Cholinergic projections to the basolateral amygdala: A combined Evans Blue and acetylcholinesterase analysis

WOOLF, N.J. AND L. L. BUTCHER. Cholinergic projections to the basolateral amygdala: A combined Evans Blue andacetylcholinesterase analysis. BRAIN RES. BULL. 8(6) 751–763, 1982.—The origins and acetylcholinesterase (AChE, EC 3.1.1.7) content of neurons projecting to the AChE-rich basolateral amygdala were studied by infusing Evans Blue (EB), a retrogradely transported fluorescent label, into that neural region and, following microscopic evaluation of labelled somata, staining the same tissue sections for AChE according to the pharmacohistochemical regimen. The following basal forebrain areas contained cells labelled with EB: the lateral preoptic area, ventral pallidum, nuclei of the diagonal band, medial septal nucleus, bed nucleus of the stria terminalis, and substantia innominata. The majority of the basal forebrain neurons projecting to the basolateral amygdala stained intensely for AChE, suggesting that they were Cholinergic. In the brainstem, EB-labelled neurons staining intensely for AChE were found less frequently, but a few were observed in the nucleus tegmenti pendunculopontis, locus ceruleus, subcerulear region, and reticular formation. Cells accumulating EB after basolateral amygdala infusion but demonstrating no, weak, or moderate AChE activity were seen in the orbitofrontal, anterior cingulate, temporal, and insular cortices; the mediodorsal, paraventricular, and parataenial nuclei of the thalamus; the periventricular gray substance; the ventromedial mesencephalic tegmentum; the lateral and compact portions of the substantia nigra; the dorsal raphe; the dorsal tegmental nucleus; and the dorsal parabrachial nucleus. On the basis of staining intensity, intracellular organization of the AChE reaction product, and previous results in the literature, we conclude that the major Cholinergic input to the basolateral amygdala derives from the basal forebrain.     

Cholinergic projections from magnocellular nuclei of the basal forebrain to cortical areas in rats

Acetylcholinesterase (AChE) and choline acetyltransferase (ChAc) activities were studied by quantitative histochemical (AChE) as well as biochemical methods (AChE, ChAc) in certain cortical brain areas in rats after stereotaxic lesions had been placed in several structures of the basal forebrain. After lesioning the magnocellular nuclei of the substantia innominata (nuc. basalis Meynert, NBM) the activities of AChE and ChAc decreased to moderate or low residual values in the ipsilateral cortical areas. This indicated that cholinergic pathways were directly linked to frontal, sensory-motor, auditory and visual cortex. After lesions of the globus pallidus the decrease in cortical AChE activity was less pronounced. Lesions of the caudate, accumbens or entopeduncular nucleus did not influence the cortical AChE activities.The results are discussed with respect to the similarity of the organization of the cholinergic projection to the cortex arising from NBM cells and the monoaminergic system which innervates the cortex. It is suggested that both neurotransmitter systems by their interaction might modulate and control cortical information processing and behavior in a manner analogous to the control of peripheral activity by the sympathetic and parasympathetic system.     

Cholinergic projections from the basal forebrain to frontal, parietal, temporal, occipital, and cingulate cortices: a combined fluorescent tracer and acetylcholinesterase analysis

The morphologies, intercellular organization, and cortical projection patterns of putative cholinergic neurons in the basal forebrain of the rat were examined by use of fluorescent tracer histology in combination with the pharmacohistochemical regimen for acetylcholinesterase (AChE). Intensity staining AChE-containing cells projecting to frontal sensorimotor (Area 10), parietal (Area 2), and temporal (Area 4) cortices were found ipsilaterally in nucleus preopticus magnocellularis, in nucleus basalis, and in association with the substantia innominata, the ansa lenticularis, and the lateral hypothalamic area; an essentially rostrocaudal topography was observed for these projections. AChE-containing pathways to cingulate (Area 29) and visual (Area 17) cortices derived from ipsilateral somata associated with the vertical and horizontal limbs of the diagonal band, nucleus preopticus magnocellularis, rostral portions of nucleus basalis, and the substantia innominata. Neurons innervating Area 29 were generally located more rostrally than those giving rise to AChE afferents to Area 17. The vast majority of cells appeared to innervate relatively discrete areas of the cortex. Evidence for collateralization was found only in neurons projecting to visual and cingulate cortices, and these represented only 3.2% of the cells providing AChE afferents to Areas 17 and 29. The basal forebrain AChE projection cells were typically large (greater than 25 micron in maximum cell body extent), and their somata were predominantly oval, with lesser proportions being fusiform or triangular. Many were organized in clusters, particularly in nucleus basalis.     

The organization of the efferent projections of the parabrachial nucleus to the forebrain in the rat: A retrograde fluorescent double-labeling study

The organization of the efferent projections of the parabrachial nucleus (PBN) to the forebrain has been investigated in the rat by means of combined injections of two fluorescent retrograde tracers: red fluorescent Evans Blue and a blue fluorescent mixture of 4′,6′-diamidino-2-phenylindol 2 HCl and primuline. First, the distributions of retrogradely labeled neurons in the PBN after bilateral injections of tracers in the central nucleus of the amygdala (CNA) was examined. The CNA on one side of the brain was injected with one of the tracers and the CNA on the opposite side of the brain was injected with the other tracer. Next, the distributions of labeled neurons were examined after bilateral ventral medial thalamus (VMT) injections. Finally, the retrograde labeling of the PBN was studied after combined ipsilateral injections of one tracer in the CNA and the other tracer in the VMT. After the various injections, characteristic distributions of populations of labeled neurons within the PBN were seen. Double-labeled neurons were present only after bilateral VMT injections. From this it was concluded that the PBN projections to the VMT in the rat are bilateral. Based on the relative distributions of populations of retrogradely labeled neurons in the PBN, it was suggested that the PBN projects primarily taste information to the VMT and mainly visceral information to the CNA. This transfer of information to the forebrain is discussed.     

Cholinergic systems in the rat brain: III. Projections from the pontomesencephalic tegmentum to the thalamus, tectum, basal ganglia, and basal forebrain

The ascending cholinergic projections of the pedunculopontine and dorsolateral tegmental nuclei, referred to collectively as the pontomesencephalotegmental (PMT) cholinergic complex, were investigated by use of fluorescent tracer histology in combination with choline-O-acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) pharmacohistochemistry. Propidium iodide, true blue, or Evans blue was infused into the anterior, reticular, mediodorsal, central medial, and posterior nuclear areas of the thalamus; the habenula; lateral geniculate; superior colliculus; pretectal/parafascicular area; subthalamic nucleus; caudate-putamen complex; globus pallidus; entopeduncular nucleus; substantia nigra; medial septal nucleus/vertical limb of the diagonal band area; magnocellular preoptic/ventral pallidal area; and lateral hypothalamus. In some animals, separate injections of propidium iodide and true blue were made into two different regions in the same rat brain, usually a dorsal and a ventral target, in order to assess collateralization patterns. Retrogradely transported fluorescent labels and ChAT and/or AChE were analyzed microscopically on the same brain section. All of the above-delimited targets were found to receive cholinergic input from the PMT cholinergic complex, but some regions were preferentially innervated by either the pedunculopontine or dorsolateral tegmental nucleus. The former subdivision of the PMT cholinergic complex projected selectively to extrapyramidal structures and the superior colliculus, whereas the dorsolateral tegmental nucleus was observed to provide cholinergic input preferentially to anterior thalamic regions and rostral portions of the basal forebrain. The PMT cholinergic neurons showed a tendency to collateralize extensively.     

Direct comparison of projections from the central amygdaloid region and nucleus accumbens shell

Certain neurochemical and connectional characteristics common to extended amygdala and the nucleus accumbens shell suggest that the two represent a single functional-anatomical continuum. If this is so, it follows that the outputs of the two structures should be substantially similar. To address this, projections from the caudomedial shell and central nucleus of the amygdala, a key extended amygdala structure, were demonstrated in Sprague-Dawley rats with different anterograde axonal tracers processed separately to exhibit distinguishable brown and blue-black precipitates. The caudomedial shell projection is strong in the ventral pallidum and along the medial forebrain bundle through the lateral preopticohypothalamic continuum into the ventral tegmental area, distal to which it thins abruptly. The central nucleus projects strongly to the bed nucleus of the stria terminalis and the sublenticular extended amygdala, but substantially to the lateral hypothalamus only at levels behind the rostral part of the entopeduncular nucleus. Innervation of the ventral tegmental area by the central amygdala is minimal, but the lateral one-third of the substantia nigra, pars compacta and an adjacent lateral part of the retrorubral field receive substantial central amygdala input. Central amygdaloid projections are robust in caudal brainstem sites, such as the reticular formation, parabrachial nucleus, nucleus of the solitary tract and dorsal vagal complex, all of which receive little input from the accumbens. The substantial differences in the output systems of the caudomedial shell of accumbens and central amygdala suggest that the two represent distinct functional-anatomical systems.     

GABAergic input to cholinergic forebrain neurons: an ultrastructural study using retrograde tracing of HRP and double immunolabeling

Amygdalopetal cholinergic neurons in the ventral pallidum were identified by combining choline acetyltransferase (ChAT) immunohistochemistry with retrograde tracing of horseradish peroxidase (HRP) following injections of the tracer in the basolateral amygdaloid nucleus. Although ChAT-positive terminals were identified in the ventral pallidum, they were never seen in contact with either immunonegative or ChAT-positive amygdalopetal neurons. In material, in which immunostaining against glutamic acid decarboxylase (GAD), the synthesizing enzyme for GABA was combined with retrograde tracing of HRP from the basolateral amygdaloid nucleus, GAD-positive terminals were seen to contact immunonegative amygdalopetal neurons. In addition, when sections of the rostral forebrain were processed, first to preserve and identify the transported HRP, and then were sequentially tested for both ChAT and GAD immunohistochemistry with the immunoperoxidase reaction for both tissue antigens, GAD-immunopositive terminals were seen to make synaptic contacts with cholinergic amygdalopetal neurons. These results suggest that amygdalopetal, presumably cholinergic, neurons receive GAD-positive terminals. In separate experiments using immunoperoxidase for ChAT and ferritin-avidin for GAD labeling, we confirmed the presence of GAD-containing terminals on cholinergic neurons. In addition, cholinergic terminals were seen in synaptic contact with GAD-positive cell bodies. These morphological studies suggest that direct GABAergic-cholinergic and cholinergic-GABAergic interactions take place in the rostral forebrain.     

Zinc-containing afferent projections to the rat corticomedial amygdaloid complex: A retrograde tracing study

The mammalian amygdaloid complex is densely innervated by zinc-containing neurons. The distribution of the terminals throughout the region has been described, but the origins of these zinc-containing fibers have not. The present work describes the origins of one major component of the zinc-containing innervation of the amygdaloid complex, namely, the component that innervates the corticomedial complex. Selective labeling of zinc-containing axons was accomplished by intracerebral microinfusion of selenium anions (SeO₃²⁻), a procedure that produces a ZnSe precipitate in zinc-containing axonal boutons with subsequent retrograde transport to the neurons of origin. After infusions of SeO₃²⁻ into combinations of cortical, medial, or amygdalohippocampal regions, retrogradely labeled zinc-containing somata were found in all amygdaloid nuclei except for the medial and central nuclei, the bed nucleus of the accessory olfactory tract, the nucleus of the lateral olfactory tract, and the anterior amygdaloid area. Extrinsic zinc-containing projections to the same amygdaloid terminal fields were found to originate from the infralimbic, cingulate, piriform, perirhinal and entorhinal cortices, and from the prosubiculum and CA1. Commissural zinc-containing projections were found to originate from the posterolateral and posteromedial cortical nuclei and from the posterior part of the basomedial nucleus. Zinc-containing neurons have been implicated in the pathophysiology of epilepsy, in cell death after seizure or stroke, and in Alzheimer's disease, all clinical conditions that involve the amygdaloid complex. Identification of the zinc-containing pathways is a prerequisite to the elucidation of zinc's role in these disorders. J. Comp. Neurol. 400:375–390, 1998. © 1998 Wiley-Liss, Inc.     

Development of basal forebrain projections to visual cortex: Dil studies in rat

We performed experiments using retrograde and anterograde labeling with DiI to examine the development of basal forebrain (BFB) projections to the visual cortex in postnatal rats. DiI placed in occipital cortex led to retrograde labeling of BFB neurons as early as postnatal day 0 (P0); labeled cells were found mainly in the diagonal band complex but also in the medial septum, globus pallidus, and substantia innominata. The retrogradely labeled BFB cells displayed remarkably well-developed dendritic arbors, even in younger animals, and showed increases in soma size, dendritic arbors, and dendritic spines over the first 2 postnatal weeks. Dil placements in the diagonal band led to anterogradely labeled axons in cortex. At early ages (P0–P1), labeled axons were largely confined to white matter. With increasing age, greater numbers of labeled axons were seen in the white matter and in deep cortical layers, and labeled axons extended into superficial layers. The leading edge of labeled fibers reached layer V of visual cortex by P2 and layer IV by P4 and were found throughout the cortical layers by P6. Numbers and densities of labeled axons in visual cortex were greater in older animals, at least through P14. The time of ingrowth of labeled BFB axons into visual cortex indicates that these afferents grow into particular cortical layers after those layers have differentiated from the cortical plate. These data indicate that basal forebrain projections arrive in occipital cortex after cortical lamination is well underway and after the entry of primary thalamocortical projections. © 1995 Wiley-Liss, Inc.     

Cholinergic limbic projections and behavioral role of basal forebrain nuclei in the rat

The purposes of the present study were to identify cholinergic non-neocortical projections of the basal forebrain and to determine the role of this region in the regulation of estrogen-dependent reproductive behaviors in the rat. Bilateral electrolytic lesions were placed in an area encompassing the horizontal limb of the diagonal band, as well as portions of the substantia innominata and magnocellular preoptic nucleus, and choline acetyltransferase (CAT) activity was assayed in microdissected brain areas seven days after lesion. Compared to sham surgery, lesions of this region significantly reduced CAT activity in the basal amygdala (34%), dorsal hippocampus (14%), cingulate cortex (25%), piriform cortex (36%), and entorhinal cortex (34%). Other limbic and midbrain structures do not appear to receive significant cholinergic innervation from this locus since no reductions in CAT were detected after bilateral lesions. These included the anterior hypothalamus, ventromedial hypothalamus, mammillary nucleus, habenula, subiculum, ventral hippocampus, insular cortex, central gray, and interpeduncular nucleus. Behaviorally, female rats with bilateral lesions of the basal forebrain displayed an unusually high incidence of rejection behavior in response to attempted mounts by stimulus male rats in sexual behavior tests. There was no effect of basal forebrain lesions on the incidence of lordosis exhibited by these females. The dissociation of rejection and lordosis suggests that distinct neural pathways mediate the occurrence of these reproductive behaviors and that rejection behavior may be regulated by basal forebrain pathways.     

The neglected constituent of the basal forebrain corticopetal projection system: GABAergic projections

At least half of the basal forebrain neurons which project to the cortex are GABAergic. Whilst hypotheses about the attentional functions mediated by the cholinergic component of this corticopetal projection system have been substantiated in recent years, knowledge about the functional contributions of its GABAergic branch has remained extremely scarce. The possibility that basal forebrain GABAergic neurons that project to the cortex are selectively contacted by corticofugal projections suggests that the functions of the GABAergic branch can be conceptualized in terms of mediating executive aspects of cognitive performance, including the switching between multiple input sources and response rules. Such speculations gain preliminary support from the effects of excitotoxic lesions that preferentially, but not selectively, target the noncholinergic component of the basal forebrain corticopetal system, on performance in tasks involving demands on cognitive flexibility. Progress in understanding the cognitive functions of the basal forebrain system depends on evidence regarding its main noncholinergic components, and the generation of such evidence is contingent on the development of methods to manipulate and monitor selectively the activity of the GABAergic corticopetal projections.     

Cholinergic innervation of the monkey amygdala: an immunohistochemical analysis with antisera to choline acetyltransferase

The organization of the cholinergic innervation of the macaque monkey amygdaloid complex was investigated by means of immunohistochemical techniques and either a polyclonal antiserum or a monoclonal antibody directed against the specific synthetic enzyme choline acetyltransferase (ChAT). Adjacent series of sections were processed histochemically for the demonstration of the degradative enzyme acetylcholinesterase (AChE) or for cell bodies with thionin. The density of ChAT immunoreactivity differed substantially among the various nuclei and cortical regions of the amygdala. In general, the distribution of ChAT immunoreactivity paralleled the pattern of AChE staining. One notable exception was the presence of AChE containing cell bodies in addition to AChE positive fibers within nearly all of the nuclear and cortical regions. In contrast, ChAT immunoreactivity was associated only with fibers and terminals. The highest density of ChAT immunoreactive fibers and terminals was consistently observed in the magnocellular subdivision of the basal nucleus. Staining was substantially less dense in the more ventrally situated parvicellular subdivision. Medially, in the adjacent accessory basal nucleus, immunoreactive fibers and terminals were densest in the magnocellular and superficial subdivisions and least prominent in the parvicellular subdivision. Of the deep nuclei, the lateral nucleus generally obtained the least ChAT immunoreactive terminals and processes. Only its more densely cellular ventrolateral portion contained appreciable fiber and terminal staining. One of the more distinctive patterns of ChAT immunoreactivity was seen in the nucleus of the lateral olfactory tract. Here, ChAT positive fibers formed pericellular basket plexuses around unstained cell bodies. This unique pattern of staining was used to delineate the boundaries of the nucleus and indicated that it is present for much of the rostrocaudal extent of the amygdala. Another region of conspicuous staining on the medial surface of the amygdala was the sulcal portion of the periamygdaloid cortex. This region, associated with the sulcus semiannularis and bordering the entorhinal cortex, consistently contained dense immunoreactivity. The central nucleus also presented a somewhat idiosyncratic pattern of ChAT staining. The lateral subdivision had a diffuse distribution of immunoreactivity in which focal patches of more densely stained terminals and occasional fine fibers were embedded. In contrast, the medial subdivision contained a larger number of thicker, stained fibers without diffuse background labeling.(ABSTRACT TRUNCATED AT 400 WORDS)     

Axonal branching in the periaqueductal gray projections to the thalamus: a fluorescent retrograde double-labeling study in the cat

The double-labeling technique based on the retrograde axonal transport of fluorescent tracers (Evans blue, EB; Fast blue, FB; Nuclear yellow, NY) was used in the cat in order to investigate the occurence of axonal branching in the periaqueductal gray (PAG) projections to some thalamic nuclei (n. ventralis postero-lateralis, VPL; n. ventralis postero-medialis, VPM; n. parafascicularis, Pf). In a first group of cats, FB and EB were injected, respectively, within the right and left VPM. In another two groups of cats, FB injections into Pf were combined with either EB or NY injections within VPL or VPM. Double-labeled neurons were found within the PAG only in the animals of the first group. The present results show that some PAG neurons project bilaterally to VPM by means of axons collaterals.     

Afferents of the lamprey striatum with special reference to the dopaminergic system: A combined tracing and immunohistochemical study

The origin of afferents to the striatum in lamprey (Lampetra fluviatilis) was studied by using fluorescein-coupled dextran-amines (FDA). Injection of FDA into the striatum retrogradely labeled several cell populations in the forebrain and the rostral rhombencephalon. No retrograde labeled cells were seen in the mesencephalon. A dopamine-specific antiserum was used to determined the distribution of dopaminergic perikarya and fibers. Many dopamine-immunoreactive (DA-ir) fibers were present throughout the brain, but the highest density of labeled fibers was in the mediobasal prosencephalon, especially in the striatum, the lateral hypothalamic area, and the neurohypophysis. Most DA-ir cells were located in the mediobasal diencephalon (preoptic region, nucleus commissurae postopticae, hypothalamus, and nucleus tuberculi posterioris). In the mesencephalon, only a few immunopositive cells were observed in the tectum opticum. In the rhombencephalon, DA-ir cells were observed in the isthmic region, dorsally to the descending trigeminal tract, and caudally to the posterior rhombencephalic reticular nucleus. The rostralmost spinal cord received many descending DA-ir fibers from the brainstem. Along the spinal cord, DA-ir neurons were also found, some of which projected to the medioventral surface, forming a prominent plexus. On the basis of double-labeling experiments, it is shown that the dopaminergic input to the striatum originates from the nucleus tuberculi posterioris. Thus, the striatum receives inputs from different structures, including a strong dopaminergic innervation from the diencephalon. Much of the dopaminergic system in Lampetra fluviatilis is basically similar to that seen in some teleosts, but it presents differences with other anamniote (elasmobranch) as well as amniote groups. J. Comp. Neurol. 386:71–91, 1997. © 1997 Wiley-Liss, Inc.     

Topographical projection of cholinergic neurons in the basal forebrain to the cingulate cortex in the rat

The cholinergic innervation of the rat's posterior cingulate cortex (Brodmann's area 29) was studied using acetylcholinesterase (AChE) histochemistry. Electrolytic lesion of the ipsilateral medial septum and diagonal band region (MS-DB) reduced the diffuse AChE staining in layers I, II, III and V of the cingulate cortex. Kainic acid lesion of the ipsilateral globus pallidus and substantia innominata area (GP-SI) abolished the dense band of AChE stain in layer IV, with small reductions of AChE stain in other layers. The results indicate that the medial cholinergic pathway from MS-DB terminates diffusely in layers I, II, III and V while the lateral cholinergic pathway from the GP-SI predominantly ends in layer IV of the posterior cingulate cortex.     

Galanin-like immunoreactivity within the primate basal forebrain: differential staining patterns between humans and monkeys.

Galanin-like immunoreactivity (GAL-ir) was examined within the basal forebrain and adjacent regions of eight young adult New World monkeys (Cebus apella), one aged Old World monkey (Macaca mulatta), and eight humans without clinical or pathological evidence of neurological disease. All monkeys demonstrated similar patterns of immunoreactive profiles characterized by a continuum of GAL-ir magnocellular neurons located within the medial septum, diagonal band nuclei, and nucleus basalis. Colocalization experiments revealed that most (greater than 90%) of GAL-ir basal forebrain neurons also expressed the receptor for nerve growth factor (NGFR), an excellent marker for primate cholinergic basal forebrain neurons. A few smaller parvicellular GAL-ir neurons were also observed within the monkey basal forebrain. In contrast, identical cytochemical experiments revealed that virtually none of the magnocellular neurons within the basal forebrain of humans were GAL-ir. Rather, a network of GAL-containing fibers and terminal-like profiles were observed encompassing the magnocellular cholinergic neurons in humans. This immunohistochemical species difference does not appear to be mediated by procedural or technical factors since human brains contained numerous GAL-ir perikarya and fibers within adjacent regions including the bed nucleus of the stria terminalis and medial hypothalamus. These data demonstrate that there is a prominent phylogenetic transformation in primates with respect to the processing of GAL-mediated information. This species difference potentially relates to the severe basal forebrain degeneration reported in human dementias and illustrates the possible need for a reevaluation of the use of monkeys as an animal model of human basal forebrain-related cognitive dysfunction.     

Apoptosis in the rat basal forebrain during development and following lesions of connections

Evidence suggests that neurotrophins are essential for the survival and phenotypic maintenance of cholinergic basal forebrain (BF) neurons. We evaluated the pattern of programmed cell death in the BF of the rat during development and after ablations of the cerebral cortex, a major target area and source of neurotrophins for BF neurons. We identified dying cells using the TUNEL (terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labelling) method and confirmed their apoptotic morphology with electron microscopy. Moreover, we demonstrated the expression of the apoptotic marker active caspase-3 in cells with features of apoptosis. TUNEL(+) cells were present in the developing BF during the first two postnatal weeks. Their frequency peaked at postnatal day (P)1 and at P5. TUNEL used in conjunction with immunofluorescence for neuronal nuclear protein (NeuN) showed that, at both peak stages, the majority of apoptotic cells were neurons. Extensive lesions of the cerebral cortex at different ages (P0, P7 and P14) did not induce significant changes in the frequency of apoptotic BF neurons. However, they resulted in alterations in the morphological phenotype of choline acetyltransferase (ChAT)-immunoreactive neurons in the BF, and a reduction in their number which was inversely proportional to the age at which the lesions were performed. We suggest that: (i) apoptosis is temporally coordinated with the morphological and neurochemical differentiation of BF neurons and the establishment of connections with their target areas; and (ii) cortical ablations do not affect the survival of BF neurons, but they influence the phenotype of cholinergic BF neurons.     

Choline acetyltransferase and acetylcholinesterase containing projections from the basal forebrain to the amygdaloid complex of the rat

The origin of the cholinergic innervation to the amygdaloid complex was investigated with the use of acetylcholinesterase (AChE) histochemistry and choline acetyltransferase (ChAT) assay of microdissected nuclei. Visualization of AChE-positive neurones in the ventral forebrain was facilitated by pretreatment of rats with 1.5 mg/kg di-isopropyl phosphofluoridate (DFP). The AChE-positive neurones in the ventral forebrain are distributed in a continuous system from the septum through the lateral preoptic area to the entopeduncular nucleus caudally. Knife cuts or kainic acid injections (1.5 microgram/l microliter) placed in the lateral preoptic area resulted in substantial depletion of the AChE and ChAT content of the amygdala nuclei. Kainic acid injections (1.5 microgram/l microliter) in the diagonal band area or cuts through the stria terminalis dorsally did not significantly modify the AChE staining or ChAT content of the amygdala (although diagonal band injections partially depleted the hippocampus of ChAT). Knife cuts severing both the so-called ventral pathway and the stria terminalis did not produce significantly greater ChAT depletion in the amygdala than those produced by the knife cuts or kainic acid injections in the lateral preoptic area. Parasagittal knife cuts undercutting the lateral pyriform cortex also failed to modify the AChE or ChAT content of the amygdala, but they depleted the undercut cortex of both ChAT and AChE; AChE-positive material accumulated ventrally and medially to the knife cut. It is suggested that the major source of the cholinergic innervation of the amygdala is the magnocellular AChE-positive neurones in the lateral preoptic area and adjacent regions of the ventral forebrain.