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Alzheimer's disease (AD) is a neurodegenerative disease that disproportionately impacts memory and the hippocampus. However, it is unclear how AD pathology influences the activity of surviving neurons in the hippocampus to contribute to the memory symptoms in AD. One well-understood connection between spatial memory and neuronal activity in healthy brains is the activity of place cells, neurons in the hippocampus that fire preferentially in a specific location of a given environment (the place field of the place cell). In the present study, place cells were recorded from the hippocampus in a recently-developed rat model of AD (Tg-F344 AD) at an age (12-20 months) at which the AD rats showed marked spatial memory deficits. Place cells in the CA2 and CA3 pyramidal regions of the hippocampus in AD rats showed sharply reduced spatial fidelity relative to wild-type (WT) rats. In contrast, spiking activity of place cells recorded in region CA1 in AD rats showed good spatial fidelity that was similar to CA1 place cells in WT rats. Oral administration of the M 1 muscarinic acetylcholine receptor agonist VU0364572 impacted place cell firing rates in CA1 and CA2/3 hippocampal regions, but did not improve the spatial fidelity of CA2/3 hippocampal place cells in AD rats. The results indicated that, to the extent the spatial memory impairment in AD rats was attributable to hippocampal dysfunction, the memory impairment was more attributable to dysfunction in hippocampal regions CA2 and CA3 rather than CA1., (© 2018 Wiley Periodicals, Inc.)

The accumulation of pathologic protein fragments is common in neurodegenerative disorders. We have recently identified in Alzheimer's disease (AD) the aggregation of the U1-70K splicing factor and abnormal RNA processing. Here, we present that U1-70K can be cleaved into an N-terminal truncation (N40K) in ∼50% of AD cases, and the N40K abundance is inversely proportional to the total level of U1-70K. To map the cleavage site, we compared tryptic peptides of N40K and stable isotope labeled U1-70K by liquid chromatography-tandem mass spectrometry (MS), revealing that the proteolysis site is located in a highly repetitive and hydrophilic domain of U1-70K. We then adapted Western blotting to map the cleavage site in two steps: (i) mass spectrometric analysis revealing that U1-70K and N40K share the same N-termini and contain no major modifications; (ii) matching N40K with a series of six recombinant U1-70K truncations to define the cleavage site within a small region (Arg300 ± 6 residues). Finally, N40K expression led to substantial degeneration of rat primary hippocampal neurons. In summary, we combined multiple approaches to identify the U1-70K proteolytic site and found that the N40K fragment might contribute to neuronal toxicity in Alzheimer's disease.

The brain consists of diverse cell types including neurons, astrocytes, oligodendrocytes, and microglia. The isolation of nuclei from these distinct cell populations provides an opportunity to identify cell-type-specific nuclear proteins, histone modifications, and regulation networks that are altered with normal brain aging or neurodegenerative disease. In this study, we used a method by which intact neuronal and non-neuronal nuclei were purified from human post-mortem brain employing a modification of fluorescence activated cell sorting (FACS) termed fluorescence activated nuclei sorting (FANS). An antibody against NeuN, a neuron specific splicing factor, was used to isolate neuronal nuclei. Utilizing mass spectrometry (MS) based label-free quantitative proteomics, we identified 1755 proteins from sorted NeuN-positive and negative nuclear extracts. Approximately 20% of these proteins were significantly enriched or depleted in neuronal versus non-neuronal populations. Immunoblots of primary cultured rat neuron, astrocyte, and oligodendrocyte extracts confirmed that distinct members of the major nucleocytoplasmic structural linkage complex (LINC), nesprin-1 and nesprin-3, were differentially enriched in neurons and astrocytes, respectively. These comparative proteomic data sets also reveal a number of transcription and splicing factors that are selectively enriched in a cell-type-specific manner in human brain.

Activation of innate and adaptive immune responses is tightly regulated, as insufficient activation could result in defective clearance of pathogens, while excessive activation might lead to lethal systemic inflammation or autoimmunity. A20 functions as a negative regulator of innate and adaptive immunity by inhibiting NF-κB activation. A20 mediates its inhibitory function in a complex with other proteins including RNF11 and Itch, both E3 ubiquitin ligases and TAX1BP1, an adaptor protein. Since NF-κB has been strongly implicated in various neuronal functions, we predict that its inhibitor, the A20 complex, is also present in the nervous system. In efforts to better understand the role of A20 complex and NF-κB signaling pathway, we determined regional distribution of A20 mRNA as well as protein expression levels and distribution of RNF11, TAX1BP1 and Itch, in different brain regions. The distribution of TRAF6 was also investigated since TRAF6, also an E3 ligase, has an important role in NF-κB signaling pathway. Our investigations, for the first time, describe and demonstrate that the essential components of the A20 ubiquitin-editing complex are present and mainly expressed in neurons. The A20 complex components are also differentially expressed throughout the human brain. This study provides useful information about region specific expression of the A20 complex components that will be invaluable while determining the role of NF-κB signaling pathway in neuronal development and degeneration., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background: The RING domain-containing protein RING finger protein 11 (RNF11) is a member of the A20 ubiquitin-editing protein complex and modulates peripheral NF-κB signaling. RNF11 is robustly expressed in neurons and colocalizes with a population of α-synuclein-positive Lewy bodies and neurites in Parkinson disease patients. The NF-κB pathway has an important role in the vertebrate nervous system, where the absence of NF-κB activity during development can result in learning and memory deficits, whereas chronic NF-κB activation is associated with persistent neuroinflammation. We examined the functional role of RNF11 with respect to canonical NF-κB signaling in neurons to gain understanding of the tight association of inflammatory pathways, including NF-κB, with the pathogenesis of neurodegenerative diseases., Methods and Results: Luciferase assays were employed to assess NF-κB activity under targeted short hairpin RNA (shRNA) knockdown of RNF11 in human neuroblastoma cells and murine primary neurons, which suggested that RNF11 acts as a negative regulator of canonical neuronal NF-κB signaling. These results were further supported by analyses of p65 translocation to the nucleus following depletion of RNF11. Coimmunoprecipitation experiments indicated that RNF11 associates with members of the A20 ubiquitin-editing protein complex in neurons. Site-directed mutagenesis of the myristoylation domain, which is necessary for endosomal targeting of RNF11, altered the impact of RNF11 on NF-κB signaling and abrogated RNF11's association with the A20 ubiquitin-editing protein complex. A partial effect on canonical NF-κB signaling and an association with the A20 ubiquitin-editing protein complex was observed with mutagenesis of the PPxY motif, a proline-rich region involved in Nedd4-like protein interactions. Last, shRNA-mediated reduction of RNF11 in neurons and neuronal cell lines elevated levels of monocyte chemoattractant protein 1 and TNF-α mRNA and proteins, suggesting that NF-κB signaling and associated inflammatory responses are aberrantly regulated in the absence of RNF11., Conclusions: Our findings support the hypothesis that, in the nervous system, RNF11 negatively regulates canonical NF-κB signaling. Reduced or functionally compromised RNF11 could influence NF-κB-associated neuronal functions, including exaggerated inflammatory responses that may have implications for neurodegenerative disease pathogenesis and progression.

Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Because the pathological changes underlying this disease can begin decades prior to the onset of cognitive impairment, identifying the earliest events in the AD pathological cascade has critical implications for both the diagnosis and treatment of this disease. We previously reported that compared to autopsy confirmed healthy control brain, expression of LR11 (or SorLA) is markedly reduced in AD brain as well as in a subset of people with mild cognitive impairment (MCI), a prodromal clinical stage of AD. Recent studies of the LR11 gene SORL1 have suggested that the association between SORL1 single nucleotide polymorphisms (SNPs) and AD risk may not be universal. Therefore, we sought to confirm our earlier findings in a population chosen solely based on clinical criteria, as in most genetic studies. Quantitative immunohistochemistry was used to measure LR11 expression in 43 cases from the Religious Orders Study that were chosen based on a final pre-mortem clinical diagnosis of MCI, mild/moderate AD or no cognitive impairment (NCI). LR11 expression was highly variable in all three diagnostic groups, with no significant group differences. Low LR11 cases were identified using the lowest tertile of LR11 expression observed across all cases as a threshold. Contrary to previous reports, low LR11 expression was found in only 29% of AD cases. A similar proportion of both the MCI and NCI cases also displayed low LR11 expression. AD-associated lesions were present in the majority of cases regardless of diagnostic group, although we found no association between LR11 levels and pathological variables. These findings suggest that the relationship between LR11 expression and the development of AD may be more complicated than originally believed.

Alzheimer's disease (AD) is a progressive neurological disorder that causes dementia and poses a major public health crisis as the population ages. Aberrant processing of the amyloid precursor protein (APP) is strongly implicated as a proximal event in AD pathophysiology, but the neurochemical signals that regulate APP processing in the brain are not completely understood. Activation of muscarinic acetylcholine receptors (mAChRs) has been shown to affect APP processing and AD pathology, but less is known about the roles of specific mAChR subtypes. In this study, we used M(1) mAChR knock-out mice (M(1)KO) to isolate the effects of the M(1) mAChR on APP processing in primary neurons and on the development of amyloid pathology in a transgenic mouse model of AD. We demonstrate that the loss of M(1) mAChRs increases amyloidogenic APP processing in neurons, as evidenced by decreased agonist-regulated shedding of the neuroprotective APP ectodomain APPsalpha and increased production of toxic Abeta peptides. Expression of M(1) mAChRs on the M(1)KO background rescued this phenotype, indicating that M(1) mAChRs are sufficient to modulate nonamyloidogenic APP processing. In APP(Swe/Ind) transgenic mice, the loss of M(1) mAChRs resulted in increased levels of brain Abeta and greater accumulation of amyloid plaque pathology. Analysis of APP metabolites in APP(Swe/Ind) brain tissue indicates that the loss of M(1) mAChRs increases amyloidogenic APP processing. These results indicate that the M(1) mAChR is an important regulator of amyloidogenesis in the brain and provide strong support for targeting the M(1) mAChR as a therapeutic candidate in AD.

Acetylcholine (ACh) regulates many key functions of the CNS by activating cell surface receptors referred to as muscarinic ACh receptors (M(1)-M(5) mAChRs). Like other mAChR subtypes, the M(4) mAChR is widely expressed in different regions of the forebrain. Interestingly, M(4) mAChRs are coexpressed with D(1) dopamine receptors in a specific subset of striatal projection neurons. To investigate the physiological relevance of this M(4) mAChR subpopulation in modulating dopamine-dependent behaviors, we used Cre/loxP technology to generate mutant mice that lack M(4) mAChRs only in D(1) dopamine receptor-expressing cells. The newly generated mutant mice displayed several striking behavioral phenotypes, including enhanced hyperlocomotor activity and increased behavioral sensitization following treatment with psychostimulants. These behavioral changes were accompanied by a lack of muscarinic inhibition of D(1) dopamine receptor-mediated cAMP stimulation in the striatum and an increase in dopamine efflux in the nucleus accumbens. These novel findings demonstrate that a distinct subpopulation of neuronal M(4) mAChRs plays a critical role in modulating several important dopamine-dependent behaviors. Since enhanced central dopaminergic neurotransmission is a hallmark of several severe disorders of the CNS, including schizophrenia and drug addiction, our findings have substantial clinical relevance.

M(1) muscarinic acetylcholine receptors (mAChRs) may represent a viable target for treatment of disorders involving impaired cognitive function. However, a major limitation to testing this hypothesis has been a lack of highly selective ligands for individual mAChR subtypes. We now report the rigorous molecular characterization of a novel compound, benzylquinolone carboxylic acid (BQCA), which acts as a potent, highly selective positive allosteric modulator (PAM) of the rat M(1) receptor. This compound does not directly activate the receptor, but acts at an allosteric site to increase functional responses to orthosteric agonists. Radioligand binding studies revealed that BQCA increases M(1) receptor affinity for acetylcholine. We found that activation of the M(1) receptor by BQCA induces a robust inward current and increases spontaneous EPSCs in medial prefrontal cortex (mPFC) pyramidal cells, effects which are absent in acute slices from M(1) receptor knock-out mice. Furthermore, to determine the effect of BQCA on intact and functioning brain circuits, multiple single-unit recordings were obtained from the mPFC of rats that showed BQCA increases firing of mPFC pyramidal cells in vivo. BQCA also restored discrimination reversal learning in a transgenic mouse model of Alzheimer's disease and was found to regulate non-amyloidogenic APP processing in vitro, suggesting that M(1) receptor PAMs have the potential to provide both symptomatic and disease modifying effects in Alzheimer's disease patients. Together, these studies provide compelling evidence that M(1) receptor activation induces a dramatic excitation of PFC neurons and suggest that selectively activating the M(1) mAChR subtype may ameliorate impairments in cognitive function.

Fas-associated factor 1 or FAF1 is a Fas-binding protein implicated in apoptosis. FAF1 is the product of a gene at PARK 10 locus on chromosome 1p32, a locus associated with late-onset PD [Hicks, A.A., Petursson, H., Jonsson, T., Stefansson, H., Johannsdottir, H.S., Sainz, J., Frigge, M.L.et al., 2002. A susceptibility gene for late-onset idiopathic Parkinson's disease. Ann Neurol. 52, 549-555.]. In the present study we investigated the role of FAF1 in cell death and in Parkinson's disease (PD) pathogenesis. FAF1 levels were significantly increased in frontal cortex of PD as well as in PD cases with Alzheimer's disease (AD) pathology compared to control cases. Changes in FAF1 expression were specific to PD-related alpha-synuclein pathology and nigral cell loss. In addition, PD-related insults including, mitochondrial complex I inhibition, oxidative stress, and increased alpha-synuclein expression specifically increased endogenous FAF1 expression in vitro. Increased FAF1 levels induced cell death and significantly potentiated toxic effects of PD-related stressors including, oxidative stress, mitochondrial complex I inhibition and proteasomal inhibition. These studies, together with previous genetic linkage studies, highlight the potential significance of FAF1 in pathogenesis of idiopathic PD.

Objective: LR11 (aka sorLA) is a multifunctional neuronal receptor that binds apolipoprotein E and interacts with amyloid precursor protein to regulate amyloidogenesis. Reduced expression of LR11, as occurs in the brains of individuals with Alzheimer's disease (AD), increases amyloidogenesis, and variants in the gene that encodes LR11, SORL1, have recently been linked to risk for late-onset AD. In this study, we sought to determine whether reduced expression of LR11 occurs early in the disease process and whether protein levels in cortical neurons are associated with clinical and pathological changes in mild cognitive impairment (MCI), a condition that may represent prodromal AD., Methods: A novel quantitative immunohistochemical approach was used to measure LR11 levels in brain tissue collected from subjects diagnosed antemortem with either no cognitive impairment, MCI, or AD from the Rush University Religious Orders Study., Results: LR11 levels in MCI were intermediate between no cognitive impairment and AD. LR11 expression was heterogeneous in MCI, forming low- and high-level LR11 subgroups. MCI subjects with low LR11 were significantly more cognitively impaired than the high LR11 subjects. We also found a significant correlation between cognitive performance and LR11 levels across all clinical groups examined. There was no association between LR11 and plaque and tangle pathology., Interpretation: Neuronal LR11 levels are reduced in prodomal AD. The correlation between LR11 expression and cognitive performance indicates that reduced LR11 levels reflect disease severity and may predict progression to AD in a subgroup of individuals with MCI.

The PARK10 locus is associated with idiopathic Parkinson disease (PD), but the responsible gene remains to be identified. Genes associated with familial PD, as well as biochemical evidence from sporadic PD and animal models, have implicated components of the ubiquitin-proteasome system in PD pathogenesis. One attractive candidate gene at the PARK10 locus is RING-Finger Protein 11 (RNF11), the deduced amino acid sequence of which predicts a RING-H2 domain common to E3 ubiquitin ligases such as parkin. To facilitate understanding of this protein and its possible role in PD, we characterized the expression and localization of RNF11 in brain. We detected RNF11 transcript and protein and provided the first direct evidence that RNF11 is expressed in brain. Immunohistochemical analysis of RNF11 protein in rat and human brain, using 2 different antibodies, corroborated the mRNA findings. Both antibodies show that RNF11 is restricted to neurons and excluded from white matter. Moreover, RNF11 is expressed by vulnerable neurons of the substantia nigra and sequestered into Lewy bodies in brains of patients with idiopathic PD. Collectively, these findings identify RNF11 as a strong candidate gene at the PARK10 locus and highlight its potential significance in the development of the common form of PD.

Mutations in DJ-1 cause autosomal recessive, early-onset Parkinson's disease (PD). The precise function and distribution of DJ-1 in the central nervous system remain unclear. In this study, we performed a comprehensive analysis of DJ-1 expression in human, monkey, and rat brains with antibodies that recognize distinct, evolutionarily conserved epitopes of DJ-1. We found that DJ-1 displays region-specific neuronal and glial labeling in human and nonhuman primate brain, sharply contrasting with the primarily neuronal expression pattern observed throughout rat brain. Further immunohistochemical analysis of DJ-1 expression in human and nonhuman primate brains showed that DJ-1 protein is expressed in neurons within the substantia nigra pars compacta and striatum, two regions critically involved in PD pathogenesis. Moreover, immunoelectron microscopic analysis revealed a selective enrichment of DJ-1 within primate striatal axons, presynaptic terminals, and dendritic spines with respect to the DJ-1 expression in prefrontal cortex. Together, these findings indicate neuronal and synaptic expression of DJ-1 in primate subcortical brain regions and suggest a physiological role for DJ-1 in the survival and/or function of nigral-striatal neurons., (Copyright 2006 Wiley-Liss, Inc.)

Several families of G protein-coupled receptors (GPCR) have been shown to activate extracellular signal-regulated kinase (ERK) in transfected cells and non-neuronal systems. However, little is known about GPCR activation of ERK in brain. Because ERK is an important component in the regulation of synaptic plasticity, in this study we examined ERK activation by three families of GPCR that respond to major neuromodulatory neurotransmitters in the hippocampus. We used an immunocytochemical approach to examine ERK activation by muscarinic acetylcholine (mAChR), metabotropic glutamate (mGluR), and beta-adrenergic (beta-AR) receptors in CA1 neurons of mouse hippocampal slices. Because these GPCR families comprise receptors coupling to each of the major heterotrimeric G proteins, we examined whether ERK activation differs according to G-protein coupling. By using immunocytochemistry, we were able to examine not only whether each family of receptors activates ERK, but also the cellular populations and subcellular distributions of activated ERK. We demonstrated that M1 mAChRs and group I mGluRs, both of which are Gq-coupled receptors, activate ERK in CA1 pyramidal neurons, although activation in response to mAChR is more robust. The G(i/o)-coupled group II mGluRs activate ERK in glia scattered throughout CA1, and Gs-coupled beta-AR receptors activate ERK in scattered interneurons. Thus, we demonstrated that GPCR coupling to Gq, G(i/o), and Gs all activate ERK in the hippocampus, although each does so with unique properties and distributions.

The long-held belief that degeneration of the cholinergic basal forebrain was central to Alzheimer's 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 (p75(NTR)), 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 p75(NTR)-immunoreactive neurons was seen in individuals with MCI (38%) and mild AD (43%). The number of p75(NTR)-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 p75(NTR) 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., (Copyright 2002 Wiley-Liss, Inc.)

The cellular and subcellular localization of muscarinic receptor proteins m1 and m2 was examined in the neostriatum of macaque monkeys by using light and electron microscopic immunocytochemical techniques. Double-labeling immunocytochemistry revealed m1 receptors in calbindin-D28k--positive medium spiny projection neurons. Muscarinic m1 labeling was dramatically more intense in the striatal matrix compartment in juvenile monkeys but more intense in striosomes in the adult caudate, suggesting that m1 expression undergoes a developmental age-dependent change. Ultrastructurally, m1 receptors were predominantly localized in asymmetric synapse-forming spines, indicating that these spines receive extrastriatal excitatory afferents. The association of m1-positive spines with lesion-induced degenerating prefronto-striatal axon terminals demonstrated that these afferents originate in part from the prefrontal cortex. The synaptic localization of m1 in these spines indicates a role of m1 in the modulation of excitatory neurotransmission. To a lesser extent, m1 was present in symmetric synapses, where it may also modulate inhibitory neurotransmission originating from local striatal neurons or the substantia nigra. Conversely, m2/choline acetyltransferase (ChAT) double labeling revealed that m2-positive neurons corresponded to large aspiny cholinergic interneurons and ultrastructurally, that the majority of m2 labeled axons formed symmetric synapses. The remarkable segregation of the m1 and m2 receptor proteins to projection and local circuit neurons suggests a functional segregation of m1 and m2 mediated cholinergic actions in the striatum: m1 receptors modulate extrinsic glutamatergic and monoaminergic afferents and intrinsic GABAergic afferents onto projection neurons, whereas m2 receptors regulate acetylcholine release from axons of cholinergic interneurons., (Copyright 2001 Wiley-Liss, Inc.)

PDZ domain-containing proteins play an important role in the targeting and localization of synaptic membrane proteins. Here, we report an interaction between the PDZ domain-containing protein PICK1 and monoamine neurotransmitter transporters in vitro and in vivo. In dopaminergic neurons, PICK1 colocalizes with the dopamine transporter (DAT) and forms a stable protein complex. Coexpression of PICK1 with DAT in mammalian cells and neurons in culture results in colocalization of the two proteins in a cluster pattern and an enhancement of DAT uptake activity through an increase in the number of plasma membrane DAT. Deletion of the PDZ binding site at the carboxyl terminus of DAT abolishes its association with PICK1 and impairs the localization of the transporter in neurons. These findings indicate a role for PDZ-mediated protein interactions in the localization, expression, and function of monoamine transporters.

The subthalamic nucleus (STN) is a key nucleus in the basal ganglia motor circuit that provides the major glutamatergic excitatory input to the basal ganglia output nuclei. The STN plays an important role in normal motor function, as well as in pathological conditions such as Parkinson's disease (PD) and related disorders. Development of a complete understanding of the roles of the STN in motor control and the pathophysiological changes in STN that underlie PD will require a detailed understanding of the mechanisms involved in regulation of excitability of STN neurons. Here, we report that activation of group I metabotropic glutamate receptors (mGluRs) induces a direct excitation of STN neurons that is characterized by depolarization, increased firing frequency, and increased burst-firing activity. In addition, activation of group I mGluRs induces a selective potentiation of NMDA-evoked currents. Immunohistochemical studies at the light and electron microscopic levels indicate that both subtypes of group I mGluRs (mGluR1a and mGluR5) are localized postsynaptically in the dendrites of STN neurons. Interestingly, pharmacological studies suggest that each of the mGluR-mediated effects is attributable to activation of mGluR5, not mGluR1, despite the presence of both subtypes in STN neurons. These results suggest that mGluR5 may play an important role in the net excitatory drive to the STN from glutamatergic afferents. Furthermore, these studies raise the exciting possibility that selective ligands for mGluR5 may provide a novel approach for the treatment of a variety of movement disorders that involve changes in STN activity.

Presenilin-1 (PS1), which is linked to familial Alzheimer's disease, participates in the proteolytic processing of Notch and amyloid-beta precursor protein (APP) by an unknown mechanism. Reports of PS1 localization to the endoplasmic reticulum (ER) and Golgi apparatus have focused attention on the early biosynthetic pathway as the site of PS1 function. However, it is unclear how Notch cleavage and APP processing events which occur at or near the cell surface are influenced by PS1. In contrast to some earlier studies, examination of endogenously expressed PS1 in PC12 cells by subcellular fractionation and immunofluorescence microscopy revealed a distribution distinct from that of ER and Golgi markers. Rather, PS1 colocalized with transferrin receptor, a marker for early endosomes. In addition, electron microscopic examination of intact vesicles immunoisolated with PS1 antibodies allowed visualization of endocytic tracer in endosomes. These findings identify an early endosomal pool of PS1 and suggest alternative mechanisms for PS1 interactions with APP and Notch., (Copyright 2000 Academic Press.)

Despite the importance of dopamine signaling, it remains unknown if the two major subclasses of dopamine receptors exist on the same or distinct populations of neurons. Here we used confocal microscopy to demonstrate that virtually all striatal neurons, both in vitro and in vivo, contained dopamine receptors of both classes. We also provide functional evidence for such colocalization: in essentially all neurons examined, fenoldopam, an agonist of the D1 subclass of receptors, inhibited both the Na+/K+ pump and tetrodotoxin (TTX)-sensitive sodium channels, and quinpirole, an agonist of the D2 subclass of receptors, activated TTX-sensitive sodium channels. Thus D1 and D2 classes of ligands may functionally interact in virtually all dopamine-responsive neurons within the basal ganglia.

The plasma membrane dopamine transporter (DAT) and the vesicular monoamine transporter (VMAT2) are essential for normal dopamine neurotransmission. DAT terminates the actions of dopamine by rapidly removing dopamine from the synapse, whereas VMAT2 loads cytoplasmic dopamine into vesicles for storage and subsequent release. Recent data suggest that perturbation of the tightly regulated balance between these two transporters predisposes the neurone to damage by a variety of insults. Most notable is the selective degeneration of DAT- and VMAT2-expressing dopamine nerve terminals in the striatum thought to underlie Parkinson's disease. DAT and VMAT2 expression can predict the selective vulnerability of neuronal populations, which suggests that therapeutic strategies aimed at altering DAT and VMAT2 function could have significant benefits in a variety of disorders.

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 Alzheimer's 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 Alzheimer's 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., (Copyright 1999 Wiley-Liss, Inc.)

Unbiased disector stereologic cell counting was applied to sections from the human substantia nigra that were immunostained by using a monoclonal antibody against the dopamine transporter (DAT). This antibody was found to penetrate the full thickness of the stained section. Quantification of the number of DAT immunostained neurons was performed in human cases stratified into three age groups, young (ages 0-49 years), middle aged (ages 50-69 years), and aged (ages 70-85 years). The number of DAT-immunoreactive nigral neurons was normalized for each case by constructing a ratio of the number of DAT-containing neurons to total number of neuromelanin-containing cells in each subject's sample. Three types of DAT nigral neurons were seen: type 1, intensely stained; type 2, lightly stained; and type 3, DAT-immunonegative neuromelanin-containing perikarya. By 50 years of age, the number of type 1 neurons decreased significantly (P < 0.0001), whereas the number of type 2 neurons increased with age (P < 0.0001). Type 3 neurons also increased with age (P < 0.01), although less robustly than type 2 neurons. Type 1 neurons decreased by 11.2% per decade, and the total number of nigral neurons (types 1-3) decreased by 6.7% per decade. Relative to the young group, there were 75% and 88% reductions in type 1 neurons in the middle-aged and aged groups, respectively. This contrasts with the 35% and 41% reductions in total number of neuromelanin-containing neurons seen in middle-aged and aged groups, respectively. The young group had significantly more type 1 neurons and fewer type 2 neurons compared with middle-aged and aged participants. Post-hoc analyses indicated that the young group had significantly fewer type 3 neurons compared with middle-aged and aged participants. These findings demonstrate an age-related reduction in the number of substantia nigra DAT-immunoreactive neurons. Therefore, insight into the mechanisms regulating the rate of DAT synthesis may aid in our understanding of the decline of DATs with aging and its functional significance.

Familial forms of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) have recently been associated with coding region and intronic mutations in the tau gene. Here we report our findings on 2 affected siblings from a family with early-onset dementia, characterized by extensive tau pathology and a Pro to Leu mutation at codon 301 of tau. The proband, a 55-year-old woman, and her 63-year-old brother died after a progressive dementing illness clinically diagnosed as Alzheimer disease. Their mother, 2 sisters, maternal aunt and uncle, and several cousins were also affected. Autopsy in both cases revealed frontotemporal atrophy and degeneration of basal ganglia and substantia nigra. Sequencing of exon 10 of the tau gene revealed a C to T transition at codon 301, resulting in a Pro to Leu substitution. Widespread neuronal and glial inclusions, neuropil threads, and astrocytic plaques similar to those seen in corticobasal degeneration were labeled with a battery of antibodies to phosphorylation-dependent and phosphorylation-independent epitopes spanning the entire tau sequence. Isolated tau filaments had the morphology of narrow twisted ribbons. Sarkosyl-insoluble tau exhibited 2 major bands of 64 and 68 kDa and a minor 72 kDa band, similar to the pattern seen in a familial tauopathy associated with an intronic tau mutation. These pathological tau bands predominantly contained the subset of tau isoforms with 4 microtubule-binding repeats selectively affected by the P301L missense mutation. Our findings emphasize the phenotypic and genetic heterogeneity of tauopathies and highlight intriguing links between FTDP-17 and other neurodegenerative diseases.

Pharmacological studies have suggested that the m2 muscarinic receptor functions as an autoreceptor in the cholinergic axons which innervate the cerebral cortex and striatum. To test this hypothesis in the macaque monkey, we used a subtype-specific antibody to the m2 muscarinic receptor. Immunoreactive cells were well visualized in the nucleus basalis, where some of these cells displayed dense m2 immunoreactivity, while others were lightly labeled. This heterogeneity of labeling intensity was not based on peculiarities of the methodology, because cholinergic cells of the striatum expressed uniformly dense m2 immunoreactivity. Concurrent labeling with choline acetyltransferase immunoreactivity proved that most of the heavily m2-labeled cells in the nucleus basalis were also choline acetyl-transferase positive. The findings demonstrate that at least 10-25% of the cholinergic neurons in the nucleus basalis of the monkey are densely m2 immunoreactive. In the striatum, concurrent labeling demonstrated that the majority, if not all, choline acetyltransferase-positive cells also contained m2 immunoreactivity. In addition, these experiments identified a population of smaller striatal cells which were m2 immunoreactive and choline acetyltransferase negative. Consecutive labeling with m2 immunoreactivity and NADPH-diaphorase histochemistry demonstrated that many of these m2-immunoreactive non-cholinergic neurons belonged to the population of nitric oxide-synthesizing medium aspiny neurons. The findings indicate that the m2 muscarinic receptor may be expressed at high levels in only a subset of cholinergic basal forebrain neurons. In contrast, m2 receptors appear to be expressed by all cholinergic cells of the striatum.

The distribution of m1-m4 muscarinic acetylcholine receptors was investigated by immunolight and electron microscopy in rat dorsal root ganglia, by using subtype-specific antibodies. The light microscopic observations show that both the intensity and the localisation of immunolabeling of the four muscarinic subtypes are different. The m2 receptor is the most represented; m2 and m4 receptor proteins are principally expressed in the small-medium type neurones, while m1 and m3 receptor proteins are more homogeneously distributed among the neuronal population of the ganglion. On the contrary, there are no relevant differences in the subcellular distribution. Ultrastructural analysis shows that immunolabeling is associated with the Nissl bodies, budding Golgi cisterns, and as far as the m2 receptor is concerned, just beneath the plasmalemma with discrete loci along the neuronal plasma membrane. Unmyelinated nerve fibres are always immunopositive for all muscarinic receptor subtypes, suggesting an active transport for all subtypes. Occasionally perineuronal satellite cells surrounding large neurones are immunopositive for the m2 receptor, while both myelinating and non-myelinating Schwann cells faintly express all muscarinic receptor subtypes. These observations, together with previous data concerning pharmacological and functional characterisation of muscarinic acetylcholine receptors in dorsal root ganglion, suggest that sensory neurones are able to respond to acetylcholine. The presence of m2 and m4 immunoreactivity in the small type neurones and in their fibres involved in nociceptive response, suggest that these receptors may participate in the modulation of transduction of noxious stimuli from the periphery to the spinal cord. m1 and m3 receptors, having a more homogeneously distribution, may be involved in transduction of different kinds of stimuli.

Mutations in the presenilin-1 gene are linked to the majority of early-onset familial Alzheimer's disease cases. We have previously shown that the expression of transforming growth factor-beta is altered in Alzheimer's patients, compared to controls. Here we examine presenilin- expression in human post-mitotic neurons (hNT cells), normal human astrocytes, and human brain tumor cell lines following treatment with three isoforms of transforming growth factor-beta, or glial cell line-derived neurotrophic factor, a member of the transforming growth factor-beta superfamily. As the NT2/D1 teratocarcinoma cell line is treated with retinoic acid to induce differentiation to hNT cells, presenilin-1 messenger RNA expression is dramatically increased. Furthermore, there is a 2-3-fold increase in presenilin-1 messenger RNA expression following treatment of hNT cells with growth factors and similar results are found by Western blotting and with immunohistochemical staining for presenilin-1 protein. However, treatment of normal human astrocytes with cytokines results in minimal changes in presenilin-1 messenger RNA and protein. Interestingly, the expression of presenilin-1 in human U87 MG astrocytoma and human SK-N-SH neuroblastoma cells is only increased when cells are treated with glial cell line-derived neurotrophic factor or transforming growth factor-beta3. These findings suggest that endogenous presenilin-1 gene expression in human neurons can be induced by growth factors present in normal and diseased brain tissue. Cytokines may play a major role in regulating expression of presenilin-1 which may affect its biological actions in physiological and pathological conditions.

Intense immunoreactivity for the m2-muscarinic receptor was found in a population of interstitial polymorphic neurons embedded within the infracortical white matter and the adjacent deep layers of the cerebral cortex. These infracortical neurons were evenly distributed throughout architectonic subdivisions of the monkey cortex except for parts of primary visual cortex where they were less numerous. A similar set of m2-immunoreactive interstitial cells was also detected in the human lateral temporal neocortex obtained at surgery. Upon electron microscopic examination, they were found to receive unlabelled synaptic inputs and displayed abundant rough endoplasmic reticulum, a prominent nucleolus, and invaginations of the nuclear membrane. Double labelling of m2 immunoreactivity and acetylcholinesterase histochemistry demonstrated that approximately 90% of the m2-positive infracortical cells were acetylcholinesterase-rich in the monkey and human brains. Conversely, the proportion of acetylcholinesterase-rich infracortical neurons that were m2-immunoreactive was over 90% in the monkey and at least 50% in the human. The concurrent visualization of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) enzyme activity with m2 immunoreactivity in the monkey and human brain showed that 85-95% of m2-immunoreactive infracortical cells were NADPH-d positive. Conversely, about 70% of NADPH-d cells contained m2 immunoreactivity. These observations provide the most convincing information to date that many of the acetylcholinesterase-rich neurons located in the infracortical white matter of the cerebral cortex are likely to be cholinoceptive. The expression of NADPH-d by these neurons suggests that they may also provide a relay through which cholinergic innervation, originating predominantly from the nucleus basalis of Meynert, could regulate the release of nitric oxide in the cerebral cortex and subjacent white matter. The degeneration of these neurons may account for at least some of the depletion of m2 receptors that has been reported in Alzheimer's disease.

Large families of genetically distinct G-protein coupled receptor subtypes mediate dopamine's (D1-D5) and acetylcholine's effects (m1-m5). A functional balance of dopamine and acetylcholine may be based in part on the differential expression of receptor subtypes by distinct neuron subpopulations. The localization of the D1 and D2 receptors, the predominant dopamine receptors in neostriatum, to distinct subpopulations of striatal projection neurons has been controversial. In addition, m4 receptor localization to specific striatal projection neuron subpopulations is also at question. To determine whether rat striatonigral neurons differentially express D1, D2, and m4 receptor proteins, we combined immunocytochemistry by using receptor subtype specific antibodies and retrograde tracing with cholera toxin-colloidal gold. D1 and m4 receptor immunoreactivity was visualized in 95% and 92% of identified striatonigral neurons, respectively. By contrast, D2 receptor immunoreactivity was visualized in only 1% of these neurons. These findings support models of basal ganglia in which D1 and D2 receptors are segregated, as well as indicate that D1 and m4 are colocalized. These cellular distributions may be important substrates for the putative DA/ACh balance that is implicated in certain movement disorders.

Intrinsic, striatal tyrosine hydroxylase-immunoreactive (TH-i) cells have received little consideration. In this study we have characterized these neurons and their regulatory response to nigrostriatal dopaminergic deafferentation. TH-i cells were observed in the striatum of both control and 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP)-treated monkeys; TH-i cell counts, however, were 3.5-fold higher in the striatum of MPTP-lesioned monkeys. To establish the dopaminergic nature of the TH-i cells, sections were double-labeled with antibodies to dopamine transporter (DAT). Immunofluorescence studies demonstrated that nearly all TH-i cells were double-labeled with DAT, suggesting that they contain the machinery to be functional dopaminergic neurons. Two types of TH-i cells were identified in the striatum: small, aspiny, bipolar cells with varicose dendrites and larger spiny, multipolar cells. The aspiny cells, which were more prevalent, corresponded morphologically to the GABAergic interneurons of the striatum. Double-label immunofluorescence studies using antibodies to TH and glutamate decarboxylase (GAD67), the synthetic enzyme for GABA, showed that 99% of the TH-i cells were GAD67-positive. Very few (<1%) of the TH-i cells, however, were immunoreactive for the calcium-binding proteins calbindin and parvalbumin. In summary, these results demonstrate that the dopaminergic cell population of the striatum responds to dopamine denervation by increasing in number, apparently to compensate for loss of extrinsic dopaminergic innervation. Moreover, this population of cells corresponds largely with the intrinsic GABAergic cells of the striatum. This study also suggests that the adult primate striatum does retain some intrinsic capacity to compensate for dopaminergic cell loss.

We have studied the source and ultrastructural characteristics of ChAT-immunoreactive fibers in the cerebellum of the rat, and the distribution of muscarinic and nicotinic receptors in the cerebellum of the rat, rabbit, cat and monkey, in order to define which of the cerebellar afferents may use ACh as a neurotransmitter, what target structures are they, and which cholinergic receptor mediate the actions of these pathways. Our data confirm and extend previous observations that cholinergic markers occur at relatively low density in the cerebellum and show not only interspecies variability, but also heterogeneity between cerebellar lobules in the same species. As previously demonstrated by Barmack et al. (1992a,b), the predominant fiber system in the cerebellum that might use ACh as a transmitter or a co-transmitter is formed by mossy fibers originating in the vestibular nuclei and innervating the nodulus and ventral uvula. Our results show that these fibers innervate both granule cells and unipolar brush cells, and that the presumed cholinergic action of these fibers most likely is mediated by nicotinic receptors. In addition to cholinergic mossy fibers, the rat cerebellum is innervated by beaded ChAT-immunoreactive fibers. We have demonstrated that these fibers originate in the pedunculopontine tegmental nucleus (PPTg), the lateral paragigantocellular nucleus (LPGi), and to a lesser extent in various raphe nuclei. In both the cerebellar cortex and the cerebellar nuclei these fibers make asymmetric synaptic junctions with small and medium-sized dendritic profiles. Both muscarinic and nicotinic receptor could mediate the action of these diffuse beaded fibers. In the cerebellar nuclei the beaded cholinergic fibers form a moderately dense network, and could in principle have a significant effect on neuronal activity. For instance, the cholinergic fibers arising in the PPTg may modulate the excitability of the cerebellonuclear neurons in relation to sleep and arousal (e.g. McCormick, 1989). Studies on the distribution of cholinergic markers in the cerebellum have proven valuable besides the issue whether cholinergic mechanism play a role in the cerebellar circuitry, because they illustrate a complexity of the cerebellar anatomy that extends beyond its regular trilaminar and foliar arrangement. For instance, AChE histochemistry has been shown to preferentially stain the borders of white matter compartments (the 'raphes', Voogd, 1967), and therefore is useful in topographical analysis of the cortico-nuclear and olivocerebellar projections (Hess and Voogd, 1986; Tan et al., 1995; Voogd et al., 1996; see Voogd and Ruigrok, 1997, this Volume). ChAT-immunoreactivity, at least in rat, appears to be a good marker to outline the morphological heterogeneity of mossy fibers, and m2-immunocytochemistry could be used to label (subpopulations of) Golgi cells, subsets of mossy fibers and, in the rabbit, a specific subset of Purkinje cells (Jaarsma et al., 1995).

Muscarinic cholinergic transmission plays an important role in modulating hippocampal activity and many higher brain functions. Many of the modulatory effects of acetylcholine on hippocampal function result from direct effects in the hippocampus or from actions on the hippocampal afferent neurons. At each site, the differential expression of a family of five distinct but related receptor subtypes governs the nature of the response. The aim of the present study was to identify the subtypes expressed in the hippocampal afferent neurons by combining retrograde tracing with immunocytochemistry. The retrograde tracer, wheat germ agglutinin conjugated to horseradish peroxidase, was injected into the hippocampus unilaterally to label afferent neurons, and was combined with muscarinic (m) acetylcholine (ACh) receptors (mAChRs) with immunocytochemistry to identify the m1-m4 subtypes expressed. The retrogradely labeled cells in the basal forebrain that contribute to the septohippocampal pathway were found to express m2, m3, and, to a lesser extent, m1. Commissural/associational pathway neurons, which were identified by retrogradely labeled cells in the ipsi- and contralateral dentate gyrus, expressed m1, m3, and m4. The retrogradely labeled cells in the entorhinal cortex of the perforant pathway expressed predominantly m1 and m3, with fewer neurons expressing m2 and m4. Raphe-hippocampal cells were found to express m1. Thus, this study provides evidence for the diversity of mAChR subtypes expressed in neurons that project to the hippocampus. The complex modulation by acetylcholine of hippocampal function, therefore, is governed not only by the variety of mAChRs expressed in the hippocampus but also by their differential expression in extrinsic hippocampal afferents.

Visual information in primates is relayed from the dorsal lateral geniculate nucleus to the cerebral cortex by three parallel neuronal channels designated the parvocellular, magnocellular, and interlaminar pathways. Here we report that m2 muscarinic acetylcholine receptor in the macaque monkey visual cortex is selectively associated with synaptic circuits subserving the function of only one of these channels. The m2 receptor protein is enriched both in layer IV axons originating from parvocellular layers of the dorsal lateral geniculate nucleus and in cytochrome oxidase poor interblob compartments in layers II and III, which are linked with the parvocellular pathway. In these compartments, m2 receptors appear to be heteroreceptors, i.e., they are associated predominantly with asymmetric, noncholinergic synapses, suggesting a selective role in the modulation of excitatory neurotransmission through the parvocellular visual channel.

Unlabelled: The flow of cortical information through the basal ganglia occurs through the so-called 'direct pathway' and 'indirect pathways'. The object of the present work was to attempt to determine whether spiny neurons in the neostriatum that give rise to the direct pathway (i.e. the striatonigral/entopeduncular pathway) and those giving rise to the indirect pathways (i.e. striatopallidal pathway) are synaptically interconnected. The approach was to carry out double immunocytochemistry at the electron microscopic level using antibodies against peptides or dopamine receptor subtypes that are selectively associated with the neurons that give rise to the direct (substance P or D1 receptors) and indirect pathways (enkephalin or D2 receptors). Sections of perfuse-fixed rat neostriatum were immunostained to reveal both substance P immunoreactivity and D2 receptor immunoreactivity or enkephalin and D1 receptor immunoreactivity, respectively. Double peroxidase methods were employed using different chromogens that were distinguishable at both the light and electron microscopic levels. In the electron microscope substance P-immunoreactive terminals were seen in synaptic contact with dendritic structures that displayed immunoreactivity for D2 receptor. Similarly, enkephalin-immunoreactive terminals were seen in synaptic contact with D1-immunoreactive dendritic structures. Thus, axon collaterals of neurons giving rise to the direct pathway form synaptic contacts with neurons that give rise to the indirect pathway and axon collaterals of neurons giving rise to the indirect pathway form synaptic contact with neurons that give rise to the direct pathway. These results indicate that the two pathways of information flow through the basal ganglia are synaptically linked at the level of the neostriatum., Keywords: spiny neurons, direct pathway,indirect pathways, rat neostriatum

The cellular and subcellular distributions of the glutamate transporter subtypes EAAC1, GLT-1, and GLAST in the rat CNS were demonstrated using anti-peptide antibodies that recognize the C-terminal domains of each transporter. On immunoblots, the antibodies specifically recognize proteins of 65-73 kDa in total brain homogenates. Immunocytochemistry shows that glutamate transporter subtypes are distributed differentially within neurons and astroglia. EAAC1 is specific for certain neurons, such as large pyramidal cortical neurons and Purkinje cells, but does not appear to be selective for glutamatergic neurons. GLT-1 is localized only to astroglia. GLAST is found in both neurons and astroglia. The regional localizations are unique to each transporter subtype. EAAC1 is highly enriched in the cortex, hippocampus, and caudate-putamen and is confined to pre- and postsynaptic elements. GLT-1 is distributed in astrocytes throughout the brain and spinal cord. GLAST is most abundant in Bergmann glia in the cerebellar molecular layer brain, but is also present in the cortex, hippocampus, and deep cerebellar nuclei.

Muscarinic m1 receptors traditionally are considered to be postsynaptic to cholinergic fibers, while m2 receptors are largely presynaptic receptors associated with axons. We have examined the distribution of these receptor proteins in the monkey cerebral cortex and obtained results that are at odds with this expectation. Using immunohistochemistry with specific antibodies to recombinant m1 and m2 muscarinic receptor proteins, we have demonstrated that both m1 and m2 receptors are prominently associated with noncholinergic asymmetric synapses as well as with the symmetric synapses that characterize the cholinergic pathways in the neocortex. At asymmetric synapses, both m1 and m2 receptor immunoreactivity is observed postsynaptically within spines and dendrites; the m2 receptor is also found in presynaptic axon terminals which, in the visual cortex, resemble the parvicellular geniculocortical pathway. In addition, m2 labeling was also found in a subset of nonpyramidal neurons. These findings establish that the m2 receptor is located postsynaptically as well as presynaptically. The association of m1 and m2 receptors with asymmetric synapses in central pathways, which use excitatory amino acids as neurotransmitters, provides a morphological basis for cholinergic modulation of excitatory neurotransmission.

The neuroanatomical location and cytological features of cholinergic neurons in the rat brain were determined by the immunocytochemical localization of the biosynthetic enzyme, choline acetyltransferase (ChAT). Perikarya labeled with ChAT were detected in four major cell groups: (1) the striatum, (2) the magnocellular basal nucleus, (3) the pontine tegmentum, and (4) the cranial nerve motor nuclei. Labeled neurons in the striatum were observed scattered throughout the neostriatum (caudate, putamen) and associated areas (nucleus accumbens, olfactory tubercle). Larger ChAT-labeled neurons were seen in an extensive cell system which comprises the magnocellular basal nucleus. This more or less continuous set of neuronal clusters consists of labeled neurons in the nucleus of the diagonal band (horizontal and vertical limbs), the magnocellular preoptic nucleus, the substantia innominata, and the globus pallidus. Labeled neurons in the pontine tegmentum were seen as a group of large neurons in the caudal midbrain, dorsolateral to the most caudal part of the substantia nigra, and extended in a caudodorsal direction through the midbrain reticular formation into the area surrounding the superior cerebellar peduncle. The neurons in this latter group constitute the pedunculopontine tegmental nucleus (PPT). An additional cluster of cells was observed medially adjacent to the PPT, in the lateral part of the central gray matter at the rostral end of the fourth ventricle. This group corresponds to the laterodorsal tegmental nucleus. Large ChAT-labeled neurons were also observed in all somatic and visceral motor nerve nuclei. The correspondence of the distribution of ChAT-labeled neurons identified by our methods to earlier immunocytochemical and acetylcholinesterase histochemical studies and to connectional studies of these groups argues for the specificity of the ChAT antibody used.

Immunohistochemical localization of choline acetyltransferase (ChAT) in cholinergic neurons has been difficult to achieve because of problems encountered in producing specific antisera. Here we describe the production and characterization of several distinct monoclonal antibodies to ChAT. Each of the monoclonal antibodies exhibits one of three general patterns of cross-species reactions; one pattern shows reactivity limited mainly to bovine ChAT, a second pattern shows reactivity only to ChAT from higher mammals including humans, and the third pattern shows reactivity to ChAT from all mammals tested. The antibodies bound specifically to two closely related bovine proteins of 68,000 and 70,000 daltons using the Western blotting technique. One of the antibodies was used to localize immunohistochemically known cholinergic structures in the rat brain, including motor neurons, basal forebrain neurons, and neostriatal neurons.