Cholinergic and non-cholinergic septohippocampal pathways

Cholinergic innervation of the hippocampus was examined in the rat by immunocytochemical localization of choline acetyltransferase immunoreactivity combined with retrograde transport of horseradish peroxidase-conjugated wheatgerm agglutinin. It was found that at least 50% of hippocampal afferents arising in the septal-diagonal band region consisted of non-cholinergic projection neurons. In addition, scattered choline acetyltransferase-immunoreactive neurons were localized to the hippocampal formation. These results indicate that: (1) the septohippocampal pathway is neither uniformly nor predominantly cholinergic; and (2) confirm that cholinergic innervation of the hippocampal formation of the rat is derived in part from intrinsic neurons.     

Colocalization of gamma-aminobutyric acid and acetylcholinesterase in rodent cortical neurons

We have previously demonstrated that neurons of the rat cerebral cortex which stain positively for acetylcholinesterase are not likely to be cholinergic since they do not colocalize with choline acetyltransferase immunoreactivity [Levey, Rye, Wainer, Mufson and Mesulam (1984) Neuroscience 9, 9-22]. These noncholinergic acetylcholinesterase-positive cells were similar in morphology to cortical neurons which localize gamma-aminobutyric acid or glutamate decarboxylase immunoreactivity. In order to investigate the possibility that the two substances may be colocalized to the same cortical neurons, gamma-aminobutyric acid immunohistochemistry and acetylcholinesterase histochemistry were combined in single sections of rat cerebral cortex. We found that 18% of gamma-aminobutyric acid-immunoreactive cortical neurons are also acetylcholinesterase-positive, and about 36% of acetylcholinesterase-positive cells are gamma-aminobutyric acid-immunoreactive. Neurons which colocalized both substances were multipolar and bipolar neurons in cortical laminae II-VI and were observed in every cortical area examined. The possibility that gamma-aminobutyric acid-immunoreactive/acetylcholinesterase-positive cortical neurons may be postsynaptic targets of cholinergic afferents to the cerebral cortex is discussed.     

Choline acetyltransferase-immunoreactive neurons in the rat entopeduncular nucleus.

Using a monoclonal antibody against choline acetyltransferase, neurons of the rat entopenduncular nucleus were found to express choline acetyltransferase immunoreactivity. These cholinergic cells were located mostly in the rostral portion of the entopeduncular nucleus with a marked decrease towards its caudal portion. To identify their target sites, a retrograde fiber tracing technique was combined with immunohistochemistry for choline acetyltransferase. After injection of wheatgerm agglutinin conjugated with horseradish peroxidase into the habenula, some of the entopedunculo-habenular cells were found to be immunoreactive for choline acetyltransferase. The cells in the peripallidal region (the substantia innominata, nucleus basalis magnocellularis and ansa lenticularis) with choline acetyltransferase immunoreactivity did not contain horseradish peroxidase. Following injection of fluorescent tracer into the frontal cerebral cortex, retrogradely labeled cells were observed in the rostral part of the entopedunucular nucleus. A majority of these entopedunculo-cortical cells exhibited choline acetyltransferase immunoreactivity, similar to the cells of the peripallidal region projecting to the neocortex. Employing two different fluorescent tracers, entopedunculo-cortical cells were shown to constitute a distinct cell population from the numerous entopedunculo-habenular cells. The present study demonstrated, in the rat entopeduncular nucleus, the presence of cholinergic neurons that projected to the neocortex and habenula.     

Choline acetyltransferase immunoreactivity in the cat cerebellum.

Choline acetyltransferase immunoreactivity was demonstrated in particular projection systems in cat cerebellum by combining immunohistochemistry, retrograde tracing and lesioning paradigms. The monoclonal antibody used in this study recognized a 68,000 mol. wt protein on immunoblots of cat cerebellum and striatum. Choline acetyltransferase immunoreactivity was localized to some neurons and varicose fibers in the cerebellar nuclei, and also to some mossy fibers and endings (rosettes), fiber plexuses around Purkinje cells, granule cells and parallel fibers in the cerebellar cortex. In addition, the presence of choline acetyltransferase-immunoreactive large cells, presumptive Golgi cells, in the granular layer was confirmed. In each cerebellar nucleus, choline acetyltransferase-immunoreactive neurons contained either large, medium-sized or small cell bodies and were distributed evenly in the entire nuclear domain. Large and medium-sized ones were frequently encountered. Choline acetyltransferase-immunoreactive mossy fibers and rosettes were most abundant in the vermal lobules I-III, VIII, IX and the simple lobule, moderately accumulated in the vermal lobules IV-VII, X, crus I and crus II, and less abundant in the paramedian lobule, paraflocculus and flocculus. Some granule cells with prominent dendritic claws and bifurcating parallel axons were immunolabeled in the entire vermis with infrequent occurrence in the remaining cortices. Following unilateral lesioning of the cerebellar nuclei with electrocoagulation or kainate injections, a reduction in number of choline acetyltransferase-immunoreactive fibers occurred ipsilaterally in the cerebellar cortex and contralaterally in the red nucleus, ventrolateral thalamic nucleus and ventroanterior thalamic nucleus. In addition, perikarya of some cerebellothalamic neurons were shown to contain choline acetyltransferase immunoreactivity. The results indicate that some nucleocortical, cerebellorubral and cerebellothalamic projections are cholinergic and that a subpopulation of cholinergic granule cell-parallel fibers exists.     

Ultrastructural relationships between choline acetyltransferase- and neuropeptide y-containing neurons in the rat striatum.

The relationships between cholinergic and neuropeptide Y-containing neuronal systems in the rat striatum were examined using a dual immunoperoxidase labelling method. These neurons were identified by their immunoreactivity to choline acetyltransferase and neuropeptide Y, respectively, and were visualized on the same sections using 3,3'-diaminobenzidine and benzidine dihydrochloride as distinct chromogens under two conditions: (i) neuropeptide Y detection by the 3,3'-diaminobenzidine diffuse brown reaction product and choline acetyltransferase detection by the benzidine dihydrochloride blue, granular reaction product; (ii) choline acetyltransferase detection by 3,3'-diaminobenzidine and neuropeptide Y detection by benzidine dihydrochloride. Although both neuropeptide Y- and choline acetyltransferase-immunoreactive cell bodies were simultaneously detected and were easily distinguishable whatever the conditions used, neuropeptide Y- and choline acetyltransferase-immunoreactive dendrites and axons could not be visualized on the same sections, since only the diaminobenzidine-labelled processes were detectable. Light microscopic observations on sections dual labelled with either method confirmed that choline acetyltransferase and neuropeptide Y immunoreactivities were localized in morphologically different populations of striatal neurons scattered throughout the striatum, choline acetyltransferase immunoreactivity being associated with large neurons and neuropeptide Y immunoreactivity with medium-sized neurons. In addition, the choline acetyltransferase-immunoreactive neurons were found to be more numerous than the neuropeptide Y-immunoreactive neurons and to be prevalent in the dorsolateral areas of the striatum, whereas neuropeptide Y-immunoreactive neurons were preferentially found in the ventromedial areas of this structure. Electron microscopic observations on sections processed under either condition revealed that choline acetyltransferase-positive terminals form synaptic contacts of the symmetrical type with neuropeptide Y-positive somata and proximal dendrites and that choline acetyltransferase-positive neurons are contacted by neuropeptide Y-positive terminals. These data show that the striatal neuropeptide Y- and choline acetyltransferase-containing neuronal systems have reciprocal synaptic interactions and provide morphological support for the hypothesis that striatal cholinergic and neuropeptide Y interneuron activities may be functionally linked.     

The cholinergic innervation of normal and transplanted superior colliculus in the rat: an immunohistochemical study

The distribution of choline acetyltransferase was determined in normal and transplanted rat superior colliculus with choline acetyltransferase immunohistochemistry. This distribution was compared to the pattern of histochemically detected acetylcholinesterase activity. To determine cholinergic input to the superficial superior colliculus, double labelling experiments combining retrograde tracing methods and choline acetyltransferase immunohistochemistry were carried out. No choline acetyltransferase-containing neurons were observed in the rat superior colliculus. A dense network of choline acetyltransferase-immunoreactive fibres and terminals was seen in the intermediate layers of the normal superior colliculus. The distribution was patchy and very similar to the pattern of acetylcholinesterase activity. Occasional fibres and terminals were seen in the deep tectal laminae. The superficial layers contained a low number of choline acetyltransferase-stained fibres and terminals but a comparatively high level of acetylcholinesterase activity. Following a unilateral injection of a tracer into the superficial superior colliculus, retrogradely labelled choline acetyltransferase-immunoreactive neurons were found in the dorsal and ventral subnuclei of the ipsilateral parabigeminal nucleus. As in the normal superior colliculus, choline acetyltransferase-positive neurons were not found in tectal transplants. However, choline acetyltransferase-immunoreactive fibres and terminals were seen in grafts but only in those which had extensive connections with the host midbrain. The pattern of staining most closely resembled that seen in the intermediate layers of the normal superior colliculus. The similar arrangement of choline acetyltransferase and acetylcholinesterase activity in the intermediate layers of normal rat superior colliculus provides further evidence for cholinergic innervation to these layers, probably originating in the dorsal and pedunculopontine tegmental nuclei. The data from the double labelling experiments indicate that the choline acetyltransferase-immunoreactive terminals observed in the superficial layers represent the terminal field of an ipsilateral cholinergic projection from the parabigeminal nucleus. Tectal grafts receive cholinergic innervation from the host. The evidence suggests that much of this input derives from the cholinergic nuclei in the brainstem tegmentum which normally project to the intermediate tectal layers.     

Separate neuronal populations of the rat globus pallidus projecting to the subthalamic nucleus, auditory cortex and pedunculopontine tegmental area.

The topographic arrangement of globus pallidus neurons sending axons to the subthalamic nucleus, auditory cortex and pedunculopontine tegmental nucleus was studied in the rat using retrograde fluorescent tracers. Neurons projecting to the subthalamic nucleus were localized in the rostral part of the globus pallidus, while neurons projecting to the auditory cortex and to the pedunculopontine tegmental nucleus were located in the caudal part. The two populations of pallidocortical and pallidotegmental neurons were also distributed in a separate manner within the caudal globus pallidus. The former neurons were large and located more ventromedially, whereas the latter were medium-sized and located more dorsolaterally. Using a retrograde fluorescent tracing technique combined with choline acetyltransferase immunofluorescence histochemistry, it was found that a vast majority of pallidocortical neurons expressed choline acetyltransferase immunoreactivity, and that pallidotegmental neurons rarely exhibited choline acetyltransferase immunoreactivity. A method of retrograde tracing with wheatgerm agglutinin conjugated with horseradish peroxidase associated to immunohistochemistry for glutamate decarboxylase confirmed the GABAergic nature of the pallidotegmental pathway. The present study revealed the independent nature of the globus pallidus neurons projecting to the subthalamic nucleus, auditory cortex and pedunculopontine tegmental nucleus. Within this cellular arrangement, the presence of functionally distinct neuronal populations at the caudal pallidal level was also identified, with large cholinergic cells innervating the neocortex and medium-sized GABAergic cells "feeding" the mesencephalic tegmentum.     

Cholinergic innervation of ferret visual system

The distribution of acetylcholinesterase and choline acetyltransferase in primary visual areas of adult pigmented ferret was determined with cholinesterase histochemistry and choline acetyltransferase immunohistochemistry. In all visual areas the distribution of acetylcholinesterase in the neuropil closely matches that of choline acetyltransferase. In the cerebral cortex acetylcholinesterase and choline acetyltransferase are associated with axons found in every cortical layer and in the white matter. Area 17, identified by Nissl architectonics and cytochrome oxidase histochemistry, is distinguished by having a relatively low density of choline acetyltransferase- and acetylcholinesterase-stained axons in layer IV. Certain cortical non-pyramidal cell types show moderate staining for acetylcholinesterase after relatively long incubations, but no choline acetyltransferase-positive cells are observed in the cortex. In the lateral geniculate nucleus and superior colliculus the levels of choline acetyltransferase and acetylcholinesterase are considerably higher than in cerebral cortex, and choline acetyltransferase-stained axons there display prominent varicosities. The distribution of choline acetyltransferase and acetylcholinesterase in the neuropil of lateral geniculate nucleus and superior colliculus of ferret shows marked laminar variation. For instance, in the lateral geniculate nucleus, the levels of acetylcholinesterase and choline acetyltransferase in the "On" sublaminae of laminae A and A1 are higher than the "Off" sublaminae. In the superficial layers of the superior colliculus the levels of choline acetyltransferase and acetylcholinesterase are highest in the stratum zonale and lowest in the stratum opticum; in the intermediate gray layer of the superior colliculus acetylcholinesterase- and choline acetyltransferase-stained fibres are distributed into dense patches. As in cortex, choline acetyltransferase-positive cell bodies are not found in the lateral geniculate nucleus or superior colliculus, and acetylcholinesterase-stained cell bodies are visible only after long incubations. Cell bodies staining positively for choline acetyltransferase are found in a satellite of the superior colliculus, the parabigeminal nucleus.     

Cholinergic neurons in the rat cerebral cortex demonstrated by immunohistochemical localization of choline acetyltransferase

The presence of cholinergic neurons in rat cerebral cortex was demonstrated by immunohistochemical localization of choline acetyltransferase, the enzyme synthesizing the neurotransmitter acetylcholine. The stained neurons were found throughout the entire cortex and were present in layers II–VI. Two morphologically d distinct classes of cholinergic neurons have been identified and compared with those neurons containing acetylcholinesterase.     

Development of cholinergic traits in the quail ciliary ganglion: expression of choline acetyltransferase-like immunoreactivity

The avian ciliary ganglion is a parasympathetic ganglion derived from the neural crest whose neurons provide cholinergic innervation to the eye. Here, we describe the time course of appearance and the morphology of cholinergic cells in the ciliary ganglion, as assessed by antibodies against choline acetyltransferase. Choline acetyltransferase immunoreactivity was first observed in 5.5-day-old quail embryos, 1 day after condensation of the ciliary ganglion. Both the intensity of choline acetyltransferase immunoreactivity and size of the choline acetyltransferase-immunoreactive cells increased with ganglionic age. By 12 days, a second population of choline acetyltransferase-immunoreactive cells, possibly corresponding to choroid neurons, was observed whose cells were smaller and less intensely stained than earlier differentiating choline acetyltransferase-immunoreactive cells. The percentage of choline acetyltransferase-immunoreactive cells was initially high, constituting approximately 50% of the total cell population. As a function of time, the proportion of cholinergic cells decreased, probably due to proliferation of non-neuronal cells and naturally-occurring cell death. Our results confirm the existence of two morphologically distinct populations of cholinergic neurons in the avian ciliary ganglion and demonstrate that these neuronal subpopulations express choline acetyltransferase immunoreactivity at different times in development. Because choroid neurons innervate their targets later than ciliary neurons, this finding is consistent with the hypothesis that target interactions regulate expression of choline acetyltransferase.     

Neurotransmitter characteristics of neurons projecting to the supramammillary nucleus of the rat

Retrograde labelling was combined with immunohistochemistry to localize neurons containing choline acetyltransferase, gamma-aminobutyric acid (GABA), glutamate, serotonin, somatostatin, Leu-enkephalin, neurotensin, and substance P-immunoreactivity in neurons projecting to the supramammillary nucleus in the rat. Injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the supramammillary nucleus resulted in retrogradely labelled neurons in the medial septal nucleus, the nuclei of the diagonal band of Broca, the infralimbic cortex, the medial and lateral preoptic nucleus, the subiculum, the laterodorsal tegmental nucleus, the compact subnucleus of the central superior nucleus and the dorsal raphe nucleus. In the medial septal nucleus and in the nuclei of the diagonal band of Broca, 80-85% of WGA-HRP- labelled neurons (30-40 per section) were also immunoreactive for choline acetyltransferase and small numbers of WGA-HRP-labelled neurons were immunoreactive for GABA, glutamate, neurotensin or substance P. In the medial preoptic nucleus, 85-90% of retrogradely labelled neurons (25-30 per section) were immunoreactive for somatostatin and a few WGA-HRP-labelled neurons displayed neurotensin-immunoreactivity. In the rostroventral part of the subiculum, small numbers of retrogradely labelled neurons were also immunoreactive for neurotensin or for glutamate. In the laterodorsal tegmental nucleus, 90% of WGA-HRP-labelled neurons (20-25 per section) were immunoreactive for choline acetyltransferase and small numbers of retrogradely labelled neurons also displayed substance P immunoreactivity. In the compact subnucleus of the central superior nucleus, 50-60% of retrogradely labelled neurons (15-20 per section) were also immunolabelled for GABA and approximately 30-40% of WGA-HRP-labelled neurons (10-12 per section) were immunoreactive for Leu-enkephalin. The compact subnucleus of the central superior nucleus also contained small numbers of retrogradely labelled neurons that displayed neurotensin immunoreactivity. In the dorsal raphe nucleus, 80-85% of WGA-HRP- labelled neurons (30-40 per section) were also immunoreactive for serotonin and small numbers of retrogradely labelled neurons displayed neurotensin or glutamate immunoreactivity. These results suggest that the multiple neurochemicals contained in ascending and descending projections to the SuM participate in complex interactions in the transmission process of SuM neurons.     

The distribution and compartmental organization of the cholinergic neurons in nucleus accumbens of the rat

In this study the distribution of the cholinergic neurons was examined in relation to the compartmental organization of nucleus accumbens. This was accomplished by charting the location of the choline acetyltransferase-immunoreactive neurons and mapping their distribution in relation to cytoarchitectural features and the patterns of acetylcholinesterase activity and enkephalin immunoreactivity. Choline acetyltransferase-containing perikarya are inhomogeneously distributed in nucleus accumbens. Their density is lowest at the rostral pole and highest, caudomedially, at the septal pole. The cells form a compact, medial column and a diffuse, lateral zone and, moreover, there are distinct gradients in their distribution. The highest numbers of immunoreactive perikarya occur within the intensely immunostained zones of choline acetyltransferase-immunoreactive neuropil in ventral and ventromedial parts of the nucleus, whereas lower numbers coincide with choline acetyltransferase-poor zones in the central part of the nucleus. Zones of intensely choline acetyltransferase-immunoreactive neuropil are largely in register with regions of high acetylcholinesterase activity in middle and caudal parts of the nucleus but do not coincide rostrally. Choline acetyltransferase-rich zones correspond to moderate enkephalin immunoreactivity in the outer shell of the nucleus, but a moderately choline acetyltransferase-immunostained matrix surrounds "patches" of intense enkephalin immunoreactivity in the core. Small aggregates of cells, which feature commonly in nucleus accumbens, seem to be avoided by both choline acetyltransferase- and enkephalin-immunoreactive zones. Choline acetyltransferase-immunoreactive processes are mostly confined by the boundaries of their respective immunoreactive zones. Few choline acetyltransferase-immunoreactive neurons lie in the enkephalin-rich patches and those that lie close to the patches show little preference in the directionality of their processes such that some cross the borders, whereas others do not. Thus, our findings show that the cholinergic elements are differentially distributed within nucleus accumbens; that these elements are compartmentally ordered; and that, in light of their limited access to other compartments, they possibly play only a minor role in intercompartmental communication.     

Topographical localization of neurons containing parvalbumin and choline acetyltransferase in the medial septum-diagonal band region of the rat

The normal morphology and distribution of parvalbumin-containing neurons (shown in a previous study to be GABAergic nerve cells) of the medial septal-diagonal band region of the adult rat brain have been studied, and the findings compared with observations on choline acetyltransferase-immunoreactive neurons. The two antigens were visualized either in the same sections using a double-label immunohistochemical procedure for the simultaneous localization of parvalbumin and choline acetyltransferase, or in immediately adjacent sections. In double-stained sections of the whole medial septal-diagonal band complex, about 34% of the total neurons showed immunoreactivity to parvalbumin; the proportion of parvalbumin-labelled neurons was slightly higher in the medial septal-vertical limb of the diagonal band region, and much lower in the horizontal limb of the diagonal band region. The distribution of parvalbumin- and choline acetyltransferase-containing neurons also varied markedly between different mediolateral subdivisions of the medial septum: about 30, 65 and 2% of the parvalbumin-immunoreactive neurons were present in the midline, medial and lateral part of the medial septum, respectively. At different rostrocaudal levels, the proportion of parvalbumin- and choline acetyltransferase-positive neurons varied in a consistent manner, and the largest number of parvalbumin-containing neurons was found at the level 1.9 mm anterior to the bregma. In the absence of reliable immunocytochemical methods for the localization of glutamate decarboxylase and GABA, parvalbumin may serve as a good marker for studying the distribution of GABAergic neurons in the medial septum-diagonal band region. Moreover, the precise maps reported in the present study of the topographic localization of parvalbumin-containing GABAergic and choline acetyltransferase-immunoreactive cholinergic nerve cells in the medial septal-diagonal band complex will serve as a useful guide in future morphological and electrophysiological studies on the septum and its efferents.     

Input from the frontal cortex and the parafascicular nucleus to cholinergic interneurons in the dorsal striatum of the rat.

Evidence derived from many experimental approaches indicates that cholinergic neurons in the dorsal striatum (caudate-putamen) are responsive to excitatory amino acids. Furthermore, evidence from physiological experiments indicate that the excitatory input is derived from the cortex and/or the thalamus. The object of the present experiment was to anatomically test whether cholinergic neurons receive cortical and/or thalamic input in the dorsal striatum using a combined anteograde tracing and immunocytochemical approach at both the light- and electron-microscopic levels. Rats received injections of the anterograde tracers Phaseolus vulgaris-leucoagglutinin or biocytin at multiple sites in the frontal cortex or parafascicular nucleus of the thalamus. Sections of the striatum were stained to reveal the anterogradely transported markers and then immunostained to reveal choline acetyltransferase immunoreactivity. The striata of these animals contained dense networks of anterogradely labelled fibres that were dispersed throughout the neuropil and interspersed with the choline acetyltransferase-immunoreactive (i.e. cholinergic) perikarya and dendrites. The anterogradely labelled fibres were often closely apposed to the choline acetyltransferase-immunoreactive neurons. Examination of electron-microscopic sections failed to demonstrate cortical terminals in synaptic contact with the cholinergic neurons even when choline acetyltransferase-immunoreactive structures were examined that had first been identified in the light microscope as having cortical terminals closely apposed to them. In these cases it was often observed that the cortical terminal, although apposed to the membrane of the labelled neurone, made synaptic contact with an unlabelled spine that was in the vicinity. In contrast to the cortical input, analysis of material that was double-stained to reveal thalamostriatal terminals and choline acetyltransferase-immunoreactive structures, revealed that the thalamostriatal terminals were often in asymmetrical synaptic contact with the perikarya and dendrites of cholinergic neurons. It is concluded that the cholinergic neurons of the dorsal striatum, like those of the ventral striatum or nucleus accumbens [Meredith and Wouterlood (1990) J. comp. Neurol. 296, 204-221] receive very little or no input from the cortex but are under a prominent synaptic control by the thalamostriatal system. Those pharmacological effects of excitatory amino acids on the cholinergic systems of the striatum are therefore presumably related to the thalamostriatal and not the corticostriatal system.     

Projections of cholinergic and non-cholinergic neurons of the brainstem core to relay and associational thalamic nuclei in the cat and macaque monkey

The projections of brainstem core neurons to relay and associational thalamic nuclei were studied in the cat and macaque monkey by combining the retrograde transport of wheat germ agglutinin conjugated with horseradish peroxidase with choline acetyltransferase immunohistochemistry. All major sensory (medial geniculate, lateral geniculate, ventrobasal), motor (ventroanterior, ventrolateral, ventromedial), associational (mediodorsal, pulvinar, lateral posterior) and limbic (anteromedial, anteroventral) thalamic nuclei of the cat were found to receive projections from cholinergic neurons located in the peribrachial area of the pedunculopontine nucleus and in the laterodorsal tegmental nucleus as well as from non-cholinergic neurons in the rostral (perirubral) part of the central tegmental mesencephalic field. Specific relay nuclei receive less than 10% of their brainstem afferents from non-cholinergic neurons located at rostral midbrain levels and receive 85-96% of their brainstem innervation from a region at midbrain-pontine junction where the cholinergic peribrachial area and laterodorsal tegmental nucleus are maximally developed. Of the total number of horseradish peroxidase-positive brainstem neurons seen after injections in various specific relay nuclei, the double-labeled (horseradish peroxidase + choline acetyltransferase) neurons represent approximately 70-85%. Three to eight times more numerous horseradish peroxidase-labeled brainstem cells were found after injections in associational (mediodorsal and pulvinar-lateral posterior complex) and diffusely cortically-projecting (ventromedial) thalamic nuclei of cat than after injections in specific relay nuclei. The striking retrograde cell labeling observed after injections in nuclei with associative functions and widespread cortical projections was due to massive afferentation from non-cholinergic parts of the midbrain and pontine reticular formation, on both ipsi- and contralateral sides. After wheat germ agglutinin-horseradish peroxidase injections in the associative pulvinar-lateral posterior complex and mediodorsal nucleus of Macaca sylvana, 45-50% of horseradish peroxidase-positive brainstem peribrachial neurons were also choline acetyltransferase-positive. While cells in the medial part of the cholinergic peribrachial area were found to project especially towards the pulvinar-lateral posterior nuclear complex in monkey, the retrograde cell labeling seen after the mediodorsal injection was mostly confined to the lateral part of both dorsal and ventral aspects of the peribrachial area.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of amino acids, neuropeptides and cholinergic neurotransmitter markers in identified projections from the mesencephalic tegmentum to the mammillary nuclei of the rat

Retrograde labelling has been combined with immunohistochemistry to localize neurons containing GABA, glutamate, choline acetyltransferase, leu-enkephalin, neurotensin and substance P-like immunoreactivity in the projection pathways from the midbrain tegmental nuclei to the mammillary nuclei in the rat. Injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the medial mammillary nucleus resulted in retrogradely labelled neurons in the ventral tegmental nucleus of Gudden, whereas injections into the lateral mammillary nucleus resulted in large numbers of retrogradely labelled neurons in the ipsilateral dorsal tegmental nucleus of Gudden and in the laterodorsal tegmental nucleus. In the ventral tegmental nucleus, moderate to small numbers of retrogradely labelled neurons were also immunolabelled for GABA and approximately ten to 18 WGA-HRP-labelled neurons per section were immunoreactive for leu-enkephalin. In addition, small numbers of WGA-HRP-labelled neurons in the principal subnucleus of the ventral tegmental nucleus were immunoreactive for Glu whereas small numbers of retrogradely labelled neurons in the compact subnucleus of the central superior nucleus displayed neurotensin-like immunoreactivity. In the ventral subnucleus of the dorsal tegmental nucleus, moderate to small numbers of retrogradely labelled neurons were also GABA-immunoreactive and approximately ten to 14 WGA-HRP labelled neurons per section were immunoreactive for leu-enkephalin. The ventral subnucleus of the dorsal tegmental nucleus also contained small numbers of retrogradely labelled neurons that displayed either glutamate or substance P-like immunoreactivity. In addition, moderate to small numbers of WGA-HRP-labelled neurons (five to 20 per section) in the laterodorsal tegmental nucleus were immunoreactive for choline acetyltransferase. These results are compatible with the possibility that tegmentomammillary projection neurons use several different neurochemicals as neurotransmitter(s) and/or neuromodulator(s).     

Cholinergic input to dopaminergic neurons in the substantia nigra: a double immunocytochemical study.

In order to determine whether the cholinergic fibres that innervate the substantia nigra make synaptic contact with dopaminergic neurons of the substantia nigra pars compacta, a double immunocytochemical study was carried out in the rat and ferret. Sections of perfusion-fixed mesencephalon were incubated first to reveal choline acetyltransferase immunoreactivity to label the cholinergic terminals and then tyrosine hydroxylase immunoreactivity to label the dopaminergic neurons. Each antigen was localized using peroxidase reactions but with different chromogens. At the light microscopic level, in confirmation of previous observations, choline acetyltransferase-immunoreactive axons and axonal boutons were found throughout the substantia nigra. The highest density of these axons was found in the pars compacta where they were often seen in close apposition to tyrosine hydroxylase-immunoreactive cell bodies and dendrites. In the ferret where the choline acetyltransferase immunostaining was particularly strong, bundles of immunoreactive fibres were seen to run through the reticulata perpendicular to the pars compacta. These bundles were associated with tyrosine hydroxylase-immunoreactive dendrites that descended into the reticulata. The choline acetyltransferase-immunoreactive fibres made "climbing fibre"-type multiple contacts with the tyrosine hydroxylase positive dendrites. At the electron microscopic level the choline acetyltransferase-immunoreactive axons were seen to give rise to vesicle-filled boutons that formed asymmetrical synaptic specializations with nigral dendrites and perikarya. The synapses were often associated with sub-junctional dense bodies. On many occasions the postsynaptic structures contained the tyrosine hydroxylase immunoreaction product, thus identifying them as dopaminergic. It is concluded that at least one of the synaptic targets of cholinergic terminals in the substantia nigra are the dendrites and perikarya of dopaminergic neurons and that in the ferret at least, the dendrites of dopaminergic neurons that descend into the pars reticulata receive multiple synaptic inputs from individual cholinergic axons.     

An atlas of the regional and laminar distribution of choline acetyltransferase immunoreactivity in rat cerebral cortex

The distribution of cholinergic fibers in rat cortex was investigated using choline acetyl-transferase immunohistochemistry. Previous studies have either shown differences in distribution, but have been limited to selected areas, or have shown no discernable differences between different cortical areas. In our study, we examined all areas of rat cortex and found that there are striking interareal and interlaminar differences in cholinergic fiber distribution. We have found that certain functionally similar cortical areas (e.g. sensory, motor, etc.) have similar patterns of cholinergic innervation and we have designated 13 general patterns of cortical cholinergic innervation. We have also compared, on an area-by-area basis, the pattern of acetylcholinesterase reactivity to that of choline acetyltransferase immunoreactivity, since acetylcholinesterase has been used for many years as a putative cholinergic marker. We found that in most cortical areas, the distribution of acetylcholinesterase-positive fibers paralleled that of choline acetyltransferase-immunoreactive fibers; however, there were some striking differences, notably primary somatosensory (the "barrelfield"), retrosplenial and cingulate cortices. In some areas, a revised concept of rat cortical organization, using cytoarchitectonics, was required. The results of this study provide a comprehensive microscopic analysis of cholinergic fiber innervation of the rat cortex. These results are discussed in relation to previous anatomical, physiological and pharmacological studies of cortical cholinergic innervation. The possible sources of this innervation are also discussed.     

Cortical projections arising from the basal forebrain: a study of cholinergic and noncholinergic components employing combined retrograde tracing and immunohistochemical localization of choline acetyltransferase

The neurochemical identity of ascending putative cholinergic pathways from the rat basal forebrain was investigated employing a method for simultaneously visualizing choline acetyltransferase immunoreactivity and retrogradely transported horseradish peroxidase-conjugated wheatgerm agglutinin. This histochemical procedure revealed three distinct populations of neurons: (1) cells which stained only for choline acetyltransferase immunoreactivity; (2) cells which stained only for retrograde tracer and (3) cells which stained simultaneously for choline acetyltransferase immunoreactivity and retrograde tracer. The results demonstrated that this projection is topographically organized and consists of both cholinergic and noncholinergic components. The relative contribution of each component varied with the telencephalic target area as follows: the olfactory bulb receives a projection from cells of the horizontal limb nucleus, 10-20% of which are cholinergic (Ch3); the hippocampal formation receives afferents from cells of the medial septal and vertical limb nuclei, 35-45% of which are cholinergic (Ch1 and Ch2); and the cortical mantle receives afferents primarily from cells within the substantia innominata-nucleus basalis complex, 80-90% of which are cholinergic (Ch4). The topographical organization of Ch4 projections is not as completely differentiated as we have previously observed in the primate.     

Cholinergic neurons of the chicken ciliary ganglion contain somatostatin

Somatostatin immunoreactivity was studied in the avian ciliary ganglion by immunocytochemistry and radioimmunoassay. Immunoreactivity was localized to small diameter cell bodies of neurons from embryos, newly-hatched and adult preparations. Immunostaining of ganglia with a mixture of antisera to substance P and monoclonal antibody to somatostatin indicated that a number of somatostatin-immunoreactive neurons were surrounded by substance P-immunoreactive boutons, which characteristically terminate on choroidal neurons. Staining with a mixture of antisera to choline acetyltransferase and antibody to somatostatin showed that the somatostatin-immunoreactive neurons were less intensely-stained for choline acetyltransferase than were the neurons lacking somatostatin immunoreactivity. Bundles of nerve fibers showing somatostatin and choline acetyltransferase immunoreactivity were found in the choroid layers of the eye. Radioimmunoassay indicated the presence of somatostatin immunoreactivity in both chick and quail ganglia; the somatostatin immunoreactivity eluted from high pressure liquid chromatography in the same positions as authentic somatostatin 14 and 28. These results show that somatostatin is contained in cholinergic choroidal neurons in the chick and quail ciliary ganglion.