Feline islands of Calleja complex: I. Cytoarchitectural organization and comparative anatomy

Cytoarchitectural analyses demonstrated that the islands of Calleja complex (ICC) is highly developed and discretely organized in the cat. The feline complex is clearly divided into morphological units, each containing a granular Callejal island and a population of satellite neurons. These ICC units change progressively in cytoarchitecture from the lateral to the medial edge of the olfactory tubercle. In particular, the islands flatten, sink into the tubercular molecular layer, and increase in cell density, while their satellite neurons increase in number and decrease in size. The lateromedial transformation was judged to take place in five stages, resulting in the successive appearance of lateral, lateral transitional, central, medial transitional, and medial ICC units. The first two unit types display prominently two additional components of the feline ICC-namely, clusters of dwarf cells and small pyramidal-like neurons constituting the densocellular layer cupping the base of lateral Callejal islands. All of the various types of ICC units contact the tubercular molecular layer via their dwarf and/or granule cell components, raising the possibility of direct olfactory input to the entire Callejal complex (apart from the isla magna). Output from the complex is presumed to arise from the satellite neurons, which are distinguished from adjoining cell populations by their close association with Callejal islands, typical chromophilic character, and relatively large size (15-42 micron in soma length). In the tubercular ICC, these neurons are most numerous immediately above Callejal islands in a fiber-rich zone continuous with the supratubercular zone and hence with the ventral pallidum. In the accumbal ICC, satellite neurons are most conspicuous in granule-cell-poor spaces within the isla magna, where many non-granular neurons are uncharacteristically small and chromophobic. The isla magna itself is unusual not only for its large size but for lateral extensions encircling a group of accumbal neurons far caudally. Such extensions are one of several indications that the isla magna is intimately associated with the nucleus accumbens. A comparative anatomical survey of the ICC in rats, cats, and macaque monkeys demonstrated a number of species differences. Of particular interest is the finding that the complex is unambiguously divided into discrete island-satellite cell units only in cats and macaques. In these species, the complex is also distinguished by a predominance of superficial islands and an especially prominent isla magna. ICC units, however, were most conspicuous in cats.     

Catecholaminergic innervation of the septal area in man: immunocytochemical study using TH and DBH antibodies

The catecholaminergic innervation of the human septal area and closely related structures has been visualized by using tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) as immunocytochemical markers. TH-like immunoreactivity with no corresponding DBH labelling was considered to be indicative of dopaminergic fibers. Catecholaminergic innervation offered the following similarities to that of rodents: moderate innervation in the medial septal division, with predominant DBH immunolabelling; dense dopaminergic innervation in the lateral septal nuclei, organized in a laminar pattern; presence of dopaminergic pericellular arrangements in the dorsal septum and bed n. of the stria terminalis; clustering of dopaminergic terminals in n. accumbens associated with a medioventral zone of DBH-like immunoreactive fibers; close overlap between dopaminergic fields and acetylcholinesterase-reactive zones in both the lateral septum and the n. of the stria terminalis. Differences with the catecholaminergic septal innervation of rodents consisted of general caudal extension of the dopaminergic fields, possibly accounted for by the vertical stretching and caudal displacement of the septal nuclei in man; complementary lateromedial topography of dopaminergic and DBH-immunoreactive inputs in the n. of the stria terminalis as opposed to their dorsoventral organization in rodents; presence of TH-immunolabelled cell group in the anterior olfactory nucleus and parolfactory cortex, which seems specific for primates. Precise topographical mapping of the catecholaminergic structures in this central region of the limbic forebrain seems to be a prerequisite for accurate tissue sampling in the biochemical investigations of pathological cases and should help in the interpretation of aminergic dysfunction in a variety of human diseases.     

The islands of Calleja complex of rat basal forebrain. III. Histochemical evidence for a Striatopallidal system

The characteristics of the islands of Calleja complex (ICC) in the basal forebrain of the rat were studied with immunohistochemistry, histofluorescence, acetylcholinesterase staining, India ink vascular perfusions, electron microscopy, and steroid autoradiography. The ICC contains clusters of granule cells and associated medium-sized and large cells in the surrounding neuropil of the olfactory tubercle and septum-nucleus accumbens interface. The ICCs were found to contain monoamine fibers (dopamine and norepi-nephrine), neuroactive peptide fibers (leu-enkephalin, met-enkephalin, substance P, cholecystokinin, luteinizing hormone-releasing hormone), acetyl-cholinesterase-containing somata and dendrites, and medium-sized and large cells that concentrate [³H] estradiol. The specific overlap and combination of putative neurotransmitters in separate compartments of the ICC suggest that these structures contain striatum- and pallidumlike components. Striatumlike regions are defined as the zone in the rim regions of the ICC and are innervated predominantly by dopamine and cholecystokinin inputs. Pallidumlike regions are defined as the synaptic zone near the medium-sized and large cells of the cap and core regions of the ICC and they are innervated predominantly by enkephalin, substance P, and gamma aminobutyric acid inputs. The morphology, connections, and neurotransmitter relationships of the ICC, therefore, resemble classical Striatopallidal systems. The additional presence of substances involved in the reproductive neuroendocrine systems (luteinizing hormone-releasing hormone, estradiol-binding cells), especially in the medial ICC, suggest that some ICC are involved in an endocrine corticostriatopallidal system. These endocrine systems resemble other neocortically and allocortically originating corticostriatopallidal systems in terms of their cell types, connections, and neurotransmitter systems. A functional role for the ICC in extrapyramidal motor systems is proposed.     

Telencephalic cholinergic system of the New World monkey (Cebus apella): morphological and cytoarchitectonic assessment and analysis of the projection to the amygdala

While the cholinergic projection from the nucleus basalis to the cortical mantle has received considerable attention, a similar projection to the magnocellular basal nucleus of the amygdala has not been studied in such detail. The present study analyzed the cholinergic basal forebrain projection to the amygdala in the Cebus apella monkey by using combined tract-tracing and immunocytochemical techniques. As a foundation for this assessment, the morphological and cytoarchitectonic organization of the cholinergic telencephalic system of the New World C. apella monkey was examined by using choline acetyltransferase (ChAT) immunocytochemistry. Although there were minor differences, the telencephalic cholinergic system of Cebus monkeys is similar to that seen in Old World nonhuman primates. ChAT-immunoreactive neurons were observed throughout the Ch1-4 regions of the basal forebrain, with subdivisions of the Ch4 region similar to those previously described (Mesulam et al., '83a). Most cholinergic neurons were hyperchromic and magnocellular; however, some neurons were parvicellular. Like most species, cholinergic neurons were also observed throughout the striatum. However, unlike in rodents, cholinergic perikarya were not observed within the cortex or hippocampus. To analyze the cholinergic fiber projections from the basal forebrain to the amygdala, monkeys received an intraamygdaloid injection of the retrograde tracer horseradish peroxidase conjugated to wheat germ agglutinin. Retrogradely labeled neurons that colocalized ChAT or acetylcholinesterase (AChE) were found predominantly in the anterolateral portion of the CH4 region. Fewer double-labeled neurons were found in the anteromedial and intermediate portion of CH4 and in the CH3 region. Neurons that exhibited retrograde labeling were only occasionally discerned in the posterior portions of the CH4 region, in the medullary laminae of the globus pallidus, or lodged within the internal capsule. These data are discussed in terms of the putative role this cholinergic input might play in cognitive processing in primates.     

Differential Oscillatory Properties of Cholinergic and Non-cholinergic Nucleus Basalis Neurons in Guinea Pig Brain Slice

Evidence has suggested that the nucleus basalis magnocellularis has the potential to influence the functional state of the cerebral cortex through topographically organized, widespread projections of the cholinergic cells in that nucleus. It has also been shown that, in addition to the cholinergic neurons, other non-cholinergic magnocellular basal forebrain neurons, some of which have been identified as gamma-aminobutyric acid-ergic, project into the cerebral cortex and thus may also participate in the modulation of its activity. We have performed a comparative study of the intrinsic rhythmic properties of immunohistochemically and morphologically characterized choline acetyltransferase (ChAT)-positive and ChAT-negative cells of the nucleus basalis by means of intracellular recordings in guinea pig brain slices. Our results demonstrate that relatively large, multipolar cholinergic and non-cholinergic neurons each display differential voltage-dependent properties that allow them to discharge rhythmically in spike bursts and spike clusters, respectively, at low frequencies (<10 Hz). Cholinergic cells display bursts of 2–4 action potentials (at -200 Hz) riding on low-threshold spikes recurring at a low frequency (<5 Hz) when depolarized from a membrane potential more negative than -55 mV and display low-frequency (<10–15 Hz) tonic firing when depolarized from a more positive level. In contrast, non-cholinergic cells fire in a unique mode, displaying non-adapting clusters of spikes interspersed with rhythmic subthreshold membrane-potential oscillations when depolarized from levels less negative than -55 mV. The spike clusters repeat rhythmically at relatively low frequencies (2–10 Hz). The intracluster spiking frequency is relatively high and coincides approximately with that of the intervening membrane-potential oscillations (-20–70 Hz). The cluster frequency of the non-cholinergic cells corresponds, in the same manner as the burst frequency of the cholinergic cells, to a delta (1–4 Hz) or theta (4–10 Hz) range of activity, whereas the intra-cluster and tonic spike frequencies of the non-cholinergic cells correspond to high beta to gamma ranges of electroencephalographic activity (19–30 Hz and 30–60 Hz, respectively). We propose that the different modes of oscillatory firing by the cholinergic and non-cholinergic basal forebrain cell populations could collectively contribute to the rhythmic modulation of slow and fast rhythms within the cerebral cortex.     

Cholinergic neurons in the rat septal complex: ultrastructural characterization and synaptic relations with catecholaminergic terminals.

Physiological and pharmacological studies have suggested that catecholamines modulate cholinergic neurons in the medial septal and diagonal band nuclei (i.e., the septal complex). Thus, the ultrastructural morphology of neurons containing choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine, and their relation to catecholaminergic terminals exhibiting immunoreactivity for the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) were examined in the rat septal complex. Dual immunoautoradiographic and peroxidase anti-peroxidase labeling methods were used to simultaneously localize antibodies raised in rabbits against TH and from rat-mouse hybridomas against ChAT in single sections. At least two types of perikarya with ChAT-immunoreactivity (ChAT-I) were observed. The first type were large (20-30 microns), elongated or round, and contained a small indented nucleus with an abundant cytoplasm and an occasional lamellar body. The second type was also either ovoid or round but was medium-sized (15-20 microns) and contained a larger indented nucleus and a smaller amount of cytoplasm than the first type. Both types of perikarya as well as dendrites with ChAT-I were surrounded by astrocytic processes apposed to most of their plasmalemmal surfaces. The distribution and types of terminal associations (i.e., asymmetric synapses, symmetric synapses and appositions which lacked a membrane specialization in the plane of section analyzed) with ChAT-labeled perikarya and dendrites were quantitatively evaluated. The majority (68% of 197) of the presynaptic terminals were unlabeled; the remaining terminals were immunoreactive for TH (25%) or ChAT (7%). All three types of terminals contacted primarily the shafts of small dendrites and more rarely ChAT-labeled perikarya and large dendrites. ChAT-labeled terminals: (1) formed associations with unlabeled perikarya and dendrites (31% of 176); (2) formed associations with perikarya and dendrites with ChAT-I (7%); (3) contacted the same unlabeled perikarya and dendrite as a TH-containing terminal (21%); (4) were in apposition to TH-labeled terminals (25%); or (5) were either in apposition to unlabeled or ChAT-labeled terminals or lacked associations with any processes. The majority of associations formed by the terminals with ChAT-I were on the shafts of small dendrites. Moreover, most of the associations formed were either symmetric synapses or appositions not separated by astrocytes in the plane of section analyzed. These findings provide cellular substrates in the septal complex (1) for sparse synaptic input relative to astrocytic investment of cholinergic neurons and (2) for direct synaptic modulation of cholinergic and non-cholinergic neurons by catecholamines and/or acetylcholine. These findings have direct relevance to catecholaminergic-cholinergic interactions and to the neuropathological basis for Alzheimer's disease.     

Cholinergic innervation of the primate hippocampal formation: II. Effects of fimbria/fornix transection

The distribution of choline acetyltransferase (ChAT)-immunoreactive and acetylcholines-terase (AChE)-positive fibers and terminals was analyzed in the hippocampal formation of macaque monkeys subjected to transection of the fimbria/fornix. Cases with either unilateral or bilateral transections were prepared, with post transection survival times ranging from 2 weeks to 1.5 years. The fimbria/fornix transection resulted in a dramatic decrease in the number of cholinergic fibers in most regions of the hippocampal formation. Some hippocampal regions. however, showed relatively greater sparing of ChAT- or AChE-positive fibers. In practically all regions of the hippocampal formation, residual AChE-positive fibers were more abundant than ChAT-immunoreactive fibers. In animals with unilateral lesions, the distribution patterns and density of AChE and ChAT staining on the side contralateral to the lesion were generally similar to those of sections from unlesioned control brains. The largest decreases in the densities of positive fibers were observed in the dentate gyrus, CA3 and CA2 fields of the hippocampus, subiculum, parasubiculum, and medial and caudal parts of the entorhinal cortex. Fibers were relatively better preserved in the rostral or uncal portion of the hippocampus and dentate gyrus and in the rostral portion of the entorhinal cortex. The presubiculum demonstrated remarkable sparing that contrasted with the almost complete loss of fibers in the parasubiculum. Interestingly, animals killed approximately 1.5 years after the fornix transection showed essentially the same pattern of fiber loss as the cases with shorter survival periods. This indicates that the residual ChAT-immunoreactive fibers, many of which reach the hippocampal formation through a ventral cholinergic pathway, are not capable of reinnervating the denervated portions of the hippocampal formation. This appears to distinguish the monkey from the rat, for which substantial sprouting and reinnervation of cholinergic fibers have been reported after similar lesions. © 1996 Wiley-Liss, Inc.     

Cholinergic neurons of the feline pontomesencephalon. II. Ascending anatomical projections

Immunoreactivity for choline acetyltransferase (ChAT) was analyzed in unoperated cats and in cats in which stereotaxic lesions were made in the pedunculopontine and laterodorsal tegmental nuclei. The fine reaction product revealed moderate to dense ChAT-immunoreactive fiber plexuses throughout the telencephalon, diencephalon, and midbrain. A pontomesencephalic origin of cholinergic innervation to virtually every nucleus of the diencephalon, as well as to various midbrain and basal telencephalic sites was indicated in the cats with lesions, in which the optical density of ChAT-immunoreactivity was significantly decreased as compared to controls. Pontomesencephalic lesions produced no changes, however, in the density of ChAT staining in the cerebral cortex, basolateral amygdala, or caudate nucleus. In addition to ChAT-positive terminal fiber arborizations which were widely distributed, cholinergic fibers-of-passage were traced in the unoperated and operated feline brains. The general course of ChAT fibers cut in cross-section was followed in successive transverse levels, and although pathways originating from the pedunculopontine nucleus demonstrated orientations in every direction, many demonstrated a rostral course. A particularly dense aggregate of ascending ChAT-positive fibers was localized in the dorsolateral sector of the pedunculopontine area which could be followed at more rostral levels into the central tegmental fields and the compact part of the substantia nigra. From the central tegmental fields, numerous ChAT-immunopositive fibers cut in cross-section continued to course rostrally in the intralaminar, reticular and lateroposterior nuclei of the thalamus, and a distinct bundle of ChAT fibers coursing dorsolaterally was observed medial to the optic tract ascending to the lateral geniculate. ChAT fibers with dorsolateral orientations were additionally observed in the zona incerta, ventral anterior thalamus, and ansa lenticularis on route to the reticular thalamus, the globus pallidus, and the substantia innominata. Pathways consisting of fibers traced from ChAT-containing cells in the laterodorsal tegmental nucleus could be traced to medial structures such as the periaqueductal gray, ventral tegmental area and dorsal raphe. Medially placed ChAT fibers were additionally followed through the ventral tegmental area, the midline thalamus, and the hypothalamus, up to the medial and lateral septal nuclei. The trajectories of the ascending cholinergic pathways from the pontomesencephalon are discussed in relation to locally generated electrophysiological responses in the cat.     

Cholinergic innervation of the human thalamus: dual origin and differential nuclear distribution.

The cholinergic innervation of the human thalamus was studied with antibodies against the enzyme choline acetyltransferase (ChAT) and nerve growth factor receptor (NGFr). Acetylcholinesterase histochemistry was used to delineate nuclear boundaries. All thalamic nuclei displayed ChAT-positive axons and varicosities. Only the medial habenula contained ChAT-positive perikarya. Some intralaminar nuclei (central medial, central lateral, and paracentral), the reticular nucleus, midline nuclei (paraventricular and reuniens), some nuclei associated with the limbic system (anterodorsal nucleus and medially situated patches in the mediodorsal nucleus) and the lateral geniculate nucleus displayed the highest density of ChAT-positive axonal varicosities. The remaining sensory relay nuclei and the nuclei interconnected with the motor and association cortex displayed a lower level of innervation. Immunoreactivity for NGFr was observed in cholinergic neurons of the basal forebrain but not in cholinergic neurons of the upper brainstem. The contribution of basal forebrain afferents to the cholinergic innervation of the human thalamus was therefore studied with the aid of NGFr-immunoreactive axonal staining. The anterior intralaminar nuclei, the reticular nucleus, and medially situated patches in the mediodorsal nucleus displayed a substantial number of NGFr-positive varicose axons, presumably originating in the basal forebrain. Rare NGFr-positive axonal profiles were also seen in many of the other thalamic nuclei. These observations suggest that thalamic nuclei affiliated with limbic structures and with the ascending reticular activating system are likely to be under particularly intense cholinergic influence. While the vast majority of thalamic cholinergic input seems to come from the upper brainstem, the intralaminar and reticular nuclei, and especially medially situated patches within the mediodorsal nucleus also appear to receive substantial cholinergic innervation from the basal forebrain.     

Cholinergic systems in the rat brain: II. Projections to the interpeduncular nucleus

The cholinergic innervation of the interpeduncular nucleus was investigated by use of fluorescent tracer histology in combination with choline-O-acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) pharmacohistochemistry. Following propidium iodide or Evans Blue infusion into the interpeduncular nucleus, brains were processed for co-localization of transported fluorescent label and ChAT and AChE. Control infusions of tracers were made into the ventral tegmental area. In order to delimit the course of putative cholinergic afferents to the interpeduncular nucleus from extra-habenular sources, knife cuts surrounding the habenular nuclei were performed. Somata containing propidium iodide that were highly immunoreactive for ChAT were found primarily in the vertical and horizontal limbs of the diagonal band, the magnocellular preoptic area, and the dorsolateral tegmental nucleus, also referred to as the laterodorsal tegmental nucleus. A few such co-labeled somata were also detected in the medial septal nucleus, substantia innominata, nucleus basalis, and pedunculopontine tegmental nucleus. A good correlation was observed between intensely-staining, AChE-containing and ChAT-positive neurons projecting to the interpeduncular nucleus from the aforementioned structures. Although the medial habenula contained numerous cells demonstrating transported label following interpeduncular infusion of fluorescent tracers, the ChAT-positivity associated with somata in that nucleus was weak compared to ChAT-like immunoreactivity in known cholinergic neurons in the basal forebrain and brainstem. Knife cuts that separated the habenular nuclei from the stria medullaris and neural regions lateral and posterior to those nuclei while leaving the fasciculus retroflexus intact resulted in a reduction of ChAT-like immunoreactivity in the medial habenular nucleus, fasciculus retroflexus, and interpeduncular nucleus. These data suggest (1) that the cholinergic innervation of the interpeduncular nucleus derives primarily from ChAT-positive cells in the basal forebrain and dorsolateral tegmental nucleus and (2) that putative cholinergic fibers having their origin in the medial habenula, if they exist, constitute a minor portion of the cholinergic input to the interpeduncular nucleus.     

Collateral sprouting in central mesolimbic dopamine neurons: Biochemical and immunocytochemical evidence of changes in the activity and distribution of tyrosine hydroxylase in terminal fields and in cell bodies of A10 neurons

The olfactory tubercle of adult rats was examined for the development of collateral sprouts from intrinsic dopaminergic axons following unilateral olfactory bulbectomy. In the ipsilateral tubercle tyrosine hydroxylase (TH) activity began to increase by 10–14 days following the lesion, gradually reaching a maximum of 125% of control (P < 0.005) by 21 days where it remained permanently elevated. The rise of TH activity in the tubercle reflected changes of the dopaminergic innervation, since dopamine-β-hydroxylase (DBH) activity was unchanged, and lesions of the dorsal noradrenergic bundle reduced DBH but not TH activity in the tubercle. By immunocytochemical staining the elevation of TH reflected an increased number and altered distribution of TH-containing processes within the olfactory tubercle. By 30 days the uptake of [³H]dopamine into synaptosomes of the olfactory tubercle was also increased to 140% of control (P < 0.05). In the dopaminergic cell bodies of the ipsilateral A10 group (which innervate the tubercle) TH activity was transiently elevated to 121% (P < 0.05) by 4 days, returning to control levels by 10 days. Histologically no change in activity was detected. The results indicate that mesolimbic dopaminergic neurons of A10 which innervate the olfactory tubercle will sprout in response to removal of a major non-dopaminergic input, that the new innervation is sustained, and that during collateral sprouting there is a transient elevation of TH activity in the uninjured cell bodies which precedes the period of axonal growth. The findings suggest that (a) the increase of TH activity in the A10 cell bodies during collateral sprouting may be a reflection of an increase in the amount of enzyme protein required for transport into the enlarging terminal fields, (b) that as in development sprouts are in place before they reach biochemical maturity, (c) the biochemical mechanisms underlying collateral sprouting of uninjured neurons are not necessarily the same as those associated with regenerative sprouting in response to axonal injury, and (d) the development and the acquisition of biochemical maturation of collateral sprouts in the CNS involves complex two-way signalling between terminal field and cell bodies. The development of collateral sprouts of dopaminergic neurons may be the cellular substrate of the development of behavioral hyperactivity and aggression produced by bilateral olfactory bulbectomy in rat.     

The islands of Calleja complex of rat basal forebrain II: Connections of medium and large sized cells

The connections of the medium (10–20 μm) and large (20–35 μm) cells of the islands of Calleja Complex (ICC) were studied in the albino rat with anterograde and retrograde transport of horseradish peroxidase (HRP) and fluorescent tracers. The medium and large size cells were found to project to the ipsilateral olfactory tubercle, ventral pallidum, septum, piriform cortex, periamygdaloid cortex, cortical nuclei of the amygdala, ventral endopiriform nucleus, lateral hypothalamic area, Forel's field H, ventral tegmental area, supramammillary complex, and nuclei gemini of the hypothalamus, midline, intralaminar and medial thalamic nuclei, and lateral habenula. Afferents of the ICC appear to include the same nuclei with the exception of the lateral habenula. In addition, the dorsal raphe projects to the ICC. These connections are consistent with the concept that the ICC is a striato-pallidal structure.     

The dopaminergic innervation of the ventral striatum in the rat: a light- and electron-microscopical study with antibodies against dopamine

Dopamine (DA) in the dorsal and ventral striatum is associated with different aspects of locomotor activity control. The ventral striatum may form an interface between the limbic system and the extrapyramidal motor system. The distribution of dopaminergic fibers in this interface position was studied in detail with a method applying antibodies against DA. Furthermore, the ultrastructural morphology of the DA fibers was examined by means of immuno-electron microscopy. The results show that DA immunoreactivity is distributed over the ventral striatum in a highly compartmentalized fashion. In the dorsal striatum few compartments were found. The DA fibers in the ventral striatum establish mainly symmetric synaptic contacts, preferably with dendritic shafts and spines. The results are discussed in relation to previous data concerning the light and electron-microscopic identification of catecholaminergic fibers in the ventral and dorsal striatum.     

Cholinergic innervation of olfactory glomeruli in the rat: An ultrastructural immunocytochemical study

The ultrastructural organization of cholinergic afferents to the rat olfactory bulb (OBI) was studied with the aid of choline acetyltransferase (ChAT) immunocytochemistry in electron microscopy. Particular attention has been paid to a subset of glomeruli characterized by a remarkably high density of cholinergic afferents. Numerous cholinergic terminals making symmetric or asymmetric synaptic contacts were observed in the periglomerular area. ChAT-labelled terminals have a diameter ranging from 0.3 to 1.5 μm and contain numerous clear agranular vesicles. Axo-somatic and axo-dendritic contacts were both observed in contact with several types of target neurons. Three types of cholinoceptive, noncholinergic neurons could be identified: periglomerular cells, superficial short-axon cells, and external tufted cells. Our results provide an anatomical substrate for the hypotheses concerning the complex effects of acetylcholine in the processing of sensory information in the olfactory bulb. © 1993 Wiley-Liss, Inc.     

Cholinergic innervation of cortex by the basal forebrain: Cytochemistry and cortical connections of the septal area,diagonal band nuclei,nucleus basalis (Substantia innominata), and hypothalamus in the rhesus monkey

The organization of projections from the cholinergic neurons of the basal forebrain to neocortex and associated structures was investigated in the rhesus monkey with the help of horseradish peroxidase transport, acetyl-cholinesterase histochemistry, and choline acetyltransferase immunohis-tochemistry. Four groups of neurons contained cholinergic perikarya and were designated as Chl-Ch4. The Ch1 group corresponds to the medial septal nucleus; about 10% of its neurons are cholinergic, and it provides a substantial projection to the hippocampus. The Ch2 group corresponds to the vertical nucleus of the diagonal band; at least 70% of its neurons are cholinergic, and it is the major source of innervation that the hippocampus and hypothalamus receive from the Chl-Ch4 complex. The Ch3 group most closely corresponds to the horizontal nucleus of the diagonal band; only 1% of its neurons can definitely be shown to be cholinergic, and it is the major source of Chl-Ch4 projections to the olfactory bulb. The Ch4 group most closely corresponds to the nucleus basalis of Meynert; at least 90% of its neurons are cholinergic, and it has projections to widespread areas of cortex and to the amygdala. In fact, the Ch4 group provides the single major source of cholinergic innervation for the entire cortical surface. In this respect, it is analogous to the raphe nuclei and to the nucleus locus coeruleus, which constitute the major sources of widespread cortical serotonergic and nor-adrenergic innervation, respectively. The extensive Ch4 group can be divided into several subdivisions. Each subdivision has a preferential set of targets for its projections even though the connection patterns contain considerable overlap. The anteromedial subdivision of Ch4 is the major source of cholinergic projections to areas on the medial aspect of the cerebral hemispheres; the anterolateral Ch4 sub-division is the major source of cholinergic projections to frontoparietal op-ercular areas and to the amygdala; the intermediate Ch4 subdivision pro-vides the major cholinergic input for a variety of dorsal prefrontal, insular, posterior parietal, inferotemporal, and peristriate areas; and the posterior subdivision of Ch4 provides the major cholinergic innervation of superior temporal and immediately adjacent areas. The basal forebrain in the human contains a cytoarchitechture analogous to that of the monkey. The Ch4 group (nucleus basalis) of the human is very extensive and can be subdivided into the same components that were identfied in the monkey brain. Pathological changes in Ch4 neurons have been described in a variety of human disease. In Alzheimer's disease, the relatively selective depression of neocortical cholinergic innervation may be closely associated with the neuronal loss in Ch4, which has also been described inthis condition. In the rhesus monkey, all types of cortical areas receive substantial projections from the hippocampus. Virtually all of this hypothalamic input into neocortex arises from acetylcholinesterase-rich neurons which lack choline acetyltransferase. The hypothalamocortical pathway is therefore acetylcholinesterase-rich but not cholinergic.     

Ultrastructural characterization of choline acetyltransferase-containing neurons in the basal forebrain of rat: evidence for a cholinergic innervation of intracerebral blood vessels

The ultrastructural morphology and vascular associations of cholinergic neurons in the horizontal limb of the nucleus of the diagonal band of Broca (nDBBhl) and amygdala of rat were determined by the immunocytochemical localization of choline acetyltransferase (ChAT), the acetylcholine biosynthetic enzyme. Within the nDBBhl peroxidase reaction product was distributed throughout the cytoplasm of selectively labeled neuronal perikarya and dendrites. Labeled perikarya were characterized by an oval cell body (7-10 microns X 17-26 microns in diameter) in which was located a large nucleus and often a prominent nucleolus. Dendrites were by far the most numerous immuno-labeled profiles in the nDBBhl. The labeled dendrites had a cross-sectional diameter of 0.4-4.6 microns and contained numerous mitochondria and microtubules. Approximately 10% of all immunolabeled dendrites received synaptic contacts from unlabeled presynaptic boutons. In contrast to the relatively large number of ChAT-labeled dendrites within the nDBBhl, ChAT-positive axons were less frequently observed and immunolabeled axon terminals were never detected. The labeled axons had an outside diameter of 0.4-1.4 micron and were myelinated. The absence or relative paucity of immunolabeled terminals in the nDBBhl indicates that most if not all of the cholinergic perikarya within this nucleus are efferent projection neurons. The nDBB is known to have widespread projections to many areas of the neocortex, hippocampus, and amygdala. In the present study we examined the amygdala and observed many ChAT-labeled axon boutons. The immunolabeled varicosities contained numerous agranular vesicles and although ChAT-positive terminals were in direct contact with unlabeled neuronal elements within the amygdala, few if any synaptic densities were detected in a single plane of section. With respect to the vasculature, immunolabeled perikarya and dendrites within the nDBBhl and axon terminals in the amygdala were often in direct apposition to blood vessels. In many instances the labeled profile was observed lying directly on the basal lamina of a capillary endothelial cell. In no instance, however, were membrane densities observed. The presence of cholinergic neuronal elements contacting the vessel wall provides morphologic evidence suggesting that the neurogenic control of cerebral vasculature is in part mediated via a cholinergic mechanism.     

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)     

Efferent connections of the substantia innominata in the rat

The efferent connections of the substantia innominata (SI) were investigated employing the anterograde axonal transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and the retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). The projections of the SI largely reciprocate the afferent connections described by Grove (J. Comp. Neurol. 277:315-346, '88) and thus further distinguish a dorsal and a ventral division in the SI. Efferents from both the dorsal and ventral divisions of the SI descend as far caudal as the ventral tegmental area, substantia nigra, and peripeduncular area, but projections to pontine and medullary structures appear to originate mainly from the dorsal SI. Within the amygdala and hypothalamus, which receive widespread innervation from the SI, the dorsal SI projects preferentially to the lateral part of the bed nucleus of the stria terminalis; the lateral, basolateral, and central nuclei of the amygdala; the lateral preoptic area; paraventricular nucleus of the hypothalamus; and certain parts of the lateral hypothalamus, prominently including the perifornical and caudolateral zones described previously. The ventral SI projects more heavily to the medial part of the bed nucleus of the stria terminalis; the anterior amygdaloid area; a ventromedial amygdaloid region that includes but is not limited to the medial nucleus; the lateral and medial preoptic areas; and the anterior hypothalamus. Modest projections reach the lateral hypothalamus, with at least a slight preference for the medial part of the region, and the ventromedial and arcuate hypothalamic nuclei. Both SI divisions appear to innervate the dorsomedial and posterior hypothalamus and the supramammillary region. In the thalamus, the subparafascicular, gustatory, and midline nuclei receive a light innervation from the SI, which projects more densely to the medial part of the mediodorsal nucleus and the reticular nucleus. Cortical efferents from at least the midrostrocaudal part of the SI are distributed primarily in piriform, infralimbic, prelimbic, anterior cingulate, granular and agranular insular, perirhinal, and entorhinal cortices as well as in the main and accessory olfactory bulbs. The cells of origin for many projections arising from the SI were identified as cholinergic or noncholinergic by combining the retrograde transport of WGA-HRP with histochemical and immunohistochemical procedures to demonstrate acetylcholinesterase activity or choline acetyltransferase immunoreactivity. Most of the descending efferents of the SI appear to arise primarily or exclusively from noncholinergic cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Aggregations of granule cells in the basal forebrain (islands of Calleja): Golgi and cytoarchitectonic study in different mammals, including man

The granule cell islands in the olfactory tubercle (islands of Calleja) and the insula magna of Calleja are present in all species examined in this study: cat, rat, mouse, rabbit, hedgehog, monkey, man, and dolphin, displaying the same basic morphology. They appear as rather undifferentiated neurons with a poorly developed dendritic tree and a short unramified axon that does not leave the island. The larger islands and the insula magna are associated with medium-sized neurons often lying in cell-sparse core regions; they probably represent the efferent component of the islands. The distribution of granule cell islands in the olfactory tubercle varies from species to species: in the cat, they are restricted to the superficial cap regions; in the hedgehog and rabbit, they lie in cap regions and in the deep polymorph layer. In the rat, they are confined mainly to the deep polymorph layer, whereas in the mouse they extend through the three layers. In most species, the lateral islands form part of the cap regions, and they may receive fibers from the lateral olfactory tract. However, the consistent relationship between dwarf cells in the cap regions and granule cells seems to be a merely topographical one. The variable location of granule cell islands indicates that they are not related to specific cell types or cell groups in the olfactory tubercle, except to the large neurons in the hilus zones, which send their dendrites into the islands. Another close and constant relationship exists between granule islands and fibers of the medial forebrain bundle. The medial islands and the insula magna are the largest and most constant aggregations of granule cells. They are present even in the dolphin, which lacks lateral islands. Medial islands and insula magna are continuous in the hedgehog and the newborn kitten and seem to belong to a medial system of granule cells that is independent from the olfactory tubercle and from olfactory fibers. Aggregations of granule cells occur also outside the olfactory tubercle and the insula magna: in the hedgehog and the rabbit, clusters lie scattered in the n. accumbens. Distribution of granule cells outside the olfactory tubercle is related to ontogenetic development: in newborn kittens, granule cells extend from the subependymal layer of the lateral ventricle, where they probably originate, to the medioventral border of the hemisphere, and also distribute throughout the n. accumbens and the ventral pallidum. Thus, the granule cell territory is initially wider, and the original distribution is maintained in some species.(ABSTRACT TRUNCATED AT 400 WORDS)