NGF mRNA is expressed by GABAergic but not cholinergic neurons in rat basal forebrain

Nerve growth factor (NGF) supports the survival and biosynthetic activities of basal forebrain cholinergic neurons and is expressed by neurons within lateral aspects of this system including the horizontal limb of the diagonal bands and magnocellular preoptic areas. In the present study, colormetric and isotopic in situ hybridization techniques were combined to identify the neurotransmitter phenotype of the NGF-producing cells in these two areas. Adult rat forebrain tissue was processed for the colocalization of mRNA for NGF with mRNA for either choline acetyltransferase, a cholinergic cell marker, or glutamic acid decarboxylase, a GABAergic cell marker. In both regions, many neurons were single-labeled for choline acetyltransferase mRNA, but cells containing both choline acetyltransferase and NGF mRNA were not detected. In these fields, virtually all NGF mRNA-positive neurons contained glutamic acid decarboxylase mRNA. The double-labeled cells comprised a subpopulation of GABAergic neurons; numerous cells labeled with glutamic acid decarboxylase cRNA alone were codistributed with the double-labeled neurons. These data demonstrate that in basal forebrain GABAergic neurons are the principal source of locally produced NGF.     

Innervation of cholinergic vestibular efferent system in vestibular periphery of rats

The innervation of cholinergic efferent fibers in the vestibular endorgans of the rats was investigated using a modified preembedding immunostaining technique of immunoelectron microscopy. A monoclonal antibody to choline acetyltransferase (ChAT) was used as a marker of cholinergic fibers. It was found that there were four types of cholinergic innervation in the vestibular endorgans of the rat: (1) cholinergic nerve endings formed axo-dendritic synapses with afferent chalice surrounding the type I sensory hair cells; (2) cholinergic nerve endings formed axo-somatic synapses with type II hair cells; (3) cholinergic fibers synapse with afferent nerve fibers and (4) a synaptic contact developed between cholinergic nerve endings. The results demonstrated that a multiform innervation of the cholinergic efferents exists in the rats vestibular periphery.     

Intrastriatal infusion of nerve growth factor after quinolinic acid prevents reduction of cellular expression of choline acetyltransferase messenger RNA and trkA messenger RNA, but not glutamate decarboxylase messenger RNA

Excitotoxic striatal lesions induced by quinolinic acid, a model for Huntington's disease, were used to test for neuroprotective actions of nerve growth factor on striatal cholinergic and GABAergic neurons. Expressions of the trkA receptor for nerve growth factor, choline acetyltransferase and glutamate decarboxylase were analysed by messenger RNA in situ hybridization in adult rats following quinolinic acid lesion (150 nmol) and daily striatal administration of nerve growth factor (1 microgram) or control protein (cytochrome C) for one week. One week after toxin administration, the numbers of cells expressing trkA or choline acetyltransferase messenger RNAs were decreased when compared with unlesioned animals. Moreover, the surviving cells showed a strong down-regulation of these messenger RNAs as deduced from grain count analysis of sections processed for emulsion autoradiography. Daily intrastriatal nerve growth factor administration for one week completely prevented the reduction in the number of cells expressing either of the two markers. Nerve growth factor treatment increased the cellular expression of choline acetyltransferase messenger RNA three times above control levels and restored the levels of trk A messenger RNA expression to control levels. In contrast to the protective effects on cholinergic cells, nerve growth factor treatment failed to attenuate the quinolinic acid-induced decrease in glutamate decarboxylase messenger RNA levels. Optical density measurements of the entire striatum on autoradiographs of brain sections from quinolinic acid-lesioned animals revealed a reduction of the glutamate decarboxylase messenger RNA-specific hybridization signal, which was unaltered by infusion of nerve growth factor or control protein. Our findings strongly suggest that in both the intact and the quinolinic acid-lesioned adult rat striatum, nerve growth factor action is confined to trk A-expressing cholinergic neurons. Striatal glutamate decarboxylase messenger RNA-expressing GABAergic neurons which degenerate in Huntington's disease are not responsive to nerve growth factor.     

Choline acetyltransferase in cochlear root neurons

Cochlear root neurons are a distinct group of cells located in the auditory nerve root in small rodents. Their transmitter is still unknown. Some of our preparations showed immunoreactivity of somata of cochlear root neurons with both polyclonal and monoclonal antibodies against choline acetyltransferase (ChAT) which, despite their very weak histochemical reaction for acetylcholinesterase (AChE), suggested that cochlear root neurons might be cholinergic. To test this, we used a radiometric assay to measure ChAT activities of rat auditory nerve root samples containing cochlear root neurons and of adjacent samples not containing them. There was no significant difference between the low mean ChAT activities of these two groups of samples. Thus, cochlear root neurons are not likely to be cholinergic.     

Serotonin1A receptors are expressed by a subpopulation of cholinergic neurons in the rat medial septum and diagonal band of Broca--a double immunocytochemical study

The possible colocalization of 5-hydroxytryptamine1A receptors and choline acetyltransferase in the same neurons of the medial septum and diagonal band of Broca was investigated using double immunocytochemical techniques, either on the same section or on adjacent thin sections of the rat brain. The presence of both antigens in the same neurons was demonstrated at the light and electron microscopic levels. The proportion of cholinergic neurons that express 5-hydroxytryptamine1A receptors was similar in the different parts of the septal complex (around 25%). By contrast, the proportion of 5-hydroxytryptamine1A receptor-positive neurons also exhibiting choline acetyltransferase immunoreactivity was much higher (40-44%) in the dorsal and ventral groups of cholinergic cells, than in the intermediate group (18%). In line with the topographical distribution of cholinergic projections, this result points out the potential involvement of 5-hydroxytryptamine1A receptors in the control of the septohippocampal cholinergic projection by serotonin. This connection might be relevant to learning and memory, and in the appearance of age-dependent or neurodegenerative cognitive deficits, which have been shown to involve alterations in both the serotoninergic and the cholinergic systems.     

Sources of p75-nerve growth factor receptor-like immunoreactivity in the rat suprachiasmatic nucleus

In mammals, the suprachiasmatic nucleus is critical for the generation of circadian rhythms and their entrainment to environmental cues. In the rat, the ventrolateral aspect of the suprachiasmatic nucleus receives a robust retinal input. This region also exhibits the most intense immunolabeling for the low-affinity nerve growth factor receptor in the forebrain. Our study was aimed at identifying the sources of this low-affinity nerve growth factor receptor immunoreactivity using immunohistochemistry combined with retrograde tract-tracing, and orbital enucleation. To determine the origin of the low-affinity nerve growth factor receptor immunoreactivity from sources extrinsic to the suprachiasmatic nucleus, unilateral injections of the retrograde tracer, Fluorogold, were made into the suprachiasmatic nucleus. Retrogradely labeled neurons that were also immunopositive for the low-affinity nerve growth factor receptor were found in both the basal forebrain and the retina. In the basal forebrain, such cells were found throughout its rostrocaudal extent, with the majority also being immunoreactive for the cholinergic marker, choline acetyltransferase. In the retina, cells retrogradely labeled with Fluorogold that were immunoreactive for low-affinity nerve growth factor receptor were located in the ganglion cell layer. Orbital enucleations were performed to confirm the findings observed following retrograde labeling in the retina. Unilateral orbital enucleations resulted in a significant reduction in low-affinity nerve growth factor receptor immunoreactivity in the contralateral suprachiasmatic nucleus compared to that seen on the ipsilateral side when examined one week post-surgery. Bilateral enucleations resulted in an equal decrease on both sides of the suprachiasmatic nucleus. Similar low-affinity nerve growth factor-like immunoreactivity was seen in the suprachiasmatic nucleus even two to four weeks after bilateral enucleations. Taken together, these findings suggest that low-affinity nerve growth factor receptors in the suprachiasmatic nucleus derive from multiple sources. While some receptors may be intrinsic to suprachiasmatic nucleus neurons, most appear to be of extrinsic origin and are located on axon terminals of basal forebrain cholinergic neurons and retinal ganglion cells.     

Sound damage and gentamicin treatment produce different patterns of damage to the efferent innervation of the chick cochlea

Both sound exposure and gentamicin treatment cause damage to sensory hair cells in the peripheral chick auditory organ, the basilar papilla. This induces a regeneration response which replaces hair cells and restores auditory function. Since functional recovery requires the re-establishment of connections between regenerated hair cells and the central nervous system, we have investigated the effects of sound damage and gentamicin treatment on the neuronal elements within the cochlea. Whole-mount preparations of basilar papillae were labeled with phalloidin to label the actin cytoskeleton and antibodies to neurofilaments, choline acetyltransferase, and synapsin to label neurons; and examined by confocal laser scanning microscopy. When chicks are treated with gentamicin or exposed to acoustic overstimulation, the transverse nerve fibers show no changes from normal cochleae assayed in parallel. Efferent nerve terminals, however, disappear from areas depleted of hair cells following acoustic trauma. In contrast, efferent nerve endings are still present in the areas of hair cell loss following gentamicin treatment, although their morphological appearance is greatly altered. These differences in the response of efferent nerve terminals to sound exposure versus gentamicin treatment may account, at least in part, for the discrepancies reported in the time of recovery of auditory function.     

Cortical input to the basal forebrain

The arborization pattern and postsynaptic targets of corticofugal axons in basal forebrain areas have been studied by the combination of anatomical tract-tracing and pre- and postembedding immunocytochemistry. The anterograde neuronal tracer Phaseolus vulgaris leucoagglutinin was iontophoretically delivered into different neocortical (frontal, parietal, occipital), allocortical (piriform) and mesocortical (insular, prefrontal) areas in rats. To identify the transmitter phenotype in pre- or postsynaptic elements, the tracer staining was combined with immunolabeling for either glutamate or GABA, or with immunolabeling for choline acetyltransferase or parvalbumin. Tracer injections into medial and ventral prefrontal areas gave rise to dense terminal arborizations in extended basal forebrain areas, particularly in the horizontal limb of the diagonal band and the region ventral to it. Terminals were also found to a lesser extent in the ventral part of the substantia innominata and in ventral pallidal areas adjoining ventral striatal territories. Similarly, labeled fibers from the piriform and insular cortices were found to reach lateral and ventral parts of the substantia innominata, where terminal varicosities were evident. In contrast, descending fibers from neocortical areas were smooth, devoid of terminal varicosities, and restricted to the myelinated fascicles of the internal capsule en route to more caudal targets. Ultrastructural studies obtained indicated that corticofugal axon terminals in the basal forebrain areas form synaptic contact primarily with dendritic spines or small dendritic branches (89%); the remaining axon terminals established synapses with dendritic shafts. All tracer labeled axon terminals were immunonegative for GABA, and in the cases investigated, were found to contain glutamate immunoreactivity. In material stained for the anterograde tracer and choline acetyltransferase, a total of 63 Phaseolus vulgaris leucoagglutinin varicosities closely associated with cholinergic profiles were selected for electron microscopic analysis. From this material, 37 varicosities were identified as establishing asymmetric synaptic contacts with neurons that were immunonegative for choline acetyltransferase, including spines and small dendrites (87%) or dendritic shafts (13%). Unequivocal evidence for synaptic interactions between tracer labeled terminals and cholinergic profiles could not be obtained in the remaining cases. From material stained for the anterograde tracer and parvalbumin, 40% of the labeled terminals investigated were found to establish synapses with parvalbumin-positive elements; these contacts were on dendritic shafts and were of the asymmetrical type. The present data suggest that corticofugal axons innervate forebrain neurons that are primarily inhibitory and non-cholinergic; local forebrain axonal arborizations of these cells may represent a mechanism by which prefrontal cortical areas control basal forebrain cholinergic neurons outside the traditional boundaries of pallidal areas.     

Technical note: a blind test of mandibular ramus flexure as a morphologic indicator of sexual dimorphism in the human skeleton

Following axotomy most medial septal neurons in the adult rat brain have dramatically reduced numbers of choline acetyltransferase (ChAT) positive neurons. Since leukemia inhibitory factor (LIF) promotes cholinergic expression in several neuronal populations, the aim of this study was to determine if LIF would continue to support cholinergic expression in axotomized medial septal neurons. Mini-osmotic pumps were used to infuse saline or LIF into the lateral cerebral ventricle. Counts of ChAT and low-affinity nerve growth factor (p75NGFR) immunostained neurons indicated that LIF-treated animals retained ChAT expression in > 90% of axotomized neurons whereas in saline-infused animals this was < 30%. Also, LIF was equally effective in maintaining p75NGFR expression levels in axotomized medial septal neurons.     

Acidic fibroblast growth factor mRNA is expressed by basal forebrain and striatal cholinergic neurons

Evidence for the importance of the basal forebrain cholinergic system in the maintenance of cognitive function has stimulated efforts to identify trophic mechanisms that protect this cell population from atrophy and dysfunction associated with aging and disease. Acidic fibroblast growth factor (aFGF) has been reported to support cholinergic neuronal survival and has been localized in basal forebrain with the use of immunohistochemical techniques. Although these data indicate that aFGF is present in regions containing cholinergic cell bodies, the actual site of synthesis of this factor has yet to be determined. In the present study, in situ hybridization techniques were used to evaluate the distribution and possible colocalization of mRNAs for aFGF and the cholinergic neuron marker choline acetyltransferase (ChAT) in basal forebrain and striatum. In single-labeling preparations, aFGF mRNA-containing neurons were found to be codistributed with ChAT mRNA+ cells throughout all fields of basal forebrain, including the medial septum/diagonal band complex and striatum. By using a double-labeling (colormetric and isotopic) technique, high levels of colocalization (over 85%) of aFGF and ChAT mRNAs were observed in the medial septum, the diagonal bands of Broca, the magnocellular preoptic area, and the nucleus basalis of Meynert. The degree of colocalization was lower in the striatum, with 64% of the cholinergic cells in the caudate and 33% in the ventral striatum and olfactory tubercle labeled by the aFGF cRNA. These data demonstrate substantial regionally specific patterns of colocalization and support the hypothesis that, via an autocrine mechanism, aFGF provides local trophic support for cholinergic neurons in the basal forebrain and the striatum.     

Induction, identification or folie à deux? Psychodynamics and genesis of Munchausen syndromes by proxy and false allegations of sexual abuse in adolescents

The substantia nigra (SN) has long been known as an important source of afferents to the pedunculopontine tegmental nucleus (PPN). However, it has not been established which of the chemospecific cell populations receive this synaptic input. We sought to address this issue by a correlative light and electron microscopic approach that combines anterograde tracing of nigral efferents with pre-embedding choline acetyltransferase (ChAT) and/or glutamate (Glu) immunohistochemistry. Following large bilateral injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) in the SN, the labeled nigrotegmental fibers were concentrated in a small area of the mesopontine tegmentum which contained very few ChAT-immunoreactive (ChAT-ir) cell bodies. However, strands of fine varicose fibers penetrated to adjacent regions of the PPN which harbored numerous cholinergic perikarya. The anterogradely labeled boutons were often seen in the proximity of ChAT-ir perikarya and dendrites, but the majority (82-93%) established symmetric synaptic junctions with noncholinergic profiles. In the pars dissipata of the PPN (PPNd), one-third of the labeled terminals synapsed onto noncholinergic perikarya and primary dendrites, while in the pars compacta of the PPN (PPNc) axosomatic synapses were rare. The possibility that the perikarya receiving a rich synaptic input from the SN are glutamatergic was tested in experiments combining anterograde transport of biotinylated tracers biocytin and dextran-amine (BDA) with glutamate immunohistochemistry. In double-labeled sections, Glu-ir perikarya within the terminal plexus of nigrotegmental fibers were surrounded by synaptic terminals. The PPNd also contained retrogradely BDA-labeled neurons which were contacted by anterogradely labeled terminals. These results indicate that although a small subpopulation of cholinergic neurons in the mesopontine tegmentum receive direct synaptic input from the SN, the primary target of nigrotegmental fibers are glutamatergic cells in the PPNd. Our results also provide ultrastructural evidence that some nigrotegmental fibers innervate pedunculonigral neurons.     

Amyloid beta-protein reduces acetylcholine synthesis in a cell line derived from cholinergic neurons of the basal forebrain

The characteristic features of a brain with Alzheimer disease (AD) include the presence of neuritic plaques composed of amyloid beta-protein (Abeta) and reductions in the levels of cholinergic markers. Neurotoxic responses to Abeta have been reported in vivo and in vitro, suggesting that the cholinergic deficit in AD brain may be secondary to the degeneration of cholinergic neurons caused by Abeta. However, it remains to be determined if Abeta contributes to the cholinergic deficit in AD brain by nontoxic effects. We examined the effects of synthetic Abeta peptides on the cholinergic properties of a mouse cell line, SN56, derived from basal forebrain cholinergic neurons. Abeta 1-42 and Abeta 1-28 reduced the acetylcholine (AcCho) content of the cells in a concentration-dependent fashion, whereas Abeta 1-16 was inactive. Maximal reductions of 43% and 33% were observed after a 48-h treatment with 100 nM of Abeta 1-42 and 50 pM of Abeta 1-28, respectively. Neither Abeta 1-28 nor Abeta 1-42 at a concentration of 100 nM and a treatment period of 2 weeks was toxic to the cells. Treatment of the cells with Abeta 25-28 (48 h; 100 nM) significantly decreased AcCho levels, suggesting that the sequence GSNK (aa 25-28) is responsible for the AcCho-reducing effect of Abeta. The reductions in AcCho levels caused by Abeta 1-42 and Abeta 1-28 were accompanied by proportional decreases in choline acetyltransferase activity. In contrast, acetylcholinesterase activity was unaltered, indicating that Abeta specifically reduces the synthesis of AcCho in SN56 cells. The reductions in AcCho content caused by Abeta 1-42 could be prevented by a cotreatment with all-trans-retinoic acid (10 nM), a compound previously shown to increase choline acetyltransferase mRNA expression in SN56 cells. These results demonstrate a nontoxic, suppressive effect of Abeta on AcCho synthesis, an action that may contribute to the cholinergic deficit in AD brain.     

Comparison of sequences for depicting bone marrow alterations in osteomyelitis applied in a low field strength magnetic resonance imaging system

Phenotypes of septal neurons, dissociated from 19-day-old fetal rat brains and then cultured in a medium containing nerve growth factor for 4 weeks, were examined using gamma-aminobutyric acid (GABA), calbindin D-28k, parvalbumin and choline acetyltransferase immunohistochemistry, and acetylcholinesterase histochemistry. There were primarily four groups of neurons identified in this septal culture: the first group (12.7% of 212 neurons examined) displayed a cholinergic, but not GABAergic, phenotype and had an average diameter of 13.6±2.7 μm (mean±S.D.); the second group (31.6%) displayed both cholinergic and GABAergic phenotypes and had a diameter of 12.2±2.8 μm; the third group (31.0%) displayed only a GABAergic phenotype and had a diameter of 10.4±2.3 μm; and the fourth group (24.7%) displayed neither a GABAergic nor cholinergic phenotype and had a diameter of 10.4±2.1 μm. Neurons in the first two groups described were significantly larger than those in the second two groups; neurons in the third and fourth groups were the same size. Calbindin D-28k was expressed in some neurons of each group (31.3%, 18.8%, 9.6% and 15.7%, respectively). These results demonstrate that septal neurons have the ability to express a variety of phenotypes when grown in vitro. This culture will be a useful tool for studying mechanisms of phenotype expression in septal neurons.     

Few cholinergic neurons in the rat basal forebrain coexpress galanin messenger RNA

The concept that galanin (GAL) is cosecreted with acetylcholine (ACh) into the ventral hippocampus is a major component of the current model delineating GAL regulation of the cholinergic memory pathways in the rat. Although GAL-immunoreactivity coexists in 50-70% of cholinergic neurons in the basal forebrain (BF) of colchicine-treated rats, the actual coexistence of these neurotransmitters in the basal state may be lower, because colchicine treatment was recently shown to both induce GAL gene expression and inhibit choline acetyltransferase (ChAT) gene expression in this brain region. We have used single and double in situ hybridization histochemistry to examine the distribution and coexistence of GAL and ChAT mRNAs in the BF of male and female rats. Compared with other forebrain regions, few GAL mRNA-expressing neurons are present within the cholinergic fields of the BF. The greatest number of GAL mRNA-expressing cells in this region are located within the nucleus of the horizontal limb of the diagonal band; but, even in this region, they represent only a small percentage (<20%) of ChAT mRNA-expressing cells. Our results indicate that few cholinergic neurons in the rat BF coexpress GAL mRNA and suggest that, in the basal state, GAL is not widely cosecreted with ACh into hippocampal memory centers.     

Cholinergic and non-cholinergic afferents of the caudolateral parabrachial nucleus: a role in the long-term enhancement of rapid eye movement sleep

A single microinjection of the cholinergic agonist carbachol into the feline caudolateral parabrachial nucleus produces an immediate increase in state-independent ipsilateral ponto-geniculooccipital waves, followed by a long-term rapid eye movement sleep enhancement lasting 7-10 days. Using retrogradely-transported fluorescent carbachol-conjugated nanospheres and choline acetyltransferase immunohistochemistry, afferent projections to this injection site for long-term rapid eye movement sleep enhancement were mapped and quantified. Six regions in the brain stem contained retrogradely-labelled cells: the raphe nuclei, locus coeruleus, laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus, parabrachial nucleus, and the pontine reticular formation. The retrogradely-labelled (rhodamine+) cells in the pontine reticular formation and pedunculopontine tegmental nucleus contributed the predominant input to the parabrachial nucleus injection site (34.3 +/- 5.3% and 28.4 +/- 5.6%, respectively), compared to the laterodorsal tegmental nucleus (5.8 +/- 3.8%), parabrachial nucleus (13.5 +/- 3.1%), raphe nuclei (12.9 +/- 2.7%), and locus coeruleus (5.1 +/- 2.4%). By comparison with findings of afferent input to the induction site for short-latency rapid eye movement sleep in the anterodorsal pontine reticular formation, the parabrachial nucleus injection site is characterized by a similar proportion of afferents, except that the raphe nuclei were found to provide more than a two-fold greater input. Retrogradely-labelled neurons quantified in these nuclear regions consisted of 21.5% double-labelled (rhodamine+/choline acetyltransferase+) cholinergic and 78.5% noncholinergic (rhodamine+/choline acetyltransferase-) cells. The pedunculopontine tegmental nucleus contributed the predominant (51.7 +/- 8.2%) cholinergic input, compared to laterodorsal tegmental nucleus (20.7 +/- 10.2%), parabrachial nucleus (23.1 +/- 7.5%), and pontine reticular formation (4.4 +/- 2.1%). A comparative analysis of the total retrogradely-labelled cells within each nuclear region which were also double-labelled showed the highest proportion in the laterodorsal tegmental nucleus (76.2 +/- 7.5%) compared to pedunculopontine tegmental nucleus (39.4 +/- 3.6%), parabrachial nucleus (37.3 +/- 2.8%), and pontine reticular formation (3.2 +/- 2.1%). These data indicate that while pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus neurons exert a powerful cholinergic influence on the injection site for long-term rapid eye movement enhancement, a major component of the afferent circuitry is non-cholinergic. Since the non-cholinergic input includes contributions from the locus coeruleus and raphe nuclei, it is probable that the caudolateral parabrachial nucleus contains cholinergic and aminergic afferent systems that participate in the long-term enhancement of rapid eye movement sleep.     

3H]acetylcholine release from rat amacrine-like neurons is inhibited by adenosine A1 receptor activation

We studied the effect of endogenous adenosine on the release of [3H]acetylcholine ([3H]ACh) in cultures enriched (96.4+/-0.4%) in rat cholinergic amacrine-like neurons, as determined by labeling with an antibody against choline acetyltransferase. A small population of these cells also contained GABA. Using these cultures we observed that both [3H]ACh release, which was largely Ca2+-dependent, and 45Ca2+ influx, evoked by depolarization with 50 mM KCl, were increased when adenosine A1 receptor activation was prevented by removal of endogenous adenosine with adenosine deaminase, or by application of the A1 receptor antagonist DPCPX. Our results indicate that, in cultured rat amacrine-like neurons, the activation of A1 receptors decreases calcium influx and, thereby, inhibits [3H]ACh release.     

Kidney cografts enhance fiber outgrowth from ventral mesencephalic grafts to the 6-OHDA-lesioned striatum, and improve behavioral recovery

Recent studies have demonstrated the presence of many different neurotrophic factors in the developing and adult kidney. Due to its production of this mixture of neurotrophic factors, we wanted to investigate whether fetal kidney tissue could be beneficial for neuritic fiber growth and/or cell survival in intracranial transplants of fetal ventral mesencephalic tissue (VM). A retrograde lesion of nigral dopaminergic neurons was performed in adult Fischer 344 male rats by injecting 6-hydroxydopamine into the medial forebrain. The animals were monitored for spontaneous locomotor activity in addition to apomorphine-induced rotations once a week. Four weeks following the lesion, animals were anesthetized and embryonic day 14 VM tissue from rat fetuses was implanted stereotaxically into the dorsal striatum. One group of animals received a cograft of kidney tissue from the same embryos in the same needle track. The animals were then monitored behaviorally for an additional 4 months. There was a significant improvement in both spontaneous locomotor activity (distance traveled) and apomorphine-induced rotations with both single VM grafts and VM-kidney cografts, with the VM-kidney double grafts enhancing the motor behaviors to a significantly greater degree. Tyrosine hydroxylase (TH) immunohistochemistry and image analysis revealed a significantly denser innervation of the host striatum from the VM-kidney cografts than from the single VM grafts. TH-positive neurons were also significantly larger in the cografts compared to the single VM grafts. In addition to the dense TH-immunoreactive innervation, the kidney portion of cografts contained a rich cholinergic innervation, as evidenced from antibodies against choline acetyltransferase (ChAT). The striatal cholinergic cell bodies surrounding the VM-kidney cografts were enlarged and had a slightly higher staining density for ChAT. Taken together, these data support the hypothesis that neurotrophic factors secreted from fetal kidney grafts stimulated both TH-positive neurons in the VM cografts and cholinergic neurons in the host striatum. Thus, these factors may be combined for treatment of degenerative diseases involving both dopaminergic and cholinergic neurons.     

Retinal cholinergic system: characterization of rat retinal acetyltransferases using specific inhibitors of choline- and carnitine-acetyltransferases

Choline acetyltransferase catalyzes the synthesis of acetylcholine from choline and acetylcoenzyme A (ACoA) in both nervous and non-nervous tissues. Carnitine acetyltransferase occurs in several tissues and transfers acetyl groups from ACoA to carnitine forming acetylcarnitine and exhibits weak choline acetyltransferase activity. Several haloacetylcholines and haloacetylcarnitines were synthesized to develop selective inhibitors of choline acetyltransferase and carnitine acetyltransferase. Acetylcholine is a transmitter for some presynaptic neurons and/or amacrine cells in retina. Selective inhibitors of choline acetyltransferase and carnitine acetyltransferase were used in the evaluation of choline acetyltransferase and carnitine acetyltransferase activities in the rat retina. Choline acetyltransferase and carnitine acetyltransferase activities were assayed by transferring of [14C]acetyl group from [14C]ACoA to choline or carnitine and estimating [14C]-acetylcholine or [14C]acetylcarnitine. This study gave the following results: (a) Bromoacetylcholine (BrACh) was a selective inhibitor of purified choline acetyltransferase (I50, 2.2 microM); (b) (R)-bromoacetylcarnitine [(R)-BrACa] was more potent for inhibiting purified carnitine acetyltransferase (I50, 4 microM) than purified choline acetyltransferase (I50, 46 microM); (c) Rat retinal sonicate gave choline acetyltransferase activity of 98 +/- 6 nmol of ACh formed/mg/10 min. When the carnitine acetyltransferase was completely inhibited by (R)-BrACa, the activity for choline acetyltransferase decreased to 47 +/- 1 nmol, and this decrease was possibly due to the formation of some [14C]acetylcholine by carnitine acetyltransferase. The net retinal choline acetyltransferase activity was 51 nmol acetylcholine/mg protein/10 min; (d) Rat retinal sonicate contained carnitine acetyltransferase activity of 102 +/- 7 nmol acetylcarnitine formed/mg protein/10 min. This was not altered by inhibition of choline acetyltransferase with BrACh. This means that choline acetyltransferase did not use carnitine as a substrate. Choline acetyltransferase and carnitine acetyltransferase activities did not change after dialysis of retinal sonicates at 4 degrees C for 24 hrs. These observations suggest that BrACh and (R)-BrACa are useful for assessing the correct values for choline acetyltransferase and carnitine acetyltransferase activities in retinal tissues.     

Nitric oxide synthase immunoreactivity in rat pontine medullary neurons

Nitric oxide synthase immunoreactivity was detected in neurons and fibers of the rat pontine medulla. In the medulla, nitric oxide synthase-positive neurons and processes were observed in the gracile nucleus, spinal trigeminal nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus, nucleus ambiguus, medial longitudinal fasciculus, reticular nuclei and lateral to the pyramidal tract. In the pons, intensely labeled neurons were observed in the pedunculopontine tegmental nucleus, paralemniscal nucleus, ventral tegmental nucleus, laterodorsal tegmental nucleus, and lateral and medial parabrachial nuclei. Labeled neurons and fibers were seen in the interpeduncular nuclei, dorsal and median raphe nuclei, central gray and dorsal central gray, and superior and inferior colliculi. Double-labeling techniques showed that a small population (< 5%) of nitric oxide synthase-positive neurons in the medulla also contained immunoreactivity to the aminergic neuron marker tyrosine hydroxylase. The majority of nitric oxide synthase-immunoreactive neurons in the dorsal and median raphe nuclei were 5-hydroxytryptamine-positive, whereas very few 5-hydroxytryptamine-positive cells in the caudal raphe nuclei were nitric oxide synthase-positive. Virtually all nitric oxide synthase-positive neurons in the pedunculopontine and laterodorsal tegmental nuclei were also choline acetyltransferase-positive, whereas nitric oxide synthase immunoreactivity was either low or not detected in choline acetyltransferase-positive neurons in the medulla. The results indicate a rostrocaudal gradient in the intensity of nitric oxide synthase immunoreactivity, i.e. it is highest in neurons of the tegmentum nuclei and neurons in the medulla are less intensely labeled. The majority of cholinergic and serotonergic neurons in the pons are nitric oxide synthase-positive, whereas the immunoreactivity was either too low to be detected or absent in the large majority of serotonergic, aminergic and cholinergic neurons in the medulla.     

Adenylyl cyclase co-distribution with the CaBPs, calbindin-D28 and calretinin, varies with cell type: assessment with the fluorescent dye, BODIPY forskolin, in enteric ganglia

The aims of the present study were: (1) to evaluate BODIPY forskolin as a suitable fluorescent marker for membrane adenylyl cyclase (AC) in living enteric neurons of the guinea-pig ileum; (2) to test the hypothesis that AC is distributed in several subpopulations of enteric neurons; (3) to test the hypothesis that the distribution of AC in the myenteric plexus is not unique to AH/Type 2 neurons. BODIPY forskolin was used to assess the co-distribution of AC in ganglion cells expressing the specific calcium-binding proteins (CaBPs), calretinin, calbindin-D28, and s-100. Cultured cells or tissues were incubated with 10 microM BODIPY forskolin for 30 min and fluorescent labeling was monitored by using laser scanning confocal microscopy. BODIPY forskolin stained the cell soma, neurites, and nerve varicosities of Dogiel Type I or II neurons. About 99% of myenteric and 27% of submucous ganglia contained labeled neurons. About 14% of myenteric and 3% of submucous glia with immunoreactivity for s-100 protein displayed BODIPY forskolin fluorescence. BODIPY forskolin differentially labeled myenteric neurons immunoreactive for calbindin-D28 (80%) and calretinin (17%). The majority (63%) of BODIPY forskolin-labeled myenteric neurons displayed no immunoreactivity for either CaBP. In submucous ganglia, the dye labeled 44.6% of calretinin-immunoreactive neurons, representing 21% of all labeled neurons; it also labeled varicose nerve fibers running along blood vessels. AC thus exists in myenteric Dogiel type II/AH neurons, enteric cholinergic S/Type 1 neurons, and other unidentified non-cholinergic S/Type 1 neurons. Our data also support the hypothesis that AC is expressed in distinct functional subpopulations of AH and S neurons in enteric ganglia, and show that BODIPY forskolin is a suitable marker for AC in immunofluorescence co-distribution studies involving living cells or tissues.