Immunocytochemical detection of acetylated alpha-tubulin and Drosophila synapsin in the embryonic crustacean nervous system.

The caridean shrimp Palaemonetes argentinus Nobili is well suited for studying developmental aspects of the crustacean nervous system due to its rapid embryonic development and short reproductive cycle. In the present paper, we demonstrate the pattern of central axonal pathways in embryos of this species by immunohistochemical detection of acetylated alpha-tubulin. Development of the neuropil was elucidated by using an antibody to a Drosophila synapsin. In the ventral nerve cord, the segmental axonal scaffold consists of the paired lateral connectives, a median connective, and the anterior and posterior commissures. Three nerve roots were found to branch off each ganglion anlage, i.e. the main segmental nerve root, a smaller posterior nerve and the intersegmental nerve. However, this pattern is different in the mandibular segment where no intersegmental nerve and only one commissure was encountered. The anterior part of the brain consists of a tritocerebral and a deutocerebral anlage as well asthe anlage of the medial protocerebrum. The latter is connected to the eyestalk via the protocerebral tract. The sequence of development of the eyestalk ganglia was demonstrated in specimens which were stained with the anti-synapsin antibody. The medulla terminalis and medulla interna are the first neuropils to appear and are still fused in early stages. Later, the medulla interna splits off the medulla terminalis. The lamina ganglionaris is the last of the eyestalk neuropils to develop. These findings prove that immunocytochemistry against acetylated alpha-tubulin and synapsin are valuable tools for studying the development of the crustacean nervous system.

Age-related toxicity in prefrontal cortex and caudate-putamen complex of gerbils (Meriones unguiculatus) after a single dose of methamphetamine.

Single, intermediate to large doses (6-60 mg/kg) of methamphetamine were applied to study the acute neurotoxic effects in developing male gerbils (up to 24 months). A sensitive silver-staining method was used to analyze the toxicity of methamphetamine by light and electron-microscopy. It was shown that treatment with the drug degraded synaptic components, as well as a small population of neurones in the caudate-putamen complex accompanied by accumulation of lysosomes in fibers and axon terminals. In juveniles, methamphetamine in doses of 25-60 mg/kg, resulted in accumulation of lysosomes, selectively in the prefrontal cortex. In young adults, only about half of these doses were sufficient to produce consistent and/or additional effects in the caudate-putamen complex. When the gerbils grew older than 8 months, treatment with drug led to accumulation of lysosomes, exclusively in the caudate-putamen, with acute doses ranging from 6 to 12 mg/kg. Acute neurotoxicity with methamphetamine has thus been induced by doses, which hitherto have been claimed to produce behavioural sensitization. Since dopamine (DA) seems the most likely transmitter to be affected, age-related differences in methamphetamine-induced neurotoxicity are discussed in relation to the background of developing DA-response systems, which are still changing in pattern during ageing.

Single doses of methamphetamine cause changes in the density of dendritic spines in the prefrontal cortex of gerbils (Meriones unguiculatus).

Single doses of methamphetamine (25 mg/kg) were administered to adult gerbils. All detectable spines were counted along defined segments of basal, apical and lateral dendrites of Golgi-impregnated pyramidal cells in the medial prefrontal cortex (layers III and V) and the parietal cortex (layer V). These two areas were selected because previous investigations had shown methamphetamine-induced neurotoxicity in prefrontal cortex but not in parietal cortex. The frequencies of dendritic spines in methamphetamine-treated gerbils were found to be significantly increased in the prefrontal cortex, while no significant change was found in the parietal cortex.

Demonstration of dopamine-immunoreactive cells in the proximal convoluted tubule of gerbil (Meriones unguiculatus) kidney.

We investigated dopamine immunoreactivity in the kidney of gerbils (Meriones unguiculatus). For that purpose a sensitive and selective antibody against glutaraldehyde-conjugated dopamine was applied. Dopamine-immunoreactive cells were found in the epithelium of the proximal convoluted tubule, where these cells revealed a typical segment-like distribution pattern. Dopamine-immunoreactive precipitates were small and concentrated at the apical pole of the labeled cells. This study has directly identified dopamine as a constituent of certain cells of the proximal convoluted tubule in gerbils. The functional significance of dopamine in these cells is discussed in relation to the present view of renal dopaminergic actions.

Demonstration of dopamine-immunoreactive cells in the gastrointestinal tract of gerbils (Meriones unguiculatus).

We investigated dopamine immunoreactivity in the small intestine of gerbils (Meriones unguiculatus), using a sensitive and selective antibody against glutaraldehyde-conjugated dopamine. Dopamine-immunoreactive cells were found within the mucosal epithelium along the entire extent of the small intestine. Labeled cells were regularly distributed in the upper half of the intestinal villi, basally attached to the basement membrane and always reaching as far as the epithelial surface. Dopamine-containing cells revealed a spindle-like shape, and both light and electron microscopic characteristics relate them to typical open-type gut endocrine cells. Thus, this current study directly identified dopamine as a probable secretory product in basal granulated cells of the small intestine. The functional significance of these cells is discussed in relation to the current view of dopaminergic actions in peripheral tissues.

Pharmacologically induced neural plasticity in the prefrontal cortex of adult gerbils (Meriones unguiculatus).

Using a selective antibody serum against glutaraldehyde-conjugated gamma-aminobutyric acid (GABA), GABAergic neurons were identified in the medial prefrontal cortex of young adult gerbils (Meriones unguiculatus) following a single non-invasive dose of methamphetamine (25 mg/kg i.p.) applied at the age of 90 days. GABA-immunoreactive profiles were electron microscopically counted in a defined test field (0.875 mm2) covering the prefrontal prelimbic area after a single dose of either methamphetamine or saline. Within 30 days following the drug challenge the density of GABAergic innervation significantly increased by about 20%. Several lines of previous investigation indicate that a single dose of methamphetamine is an appropriate stimulus to cause selective autotoxic destruction of certain prefrontal dopamine fibres due to drug-induced hyperactivation. There is further indication of postsynaptic and transneuronal neuroplasticity since the densities of dendritic spines on prefrontal pyramidal cells went through a significant sequence of post-drug gain and loss. These structural dynamics resemble typical alterations seen after classical mechanical or chemical lesioning in other regions of the brain. The present results on drug-induced reactive neuroplasticity are discussed together with the current understanding of stimulus-induced adaptive reorganization in the mammalian central nervous system.

Indication of methamphetamine-induced reactive synaptogenesis in the prefrontal cortex of gerbils (Meriones unguiculatus).

A single dose of methamphetamine (25 mg/kg i.p.) was administered to young adult gerbils (Meriones unguiculatus) aged 90 days and the number of spices was determined along 40-microns segments of basal, lateral and apical dendrites of pyramidal cells in layers III and V of the prefrontal cortex, after 1.5, 7, 20 and 30 days. The density of spines rapidly increased by more than 80% within 7 days after drug challenge, and subsequently returned to the original normal values within about 2 weeks. Thirty days after drug administration the density of dendritic spines was slightly, but significantly, less than control values (about 5%). The density of spines was likewise affected in layer III and V neurones, irrespective of the spatial domain of their dendritic ramifications. Since several lines of investigation indicate that methamphetamine can cause the destruction of dopaminergic nerve terminals in the mammalian forebrain, the present results are discussed against the background of current concepts about reactive synaptic reorganization and adaptive remodelling of neural circuits in the central nervous system.

A single dose of methamphetamine in neonatal gerbils affects adult prefrontal gamma-aminobutyric acid innervation.

A single non-invasive dose of methamphetamine (50 mg/kg i.p.) was administered to neonatal male gerbils (Meriones unguiculatus) aged 14 days. At the age of postnatal day 90, the gamma-aminobutyric acid (GABA) immunoreactive profiles were electron microscopically quantified in the prelimbic area of the prefrontal cortex and compared with those of saline-treated controls. This early solitary drug challenge resulted in adult GABAergic innervation densities which were approximately 40% above those of saline-treated controls.

Epigenetic factors differentially influence postnatal maturation of serotonin (5-HT) innervation in cerebral cortex of gerbils: interaction of rearing conditions and early methamphetamine challenge.

The effects of disjunctive environmental deprivation combined with a single methamphetamine (MA) challenge on postnatal maturation of the serotonin (5-HT) innervation pattern in cerebral cortex of gerbils were studied. Gerbils were assigned to either enriched (ER) or impoverished (IR) environmental rearing conditions. On postnatal day 110, 5-HT was immunostained. The 5-HT innervation pattern of the brain was qualitatively evaluated and provided in graphic form. The densities of 5-HT fibres were quantified in areas of prefrontal, insular, frontal, parietal, and entorhinal cortices of the right hemisphere using digital image analysis. The early MA challenge led to an overshoot of the fibre density in medial and orbital prefrontal cortex and entorhinal cortex of ER animals. IR animals mostly resisted MA effects except of a restraint of the innervation of the insular cortex. In comparison to enriched rearing, restricted rearing caused overshoot maturation of 5-HT innervation in insular and entorhinal cortices. The present data provide evidence for a region-specific postnatal vulnerability of the maturing 5-HT innervation, namely in association cortices. In contrast, both sensory and motor cortices showed no significant changes at all. The results are discussed in context with previously presented findings of alterations of the cortical dopamine innervation depending on both epigenetic factors. In conclusion, both experimental variables together give new insight into raphe-cortical plasticity that may contribute to a better understanding of the role of 5-HT fibre systems in structural maturation of the cortex. Postnatal environment may be involved in individual vulnerability of a variety of mental disorders during adolescence and aging.

Dopamine and the regulation of cell proliferation in gerbil (Meriones unguiculatus) pyloric mucosa.

The epithelium of the digestive system mucosa consists of a highly dynamic cell population. The conditions under which mitotic activity in the gastrointestinal epithelium is regulated is as yet poorly understood. Nevertheless, it is assumed that some biogenic amines might be involved. Having demonstrated that dopaminergic cells occur in the stomach of gerbils (Meriones unguiculatus), in the present study we examined the influence of dopamine antagonist haloperidol on the proliferation of epithelial cells in the mucosa of the stomach. Proliferating cells were detected immunocytochemically and quantified after in-vivo labeling with 5-bromo-2'-desoxyuridine in both haloperidol- and saline-treated animals. The results show that acute doses of haloperidol significantly increases the proliferation rate in the pyloric mucosa, suggesting that dopamine plays a probable modulatory role in the regulation of mitotic activity. These findings are discussed with regard to the role of paraneurons in regulating epithelial mitosis.

Demonstration of dopamine immunoreactivity in open and closed type endocrine cells of gerbil (Meriones unguiculatus) stomach.

Dopamine-immunoreactive cells were identified in the stomach of gerbils using a selective antibody against glutaraldehyde-conjugated dopamine. In the pyloric area dopamine-containing cells were exclusively localized in the basal part of the mucous membrane, whereas in the fundic area dopaminergic cells were seen throughout the basodistal extent of the mucous membrane. Dopamine-containing cells belong to the class of closed and open type endocrine cells and were found in the fundic area and in the pyloric area respectively. These findings are discussed along with the likely modulatory function of dopamine in peripheral tissues.

A developmental study of serotonin-immunoreactive neurons in the larval central nervous system of the spider crab Hyas araneus (Decapoda, Brachyura).

Larval development in crabs is characterized by a striking double metamorphosis in the course of which the animals change from a pelagic to a benthic life style. The larval central nervous system has to provide an adequate behavioural repertoire during this transition. Thus, processes of neuronal reorganization and refinement of the early larval nervous system could be expected to occur in the metamorphosing animal. In order to follow identified sets of neurons throughout metamorphosis, whole mount preparations of the brain and ventral nerve cord of laboratory reared spider crab larvae (Hyas araneus) were labelled with an antibody against the neurotransmitter serotonin. The system of serotonin-immunoreactive cell bodies, fibres and neuropils is well-developed in newly hatched larvae. Most immunoreactive structures are located in the protocerebrum, with fewer in the suboesophaegeal ganglia, while the thoracic and abdominal ganglia initially comprise only a small number of serotonergic neurons and fibres. However, there are significant alterations in the staining pattern through larval development, some of which are correlated to metamorphic events. Accordingly, new serotonin-immunoreactive cells are added to the early larval set and the system of immunoreactive fibres is refined. These results are compared to the serotonergic innervation in other decapod crustaceans.

Association between body weight of newborn rats and density of serotonin transporters in the frontal cortex at adulthood.

Persisting alterations in monoaminergic innervation patterns have been observed following various environmental manipulations and neuro-psychopharmacological treatments during fetal or early postnatal life. The present study investigates the question how differences in initial growth conditions at birth might interfere with subsequent development of both serotonergic and noradrenergic innervation in the rat frontal cortex (FC) and brain stem. For this purpose, newborn rat littermates were divided into two groups, a low and a high birth weight group, and the densities of both serotonin (5-HT) and norepinephrine (NE) transporters in the FC and brain stem were analyzed at adulthood. 5-HT transporter density in the FC was significantly higher in the high birth weight group as compared with the low birth weight group. No significant differences were observed between both groups in the density of 5-HT transporters in the brain stem and in the densities of NE transporters in FC and brain stem. It is discussed that differences in birth weight may affect the postnatal development of 5-HT projections to the frontal cortex.

Naturally occurring synapse degradation in the developing cerebellum of the mallard (Anas Platyrhynchos) and the Peking duck (Forma Domestica).

In order to analyze remodelling of junctions as component of adaptive mechanism in the developing avian cerebellum, a silver-staining technique was applied which is highly sensitive towards the detection of lysosomal aggregations (LA) in degrading synapses. LA was quantified in ducks at incubation ages from day 21 to 28 and from hatching up to 32 days. In the mallard, LA increased gradually throughout the whole investigation period, while in the Peking duck LA showed a dramatic increase in the perihatching period followed by the beginning of a decline at about 3 weeks of age. In both species LA occurred initially in the superficial molecular layer prehatching followed by a succession of degradative events down to the granular layer. LA dynamics thus coincide with the outside-in proliferation and differentiation of granule cells during advanced cortical histogenesis. Light microscopy of Epon sections impregnated with silver indicated that granule and Purkinje cell contacts were the center of LA dynamics. Electron microscopy confirmed that synapse degradation occurred predominantly with presynaptic elements of climbing, parallel and mossy afferents. As illustrated by the structural dynamics, maturation of cerebellar circuitry passes through a period of unbalanced synaptogenesis posthatching. Remarkably, the ongoing "critical period" of synaptogenesis in the mallard provides the opportunity for more differentiated behavioral pattern formation in the wild as compared to the domestic duckling.

Neurogenesis in the developing crab brain: postembryonic generation of neurons persists beyond metamorphosis.

A considerable amount of information is available about the structure and function of the central nervous system in adult crustaceans. However, little effort has been directed toward understanding embryonic and larval neurogenesis in these animals. In the present study we recorded neurogenesis in the brain of laboratory-reared larvae of the spider crab Hyas araneus. Proliferating cells were detected immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine. This method has already been used to study the proliferation of neuroblasts in the ventral nerve cord of spider crab larvae. In the brain, a set of mitotically highly active neuroblasts was found in newly hatched zoea 1 larvae. These neuroblasts are individually identifiable due to their position and therefore a schematic map of the cerebral neuroblasts could be established. The number of active neuroblasts is high from hatching throughout the molt to the zoea 2. This proliferative action then decreases dramatically and has ceased at the time of first metamorphosis toward the megalopa larva. However, many ganglion mother cells born by unequal division of neuroblasts then go through their final division throughout the subsequent megalopa stage. In the brain, all mitotic activity has ceased at the time of second metamorphosis with the exception of a cluster of labeled nuclei within the olfactory lobe cells. In this cluster, the generation of neurons persists beyond the second metamorphosis into the crab 1 stage. Meanwhile, the neuropil volume of the olfactory lobes increases 10-fold from hatching to the crab 1. These results are discussed with regard to reports on neuronal proliferation during adult life in insects and rodents.

A novel pharmacological concept in an animal model of psychosis.

OBJECTIVE: We have analysed pharmacologically induced perturbation of functional and structural neurogenesis in the prefrontal cortex (PFC) and hippocampus. METHOD: Juvenile gerbils received a single dose of methamphetamine (METH, 50 mg/kg, i.p.). In adults the following parameters were quantitatively investigated: prefrontal dopaminergic and GABAergic innervation densities (immunocytochemistry), morphogenesis of pyramidal cells (Golgi), dentate granule cell proliferation (BrdU-labelling), working memory and behavioural inhibition (delayed response, open-field). RESULT: A single challenge of METH continuously suppresses granule cell proliferation in adult gerbils and initiates rewiring of neuronal networks in the PFC which run concurrently with the development of severe deficits in PFC-related behaviours. CONCLUSION: It appears that a continuous remodelling of neuronal circuits is an inherent property of the brain, the biological significance of which seems to be to ascertain adaptive interaction between brain and environment. Learning more about drug-induced neuronal reorganization might be basic for understanding the genesis of psychotic conditions in the brain. This presentation is based both on own research and on a review of the literature.

Ontogeny of PFC-related behaviours is sensitive to a single non-invasive dose of methamphetamine in neonatal gerbils (Meriones unguiculatus).

A single dose of methamphetamine (50 mg/kg; i.p.) was administered to neonatal male gerbils (Meriones unguiculatus) aged 14 days, and adult prefrontal cortex (PFC)-related behaviours were analysed and compared with saline-treated controls at the age of postnatal day 90. For that purpose, animals were tested for open-field activities and y-maze delayed alternation. This solitary and non-invasive drug challenge, which has recently been found to initiate serious restraint in maturation of the mesoprefrontal dopamine (DA)-system (Dawirs et al., 1994), induces a significant delayed alternation impairment as well as significant increases in open-field motor activity and emotionality. Since an undisturbed development of the prefrontal DA-innervation seems to be a precondition for the maturation of normal PFC-related behaviours, a single early methamphetamine impact may be a suitable animal model for further investigation of structural and functional aspects of non-invasively induced behavioural deficits in rodents. The present results are discussed with regard to the assumption that hypofunctional mesoprefrontal DA-systems might be basic to schizophrenic behaviours in man.

Social environment alters both ontogeny of dopamine innervation of the medial prefrontal cortex and maturation of working memory in gerbils (Meriones unguiculatus).

Male gerbils (Meriones unguiculatus) were bred and reared either grouped under enriched environmental conditions or isolated under impoverished environmental conditions. The objective of the present study was to examine the influence of social environment on structural and functional ontogeny of the medial prefrontal cortex (mPFC). In this respect, we investigated the maturation of both prefrontal dopamine (DA) innervation and working memory. For that purpose, at the age of postnatal day 90, prefrontal DA fibers were stained immunocytochemically using an antibody against glutaraldehyde-conjugated DA and innervation density was determined by means of a computer controlled program for image analysis. In order to evaluate environmental effects on working memory, 90-day-old gerbils were tested for y-maze delayed alternation. It was found that, isolation produced a significant and severe restraint of the maturation of prefrontal DA innervation, leading to fiber densities which were 56% below those in group-reared gerbils. Isolation also induced a significant impairment of delayed alternation performance on the y-maze indicating that obvious deficits in working memory had developed under restricted rearing conditions. The present results are discussed with regard to activity-dependent postnatal maturation of the cortex and adaptive neuroplasticity.

The postnatal maturation of dopamine innervation in the prefrontal cortex of gerbils (Meriones unguiculatus) is sensitive to an early single dose of methamphetamine. A quantitative immunocytochemical study.

Dopamine (DA)-immunoreactivity was investigated in the prefrontal cortex (PFC) of 90 day old adult male gerbils (Meriones unguiculatus) after they had received a single dose of either methamphetamine (50 mg/kg; i.p.) or saline at the age of postnatal day 14. For that purpose, a selective and sensitive antibody directed against glutaraldehyde-conjugated dopamine was applied. All detectable fragments of dopamine-immunoreactive fibres were identified in consecutive frontal sections of the pregenual prefrontal cortex, and their total numbers and total length were determined in the medial (mPFC) and orbital prefrontal cortex (oPFC). The results indicate that a single application of methamphetamine during early postnatal development caused a significant and severe restraint of the subsequent maturation of the prefrontal dopamine-innervation. Although, on postnatal day 14, the total dopamine-immunoreactivity had only attained about 4% (mPFC) and 7% (oPFC) of the regular adult values, this solitary pharmacological challenge entailed final adult innervation densities which were about 38% (mPFC) and 50% (oPFC) below those of the controls. Considering the pivotal role which mesoprefrontal dopaminergic afferents play in morphogenesis and regular functioning, the present results are discussed with current understanding of structural and functional plasticity during maturation of the prefrontal cortex.

Adult treatment with haloperidol increases dentate granule cell proliferation in the gerbil hippocampus.

Male gerbils were bred and reared grouped under enriched semi-natural environmental conditions. The objective of the present study was to examine the influence of an acute treatment with the neuroleptic haloperidol on adult granule cell neurogenesis in the hippocampus. For that purpose, at the age of postnatal day 90 adult animals received 4 challenges of either haloperidol (5 mg/kg, i.p.) or saline. Proliferation of granule cells was identified by in-vivo labeling with 5-bromo-2'-desoxyuridine (BrdU) which was applied 1 hour after the final dose of haloperidol. BrdU-labeled granule cell nuclei were identified in consecutive horizontal slices along the mid-septotemporal axis of the hippocampus and light-microscopically quantified 7 days after the BrdU-labeling. It was found that in both saline- and haloperidol-treated animals there was a highly significant spatial septotemporal gradient in granular cell proliferation with numbers of BrdU-labeled cells gradually declining from the septal towards the temporal pole. The acute treatment with haloperidol stimulated granule cell proliferation by about 75% and the septotemporal gradient of mitotic activity became significantly enhanced. The present results are discussed with regard to known factors regulating cell proliferation in the hippocampus and other cell systems.