Monoamine containing neurons in the pigeon brain stem and their terminal fields in the telencephalon

Cellular localization of monoamines in the upper brain stem and distribution of their telencephalic terminal fields were studied in pigeons by fluorescence histochemistry. Two distinct cellular types were identified: one containing primary catecholamines (NE or DA), the other, 5-HT. In the telencephalon numerous DA axons were identified in the paleostriatum augmentatum and in the lobus paraolfactorius. The noradrenergic fibers were mainly concentrated in the Wulst regions which receive visual afferents from the dorsolateral thalamus.     

Passive avoidance learning in the young chick results in time- and locus-specific elevations of alpha-tubulin immunoreactivity.

A monoclonal antibody was used to examine changes in immunoreactivity of the cytoskeletal protein, alpha-tubulin, following passive avoidance learning in day-old chicks. Postmitochondrial fractions (16,000 g supernatants) were prepared from specific forebrain loci taken at several time points after training and assayed with the anti-alpha-tubulin antibody, YL1/2. Of the regions examined, elevations in the titre of YL1/2 were found in the left intermediate hyperstriatum ventrale 1 h, 6 h and 24 h following training, in the left lobus parolfactorius 1 h following training and in the right lobus parolfactorius 6 h and 24 h following training. No training-related changes were detected in a third forebrain region, the paleostriatum augmentatum. These results regarding the cellular dynamics of memory formation in the chick confirm and expand on earlier findings from our laboratory.     

Receptor Autoradiography as a Tool for the Study of the Phylogeny of the Basal Ganglia

Abstract

Recent studies on the neurotransmitter organization of the basal ganglia and forebrain in lower vertebrates suggest that, in contrast to the old concepts of the phylogeny of the brain, there are many similarities between the chemical organization of the brain throughout evolution. By examining neurotransmitter receptors using in vitro autoradiography we have attempted to further our understanding of the evolution of the brain. Receptors enriched in different parts of the basal ganglia in mammals appear to be also enriched in the homologous areas in lower vertebrates. Thus, for example, dopamine and muscarinic receptors, but not serotonin-1A, are enriched in the paleostriatum augmentatum while GABA/benzo-diazepine receptors are enriched in the paleostriatum primitivum corresponding with their localization to the caudate-putamen and globus pallidus respectively. Our results support the concept of a more conservative evolution of the vertebrate brain and demonstrate the usefulness of receptor autoradiography in the understanding of brain evolution.     

Indoleamines and the eccentric cells of the Limulus lateral eye

The lateral eye of the horseshoe crab, Limulus polyphemus, was studied by fluorescence microscopy according to Falck and Hillarp and microspectrofluorometry for identifying neuronal monoamines. After the formaldehyde treatment, the eccentric cells and their axons have a yellowish, rapidly fading fluorescence, such as is seen with 5-hydroxytryptamine. The microspectrofluorometric analysis was compatible with the fluorescence being caused by an indole, which could not be definitely identified, however. The eccentric cells have the ability to accumulate indoleamines such as 5-hydroxytryptamine, 6-hydroxytryptamine and 5,6-dihydroxytryptamine. Their axons were best demonstrated after being loaded with 6-hydroxytryptamine. Characteristic varicose terminals were seen in the neuropil, often arranged in clusters. Other terminals, possibly originating from the eccentric cells, were also seen among the pigment cells in the basal part of the ommatidia.     

DOPAMINE-CONTAINING VASOMOTOR NERVES IN THE DOG KIDNEY

Abstract— The dopamine (DA) content of the canine renal cortex is greater than can be attributed to its presence in noradrenergic axons only. Most of the excess DA is in the outer part of the cortex. By fluorescence histochemistry numerous catecholamine-containing axons are seen to be associated with renal cortical arteries and arterioles. The fluorescence is abolished following treatment of animals with 6-hydroxydopamine or with reserpine, but is restored to some axons if reserpine is followed by systemic administration of 1-DOPA. This procedure does not restore fluorescence to atrial noradrenergic axons after reserpine-induced depletion. Pretreatment of animals with guanethidine abolishes axonal fluorescence and depletes tissue NA and DA from atrium, but in renal cortex the tissue DA level is little affected and some fluorescent axons remain. These results are discussed in the light of previous functional evidence for dopaminergic autonomic axons in the canine kidney.     

Forebrain projections of the pigeon olfactory bulb

The olfactory system of the pigeon (Columba livia) was examined. Our electrophysiological and experimental neuroanatomical (Fink-Heimer technique) data showed that axons from the olfactory bulb terminated in both sides of the forebrain. The cortex prepiriformis (olfactory cortex), the hyperstriatum ventrale and the lobus parolfactorius comprised the uncrossed terminal field. The crossed field included the paleostriatum primitivum and the caudal portion of the lobus parolfactorius, areas which were reached through the anterior commissure. In this report the relationships between areas that receive olfactory information and the possible roles that olfaction plays in the birds' behavior are discussed.     

Distribution of intrinsic nerve cell bodies and axons which take up aromatic amines and their precursors in the small intestine of the guinea-pig

Summary The sites of uptake, decarboxylation and retention of 1-dopa and the uptake and retention of dopamine and 6-hydroxytryptamine in the small intestine of the guinea-pig have been localised histochemically with a fluorescence technique for arylethylamines. In segments of ileum from untreated guinea-pigs only noradrenergic axons are fluorescent; these axons were eliminated by surgical denervation (crushing nerves running to the intestine through the mesentery) or by chemical denervation with 6-hydroxydopamine. In denervated segments of ileum, cell bodies and processes of intrinsic neurons become fluorescent after the injection of 1-dopa, dopamine or 6-hydroxytryptamine and the inhibition of monoamine oxidase, as do cells of Brunner's glands and Paneth cells. About 11% of the nerve cell bodies in the submucous plexus and 0.4% of those in the myenteric plexus become fluorescent. Varicose intrinsic axons which take up amines are found amongst the nerve cell bodies of the myenteric and submucous plexuses. They also ramify in the principal connections of the plexuses, in the tertiary strands of the myenteric plexus, in the deep muscular plexus and contribute sparse supplies of axons to arterioles in the submucosa and to the lamina propria of the mucosa. The axons are resistant to the degenerative actions of 6-hydroxydopamine.It is suggested that the intrinsic amine handling axons are more likely to utilise an indolamine related to 5-hydroxytryptamine than they are to utilise a catecholamine as a neurotransmitter.     

Distribution of gastrin-releasing peptide immunoreactivity in the brain of the collared dove (Streptopelia decaocto)

The distribution of immunoreactivity after applying an antibody against gastrin-releasing peptide (GRP) was studied in the brain of the collared dove (Streptopelia decaocto). In the forebrain GRP-immunoreactive (GRP-ir) cells were found in the hyperstriatum accessorium, medial and lateral parts of the neostriatum, corticoidea dorsolateralis and temporoparieto-occipitalis areas, hippocampus, pre- and parahippocampal areas and prepiriform cortex. In the brainstem, GRP-ir cells were restricted mainly to the substantia nigra and ventral tegmental nucleus. Areas with densely packed GRP-ir clusters of varicosities were the medial intermediate hyperstriatum ventrale and lateral septal nucleus; dense GRP-ir neuropil was found in the parolfactory lobe, and in the dorsal half of the intermediate and caudal archistriatum. The ventral lamina medullaris contained many GRP-ir fibers. Forebrain areas devoid of immunoreactivity were the basal nucleus, ectostriatum, rostral archistriatum, most of the paleostriatum augmentatum and the lateral bed nucleus of the stria terminalis. Moderate densities of GRP-ir elements were found in the other telencephalic areas and further in, among others, the preoptic and hypothalamic region, ventral area of Tsai, cerulean nuclei, parabrachial complex, dorsal glossopharyngeal and vagus motor nuclei and medial nuclei of the solitary complex. The observations are compared with data from the literature and the implications for the definition of specific centers within the avian brain are discussed, with emphasis on systems with a role in visceral and motivational functions and in learning.     

Spectral analyses of extrinsic fluorescence of the nerve membrane labeled with aminonaphthalene derivatives

A fluorescence spectrophotometer was constructed to determine the emission spectrum of a nerve labeled with various fluorochromes. Using this spectrophotometer, the spectra of 2-p-toluidinylnaphthalene 6-sulfonate (2,6-TNS) and other aminonaphthalene derivatives in squid giant axons were determined at the peak of nerve excitation, as well as in the resting state of the axons. During nerve excitation the fluorescent light deriving from the 2,6-TNS-stained nerve undergoes a transient change in intensity. The spectrum of the light contributing to this change in intensity was found to be much narrower and sharper than the fluorescent spectrum of the light arising from labeled axons at rest. This narrow and sharp spectrum is interpreted as being derived from a transient variation in the polarity of the 2,6-TNS binding sites in the axon. In the Appendix, the results of a physicochemical investigation into the factors affecting the fluorescence of 2,6-TNS in vitro are described.     

Innervation of the intestine in the bivalve mollusc Chione stutchburyi

Summary The innervation of the gut of the venerid bivalve mollusc, Chione stutchburyi, has been examined by fluorescence histochemistry, electron microscopy and autoradiography. Specific green and yellow varicose fluorescent fibres indicate the presence of dopaminergic and serotonergic axons, respectively. Three different types of axons can be distinguished by the morphological characteristics of their vesicles. Type I axons contain predominantly small granular vesicles (average diameter 65 nm), Type II axons possess large granular vesicles (average diameter 100 nm) and Type III axons contain large opaque vesicles (average diameter 150 nm). The granular vesicles in both Types I and II axons react positively to dichromate, and their granularity is reduced by reserpine indicating that they are monoaminergic. Only Type I axons accumulate tritiated dopamine and are selectively damaged by 6-hydroxydopamine. It is concluded that Type I axons are dopaminergic. Type II axons are serotonergic: they alone take up tritiated 5-hydroxytryptamine, and 5,7-dihydroxytryptamine selectively causes degenerative changes in these axons. Type III axons contain an unidentified neurotransmitter substance. The large opaque vesicles of these axons do not react to dichromate and are unaffected by reserpine, 6-hydroxydopamine or 5,7-dihydroxytryptamine.     

Fluorescence of squid axon membrane labelled with hydrophobic probes

Summary Extrinsic fluorescence changes in squid giant axons were examined under a variety of experimental conditions using 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and other fluorescent probes. Measurements of the degree of polarization of the fluorescent light (with the axis of the polarizer parallel to the longitudinal axis of the axon) indicated that the class of the TNS molecules in the axon membrane which participate in production of fluorescence signals have a definite orientation with their absorption and emission oscillators directed parallel to the long axis of the axon. Rectangular depolarizing voltage pulses produced a transient decrease in the fluorescent intensity, of which the early component is correlated tentatively with the rise in the membrane conductance. In response to hyperpolarizing pulses, there was an increase in fluorescence intensity which may be explained in terms of increased incorporation of TNS into the ordered structure in the membrane. Hyperpolarizing responses in KCl depolarized axons were accompanied by a change in fluorescent intensity. Tetrodotoxin appeared to suppress the initial component of the fluorescence signal produced by depolarizing clamping pulses. The technique for detecting these fluorescence changes and the physico-chemical properties of TNS are described in some detail.     

A histofluorescence study of events accompanying accumulation and migration of norepinephrine within locally cooled nerves

Glyoxylic acid was used to induce fluorescence in sections of rabbit sciatic nerve. In fresh nerves treated with this agent there were scattered finely beaded axons with a weak blue-green fluorescence. During local cooling, blue—green fluorescence accumulated steadily at the proximal boundary of the cooled region but never at its distal boundary. This accumulation gave rise to dilated axons that often swelled into brilliantly fluorescent balloon-like structures up to 10 μm in diameter. Axonal fluorescence was probably specific for norepinephrine, being enhanced by inhibition of the metabolism and diminished by inhibition of the synthesis or storage of this neurotransmitter. After local cooling of nerves for 1.5 hr, specific fluorescence was confined within 0.8 mm of the cooled region. Rewarming led to rapid removal of fluorescence from the cooled region and to disappearance of most of the balloon-like swellings. Simultaneously, rewarming caused brightly fluorescent fibers that were neither dilated nor swollen to appear in distal regions of nerve. As this wave of fluorescence migrated distally with increasing duration of rewarming, it was spread over increasingly broad regions of nerve, which suggests that axonal transport of norepinephrine may involve some kind of dispersive process.     

Axon fasciculation and differences in midline kinetics between pioneer and follower axons within commissural fascicles

Early neuronal scaffold development studies suggest that initial neurons and their axons serve as guides for later neurons and their processes. Although this arrangement might aid axon navigation, the specific consequence(s) of such interactions are unknown in vivo. We follow forebrain commissure formation in living zebrafish embryos using timelapse fluorescence microscopy to examine quantitatively commissural axon kinetics at the midline: a place where axon interactions might be important. Although it is commonly accepted that commissural axons slow down at the midline, our data show this is only true for leader axons. Follower axons do not show this behavior. However, when the leading axon is ablated, follower axons change their midline kinetics and behave as leaders. Similarly, contralateral leader axons change their midline kinetics when they grow along the opposite leading axon across the midline. These data suggest a simple model where the level of growth cone exposure to midline cues and presence of other axons as a substrate shape the midline kinetics of commissural axons.     

Distribution of immunoreactive mesotocin and vasotocin in the brain and pituitary of chickens

The distribution of vasotocin and mesotocin in the pituitary and central nervous system in male chickens was determined using radioimmunoassays. Neither peptide was detected in the pineal. Mesotocin, but not vasotocin, was detected in the cerebellum. Both peptides were found in the septal area, archistriatum, paleostriatum, optic lobe, anterior, medial and posterior hypothalamus, midbrain, pons, medulla oblongata, and the anterior and posterior pituitary. Equal amounts of the 2 peptides were present in the septal area, archistriatum and anterior hypothalamus whereas vasotocin was more abundant (2- to 10-fold) in the paleostriatum, optic lobe, midbrain, and pituitary. The amount of mesotocin was about twice that of vasotocin in the medulla oblongata and the medial and posterior hypothalamus. The wide distribution of vasotocin and mesotocin in extrahypothalamic sites in the central nervous system suggests that the peptides may, as in mammals, have a role in a variety of autonomic and endocrine regulatory processes in chickens.     

The ultrastructure and distribution of 5-HT-containing neurons in the intestine of a teleost fish,Aldrichetta forsteri

Summary The intestine of Aldrichetta forsteri was examined ultrastructurally for evidence of 5-HT-containing intrinsic neurons. A population of nerve cell bodies and processes characterized by the presence of amine-containing vesicles was found after the destruction of adrenergic processes with 6-hydroxydopamine. The distribution of the neurons matched that of the 5-HT-containing neurons previously seen with formaldehyde-induced fluorescence and 5-HT immunohistochemistry in A. forsteri and other teleosts. The axons made close contacts with the circular smooth muscle layer and the muscularis mucosae. The axons also lay exposed to the epithelial cells of the mucosa but did not make contact with neurons or with the longitudinal smooth muscle layer.     

Fine structure and fluorescence morphology of adrenergic nerves after 6-hydroxydopamine in vivo and in vitro

Summary In parallel fine structural, fluorescence histochemical and biochemical experiments the effect of 6-OH-DA administered in vivo and in vitro on the adrenergic nerves in the mouse iris was studied. As seen in the electron microscope, in vivo administration of 6-OH-DA causes a selective, rapid degeneration of the adrenergic axon terminals similar to that found after axotomy, whereas the cholinergic nerves are unaffected at all time intervals studied. Already 1 hr after the injection of 6-OH-DA the axonal enlargements swell and the size of the dense core of the granular vesicles is strongly reduced. Since the NA stores are almost completely depleted at this time interval, the small core present may be due to a reaction between 6-OH-DA and the fixative. From 2–4 hr after the injection increasing numbers of axonal enlargements with a high electron density are observed in the Schwann cell cytoplasm, which later are digested and completely absent about 48–72 hr after the 6-OH-DA injection. During the following weeks adrenergic axons reappear. This time course of degeneration obtained is considerably faster than that seen after axotomy in other studies. After incubation in 6-OH-DA containing media similar changes were observed in the axonal enlargements, starting already after 30 min of incubation. At this time-point there is a considerable reduction of endogenous NA and a severe damage of the membrane pump uptake mechanism. Incubation with 6-OH-DA and subsequent rinsing for 2 hr caused marked changes, including partly swelling of axons and partly shrinking of the axons into electron dense bodies.The fluorescence histochemical and biochemical results are in good agreement with the ultrastructural studies demonstrating a rapid loss of NA from the adrenergic nerve terminals and main axons and a long lasting depletion of the NA, with a gradual recovery to 75% 6 weeks after the injection.The investigation has been supported by research grants from the Swedish Medical Research Council (14X-2295, 14X-2887 and 04X-3881) Karolinska Institutet, Magnus Bergvalls and Carl-Berthel Nathorst Stiftelser. For generous supplies of drugs we are indebted to the following companies: AB Hässle (6-OH-DA, through Dr H. Corrodi), Pfizer (Niamid^®), Ciba (Serpasil^®). The skilful technical assistance of Miss Bodil Flock, Mrs Waltraut Hiort and Mrs Eva Lindqvist is gratefully acknowledged.     

A stimulatory effect by nerve growth factor on the regrowth of adrenergic nerve fibres in the mouse peripheral tissues after chemical sympathectomy with 6-hydroxydopamine

Summary Systemically administered Nerve Growth Factor (NGF) had a strong stimulating effect on the regeneration of fully developed adrenergic neurons in the peripheral tissues of the mouse after axotomy induced by 6-hydroxydopamine. The NGF stimulation was investigated at 9 and 21 days after the 6-hydroxydopamine injection, and was observed fluorescence histochemically as an increase in number, length, and thickness of the outgrowing adrenergic fibre bundles, in the extent and abundance of the terminal ramifications of the regrowing fibres, and in their fluorescence intensity. This increase in the regrowth of the lesioned adrenergic axons was paralleled by strong and significant increases in the recovery of endogenous noradrenaline in several peripheral tissues. The present findings demonstrate a sensitivity of fully developed adrenergic neurons to NGF during axonal regeneration, and it is suggested that NGF might play a normal physiological role in this process.     

Identification and characterization of zebrafish semaphorin 6D

The semaphorin gene family contains a large number of secreted and transmembrane proteins; some function as repulsive and attractive cues of axon guidance during development. Here, we report cloning and characterization of zebrafish transmembrane semaphorin gene, semaphorin 6D (sema6D). Sema6D is expressed predominantly in the nervous system during embryogenesis, as determined by in situ hybridization. We also found that Sema6D binds Plexin-A1 in vitro, but not other Plexins. It induces the repulsion of dorsal root ganglion axons, but not sympathetic axons. Consequently, Sema6D might use Plexin-A1 as a receptor to repel specific types of axons during development.     

A histofluorescence study of events accompanying accumulation and migration of norepinephrine within locally cooled nerves.

Glyoxylic acid was used to induce fluorescence in sections of rabbit sciatic nerve. In fresh nerves treated with this agent there were scattered finely beaded axons with a weak blue-green fluorescence. During local cooling, blue-green fluorescence accumulated steadily at the proximal boundary of the cooled region but never at its distal boundary. This accumulation gave rise to dilated axons that often swelled into brilliantly fluorescent balloon-like structures up to 10 microgram in diameter. Axonal fluorescence was probably specific for norepinephrine, being enhanced by inhibition of the metabolism and diminished by inhibition of the synthesis or storage of this neurotransmitter. After local cooling of nerves for 1.5 hr, specific fluorescence was confined within 0.8 mm of the cooled region. Rewarming led to rapid removal of fluorescence from the cooled region and to disappearance of most of the balloon-like swellings. Simultaneously, rewarming caused brightly fluorescent fibers that were neither dilated nor swollen to appear in distal regions of nerve. As this wave of fluorescence migrated distally with increasing duration of rewarming, it was spread over increasingly broad regions of nerve, which suggests that axonal transport of norepinephrine may invole some kind of dispersive process.     

Chemical degeneration of indolamine axons in rat brain by 5,6-dihydroxytryptamine

Summary Evidence has been obtained by electron microscopy of a direct cytotoxic effect of intraventricularly administered 5,6-dihydroxytryptamine (5,6-DHT) on unmyelinated axons in the rat brain. Ultrastructural signs of axonal damage were observed in areas rich in indolamine nerve terminals as early as 2 hrs after injection. By 6–24 hrs, characteristic and more dramatic signs of degeneration developed, involving coalescence of all axonal constituents—often in combination with a uniform osmiophilic impregnation of the axoplasm—accompanied by engulfment of the dystrophic structures by glial processes. During the next five days, the degenerating axons and axon terminals appeared to be removed by glial cell phagocytosis, whose equivalents were the inclusion of axonal residues into membrane-bound lysosome-like bodies. Concomitantly, there was a progressively increasing number of extremely large and dilated axons in all regions analysed. These axonal swellings, which have an ultramorphology similar to that of dilated stumps of mechanically severed monoamine axons, correspond most probably to proximal, dilated portions of drug-damaged axons.The present results, in combination with biochemical and fluorescence microscopical data, indicate that within a proper dose range the 5,6-DHT-induced degeneration is largely restricted to indolamine axons and axon terminals. However, unselective effects on other unmyelinated axons, on myelin, and on glial cells were observed in narrow subependymal zones close to the lateral ventricles, i.e. close to the injection cannula.Supported by grants from the Deutsche Forschungsgemeinschaft.Supported by grants from the National Institutes of Health, USPHS (NS-06701-06) and from the Swedish Medical Research Council (grants No. B72-14X-712-07B and B72-14X-56-08B).