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.

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.

Postnatal rearing conditions influence ontogeny of adult dopamine transporter (DAT) immunoreactivity of the striatum in gerbils.

In the present study, the influence of postnatal rearing conditions on the structural maturation of the striatum of adult male gerbils (Meriones unguiculatus) was investigated. For that purpose, animals were bred and reared either grouped in an object-filled environment (EC) or isolated under restricted environmental conditions (IC). At the age of postnatal day 90, dopamine fibers were stained immunocytochemically using an antibody against the dopamine transporter (DAT). Innervation density was determined along the entire rostrocaudal axis of the ventromedial and dorsolateral part of the striatum. As a result, restricted rearing produced a significant restraint of the maturation of striatal dopamine (DA) innervation, leading to adult fiber densities which were approximately 9% below those in semi-naturally reared gerbils. Results are discussed with structural and functional alterations observed in the brain of IC animals.

An early methamphetamine challenge suppresses the maturation of dopamine fibres in the nucleus accumbens of gerbils: on the significance of rearing conditions.

The effect of a single early methamphetamine (MA) challenge on postnatal maturation of the nucleus accumbens (NAC) was studied. Therefore, male gerbils received a single dose of MA (50 mg/kg, i.p.) on postnatal day 14. At the age of postnatal day 90, dopamine fibres were stained immunocytochemically and innervation density was determined in several test fields along the rostrocaudal extent of both core and shell of the NAC. Since we already know that the differential environment can alter ontogeny of dopamine innervation in the prefrontal cortex of gerbils, in the present study we investigated whether probable drug effects may be influenced by rearing conditions. For that purpose, animals were bred and reared either isolated in standard laboratory cages or grouped in an object-filled environment. The results showed that a single early MA challenge significantly alters maturation of dopamine fibre innervation in both subregions of the NAC. In seminaturally reared gerbils the drug challenge caused dopamine fibre densities which were about 54% below those of saline-treated controls in both the shell and core. However, in animals from restricted rearing this MA-induced effect was more pronounced in the core (-43%) but not significant in the shell (-14%). In conclusion, an early MA challenge caused a significant restraint of adult dopamine fibre density developing in the NAC postnatally. Additionally, rearing conditions significantly interfered with drug-induced alterations in maturation of dopaminergic innervation pattern of the NAC. The present results are discussed with recent findings on MA-induced impairment of prefrontal dopamine innervation and further reactive morphogenetic effects caused by the drug. In this respect, functional interactions between the prefrontal cortex and NAC are specifically considered.

Differential influence of rearing conditions and methamphetamine on serotonin fibre maturation in the dentate gyrus of gerbils (Meriones unguiculatus).

Environmental experience and drugs are two parameters that affect the maturation of neurotransmitter systems. The influence of impoverished rearing (IR) versus enriched rearing (ER) was compared in conjunction with postnatal methamphetamine (MA) treatment. The densities of immunostained 5-HT fibres were quantified in septal and temporal regions of the hippocampal dentate gyrus (DG) in young adult gerbils. In the IR group, 5-HT fibre densities were significantly increased in the molecular, granular and polymorphic layers of the DG in the temporal plane. After postnatal MA treatment, the 5-HT fibre density in the ER group reached a level equivalent to that of the IR group in nearly all respects. Under IR conditions, the pharmacological intervention significantly increased the maturation of fibre densities in septal layers only in the right hemisphere with no significant alterations in the left hemisphere and in temporal regions of either hemisphere. According to our previous studies on hippocampal neurogenesis, adaptations of 5-HT fibre densities partly proved to be positively correlated to cell proliferation rates for each of the specific conditions. Thus, the induced MA sensitivity, caused by pharmacological intervention at day 14, was manifested as direct interaction of 5-HT fibre maturation and cell proliferation in dependence of environmental factors. Both IR and MA together give us a better understanding of raphe-hippocampal plasticity and offer new perspectives for pharmacological studies on the 5-HT participation in mental disorders.

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.

Postnatal maturation of prefrontal pyramidal neurones is sensitive to a single early dose of methamphetamine in gerbils (Meriones unguiculatus).

The effect of a single methamphetamine application on postnatal maturation of the prefrontal cortex was studied using pyramidal cell morphology and spine density as parameters of systemic plasticity. Male gerbils were injected a single dose of methamphetamine (METH, 50mg/kg, i.p.) on postnatal day 14. On postnatal day 90, prefrontal cortices of METH-treated animals and saline-treated controls were processed for Golgi-staining. Dendritic arbours of layer III and V pyramidal neurones were measured to describe pyramidal cell morphology, and segmental spine counts were carried out. The results showed that a single postnatal METH-challenge significantly alters morphological differentiation of pyramidal cells towards adulthood. Cells from METH-treated animals showed a higher total dendritic length based on longer segments between subsequent dendritic branchings, with only the apical stem dendrite being shorter in METH-treated than in control subjects. The branching rate was slightly but not significantly increased in METH-treated animals. Nevertheless, spine density was significantly increased on all types of dendrites, with apical dendrites of both layers III and V showing the highest drug-induced progression of about 50% compared to control values. The present results are discussed with regard to probable clues they may provide for investigating neurobiological principles of psychotic behaviour in an animal model.

Differential environment alters ontogeny of dopamine innervation of the orbital prefrontal cortex in gerbils.

In the present study, the influence of postnatal environmental conditions on the structural ontogeny of the orbital prefrontal cortex of adult gerbils (Meriones unguiculatus) was examined. The animals were bred and reared either isolated in standard laboratory cages or grouped in an object-filled environment. At the age of postnatal day 90, dopamine fibers were stained immunocytochemically and innervation density was determined in the orbital prefrontal cortex. By comparison, restricted rearing produced a restraint of the subsequent maturation of orbital prefrontal dopamine innervation, leading to adult fiber densities that were approximately 38% below those in seminaturally reared gerbils. Results are discussed in terms of activity-dependent postnatal maturation of the cortex and adaptive neuroplasticity with regard to previously published data concerning diminished dopamine innervation in the medial prefrontal cortex (Winterfeld et al. [1998]

Granule cell proliferation and axon terminal degradation in the dentate gyrus of gerbils (Meriones unguiculatus) during maturation, adulthood and aging.

The objective of the present study was to examine both naturally occurring degrading events in axon terminals of the dentate gyrus and granule cell proliferation in the dentate gyrus of gerbils (Meriones unguiculatus) throughout postnatal life. For that purpose, (1) a selective silver staining technique was applied to analyze neuronal lysosome accumulation (LA), indicating synaptic degradation during development. LA was quantified by counting silver grains in the inner third and outer two thirds of the molecular layer, granular layer, subgranular layer and the hilus of the dentate gyrus. (2) Proliferation of granule cells was identified by in-vivo labeling with 5-bromo-2'-desoxyuridine (BrdU). BrdU-labeled granule cell nuclei were identified in consecutive horizontal slices along the mid-septotemporal axis of the hippocampus and light-microscopically quantified 4h after the BrdU-labeling. It was found (1) that in young animals LA significantly increased within all layers and reached adult levels after about 3 months. During subsequent development LA kept on this level throughout aging with highest values within the inner molecular layer. (2) There was a highly significant temporal gradient in granule cell proliferation with numbers of BrdU-labeled cells exponentially declining during juvenile life. Nevertheless, granule cell proliferation occurred throughout adult life and aging. The present results are discussed (1) with concepts of ongoing neuroplasticity and remodeling of neuronal networks in the developing and adult brain, and (2) with regard to pharmacologically induced neuromorphogenesis.

Adult treatment with methamphetamine transiently decreases dentate granule cell proliferation in the gerbil hippocampus.

The objective of the present study was to examine whether acute treatment with the recreational drug methamphetamine influences adult granule cell proliferation in the dentate gyrus of the hippocampus. For that purpose, at the age of postnatal day 90 adult male gerbils (Meriones unguiculatus) received a single dose of either methamphetamine (25 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 either simultaneously with methamphetamine or 36 h after administration of the drug. 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 methamphetamine-treated animals there was a highly significant spatial septotemporal gradient in granule cell proliferation with numbers of BrdU-labeled cells gradually declining from the septal towards the temporal pole. The acute treatment with methamphetamine suppressed granule cell proliferation by about 28% and the septotemporal gradient of mitotic activity became significantly attenuated. It was further found that 36 h after the drug challenge granule cell proliferation rates had been restored almost to the control values along the whole septotemporal axis of the hippocampus. The present results are discussed with regard to (1) pharmacological regulation of neurogenesis in the hippocampus and (2) probable clues they may provide for both understanding the biological correlates of psychotic disorders and evolution of future concepts in neuropharmacological intervention.

A single neonatal dose of methamphetamine suppresses dentate granule cell proliferation in adult gerbils which is restored to control values by acute doses of haloperidol.

A single non-invasive dose of methamphetamine (50 mg/kg; i.p.) was administered to neonatal male gerbils (Meriones unguiculatus) aged 14 days. The first objective of the present study was to examine whether this early drug challenge, which has been shown to induce suppressive postnatal maturation of prefrontal dopamine (DA) innervation (Dawirs et al., 1994), interferes with adult granule cell proliferation in the dentate gyrus. Proliferation of granule cells was identified by in-vivo labeling with 5-bromo-2'-desoxyuridine (BrdU). BrdU-labeled granule cell nuclei were identified in consecutive horizontal sections along the mid-septotemporal axis of the hippocampus and light-microscopically quantified 7 days after BrdU-labeling. It was found that a single neonatal dose of methamphetamine was a stimulus strong enough to significantly attenuate adult granule cell proliferation. This effect was clearly lateralized with significant suppression of mitotic activity becoming apparent solely in the left dentate gyrus (-34%). The second objective of the present study was to examine whether acute doses of haloperidol, which have been found to stimulate granule cell proliferation in healthy adult animals (Dawirs et al., 1988), might restore mitotic activity to control values. For that purpose, at the age of postnatal day 90 adult animals which had been challenged with methamphetamine as juveniles received 4 doses of haloperidol (5 mg/kg; i.p.). Proliferation of granule cells was identified by BrdU-labeling. It was found that this neuroleptic treatment acutely restored granule cell proliferation rates to control values. The present results are discussed with regard to (1) factors, regulating mitotic activity in the hippocampus and (2) probable clues they may provide for understanding the neurobiological basis of psychotic behavior.

A new look at embryonic development of the visual system in decapod crustaceans: neuropil formation, neurogenesis, and apoptotic cell death.

In recent years, comparing the structure and development of the central nervous system in crustaceans has provided new insights into the phylogenetic relationships of arthropods. Furthermore, the structural evolution of the compound eyes and optic ganglia of adult arthropods has been discussed, but it was not possible to compare the ontogeny of arthropod visual systems, owing to the lack of data on species other than insects. In the present report, we studied the development of the crustacean visual system by examining neurogenesis, neuropil formation, and apoptotic cell death in embryos of the American lobster, Homarus americanus, the spider crab, Hyas araneus, and the caridean shrimp, Palaemonetes argentinus, and compare these processes with those found in insects. Our results on the patterns of stem cell proliferation provide evidence that in decapod crustaceans and hemimetabolous insects, there exist considerable similarities in the mechanisms by which accretion of the compound eyes and growth of the optic lobes is achieved, suggesting an evolutionary conservation of these mechanisms.

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.

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.

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.

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.

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.

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.

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.

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.