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.     

Somatostatin immunoreactivity in quail pterygopalatine ganglion

In the ciliary ganglion of the chicken and quail, somatostatin (SOM) is an exclusive marker for parasympathetic postganglionic neurons innervating the choroid. A second parasympathetic pathway projecting to the choroid originates from the pterygopalatine ganglion. The aim of this study was to investigate SOM immunoreactivity in the pterygopalatine ganglion of the Japanese quail (Coturnix coturnix japonica) and on neurons within the choroid, the intrinsic choroidal neurons (ICN). We did so using immunohistochemistry and subsequent light, electron and confocal laser scanning microscopy. Pterygopalatine neurons were characterized by nNOS-immunohistochemistry or NADPH-diaphorase cytochemistry. SOM immunoreactivity was absent in the perikarya, but neurons were densely surrounded by SOM-positive nerve fibres. Electron microscopy revealed that these fibres formed contacts with and without membrane specializations on pterygopalatine neurons. In the choroid, neuronal nitric-oxide synthase (nNOS)-immunoreactive ICN were likewise closely apposed by SOM-immunoreactive nerve fibres, as revealed by confocal microscopy. There was no detectable co-localization of the markers. In the absence of tracing studies, it is open to speculation whether SOM immunoreactivity originates from preganglionic fibres of the superior salivatory nucleus, postganglionic fibres of the ciliary ganglion or fibres of the brainstem via as yet unknown pathways. SOM may regulate the production of NO in pterygopalatine neurons and ICN, respectively, and is therefore involved in neuronal circuits regulating ocular homeostasis.     

Neuronal ultrastructure and somatostatin immunolocalization in the ciliary ganglion of chicken and quail

Summary Ciliary and choroid neurons of the avian ciliary ganglion innervate different targets in the eye bulb. By light microscopic immunocytochemistry, somatostatin (SOM) has been localized to a subset of ganglionic neurons believed to be, for the most part, choroid neurons. Although several studies have been published on the physiology, afferent and efferent innervation, and response to experimental injury of this population of cells, their morphological features are still unclear. This has led us to perform a fine structural and immunocytochemical study on the ciliary ganglia of adult chickens and quails to provide the first thorough characterization of the choroid neurons and to analyze whether or not they can be unequivocally identified by expression of SOM. Here, we show that standard and immuno-electron microscopy provide firm criteria for the distinction of ciliary and choroid neurons, whose populations overlap in cell size and territory of distribution. The satellite cell sheaths form compact myelin lamellae around ciliary neurons and flattened processes around choroid neurons. Moreover, ciliary neurons are innervated by a larger number of boutons than choroid neurons. Chicken ciliary neurons are invested by boutons only over one pole of the cell body, while their quail counterparts have an almost complete shell of presynaptic boutons over the entire cell body. Ciliary neurons form mixed synaptic junctions (chemical and electrical), while choroid neurons form only chemical synapses. Crest synapses are present in ciliary neurons of both species. Nematosomes occur in both ciliary and choroid neurons. Choroid neurons contain a larger complement of large dense core vesicles than ciliary neurons and their Golgi apparatuses are more prominent. In the light microscope, somatostatin-immunostaining appears noticeably different in the two species: mostly granular in the chicken and skein-shaped in the quail. Immuno-electron microscopy reveals that somatostatin-like immunoreactivity is localized to Golgi apparatus and large dense core vesicles. Somatostatin is expressed by all the choroid neurons, but not by the ciliary neurons. This neuropeptide is, therefore, a true cell population marker.     

Immunohistochemical identification of cholinergic neurons in the myenteric plexus of guinea-pig small intestine.

It is well established that acetylcholine is a neurotransmitter at several distinct sites in the mammalian enteric nervous system. However, identification of the cholinergic neurons has not been possible due to an inability to selectively label enteric cholinergic neurons. In the present study an immunohistochemical method has been developed to localize choline acetyltransferase, the synthetic enzyme for acetylcholine, in order that cholinergic neurons can be visualized. The morphology, neurochemical coding and projections of cholinergic neurons in the guinea-pig small intestine were determined using double-labelling immunohistochemistry. These experiments have revealed that many myenteric neurons are cholinergic and that they can be distinguished by their specific combinations of immunoreactivity for neurochemicals such as calretinin, neurofilament protein triplet, substance P, enkephalin, somatostatin, 5-hydroxytryptamine, vasoactive intestinal peptide and calbindin. On the basis of their previously described projections, functional roles could be attributed to each of these populations. The identified cholinergic neurons are: motorneurons to the longitudinal muscle (choline acetyltransferase/calretinin); motorneurons to the circular muscle (choline acetyltransferase/neurofilament triplet protein/substance P, choline acetyltransferase/substance P and choline acetyltransferase alone); orally directed interneurons in the myenteric plexus (choline acetyltransferase/calretinin/enkephalin); anally directed interneurons in the myenteric plexus (choline acetyltransferase/somatostatin, choline acetyltransferase/5-hydroxytryptamine, choline acetyltransferase/vasoactive intestinal peptide); secretomotor neurons to the mucosa (choline acetyltransferase/somatostatin); and sensory neurons mediating myenteric reflexes (choline acetyltransferase/calbindin). This information provides a unique opportunity to identify functionally distinct populations of cholinergic neurons and will be of value in the interpretation of physiological and pharmacological studies of enteric neuronal circuitry.     

Differential regulation of peptide and catecholamine characters in cultured sympathetic neurons

Mechanisms regulating peptidergic, noradrenergic and cholinergic development were compared in dissociated cell cultures of neonatal rat sympathetic ganglia. The majority of cultured neurons contained at least two neurotransmitters and many neurons contained three or more. These studies were undertaken to determine whether co-existing transmitters were co-ordinately regulated by the environment. Co-culture of sympathetic neurons with ganglion non-neuronal cells increased substance P and choline acetyltransferase activity but decreased somatostatin and tyrosine hydroxylase activity. Conversely, elimination of non-neuronal cells virtually abolished neuronal expression of substance P and choline acetyltransferase and increased somatostatin and tyrosine hydroxylase. Consequently, under these conditions, somatostatin and tyrosine hydroxylase were similarly regulated, whereas substance P was associated with choline acetyltransferase. By contrast, stimulation of adenylate cyclase or treatment with membrane-permeable adenosine 3',5'-phosphate analogs increased tyrosine hydroxylase and decreased choline acetyltransferase, but had no effect on substance P or somatostatin levels. Moreover, potassium- or veratridine-induced membrane depolarization increased tyrosine hydroxylase but decreased substance P, somatostatin and norepinephrine levels. However, inhibition of neurotransmitter release with magnesium or calcium-free medium prevented the decrease in norepinephrine levels but not the decrease in substance P and somatostatin. Consequently, the effects of membrane depolarization on peptide levels cannot be ascribed to release and subsequent depletion of substance P and somatostatin and must result from decreased net synthesis (synthesis minus catabolism) of the transmitters. Nerve growth-factor treatment also differentially regulated transmitter metabolism; nerve growth factor increased protein-specific activities of tyrosine hydroxylase and choline acetyltransferase but did not increase the protein-specific content of substance P and somatostatin. Quantitative transmitter expression was also influenced by neuron density; increasing density elevated substance P and choline acetyltransferase activity but decreased somatostatin and tyrosine hydroxylase activity per neuron. Finally, culture of sympathetic neurons in a defined (serum-free) medium also altered some but not all traits, decreasing substance P, somatostatin and choline acetyltransferase without any change in tyrosine hydroxylase.(ABSTRACT TRUNCATED AT 400 WORDS)     

BK-Type K(Ca) channels in two parasympathetic cell types: differences in kinetic properties and developmental expression

The intrinsic electrical properties of identified choroid and ciliary neurons of the chick ciliary ganglion were examined by patch-clamp recording methods. These neurons are derived from a common pool of mesencephalic neural crest precursor cells but innervate different target tissues and have markedly different action potential waveforms and intrinsic patterns of repetitive spike discharge. Therefore it is important to determine whether these cell types express different types of plasma membrane ionic channels, and to ascertain the developmental stages at which these cell types begin to diverge. This study has focused on large-conductance Ca(2+)-activated K(+) channels (K(Ca)), which are known to regulate spike waveform and repetitive firing in many cell types. Both ciliary ganglion cell types, identified on the basis of size and somatostatin immunoreactivity, express a robust macroscopic K(Ca) carried by a kinetically homogeneous population of large-conductance (BK-type) K(Ca) channels. However, the kinetic properties of these channels are different in the two cell types. Steady-state fluctuation analyses of macroscopic K(Ca) produced power spectra that could be fitted with a single Lorentzian curve in both cell types. However, the resulting corner frequency was significantly lower in choroid neurons than in ciliary neurons, suggesting that the underlying K(Ca) channels have a longer mean open-time in choroid neurons. Consistent with fluctuation analyses, significantly slower gating of K(Ca) channels in choroid neurons was also observed during macroscopic activation and deactivation at membrane potentials positive to -30 mV. Differences in the kinetic properties of K(Ca) channels could also be observed directly in single-channel recordings from identified embryonic day 13 choroid and ciliary neurons. The mean open-time of large-conductance K(Ca) channels was significantly greater in choroid neurons than in ciliary neurons in excised inside-out patches. The developmental expression of functional K(Ca) channels appears to be regulated differently in the two cell types. Although both cell types acquire functional K(Ca) at the same developmental stages (embryonic days 9-13), functional expression of these channels in ciliary neurons requires target-derived trophic factors. In contrast, expression of functional K(Ca) channels proceeds normally in choroid neurons developing in vitro in the absence of target-derived trophic factors. Consistent with this, extracts of ciliary neuron target tissues (striated muscle of the iris/ciliary body) contain K(Ca) stimulatory activity. However, K(Ca) stimulatory activity cannot be detected in extracts of the smooth muscle targets of choroid neurons.     

Leukaemia inhibitory factor prevents loss of p75-nerve growth factor receptor immunoreactivity in medial septal neurons following fimbria-fornix lesions.

Transection of the fimbria-fornix leads to retrograde degeneration of axotomized septal cholinergic neurons as manifested by loss of choline acetyltransferase and low-affinity nerve growth factor receptor (p75NGFR) immunoreactivity. Nerve growth factor administered into cerebral ventricles at the time of axotomy can prevent these changes, while ciliary neurotrophic factor can prevent the loss of p75NGFR immunostaining. Leukaemia inhibitory factor shares structural homologies with ciliary neurotrophic factor and has similar actions in the nervous system. Both proteins share the same signalling pathways, which involve the interleukin-6 transducing receptor components leukaemia inhibitory factor receptor beta and gp130. In this study, we compared the effects of leukaemia inhibitory factor, ciliary neurotrophic factor and nerve growth factor, administered into cerebral ventricles, on p75NGFR and choline acetyltransferase immunoreactivity in septal neurons after fimbria-fornix transection. We found that leukaemia inhibitory factor, like ciliary neurotrophic factor, prevents the loss of p75NGFR-stained medial septal neurons after fimbria-fornix axotomy, without maintaining choline acetyltransferase expression in these neurons. In addition, p75NGFR-immunostained neurons had significantly smaller mean diameter after axotomy in leukaemia inhibitory factor- and ciliary neurotrophic factor-treated animals as compared with either nerve growth factor-treated or unlesioned animals. These findings suggest that both leukaemia inhibitory factor and ciliary neurotrophic factor can prevent the axotomy-induced cell death of septal cholinergic neurons, but that, in contrast to nerve growth factor, these growth factors do not maintain the expression of choline acetyltransferase or the normal neuronal size of these injured neurons.     

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.     

Immunohistochemical evidence that acetylcholine and glycine exist in different populations of GABAergic neurons in lamina III of rat spinal dorsal horn.

Pre-embedding immunohistochemistry with monoclonal antibody to choline acetyltransferase was combined with post-embedding immunohistochemistry with antisera to GABA and glycine in order to study the pattern of coexistence of GABA, glycine and acetylcholine in neurons in lamina III of rat spinal dorsal horn. Of 50 neurons which were choline acetyltransferase immunoreactive, 47 showed GABA-like immunoreactivity and none were immunoreactive with antiserum to glycine, despite the fact that glycine is thought to be present in the majority of GABAergic neurons in lamina III. This suggests that while acetylcholine and glycine can both coexist with GABA in lamina III neurons, they are present in different populations of GABAergic cells. Taken together with recent ultrastructural evidence concerning the synaptic connections of glycinergic and cholinergic structures in the dorsal horn, this suggests that there are functional differences between neurons which contain GABA and glycine and those which contain GABA and acetylcholine.     

Nitrergic and VIPergic neurons in the choroid and ciliary ganglion of the duck Anis carina

Immunohistochemistry for neuronal nitric oxide synthase (nNOS) and vasoactive intestinal peptide (VIP), and NADPH diaphorase histochemistry, were applied to investigate neurons in the choroid and the ciliary ganglion of the muscovy duck Anis carina. Up to 1000 neurons in the choroid stained for NADPH diaphorase and showed virtually complete colocalization for nNOS immunoreactivity. Almost all of them co-stained for VIP, while about 90% of VIP immunoreactive cell bodies showed colocalization for nNOS. Two-thirds of the neurons were located, mostly singly, at nodes of a widemeshed nerve plexus in the suprachoroid and were only rarely grouped in ganglia of up to 3 neurons. Numerous varicose nNOS/NADPH-diaphorase-positive nerve fibers were seen around large arterial blood vessels. These fibers derived mainly from paravascular cell bodies that represented about one-third of all choroidal neurons and also displayed costaining for nitrergic markers and VIP. Colocalization of nNOS/NADPH-d and VIP could be demonstrated in most of the perivascular fibers, while slightly more VIP-positive axons in the suprachoroid plexus did not costain for nNOS/NADPH-d. Small-caliber blood vessels and those localized in the choriocapillaris were not endowed with VIP/nNOS/NADPH-diaphorase-positive fibers. A few reactive neuronal cell bodies were also found in ciliary nerves, while most ciliary axons were unstained. In the ciliary ganglion a small subpopulation of neurons showed VIP/nNOS/NADPH-diaphorase colocalization. There were also nNOS/ NADPH-d-positive cap-like terminals on ciliary ganglion cells. The presence of VIP/nNOS/NADPH-diaphorase positive neurons and nerve fibers in both the choroid and ciliary ganglion, and in the choroidal perivascular plexus, indicates peripheral nitrergic and VIPergic control of blood flow in the choroid of the duck.     

Nitrergic and VIPergic neurons in the choroid and ciliary ganglion of the duck Anis carina

Immunohistochemistry for neuronal nitric oxide synthase (nNOS) and vasoactive intestinal peptide (VIP), and NADPH diaphorase histochemistry, were applied to investigate neurons in the choroid and the ciliary ganglion of the muscovy duck Anis carina. Up to 1000 neurons in the choroid stained for NADPH diaphorase and showed virtually complete colocalization for nNOS immunoreactivity. Almost all of them co-stained for VIP, while about 90% of VIP immunoreactive cell bodies showed colocalization for nNOS. Two-thirds of the neurons were located, mostly singly, at nodes of a widemeshed nerve plexus in the suprachoroid and were only rarely grouped in ganglia of up to 3 neurons. Numerous varicose nNOS/NADPH-diaphorase-positive nerve fibers were seen around large arterial blood vessels. These fibers derived mainly from paravascular cell bodies that represented about one-third of all choroidal neurons and also displayed costaining for nitrergic markers and VIP. Colocalization of nNOS/NADPH-d and VIP could be demonstrated in most of the perivascular fibers, while slightly more VIP-positive axons in the suprachoroid plexus did not costain for nNOS/NADPH-d. Small-caliber blood vessels and those localized in the choriocapillaris were not endowed with VIP/nNOS/NADPH-diaphorase-positive fibers. A few reactive neuronal cell bodies were also found in ciliary nerves, while most ciliary axons were unstained. In the ciliary ganglion a small subpopulation of neurons showed VIP/nNOS/NADPH-diaphorase colocalization. There were also nNOS/ NADPH-d-positive cap-like terminals on ciliary ganglion cells. The presence of VIP/nNOS/NADPH-diaphorase positive neurons and nerve fibers in both the choroid and ciliary ganglion, and in the choroidal perivascular plexus, indicates peripheral nitrergic and VIPergic control of blood flow in the choroid of the duck.     

Quantitative study of neuronal degeneration induced by Ricinus toxin and crush of postganglionic nerves in the ciliary ganglion of quail.

The effects of Ricinus toxin on the neurons of the ciliary ganglia were investigated in the quail. The neuronal death and the morphological alterations of the ganglionic cells were assessed following injection of the toxin in the anterior chamber of the eye or after application of the toxin on the postganglionic nerves at a crush site. A 45% loss of choroid neurons without loss of ciliary neurons was observed after postganglionic nerve crush alone. Injection of the toxin in the anterior chamber of the eye led to a selective loss of ciliary neurons (38%). Application of the toxin to the crushed postganglionic nerves led to a loss from both neuronal populations (40% of total neurons). This work indicates that different procedures result in selective lesion of the different neuronal populations in the ciliary ganglion.     

Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat

The localization of two small peptides, somatostatin and substance P, has been studied with the indirect immunofluorescence technique. Both peptides were present in small neuronal cell bodies in spinal ganglia, in fibers in the dorsal horn of the spinal cord and in fibers in the intestinal wall. By comparing consecutive sections incubated with antisera to somastostatin and to substance P respectively, it was established that somatostatin, or somatostatin-like immunoreactivity and substance P, or substance P-like immunoreactivity are present in different cells. This is possibly indicated also by a somewhat differential distribution of the immunoreactive fibers in the dorsal horn: the highest concentration of somatostatin-positive fibers was observed in lamina II, whereas abundant substance P-positive fibers were present also in lamina I. Furthermore, numerous substance P-, but no somatostatin-positive fibers, were found around the central canal and in the ventral horns. In the intestinal wall more substance P-positive than somatostatin-positive fibers were seen.The present results indicate that two subpopulations of primary sensory neurons exist, one containing somatostatin, or somatostatin-like immunoreactivity, and the other containing substance P, or substance P-like immunoreactivity.     

A group of cortical interneurons expressing mu-opioid receptor-like immunoreactivity: a double immunofluorescence study in the rat cerebral cortex

mu-Opioid receptor-expressing neurons in the rat cerebral neocortex were characterized by an immunolabeling method with an antibody to a carboxyl terminal portion of the receptor. They were small, bipolar, vertically elongated, non-pyramidal neurons, and scattered mainly in layers II-IV. We examined chemical characteristics of mu-opioid receptor-expressing neocortical neurons by the double immunofluorescence method. Almost all neuronal cell bodies expressing mu-opioid receptor-like immunoreactivity showed immunoreactivity for GABA, suggesting that they were cortical inhibitory interneurons. mu-Opioid receptor-immunoreactive neurons were further studied by the double staining method with markers for the subgroups of cortical GABAergic neurons. Immunoreactivities for vasoactive intestinal polypeptide, corticotropin releasing factor, choline acetyltransferase, calretinin and cholecystokinin were found in 92, 79, 67, 35 and 35% of mu-opioid receptor-immunoreactive cortical neurons, respectively. In contrast, less than 10% of mu-opioid receptor-immunoreactive neurons showed immunoreactivity for parvalbumin, calbindin, somatostatin, neuropeptide Y or nitric oxide synthase. Moreover, mu-opioid receptor-immunoreactive neurons very frequently exhibited preproenkephalin immunoreactivity, but not preprodynorphin immunoreactivity.The present results indicate that mu-opioid receptor-expressing neurons belong to a distinct subgroup of neocortical GABAergic neurons, because vasoactive intestinal polypeptide, corticotropin releasing factor, choline acetyltransferase, calretinin and cholecystokinin have often been reported to coexist with one another in single neocortical neurons. Methionine-enkephalin, which is a major product of the preproenkephalin gene, is known to be one of the most potent endogenous ligands for mu-opioid receptor. Thus, the expression of mu-opioid receptor in preproenkephalin-producing neurons suggested that mu-opioid receptor serves as an autoreceptor for the subpopulation of GABAergic interneurons at a single-neuron or population level.     

The coexistence of neuropeptides in feline sensory neurons

The coexistence of immunoreactivity to the peptides substance P, bombesin, calcitonin gene-related peptide and somatostatin has been determined in single, lumbar and sacral dorsal root ganglion cells in the cat. Colchicine pretreated L7 and S1 dorsal root ganglia were embedded in wax and cut into 5 microns sections. Groups of four, serially adjacent sections were reacted with antisera to one of four peptides using avidin-biotin immunocytochemistry. It was thus possible to determine the coincidence of the four peptides in single cell bodies by examining the immunoreactivity in a ganglion cell in one section and then locating the same cell in three adjacent sections. As a comparison, this procedure was repeated on a different population of ganglion cells using antiserum to substance P, bombesin and calcitonin gene-related peptide only. The results indicate that different combinations of three or four peptides may occur in single, small diameter sensory neurons in the cat. It would appear that immunoreactivity to bombesin and/or calcitonin gene-related peptide coexists with immunoreactivity to substance P in some dorsal root ganglion cells. However, immunoreactivity to each of these peptides was also found to occur alone in single cells. Immunoreactivity to calcitonin gene-related peptide but not to the other three peptides was found to occur in some medium-sized cell bodies (up to 70 microns). Somatostatin-like immunoreactivity was found to have a high level of coexistence with substance P-like immunoreactivity in cells which contained immunoreactivity to these two peptides only. Immunoreactivity to all the four peptides tested was found to occur in 18-26% of ganglion cells which contained at least one peptide.     

α-Bungarotoxin-acetylcholine receptors in the chick ciliary ganglion during development

[¹²⁵I]α-Bungarotoxin binds in a saturable and practically irreversible fashion to membrane-associated receptors in the ciliary ganglion of the chick embryo and young chick. Nicotinic but not muscarinic ligands are potent inhibitors of toxin binding. Thus, the binding site for toxin in the ganglion appears to have the properties of a nicotinic acetylcholine receptor. The ontogeny of the receptors was studied from day 7in ovo to day 70 after hatching. When expressed per ganglion, the developmental pattern of the receptors may be divided into three stages: a peak of high receptor level (days 7–15in ovo) with a maximum at day 12in ovo, a period of constant level (days 15in ovo to day 8 after hatching) and a second increase in receptor number (days 8–50 after hatching).Light-microscope autoradiography suggests that different developmental changes in receptor distribution occur in the two neuronal populations of the ganglion (i.e. the ciliary and choroid neurons). While choroid neurons show an adult-like receptor distribution from late embryonic stages, the ciliary neurons do not fully develop their distribution pattern until after hatching.These findings are discussed in relation to morphological and physiological events which are known to occur in the ganglion during development, in particular those concerning synaptic transmission.     

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.     

Neurochemical characteristics of aluminum-induced neurofibrillary degeneration in rabbits

Aluminum-induced neurofibrillary degeneration in rabbits is known to affect particular populations of neurons. The neurotransmitter alterations which accompany aluminum neurofibrillary degeneration were examined in order to assess how closely they mimic those of Alzheimer's disease. There was a significant reduction in choline acetyltransferase activity in entorhinal cortex and hippocampus as well as significant reductions in cortical concentrations of serotonin and norepinephrine in the aluminum-treated rabbits. Significant reductions in glutamate, aspartate and taurine were found in frontoparietal and posterior parietal cortex. Concentrations of GABA were unchanged in cerebral cortex. Both substance P and cholecystokinin immunoreactivity were significantly reduced in entorhinal cortex but there were no significant changes in somatostatin, neuropeptide Y and vasoactive intestinal polypeptide. The five neuropeptides were unaffected in striatum, thalamus, cerebellum and brainstem. Neurochemical changes were found in the regions with the most neurofibrillary degeneration while regions with little or no neurofibrillary degeneration were unaffected. The reductions in choline acetyltransferase activity, serotinin and noradrenaline suggest that some neuronal populations preferentially affected in Alzheimer's disease are also affected by aluminum-induced neurofibrillary degeneration; however, the cortical somatostatin deficit which is a feature of Alzheimer's disease is not replicated in the aluminum model.     

Concurrent visualization of choline acetyltransferase-like immunoreactivity and retrograde transport of neocortically injected markers in basal forebrain neurons of cat and rat

Magnocellular neurons in the basal forebrain of rats and cats were retrogradely labeled with Fast Blue or horseradish peroxidase injected into the neocortex. Using antisera against choline acetyltransferase (ChAT) a direct double-labeling technique was carried out and it was demonstrated that retrogradely transported markers and ChAT-like immunoreactivity occur within the same neurons. These findings strongly support the cholinergic nature of basal forebrain projection to the neocortex.     

Substance P immunoreactivity in the rat interpeduncular nucleus: synaptic interactions between substance P-positive profiles and choline acetyltransferase- or glutamate decarboxylase-immunoreactive structures.

The subnuclear and synaptic distribution of substance P immunoreactivity was examined in the rat interpeduncular nucleus at the light and electron microscope level. The nucleus possessed a prominent substance P-immunoreactive axonal plexus in the lateral and dorsomedial subnuclei, and in the dorsal cap of the rostral subnucleus. The density of substance P-immunoreactive axons in the remaining subnuclear divisions was sparse to moderate. Terminals of immunoreactive axons contained spherical vesicles and formed asymmetric contacts on dendritic processes exclusively. Immunoreactive neurons, restricted to the rostral subnucleus, possessed long, sparsely branched dendrites. Unlabelled terminals containing either spherical or pleomorphic vesicles contacted substance P-immunoreactive dendritic profiles. Axodendritic and axosomatic synapses containing substance P immunoreactivity pre- and postsynaptically were not observed. Ultrastructural evidence for synaptic relationships between substance P-containing profiles and those containing either choline acetyltransferase or glutamate decarboxylase was obtained by means of double antigen immunohistochemistry. Terminals of fasciculus retroflexus axons stained for choline acetyltransferase immunoreactivity formed asymmetric synaptic contacts with substance P-immunoreactive dendritic profiles. Few substance P-positive dendrites in the rostral subnucleus received terminals possessing glutamate decarboxylase activity. Unlabelled terminals containing either spherical or pleomorphic vesicles contacted substance P- and glutamate decarboxylase-immunoreactive dendritic profiles simultaneously. Terminals possessing either substance P or glutamate decarboxylase immunoreactivity formed synaptic contacts with dendritic processes of neurons in the lateral subnucleus. Many of the neurons within this subnuclear division contained glutamate decarboxylase. This study provides direct evidence of synaptic relationships between choline acetyltransferase-immunoreactive axons and substance P-immunoreactive dendritic profiles, and between substance P-positive axons and glutamate decarboxylase-immunoreactive dendrites. These findings reveal that two types of transmitter-specific axons of the fasciculus retroflexus innervate neuronal populations of the interpeduncular nucleus stained immunohistochemically for either substance P or glutamate decarboxylase.