Amine-containing neurons in the brain of Lymnaea stagnalis: distribution and effects of precursors

Glyoxylic acid-induced fluorescence in whole-brain preparations of the central nervous system of the freshwater pond snail, Lymnaea stagnalis, was used to map the distribution of serotonin-and dopamine-containing neurons. Serotonin and dopamine were easily distinguishable by differences in color of fluorescence. Serotonin-containing neurons were consistently found in the cerebral, pedal, right parietal and visceral ganglia. Dopamine-containing neurons were found in the pedal, and buccal ganglia. Prior incubation of brains in 5-hydroxytryptophan (5-HTP), the immediate precursor to serotonin, produced serotonin-like fluoresence in neurons which do not normally fluoresce. These neurons thus probably possess specific uptake mechanisms for 5-HTP. Since 5-HTP itself fluoresces yellow, the glyoxylic acid technique cannot determine if these neurons contain the enzyme aromatic amino acid decarboxylase, which converts 5-HTP to serotonin, or merely fluoresce because of the 5-HTP taken into the cells.     

Distribution of retrogradely primulene-labeled dopamine-containing neurons forming spinal projections

The distribution of primuline-labeled dopamine-containing neurons in the rat forebrain was investigated by combining catecholamine fluorescence and retrograde neuronal labeling techniques following injection of fluorochrome into the upper thoracic spinal cord. It was found that only diencephalic neurons of catecholamine-containing group All, located in the dorsal hypothalamus and the caudal thalamus send out direct projections to the spinal cord. Cells of nonidentified transmitter category were also primuline-labeled in this area. An average of 173±4 catecholamine-containing neurons were revealed in group All, of which 86 were retrogradely labeled with primuline. Numbers of labeled dopamine-containing neurons increased, traveling in a rostro-caudal direction. The functional role which may be played by the dopaminergic diencephalo-spinal neuronal system described is discussed.S. V. Kurashov Medical Institute, Ministry of Public Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 19, No. 6, pp. 771–779, November–December, 1987.     

Dopamine in a molluscan nervous system: Synthesis and fluorescence histochemistry

The nervous system of the pond snail, Helisoma trivolvis, was investigated for its ability to synthesize and accumulate ³H-catecholamines from ³H-tyrosine. ³H-Dopamine, but not ³H-norepinephrine, was synthesized by several ganglia. The highest accumulations were found in the cerebral, pedal, and buccal ganglia. The Falck-Hillarp and glyoxylic acid fluorescence histochemical techniques were applied to the buccal ganglia to visualize dopamine-containing cells. Fluorescing cells were found on both dorsal and ventral sides of the ganglion. Peripheral nerves of the buccal ganglia also displayed catecholamine fluorescence and accumulated ³H-dopamine. However, no ³H-dopamine synthesis occurred in the cerebral-buccal connectives, which connect the buccal ganglia with the rest of the central nervous system. Therefore, we conclude that there is a dopaminergic system intrinsic to the buccal ganglia and their peripheral targets.     

A rapid, simple and sensitive method for the demonstration of central catecholamine-containing neurons and axons by glyoxylic acid-induced fluorescence. II. A detailed description of methodology.

The glyoxylic acid-induced fluorescence method for localization of brain catecholamine neurons has been modified. Fluorescence is developed rapidly in cryostat sections of brains fixed by perfusion with 0.5% depolymerized paraformaldehyde and 2.0% glyoxylic acid. Since neither freeze drying nor vibratome sectioning is required, total processing time can be less than 1 hr. Both perikarya and fine varicose axons of norepinephrine- and dopamine-containing neurons can be seen throughout the neuroaxis. The modified technique retains good cytologic integrity and may provide a useful alternative for methods combining histochemical approaches.     

Cytochemical dopamine visualization as a method for estimation of globular actin content in cytosol of living cells

The possibility to reveal globular (G-) actin in cell cytosole by means of microscopy has been studied. The applicability of this method was in particular evaluated for diagnostics of malignant cells, whose main pathocytological feature is an anomalously high content of G-actin in cytosol. The cells of a common origin but with different states of cytosolic actin were analyzed by means of cytochemical reaction for biogenic amines using Falck-Hillarp method after 40-h incubation of the cells in dopamine-containing cultivation medium. Mouse embryo cell line BALB/3T3, clone A31 with differentiated actin cytoskeleton were used as a control cell line. The same cells infected with pathogenic virus SV-40 (cell line 3T3B-SV40) exhibited a malignant phenotype; their cytosol mainly consisted of G-actin. Manifold increase in fluorescence intensity of cytosol and karyoplasms, the loci with the highest G-actin concentration, was revealed in malignant cells in comparison with their healthy prototype. Thus, it was shown that G-actin of malignant cells is a diagnostic target for dopamine, which, as it was earlier shown, penetrates into the cytosol, polymerizes G-actin, incorporating into the filaments as integral component in the 100:1 ratio, and thus fluorescently labels G-actin due to conversion into isoquinoline by reaction with formaldehyde. Besides, dopamine exhibited a strong cytotoxicity that considerably reduced the viability of malignant cells. The data suggest that the content of Gactin in cytosol of living cells can be quantitatively estimated by fluorescence intensity of cytosol following incubation of the cells in dopamine-containing medium.     

Application of the glyoxylic acid method to Vibratome sections for the improved visualization of central catecholamine neurons

Summary The glyoxylic acid fluorescence histochemical method for the visualization of neuronal monoamines has been applied to fresh or glyoxylic acid-perfused brain tissue, sectioned with the Vibratome intrument. This technique demonstrates the central noradrenaline and dopamine neuron systems with a sensitivity and richness in details that is superior to the standard Falck-Hillarp formaldehyde method, as observed in the following three ways: First, the entire axon, including the non-terminal portions, became fluorescent; second, more extensive terminal systems were detected in certain brain regions ; third, due to the absence of diffusion, the delicate dopamine-containing fibres in e. g. the caudate nucleus and the median eminence had a distinct fluorescence. It is concluded that the glyoxylic acid method applied to Vibratome sections should be ideal for precise and detailed neuroanatomical studies on central catecholamine neuron systems.     

Functional reinnervation of the neostriatum in the adult rat by use of intraparenchymal grafting of dissociated cell suspensions from the substantia nigra

Summary Dissociated cell suspensions were prepared from the substantia nigra of 15–17 day-old rat embryos and grafted via an intraparenchymal injection into the depth of the neostriatum of adult recipient rats. The survival and fibre outgrowth of the dopamine-containing neurones in the implants were studied by fluorescence histochemistry, and the functional capacity of the grafts was monitored by repeated testing of the amphetamine-induced turning behaviour of the implanted rats.Before transplantation the target neostriatum of the recipient rats was denervated of its normal dopaminergic innervation by an injection of 6-hydroxydopamine into the ipsilateral nigrostriatal dopamine pathway. The completeness of the denervation was ascertained by measurement of the intensity of the amphetamine-induced turning response. After injection of the dissociated cells large numbers of dopamine-containing neurones were found in clusters at the site of injection as well as scattered in the apparently intact neostriatal tissue up to a distance of about 0.5 mm from the site of injection. Extensive dopamine-containing fibre networks had developed around the implant. These newly formed fibres, which were most abundant around the cell clusters at the injection site, extended in a loose network into large areas of the initially denervated caudate-putamen. In all animals with surviving dopamine neurones the amphetamine-induced turning response was reduced, and in the most extensively reinnervated cases even reversed, within 3–5 weeks after transplantation. This strongly suggests that the implanted dopamine neurones are capable of restoring dopaminergic neurotransmission in the denervated neostriatum, probably via reinnervation of the denervated neostriatal tissue.The use of dissociated brain tissue preparations thus permits reliable intraparenchymal grafting of neurones to plausibly any desired site within the central nervous system, and should open entirely new possibilities for investigation of neuronal growth dynamics and functional reconstruction of damaged brain circuits, perhaps even in brains of larger mammals.     

Identification of epinephrine and phenylethanolamine N-methyltransferase activity in rat retina

Epinephrine (E) and phenylethanolamine N-methyltransferase (PNMT) are endogenous to the rat retina. The retinal enzyme shows substrate specificity and inhibitor sensitivity similar to the PNMT of brain. The E system in the retina may be part of a functional adrenergic system, because amine metabolism of dopamine-containing amacrine cells is inhibited by alpha 2 agonists and stimulated by alpha 2 antagonists.     

Dopaminergic innervation ofAplysia gill muscle

1. The fluorescence histochemical method of Hillarp and Falck was employed to investigate the distribution of dopamine in the gill of Aplysia. 2. Dopamine-containing nerve fibers and varicosities were found in close association with the lateral and medial external pinnule muscles, the circular and longitudinal muscles of the afferent vessel, and the circular muscles of the efferent vessel. 3. Other large, dopamine-containing, intensely fluorescent processes were observed lining the vascular sinuses of the gill and within the muscle bundles themselves. 4. Our findings support the hypothesis that dopamine is a neuromuscular transmitter in the gill. However, the data also suggest that dopamine may play a hormonal role as well.     

Sclerotization of the perisarc of the calyptoblastic hydroid, Laomedea Flexuosa 1. The identification and localization of dopamine in the hydroid

With histochemical methods, the perisarc and a certain cell type in Laomedea flexuosa have been shown to contain a catecholamine. The only catecholamine detected in methanolic extracts of the hydroid is dopamine. It is thought to be transferred from spherical inclusions in these cells to the perisarc and to be involved in sclerotization. The dopamine-containing cells appear to differentiate in specific regions of the colony and migrate out to all other regions by active amoeboid movement between ectodermal epithelial cells. The rivets (or desmocytes) contain an unidentified phenolic substance and may also be sclerotized structures.     

Electrophysiological evidence for a dopaminergic action of LSD: depression of unit activity in the substantia nigra of the rat.

LSD (25–50 μg/kg, i.v.) significantly decreased the firing rate of 78% of the dopamine-containing neurons in the substantia nigra of chloral hydrate anesthetized rats. In a subgroup of neurons (22%), LSD either had no clear effect or caused a slight excitation. On the other hand, brom-LSD (100 μg/kg, i.v.), a non-hallucinogenic congener of LSD, had no effect on 71% of dopaminergic cells and slightly reduced the firing rate with 29% of the units. Pretreatment with haloperidol (0.1 mg/kg) blocked the inhibitory effects of LSD, and haloperidol injected following LSD reversed its depressive effects. Non-dopaminergic neurons in the region of the substantia nigra typically showed large increases in firing rate in response to LSD administration. The inhibitory effects of LSD on dopamine-containing neurons are probably not attributable to the serotonergic properties of LSD, since 5-methoxy N,N dimethyltryptamine (25–100 μg/kg), which has central serotonergic properties similar to those of LSD, produced exclusively excitatory effects on the firing rate of dopaminergic cells. These electrophysiological results are consistent with recent behavioral and neurochemical data which suggest that LSD can act as a dopamine agonist in the CNS.     

Development of dopamine-containing neurons and dopamine uptake in embryos of Hirudo medicinalis

The embryonic development of neurons which contain or take up dopamine was studied with glyoxylic acid histofluorescence in Hirudo medicinalis. Beginning at the time of the formation of the tail ganglion, one pair of dopamine-containing neurons was stained per segmental ganglion. The normal outgrowth of the cell bodies into the anterior roots was prevented in isolated and cultured chains of embryonic ganglia. Preincubation of intact embryos in dopamine led to the staining of additional neurons at certain developmental stages. These neurons presumably are the precursors of serotonin-containing cells, which have a temporary capability of taking up and storing dopamine.     

Cytological features of serotonin-containing neurons and their processes in the retina of the carp (Cyprinus carpio). An immunohistochemical study using flat-mount preparations

The morphological features of serotonin-containing neurons (SCNs) and their processes in the retina of the carp (Cyprinus carpio) were immunohistochemically studied by applying a modified peroxidase-antiperoxidase technique using flat-mount preparations. The somata of immunoreactive SCNs were mostly located in the innermost part of the inner nuclear layer (INL). These cells were distributed at a density of 64.8 cells/mm2, this being similar to the density of dopamine-containing interplexiform cells. The processes of the SCNs ramified successively into two finer branches, eventually forming a broad, extensive network in the thin layer just subjacent to the plane of the somata of the SCNs. Processes originating from neighboring SCNs exhibited cytoplasmatic continuity with each other at the lightmicroscope level. Due to their location and cytological features, these SCNs appeared to correspond to amacrine cells.     

Activation by dopamine of patterned motor output from the buccal ganglia of Helisoma trivolvis

The buccal ganglia of the snail, Helisoma trivolvis, contain an intrinsic system of dopamine-containing neurons (Trimble, Barker, and Bullard, 1983). Dopamine, when bath applied to the isolated buccal ganglia, activates patterned motor output in a dose-dependent fashion. Haloperidol blocks the activating effect of dopamine, but the similar activation evoked by serotonin is not blocked by haloperidol. We suggest that there are two separate mechanisms for activating patterned motor output from the buccal ganglia. One is serotonergic, emanating from identified cerebral ganglion cells (Granzow and Kater, 1977), while the other is dopaminergic, involving neurons intrinsic to the buccal ganglia.     

Oscillatory potentials of the cat retina: effects of adrenergic drugs

When reserpine was used as a monoaminergic depletion agent, a diminution or suppression of certain components of the oscillatory potentials of the electroretinogram of the cat was observed. Administration of L-Dopa to previously reserpinized cats restored the altered OP components. The possible interference of systematic disturbances provoked by the drugs was studied by means of artificial variations of blood pressure. Results reported in this paper suggest that reserpine and L-Dopa are acting on catecholamine-dependent retinal structures, probably the dopamine-containing population of amacrine cells. An hypothesis is advanced concerning the possible role of tangential structures in the generation of the oscillatory potentials of the retina.     

Glucose potentiates haloperidol-induced catalepsy

Treatment of rats with D-glucose greatly potentiates the cataleptic response to haloperidol. Neither L-glucose, which is not metabolized, nor saline administration alters haloperidol-induced catalepsy. These results are discussed in relation to our previous observation that D-glucose administration suppresses the firing of central dopamine-containing neurons.     

N-methyl-4-phenylpyridine (MMP+) together with 6-hydroxydopamine or dopamine stimulates Ca2+ release from mitochondria

The nigrostriatal neurotoxin N-methyl-1,2,3,6-tetrahydropyridine (MPTP) causes Parkinsonism in humans and laboratory animals. MPTP neurotoxicity is dependent on its oxidation to N-methyl-4-phenylpyridine (MPP+). The mechanism by which MPP+ causes destruction of dopamine-containing nigrostriatal cells is unknown. Here we show that MPP+ but not MPTP is taken up by energized mitochondria. MPP+ in the presence of dopamine and particularly of 6-hydroxydopamine stimulates Ca2+ release from mitochondria. Ca2+ release is accompanied by hydrolysis of intramitochondrial pyridine nucleotides. Our findings suggest that the MPTP-induced model of Parkinson's disease may be due to a disturbed Ca2+ homeostasis in dopamine neurons.     

Striatal opiate receptors: Pre- and postsynaptic localization

Autoradiographic localization of opiate receptors in the rat striatum with specifically bound ³H-diprenorphine reveals relatively small, high density clusters of receptors, a high density band of receptors along the striatal-callosal border, and a lower density of receptors spread over the remainder of the neuropil. Placement of kainate lesions resulted in a 94% loss of receptors in the clusters and a smaller loss in other areas. Removal of the dopamine-containing input to the striatum by placement of medial forebrain bundle lesions or by intrastriatal injection of 6-hydroxydopamine resulted in a greater depletion of receptors in the non-cluster areas compared to cluster areas. In repeated biochemical and autoradiographic studies, no change was found after either decortication or placement of thalamic lesions. It is concluded that the bulk of striatal opiate receptors are localized postsynaptically on intrinsic striatal neurons and their processes with the bulk of the remaining receptors localized to the dopamine-containing axons and terminals.     

Cytological features of serotonin-containing neurons and their processes in the retina of the carp (Cyprinus carpio)

Summary The morphological features of serotonin-containing neurons (SCNs) and their processes in the retina of the carp (Cyprinus carpio) were immunohistochemically studied by applying a modified peroxidase-antiperoxidase technique using flat-mount preparations. The somata of immunoreactive SCNs were mostly located in the innermost part of the inner nuclear layer (INL). These cells were distributed at a density of 64.8 cells/mm², this being similar to the density of dopamine-containing interplexiform cells. The processes of the SCNs ramified successively into two finer branches, eventually, forming a broad, extensive network in the thin layer just subjacent to the plane of the somata of the SCNs. Processes originating from neighboring SCNs exhibited cytoplasmatic continuity with each other at the lightmicroscope level. Due to their location and cytological features these SCNs appeared to correspond to amacrine cells.In honour of Prof. P. van Duijn     

Blastomere ablation and the developmental origin of identified monoamine-containing neurons in the leech

Ablation of different identifiable blastomeres of the early embryo of the leech Helobdella triserialis was found to lead to the absence of different sets of segmentally iterated monoamine-containing neurons in subsequent development. Thus the ablation of one of the paired N ectoteloblasts leads to the absence of one member of each of the three bilateral pairs of serotonin-containing neurons (one of which is the Retzius cell) from each segmental ganglion. The ablation of one of the paired OP blastomeres (precursors of the paired O and P ectoteloblasts) leads to the absence of one member of each of the two bilateral pairs of lateral dopamine-containing neurons that lie in the body wall of each segment. And the ablation of one of the paired Q ectoteloblast leads to the absence of one member of the bilateral pair of medial dopamine-containing neurons that lie in the body wall of each segment. These results suggest that each of these sets of monoamine-containing neurons is derived from a particular blastomere. Upon ablation of that blastomere the set does not develop from any other source.