described for acetylcholinesterase. A similar increase in enzyme activity is also observed when ChAT is measured in dorsal root ganglia explants and in dissociated cells in culture. The study of ChAT activity in cultured cells shows an increase over a period of 3 days, thus ruling out the hypothesis that motor fibers, still associated to the ganglia, may represent a possible source of the enzyme. Immunostaining of whole ganglia or cultured cells shows that ChAT immunoreactivity is not restricted to a specific neuronal subpopulation but appears as a common marker of sensory neurons. High affinity choline uptake, blocked by hemicholinium, is present in sensory neurons cultured from E7 dorsal root ganglia. Observations on cultured neurons from later stages (E18) indicate that choline transport is not a transient property of sensory neurons. These observations show a similar pattern of expression of several cholinergic markers during development. Such a pattern is maintained at significant levels also in mature ganglia. © 1994 Wiley-Liss, Inc.     

Distributions of choline acetyltransferase and acetylcholinesterase activities in the retinal layers of the red-tailed hawk and road runner.

The activities of choline acetyltransferase and acetylcholinesterase were assayed in submicrogram samples from layers of red-tailed hawk and road runner retina. Both enzyme activities were concentrated in and near the inner plexiform layer. Within the inner plexiform layers of both species, activities of each enzyme were concentrated in two bands, one in each half of this layer. Little choline acetyltransferase activity was found superficial to the middle third of the inner nuclear layer. The distributions of acetylcholinesterase activities corresponded well to those of choline acetyltransferase, except in the outer plexiform layer and the outer margin of the inner nuclear layer of the hawk. These distributions of enzyme activities indicate that populations of amacrine cells in the retinae of these species are cholinergic. In addition to these same cells and presumably cholinoceptive amacrine and ganglion cells, acetylcholinesterase activity in the hawk was associated with a population of horizontal cells that may be unrelated to synaptic cholinergic neurotransmission. Choline acetyltransferase activities associated with amacrine somata and processes were about four times greater in the hawk than in the road runner, suggesting important differences in the density and function of cholinergic elements between species. Possible synaptic relationships in the inner plexiform layer consistent with the interspecies differences in enzyme activities are considered.     

Immunohistochemical localization of choline acetyltransferase in rabbit forebrain

uinea pig antiserum specific to the purified bovine choline acetyltransferase was used to demonstrate the localization of this enzyme in rabbit forebrain by the peroxidase-antiperoxidase immunohistochemical method. Choline acetyltransferase was localized in olfactory bulb, olfactory tract, olfactory tubercle, piriform cortex, septum, diagonal band, basal ganglia, thalamus, hypothalamus, subthalamus, habenula, cerebral cortex, hippocampal region, corpus callosum, internal capsule, fornix, longitudinal striae and other areas. The findings reflect the distribution of cholinergic axons and, possibly, their terminals. These observations correlate well with biochemical determinations of choline acetyltransferase and with previously proposed cholinergic pathways.     

Cholinergic innervation of the song control nuclei by the ventral paleostriatum in the zebra finch: a double-labeling study with retrograde fluorescent tracers and choline acetyltransferase immunohistochemistry

During the sensitive period of song learning, the content of acetylcholine and the enzyme activity of choline acetyltransferase (ChAT) increase remarkably in the song control nuclei of a young male zebra finch. Cholinergic fibers innervate the two main song control nuclei of the forebrain: the higher vocal center (HVC) and the robust nucleus of the archistriatum (RA). The present study combines the retrograde tracer, Fluoro-Red (FRe), with ChAT immunohistochemistry. The results indicate that the cholinergic fibers which innervate the RA and HVC originate from the ventral paleostriatum (VP) in the basal forebrain, and that there is an anterior-posterior topography in the location of the cholinergic neurons in the VP that project to the HVC and RA, although there are a few neurons which project to both nuclei. These findings suggest that the VP is homologous to the nucleus basalis of Meynert of the basal forebrain cholinergic system of mammals which is associated with learning and memory processes, and that the cholinergic neurons in the VP play an important role in avian song learning.     

Choline acetyltransferase immunoreactivity suggests that ganglion cells in the goldfish retina are not cholinergic

Published evidence that ganglion cells in the retinae of nonmammalian species are cholinergic is strong but indirect. In this paper we report results of attempts to demonstrate choline acetyltransferase immunoreactivity in ganglion cells of goldfish retina using two different antisera against choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme. We obtained ChAT-immunoreactive staining of amacrine and displaced amacrine cells in the retina and type XIV cells in the tectum, but we obtained no direct immunocytochemical evidence that ganglion cells in the goldfish retina are cholinergic. Thus, ganglion cells identified by retrograde transport of propidium iodide were never ChAT-immunoreactive. Intraocular injections of colchicine did not result in the appearance of a population of ChAT-immunoreactive neurons in the ganglion cell layer. ChAT-immunoreactive axons were not observed in intact, ligated, or transected optic nerves. And finally, the ChAT immunoreactivity of cells and fibers in the optic tectum was unaffected by deafferentation. These experiments provide no positive evidence that any ganglion cells in goldfish retina contain the acetylcholine-synthesizing enzyme, ChAT. While it is possible that our method is too insensitive to detect the enzyme in ganglion cell somata or too specific to recognize the form of ChAT present in these cells, the fact that we can stain putatively cholinergic retinal amacrine cells and tectal neurons makes these alternative explanations improbable. We conclude that it is unlikely that any of the ganglion cells in the retina are cholinergic and that alternative explanations should be sought for previously published results that suggest that they are.     

Immunohistochemical localization of choline acetyltransferase in rabbit spinal cord and cerebellum

Guinea pig antiserum specific for purified bovine choline acetyltransferase has been shown to cross-react with rabbit enzyme. We used the peroxidase-antiperoxidase immunohistochemical method to demonstrate the localization of choline acetyltransferase in formalin-fixed and paraffin-embedded sections of rabbit spinal cord and cerebellum. In the spinal cord, in agreement with our and others' previous results using immunofluorescent techniques, choline acetyltransferase was found in the cell bodies of the ventral horn motor neurons. In the cerebellum, choline acetyltransferase was localized exclusively in the mossy fibers and the glomeruli of the cerebellar folia. The immunohistochemical findings in the cerebellum reveal the morphological detail of cholinergic axons and their terminals. The results are consistent with published biochemical data on the cerebellar distribution of choline acetyltransferase.     

Neuronal versus endothelial origin of vasoactive acetylcholine in pial vessels

Functional pial vessels denuded in situ of the endothelial cell layer exhibit a markedly decreased choline uptake capacity (−53%) but integrally preserved choline acetyltransferase (ChAT) activity and acetylcholine (ACh) release mechanisms. These studies demonstrate that endothelial cells possess a specific choline uptake system. However, the unimpaired ChAT activity is denuded pial vessels implies that the endothelial pool of choline is not significantly metabolized into ACh. In spite of possible differences in the mechanisms that govern release processes in endothelial and neuronal elements, taken together the findings of the present study suggest that the ACh released following depolarization of pial blood vessels originates predominantly from cholinergic perivascular nerve terminals.