A monoclonal antibody that induces neuronal apoptosis binds a metastasis marker.

The cell surface molecules controlling apoptosis in cortical neurons are largely unknown. A monoclonal antibody was derived that induces cultured neocortical neurons to undergo apoptosis. A Fab fragment of the antibody, however, lacked the ability to induce cell death. The antigen was purified, and characterized by compositional analysis, fast atom bombardment (FAB) mass spectrometry, sequential exoglycosidase treatments, methylation analysis, and (1)H-nuclear magnetic resonance spectroscopy, proving to be isoglobotetraosylceramide (IsoGb4). IsoGb4 has been shown previously to be a metastasis marker, antibodies against which block metastases in a mammary adenocarcinoma model (S. A. Carlsen et al., Cancer Res., 53: 2906-2911, 1993). Addition of the purified antigen to cells lacking this glycolipid demonstrated that it is capable of functioning as a portable apoptosis-transducing molecule. Intracellular ceramide levels were increased after the treatment with the apoptosis-inducing antibody, but the membrane sphingomyelin level remained unchanged. Fumonisin B1 inhibited both the ceramide increase and the apoptosis induced via IsoGb4, which indicated that the ceramide synthase pathway is likely to be involved in apoptosis induction by IsoGb4.

Overexpression of neurotrophin receptor p75 contributes to the excitotoxin-induced cholinergic neuronal death in rat basal forebrain.

Both excitotoxicity and altered trophic factor support have been implicated in the pathogenesis of Alzheimer's disease. To determine whether stimulation of p75, the low-affinity receptor for nerve growth factor, contributes to the excitotoxin-induced apoptotic death of cholinergic neurons, we examined the effect of unilateral kainic acid (KA; PBS vehicle, 1.25, 2.5 and 5.0 nmol) administration into rat basal forebrain on neuronal loss and p75 expression. KA (2. 5 nmol) destroyed 43% of Nissl-stained neurons and 70% of choline acetyltransferase (ChAT)-positive neurons 5 days after injection. Agarose gel electrophoresis revealed that KA (2.5 nmol) induced local internucleosomal DNA fragmentation after 6-48 h. Immunohistochemical analysis further showed that KA (2.5 nmol) augmented p75 immunoreactivity at a time when terminal transferase-mediated deoxyuridine trophosphate (d-UTP)-digoxigenin nick end labeling (TUNEL)-positive nuclei were increased. Many fragmented nuclei were co-labeled with ChAT antibody. The chronic administration of anti-rat p75 or the protein synthesis inhibitor, cycloheximide, but not anti-human p75, substantially reduced the KA-induced destruction of cholinergic neurons and the induction of internucleosomal DNA fragmentation. Anti-rat p75, but not cycloheximide, also reversed the spatial memory impairment produced by KA. These findings suggest that overexpression of p75 contributes to the excitotoxin-induced death of rat basal forebrain cholinergic neurons by an apoptotic-like mechanism.

Neurotrophin dependence domain: a domain required for the mediation of apoptosis by the p75 neurotrophin receptor.

The mechanisms underlying neurotrophin dependence, and cellular dependent states in general, are unknown. We show that a 29 amino acid region in the intracellular domain of the common neurotrophin receptor, p75NTR, is required for the mediation of apoptosis by p75NTR. Furthermore, contrary to results obtained with Fas, monomeric p75NTR is required for apoptosis induction, whereas multimerization inhibits the pro-apoptotic effect. Within the 29-residue domain required for apoptosis induction by p75NTR, a 14-residue region is sufficient as a peptide inducer of apoptosis. This 14-residue peptide requires the positively charged carboxyterminal residues for its effect on cell death, and these same residues are required by the full-length p75NTR. These studies define a novel type of domain that mediates neurotrophin dependence, and suggest that other cellular dependent states may be mediated by proteins displaying similar domains.

Distribution of the vesicular transporter for acetylcholine in the rat central nervous system.

In order to develop another selective marker for cholinergic cell bodies and fibres, we have raised a highly specific polyclonal antibody against a peptide derived from the C-terminus of a recently cloned putative vesicular acetylcholine transporter. This antibody recognizes the vesicular acetylcholine transporter protein on western blots of membranes from transfected monkey fibroblast COS cells as well as from various rat brain regions but not from untransfected COS cells or rat liver. In separate mapping studies, the antibody was found to stain cell bodies and fibres in all of the regions of the nervous system known to be cholinergic, including (i) the various nuclei of the basal nuclear complex and their projections to the hippocampus, amygdala, and cerebral cortex, (ii) the caudate-putamen nucleus, accumbens nucleus, olfactory tubercle, and islands of Calleja complex, (iii) the medial habenula, (iv) the mesopontine cholinergic complex and its projections to the thalamus, extrapyramidal motor nuclei, basal forebrain, cingulate cortex, raphe and reticular nuclei, and some cranial nerve nuclei, and (v) the somatic motor and autonomic nuclei of the cranial and spinal nerves. In many of these cholinergic neurons, it is possible to detect immunoreactivity for the vesicular acetylcholine transporter in proximal portions of processes and their branches, as well as in numerous puncta in close association with them. Some of these puncta are large and surround cell bodies and processes of neurons in several regions, including the somatic motor neurons of cranial nerve nuclei in the brainstem and in the ventral horn of the spinal cord. Double immunofluorescence studies indicated that neurons positive for the vesicular acetylcholine transporter also stained for the biosynthetic enzyme of acetylcholine, choline acetyltransferase. We conclude that antibody against the C-terminus of the putative vesicular acetylcholine transporter provides another marker for cholinergic neurons that, unlike in situ hybridization procedures, labels terminals as well as cell bodies. Therefore this antibody has the potential to reveal changes in number and morphology of cholinergic cell bodies and their terminal varicosities that occur in both physiologic and pathologic conditions.

Absence of p75NTR causes increased basal forebrain cholinergic neuron size, choline acetyltransferase activity, and target innervation.

Emerging evidence suggests that the p75 neurotrophin receptor (p75NTR) mediates cell death; however, it is not known whether p75NTR negatively regulates other neuronal phenotypes. We found that mice null for p75NTR displayed highly significant increases in the size of basal forebrain cholinergic neurons, including those that are TrkA-positive. Cholinergic hippocampal target innervation also was increased significantly. Activity of the cholinergic neurotransmitter synthetic enzyme choline acetyltransferase (ChAT) was increased in both the medial septum and hippocampus. Upregulation of these cholinergic features was not associated with increased basal forebrain or hippocampal target NGF levels. In contrast, striatal cholinergic neurons, which do not express p75NTR, showed no difference in neuronal number, size, or ChAT activity between wild-type and p75NTR null mutant mice. These findings indicate that p75NTR negatively regulates cholinergic neuronal phenotype of the basal forebrain cholinergic neurons, including cell size, target innervation, and neurotransmitter synthesis.

Deficient LAR expression decreases basal forebrain cholinergic neuronal size and hippocampal cholinergic innervation.

A role in neural development for protein tyrosine phosphatase (PTPase) receptors has been suggested by the finding of aberrant neurite outgrowth in Drosophila mutants lacking functional leukocyte common antigen-related (LAR) PTPase receptors; however, PTPase functions in the mammalian nervous system remain to be established. In transgenic mice containing a gene trap in the LAR gene, only trace expression of full-length LAR transcripts was found. In these mice, the size of basal forebrain cholinergic neurons was significantly reduced and cholinergic innervation of the dentate gyrus was markedly decreased. These findings constitute the first demonstration of an aberrant neuronal phenotype in a mammalian PTPase mutant and support the hypothesis that LAR-type PTPase receptors function to establish and/or maintain neuronal networks.

Differential distribution of the putative vesicular transporter for acetylcholine in the rat central nervous system.

The organization and distribution of the mRNA for the putative vesicular transporter for acetylcholine (VAChT) was studied in the rat brain by use of digoxigenin-labeled riboprobes and in situ hybridization technology. Signal was observed in all neural regions deduced to contain cholinergic somata on the basis of previous histochemical investigations employing choline acetyltransferase riboprobes and prior immunocytochemical studies with antibodies against choline acetyltransferase. It was absent in areas believed to contain no cholinergic neurons. Anti-sense riboprobes hybridized to the mRNA for the putative VAChT: (a) the projection neurons of the various nuclei of the basal nuclear complex, (b) the local circuit cells of the dorsal and ventral striata, (c) the projection neurons of the mesopontine complex, (d) perikarya in the ventral 2/3 of the medial habenula, (e) the somatic motor and autonomic cells of cranial nerves 3-7 and 9-12, as well as perikarya in the dorsal and ventral cochlear nuclei presumably giving rise to efferent fibers of cranial nerve 8, and (f) the alpha-motor and gamma-efferent motor neurons of the spinal cord. In addition, the mRNA for the VAChT was found in a few somata, probably ectopically located cells of the basal nuclear complex, in the internal capsule, central nucleus of the amygdala, entopeduncular nucleus, and zona incerta. It was also detected in some cell bodies in the reticular part of the substantia nigra, probably the rostral extension of the mesopontine complex, in the parabigeminal nucleus, and around the central canal in the spinal cord but not in cortical, hippocampal, and cerebellar perikarya. It is concluded that, like choline acetyltransferase, the mRNA for the putative acetylcholine vesicular transporter is another specific marker for neurons utilizing acetylcholine as a neurotransmitter. Further investigations of that transporter could have important implications for various diseases involving cholinergic systems, such as Alzheimer's disease.

Trophic-factor modulation of cortical acetylcholinesterase reappearance following transection of the medial cholinergic pathway in the adult rat.

Laminar patterns of cortical acetylcholinesterase (AChE) activity are reestablished in the adult, pharmacologically unmanipulated rat following axotomy of the medial cholinergic pathway. The extent to which trophic and/or growth promoting or inhibiting agents modulate AChE fiber reappearance is not fully understood. Such studies, however, would further clarify possible roles for these agents in neuronal plasticity in response to injury, as well as in plastic processes associated with normative functions. In the present experiments, we explored trophic modulation by intracortically infusing nerve growth factor (NGF) or somatostatin into cingulate cortex at a site distal to transection of the medial cholinergic pathway. Comparisons were made with sham-operated or noninfused transected controls, as well as with transected animals infused with renin or antibodies against NGF. Administration began 2 days after axotomy and continued at successive 3-day intervals for 4 weeks. It was found that, proximal to the lesion site, NGF increased and somatostatin decreased optical density of AChE; the number of AChE-containing fibers was unaltered compared to controls. Distal to the knife cut, both NGF and somatostatin increased number of AChE fibers but did not alter overall AChE optical density. Nonetheless, NGF produced an increase in the number of intensely staining puncta both proximal and distal to the cut. Neither renin nor anti-NGF antibodies produced statistically significant effects on optical density or number of fibers at any cortical locus studied. We conclude that NGF and somatostatin have opposite effects on the expression of AChE: whereas NGF increases AChE levels, somatostatin inhibits AChE accumulation in proximal fibers, perhaps by actions on synthesis or transport. Fiber proliferation, which only occurred distally, was affected positively by both NGF and somatostatin, indicating that neurite-promoting effects produced by both agents are confined to tissue regions where neurite extension is stimulated by axotomy. Increases in AChE-positive puncta produced by NGF, however, were not confined to regions of fiber proliferation.

Expression of the low-affinity nerve growth factor receptor enhances beta-amyloid peptide toxicity.

The low-affinity nerve growth factor receptor (NGFR) p75NGFR induces apoptosis in the absence of nerve growth factor (NGF) binding but enhances neural survival when bound by NGF. Basal forebrain cholinergic neurons express the highest levels of p75NGFR in the adult human brain and are preferentially involved in Alzheimer disease, raising the question of whether there may be a functional relationship between the expression of p75NGFR and basal forebrain cholinergic neuronal degeneration in Alzheimer disease. The expression of p75NGFR by wild-type and mutant PC12 cells potentiated cell death induced by beta-amyloid peptide. NGF binding to p75NGFR inhibited the toxicity of beta-amyloid peptide, whereas NGF binding to TrkA, the high-affinity NGFR, enhanced it. These results suggest a possible link between beta-amyloid peptide toxicity and preferential degeneration of cells expressing p75NGFR.

Involvement of free oxygen radicals in beta-amyloidosis: an hypothesis.

Compelling evidence suggests that cerebral deposition of aggregating beta-amyloid protein may trigger the neurodegenerative cascades of Alzheimer's disease, Down syndrome, and, to a lesser degree, normal aging. We propose further that free oxygen radicals are critically involved in beta-amyloidosis. Apart from the established role of free radicals in other amyloidoses, our proposal is consistent with a large number of findings. Among these are (a) the salient relationship of Alzheimer's disease with aging and the increase in free oxygen radical liberation with advancing age; (b) biochemical and analytic epidemiologic evidence that free radical formation is increased in the disorder; (c) preliminary evidence that quenching free radicals slows the clinical progression of Alzheimer's disease; (d) the early and invariable beta-amyloid accumulation in trisomy 21, a syndrome associated with elevated free radical activity and with concomitant high levels of beta-amyloid precursor protein; (e) other factors that may be associated with increased liberation of free oxygen radicals and deposition of beta-amyloid protein. Possible mechanisms by which free radicals might modulate beta-amyloidosis are discussed.

Induction of apoptosis by the low-affinity NGF receptor.

Nerve growth factor (NGF) binding to cellular receptors is required for the survival of some neural cells. In contrast to TrkA, the high-affinity NGF receptor that transduces NGF signals for survival and differentiation, the function of the low-affinity NGF receptor, p75NGFR, remains uncertain. Expression of p75NGFR induced neural cell death constitutively when p75NGFR was unbound; binding by NGF or monoclonal antibody, however, inhibited cell death induced by p75NGFR. Thus, expression of p75NGFR may explain the dependence of some neural cells on NGF for survival. These findings also suggest that p75NGFR has some functional similarities to other members of a superfamily of receptors that include tumor necrosis factor receptors, Fas (Apo-1), and CD40.

Reestablishment of laminar patterns of cortical acetylcholinesterase activity following axotomy of the medial cholinergic pathway in the adult rat.

Little is known about injury-induced restructuring of cholinergic neurons projecting from the basal forebrain to nonhippocampal loci in the mature mammalian brain. In an attempt to address this issue further, we made unilateral knife cuts in the cingulate cortex of adult rats and assessed the rates and extents of fiber regeneration. Among the fibers interdicted by these transections were axons of the medial cholinergic pathway, which originates prominently in the diagonal band nuclei and projects to medial frontal, cingulate, and occipital cortices. Immediately following the lesion, acetylcholinesterase (AChE)-containing fibers proximal to the transection demonstrated increased enzyme activity that remained elevated for at least 12 weeks before returning to control values within 9 months. Loss of distal cholinesterase staining was seen from 1-2 weeks postaxotomy, with partial restoration of enzyme activity within 4 weeks. By 12 weeks, virtually complete restitution of laminar patterns of AChE innervation had occurred in the cortex posterior to the knife cut. At least part of this reinnervation was attributable to fibers growing or elongating across the glial scar, with the degree of regrowth being correlated positively with the rapidity and magnitude of wound closure. Sprouting from the cut ends of axons, as well as collateral growth of those and uncut adjacent fibers, are likely sources of fibers contributing to the restoration of normal laminar profiles of AChE activity.

Differential visualization of cholinesterasic neuronal somata and fibers by use of modifications of acetylcholinesterase pharmacohistochemistry.

We modified existing acetylcholinesterase (AChE) histochemical techniques to visualize cholinergic neuronal somata and processes in the same rat brain. The AChE staining procedure of Karnovsky and Roots, combined with diisopropylfluorophosphate (DFP) pre-treatment, have previously allowed visualization of neuronal somata. Fibers have been visualized by nickel-diaminobenzidine (DAB)-hydrogen peroxide histochemical intensification. We combined novel modifications of these techniques to visualize cell bodies and fibers simultaneously. Maximal staining of neuronal somata occurs with a 1:1 dilution of both the Karnovsky-Roots medium and the nickel-DAB solution; diluting the Karnovsky-Roots medium 1:10 and increasing the concentration of the nickel-DAB solution twofold allows visualization of fibers previously unstainable with DFP pre-treatment and the classical AChE protocol. It is now possible to visualize both neuronal somata and fibers in adjacent sections of the same brain, in a quick and inexpensive manner.

Organization of central cholinergic neurons revealed by combined in situ hybridization histochemistry and choline-O-acetyltransferase immunocytochemistry.

Digoxigenin-labeled riboprobes and in situ hybridization of choline-O-acetyltransferase mRNA, both alone and in combination with immunohistochemical procedures for the synthetic enzyme of acetylcholine, were used to map the topography of putative cholinergic neurons in the rat central nervous system. Only the anti-sense riboprobe yielded specific labeling, which was absent in brain sections processed with sense riboprobe. Telencephalic neurons demonstrating the mRNA for choline-O-acetyltransferase and choline-O-acetyltransferase-like immunoreactivity were found in the caudate-putamen nucleus, nucleus accumbens, olfactory tubercule, Islands of Calleja complex, medial septal nucleus, vertical and horizontal limbs of the diagonal band, substantia innominata, nucleus basalis, and nucleus of the ansa lenticularis, as well as occasionally in the amygdala. Neurons in the cerebral cortex, hippocampus, and primary olfactory structures did not demonstrate hybridization signal, even though some cells in those areas were observed to exhibit choline-O-acetyltransferase-like immunopositivity. Thalamic cells were devoid of hybrido- and immunoreactivity, with the exception of several neurons located primarily in the ventral two-thirds of the medial habenula. A few cell bodies labeled with riboprobe and co-localizing choline-O-acetyltransferase-like immunopositivity were found in the lateral hypothalamus, caudal extension of the internal capsule, and zona incerta. Neurons in the pedunculopontine and laterodorsal tegmental nuclei evinced moderate hybridization signal, whereas cells of the parabigeminal nucleus were very weakly reactive. In contrast, motor neurons of the cranial nerve nuclei demonstrated high levels of choline-O-acetyltransferase mRNA and choline-O-acetyltransferase-like immunoreactivity. Putative cholinergic somata in the ventral horns and intermediolateral cell columns of the spinal cord and around the central canal were also labeled with riboprobe. It is concluded that hybridocytochemistry with digoxigenin-labeled riboprobes confirms the existence of cholinergic neurons in most of the neural regions believed to contain them on the basis of acetylcholinesterase pharmacohistochemistry and choline-O-acetyltransferase immunocytochemistry, with the prominent exceptions of the cerebral cortex, hippocampus, olfactory bulb, anterior olfactory nucleus, and caudal raphe nuclei, which apparently do not possess neurons expressing detectable levels of the mRNA for the synthetic enzyme of acetylcholine.

Dopaminergic drugs reverse the impairment of radial-arm maze performance caused by lesions involving the cholinergic medial pathway.

Pharmacological studies have shown that both cholinergic and dopaminergic transmitter systems are crucial for optimal choice accuracy in the radial-arm maze and that these systems interact in a complex fashion. Lesion studies have provided evidence that the basal nuclear complex of the forebrain, the origin of cholinergic projections to the cerebral mantle, may be critical for the cholinergic modulation of learning and memory. We have shown that knife-cut lesions of the medial cholinergic pathway significantly impair radial-arm maze choice accuracy performance. The current study examined the effectiveness of D1 and D2 ligands in counteracting this lesion-induced deficit. The adverse effects of medial cholinergic pathway lesions were diminished or reversed by daily treatment with a D1 agonist (SKF 38393), a D2 agonist (LY 171555) or a D1 antagonist (SCH 23390), but were not affected by treatment with a D2 antagonist (raclopride). The three beneficial treatments have previously been found to attenuate the adverse effects of nictonic or muscarinic blockade on choice accuracy performance in the radial-arm maze. The finding that these dopaminergic drugs ameliorate the memory deficit caused by lesions involving the cholinergic medial pathway suggests the importance of interactions between cholinergic and dopaminergic systems in radial-arm maze performance. These results may provide leads for the development of novel therapeutic approaches for treating human disorders thought to result from cholinergic hypofunction.

Cat 'P300' and cholinergic septohippocampal neurons: depth recordings, lesions, and choline acetyltransferase immunohistochemistry.

The role of septohippocampal circuits in the generation of the P300 response in cats (n = 12) was explored in a series of depth recording, tract-tracing and lesion experiments. Systematic mapping of the hippocampus in 1-mm increments from rostral to caudal extent revealed large positive potentials, greater in amplitude to rare than to frequent stimuli, within the 200-500 ms range. Each map revealed maximal amplitude responses at diverse, widely distributed hippocampus loci. Furthermore, these electrical responses displayed polarity inversion within the hippocampus that was generally localized to the pyramidal cell layer; polarity inversion was also observed in the adjacent entorhinal cortex and amygdala. Injections of propidium iodide, a tract-tracing agent, into these inversion sites resulted in retrograde labeling of small clusters of choline acetyltransferase (ChAT)-positive neurons in the medial septal nucleus and vertical limb of the diagonal band. Aspiration lesions that bilaterally destroyed large amounts of caudal hippocampus from stereotaxic levels A4 to A1 resulted in a decreased number of cells expressing ChAT in the rostral basal nuclear complex. In only 2 cats was the preoperative presence of a significant vertex P300 absent postoperatively. In the majority of cases (5 of 8 animals), hippocampal aspiration produced an enhancement of the preoperative P300 potential. We conclude that cholinergic mechanisms are importantly, albeit not exclusively, involved in the mediation of P300 potentials in cats. Neurons mediating P300 responses appear to be organized in diverse clusters of septal and diagonal band cells. These septal cells may facilitate, and in turn be facilitated or inhibited as a function of hippocampal, or other, allocortical feedback loops.

Cholinergic neurons in the rat central nervous system demonstrated by in situ hybridization of choline acetyltransferase mRNA.

Digoxigenin-labeled RNA probes and in situ hybridization histochemistry were used to examine choline acetyltransferase gene expression in the rat central nervous system. Hybridization signal was present only in brain sections processed with the antisense riboprobe. The sense probe did not yield labeling, further validating the specificity of tissue reactivity. Telencephalic neurons containing the mRNA for the cholinergic synthetic enzyme were found in the caudate-putamen nucleus, nucleus accumbens, olfactory tubercule, islands of Calleja complex, medial septal nucleus, vertical and horizontal limbs of the diagonal band, substantia innominata, nucleus basalis, and nucleus of the ansa lenticularis. Some somata evincing hybridization signal were observed in the anterior amygdalar area, and an occasional such cell was seen in the basolateral and central amygdalar nuclei. Neurons in the cerebral cortex, hippocampus, and primary olfactory structures did not demonstrate hybridocytochemically detectable amounts of choline acetyltransferase mRNA. Thalamic cells were devoid of reactivity, with the exception of several neurons located primarily in the ventral two-thirds of the medial habenula. A few somata labeled with riboprobe were found in the lateral hypothalamus, caudal extension of the internal capsule, and zona incerta. Neurons in the pedunculopontine and laterodorsal tegmental nuclei were moderately reactive, whereas cells of the parabigeminal nucleus exhibited a very weak hybridization signal. No somata in the brainstem raphe nuclei, including raphe obscurus and raphe magnus, were observed to bind riboprobe. In contrast, motor neurons of the cranial nerve nuclei demonstrated relatively large amounts of choline acetyltransferase mRNA. Putative cholinergic somata in the ventral horns and intermediolateral cell columns of the spinal cord were also labeled with riboprobe, as were a few cells around the central canal. We conclude that hybridocytochemistry with digoxigenin-labeled riboprobes confirms the existence of cholinergic neurons (i.e. those that synthesize and use acetylcholine as a neurotransmitter) in most of the neural regions deduced to contain them on the basis of previous histochemical and immunocytochemical data. Notable exceptions are the cerebral cortex and hippocampus, which do not possess neurons expressing detectable levels of choline acetyltransferase mRNA.

Prolongation of magnetic resonance T2 time in hippocampus of human patients marks the presence and severity of Alzheimer's disease.

Spin-spin relaxation time (T2) was measured in the hippocampal formation, thalamus, and cortical white matter in 13 patients with probable Alzheimer's disease (AD), 11 elderly normal individuals, 23 healthy young persons, and 9 subjects diagnosed with multi-infarct dementia. A 0.04 tesla magnetic resonance scanner was used. Hippocampal T2 values for all AD patients exceeded those of any non-AD individual, regardless of age or dementia due to infarction. Further, these T2 values were highly correlated (+ 0.96) with the severity of functional and cognitive impairment of the AD patients. This T2 prolongation was not observed at the other sites examined. These results suggest that hippocampal T2 prolongation may provide a specific marker by which AD pathology can be detected, characterized, and followed in vivo.

Postnatal development of cholinergic neurons in the rat: I. Forebrain.

The postnatal development of cholinergic projection and local-circuit neurons in the rat forebrain was examined by use of choline acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) histochemistry. Although regional nuances were apparent, a general trend emerged in which cholinergic projection neurons in the basal nuclear complex (i.e., medial septal nucleus, vertical and horizontal diagonal band nuclei, magnocellular preoptic field, substantia innominata, nucleus basalis, and nucleus of the ansa lenticularis) demonstrated ChAT-like immunoreactivity earlier in postnatal development than intrinsically organized cholinergic cells in the caudate-putamen nucleus and nucleus accumbens, although this disparity was less apparent for local circuit neurons in the olfactory tubercle and Islands of Calleja complex. Ontologic gradients of enzyme expression also existed in some regions. A lateral to medial progression of ChAT and AChE appearance was observed as a function of increasing postnatal age in the nucleus accumbens and rostral caudate-putamen nucleus. By comparison, a rostrocaudal gradient of expression of ChAT-like immunoreactivity was apparent within the basal nuclear complex. Moderate to intense ChAT positivity, for example, appeared first in the medial septal nucleus. Furthermore, compared to more caudal regions, a greater proportion of AChE-positive neurons in rostral aspects of the basal forebrain expressed ChAT immunoreactivity on postnatal day 1, a difference that was no longer present by postnatal day 5. Cholinergic neurons in all forebrain regions also underwent an initial stage of progressive soma and proximal-dendrite hypertrophy, which peaked during the third postnatal week, followed by a period of cell-body and dendritic shrinkage that persisted into the fifth postnatal week when adult configurations were reached. These soma and dendritic size increases and decreases were not correlated with the magnitude of postnatal ChAT expression, which increased progressively until adult levels were attained approximately by the third to fifth weeks after birth. Expression of AChE in putative cholinergic neurons appeared to precede that of ChAT, especially in the caudate-putamen complex. Staining intensity of AChE also incremented earlier than that of ChAT.