Michelle L. Gilmor

Expression of the putative vesicular acetylcholine transporter in rat brain and localization in cholinergic synaptic vesicles

A cholinergic locus has recently been identified consisting of a unique mammalian genomic arrangement containing the genes for choline acetyltransferase (ChAT) and a putative vesicular acetylcholine transporter (VAChT). Although transcripts for ChAT and VAChT protein have been localized in cholinergic neurons, little is known about the encoded VAChT protein. Here we describe production of highly specific rabbit polyclonal antibodies, generated using a VAChT C- terminus/glutathione-S-transferase fusion protein, and immunological characterization of the native VAChT protein. These antibodies specifically recognized full-length recombinant VAChT expressed in transfected HeLa cells by Western blotting, with the prominent immunoreactive band at 55 kDa. In rat brain homogenates, a single VAChT- immunoreactive band of approximately 70 kDa was predominant in known areas of cholinergic innervation, including striatum, cortex, hippocampus,and amygdala. Light microscopic immunocytochemistry revealed reaction product in cholinergic cell groups but not in noncholinergic areas. More significantly, immunoreactivity was also concentrated in axonal fibers in many regions known to receive prominent cholinergic innervation, such as cerebral cortex, hippocampus, amygdala, striatum, several thalamic nuclei, and brainstem regions. Electron microscopy using immunoperoxidase revealed that VAChT was localized in axon terminals, and using more precise immunogold techniques, to synaptic vesicles. In VAChT-positive perikarya, the immunogold particles were localized to the cytoplasmic face of the Golgi complex. These findings confirm that VAChT protein is expressed uniquely in cholinergic neurons, concentrated in synaptic vesicles, and at least for the C terminus, topologically oriented as predicted by models.

Preservation of nucleus basalis neurons containing choline acetyltransferase and the vesicular acetylcholine transporter in the elderly with mild cognitive impairment and early Alzheimer's disease

Immunocytochemistry for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) was used to examine the expression of these linked cholinergic markers in human basal forebrain, including cases with early stages of Alzheimers disease (AD). Previous neurochemical studies have measured decreased ChAT activity in terminal fields, but little change or even increased levels of VAChT. To determine total cholinergic neuron numbers in the nucleus basalis of Meynert (nbM), stereologic methods were applied to tissue derived from three groups of individuals with varying levels of cognition: no cognitive impairment (NCI), mild cognitive impairment (MCI), and early‐stage Alzheimers disease (AD). Both markers were expressed robustly in nucleus basalis neurons and across all three groups. On average, there was no significant difference between the number of ChAT‐ (210,000) and VAChT‐ (174,000) immunopositive neurons in the nbM per hemisphere in NCI cases for which the biological variation was calculated to be 17%. There was approximately a 15% nonsignificant reduction in the number of cholinergic neurons in the nbM in the AD cases with no decline in MCI cases. The number of ChAT‐ and VAChT‐immunopositive neurons was shown to correlate significantly with the severity of dementia determined by scores on the Mini‐Mental State Examination, but showed no relationship to apolipoprotein E allele status, age, gender, education, or postmortem interval when all clinical groups were combined or evaluated separately. These data suggest that cholinergic neurons, and the coexpression of ChAT and VAChT, are relatively preserved in early stages of AD. J. Comp. Neurol. 411:693–704, 1999.

Loss of basal forebrain P75NTR immunoreactivity in subjects with mild cognitive impairment and Alzheimer's disease

The long‐held belief that degeneration of the cholinergic basal forebrain was central to Alzheimers disease (AD) pathogenesis and occurred early in the disease process has been questioned recently. In this regard, changes in some cholinergic basal forebrain (CBF) markers (e.g. the high affinity trkA receptor) but not others (e.g., cortical choline acetyltransferase [ChAT] activity, the number of ChAT and vesicular acetylcholine transporter‐immunoreactive neurons) suggest specific phenotypic changes, but not frank neuronal degeneration, early in the disease process. The present study examined the expression of the low affinity p75 neurotrophin receptor (p75NTR), an excellent marker of CBF neurons, in postmortem tissue derived from clinically well‐characterized individuals who have been classified as having no cognitive impairment (NCI), mild cognitive impairment (MCI), and mild AD. Relative to NCI individuals, a significant and similar reduction in the number of nucleus basalis p75NTR‐immunoreactive neurons was seen in individuals with MCI (38%) and mild AD (43%). The number of p75NTR‐immunoreactive nucleus basalis neurons was significantly correlated with performance on the Mini‐Mental State Exam, a Global Cognitive Test score, as well as some individual tests of working memory and attention. These data, together with previous reports, support the concept that phenotypic changes, but not frank neuronal degeneration, occur early in cognitive decline. Although there was no difference in p75NTR CBF cell reduction between MCI and AD, it remains to be determined whether these findings lend support to the hypothesis that MCI is a prodromal stage of AD. J. Comp. Neurol. 443:136–153, 2002.

Localization of M2 muscarinic acetylcholine receptor protein in cholinergic and non-cholinergic terminals in rat hippocampus

The muscarinic receptor family (M(1)-M(4)) mediates cholinergic modulation of hippocampal transmission. Pharmacological and physiological studies have indicated that a presynaptic receptor on cholinergic terminals plays a key role in regulating ACh release, although the molecular identity of this subtype is uncertain. In this study, the localization of the M(2) receptor is described in detail for the pyramidal cell layer in the CAl region of the hippocampus. Electron microscopic analysis of M(2) immunoreactivity in this area revealed mainly presynaptic expression of this subtype. Double-labeling experiments using antibodies to M(2) and to the vesicular acetylcholine transporter, a novel, specific marker of cholinergic terminals, were used to investigate the nature of these presynaptic receptors. These studies have revealed that M(2) is located in cholinergic and non-cholinergic terminals. This is the first direct anatomical evidence that suggests that M(2) may indeed function as a cholinergic autoreceptor in the hippocampus. The distribution of the M(2) receptor in non-cholinergic terminals also suggests functional roles for M(2) as a presynaptic heteroreceptor.

Differential presynaptic and postsynaptic expression of m1–m4 muscarinic acetylcholine receptors at the perforant pathway/granule cell synapse

A family of muscarinic acetylcholine receptor proteins mediates diverse pre- and postsynaptic functions in the hippocampus. However the roles of individual receptors are not understood. The present study identified the pre- and postsynaptic muscarinic acetylcholine receptors at the perforant pathway synapses in rat brain using a combination of lesioning, immunocytochemistry and electron microscopic techniques. Entorhinal cortex lesions resulted in lamina-specific reductions of m2, m3, and m4 immunoreactivity in parallel with the degeneration of the medial and lateral perforant pathway terminals in the middle and outer thirds of the molecular layer, respectively. In contrast, granule cell lesions selectively reduced m1 and m3 receptors consistent with degeneration of postsynaptic dendrites. Direct visualization of m1-m4 by electron microscopic immunocytochemistry confirmed their differential pre- and postsynaptic localizations. Together, these findings provide strong evidence for both redundancy and spatial selectivity of presynaptic (m2, m3 and m4) and postsynaptic (m1 and m3) muscarinic acetylcholine receptors at the perforant pathway synapse.

Coordinate Expression of the Vesicular Acetylcholine Transporter and Choline Acetyltransferase Following Septohippocampal Pathway Lesions

Abstract: The gene for the vesicular acetylcholine transporter (VAChT) was recently cloned and found to be located within a 5′ noncoding intron of the gene for choline acetyltransferase (ChAT). There appear to be several shared and unique promoters for each gene, suggesting that control of expression of these two genes can be either coordinated or independent. Two lesions, axotomy and immunotoxin, directed at the well defined septohippocampal cholinergic pathway were used to determine VAChT and ChAT protein expression in the degenerating terminal fields in the hippocampus and the cell bodies of the medial septum nucleus after injury. Two weeks after lesioning, decreases of up to 90% in VAChT were found in the affected hippocampus by immunoblotting and immunocytochemistry, similar to ChAT activity. The number of VAChT‐ and ChAT‐immunopositive neurons in the medial septum decreased by up to 95%. Eight weeks following axotomy, the number of VAChT‐ and ChAT‐immunopositive neurons had increased to almost 50% in fimbria‐fornix‐lesioned animals, indicating coordinate reexpression of both cholinergic markers in recovered neurons. There was no recovery of either VAChT or ChAT immunoreactivity after the irreversible immunotoxin lesions. Thus, with use of immunological techniques, there appears to be coordinate expression of VAChT and ChAT in the septohippocampal pathway following either unilateral fimbria‐fornix or bilateral immunotoxin lesion.

Generation of transporter-specific antibodies.

Publisher Summary This chapter describes the steps involved in generating fusion protein antibodies, including construction of the expression plasmid, production and purification of the fusion protein containing a portion of the transporter, immunization of animals for antisera production, and purification and characterization of antibodies. Many of these procedures can be employed in the production and characterization of antipeptide antibodies. Neurotransmitter transporters play an integral role in the regulation of synaptic and intracellular neurotransmitter levels. Various methods have been employed to study the biology of these transporters. There are two distinct methods for producing transporter-specific antibodies. The first method—anti-peptide antibody generation—uses a relatively short peptide as the target sequence. The production of anti-peptide antibodies has become very common with numerous companies and universities providing services to produce both the peptides and antibodies.

UNIT 5.7 Production of Antisera Using Fusion Proteins

The use of fusion proteins for the production of antisera allows specific areas of proteins to be targeted as epitopes and facilitates the purification of the antisera. This unit first describes the use of standard molecular biological techniques to construct a fusion‐protein expression plasmid by inserting a region of cDNA into a pGEX vector. Next, E. coli are transformed with the plasmid and induced to generate fusion protein. Also described is the purification of the soluble fusion protein, which is necessary for immunization and other subsequent procedures. This is accomplished by taking advantage of the GST fusion tags affinity for glutathione. The purified fusion protein is then used to immunize animals, and antisera from these animals are then purified using affinity columns. Support protocols describe the construction and calibration of affinity columns for purifying antibodies using soluble fusion proteins, the use of insoluble fusion proteins for animal immunization, and preparation of affinity columns for purifying antibodies using insoluble fusion proteins.