Developmental pattern changes of prefrontal efferents in the juvenile gerbil (Meriones unguiculatus).

Previous findings of our group showed that early traumatisation leads to a dysfunctional organisation of prefrontocortical efferents in adulthood. To identify vulnerable time windows during maturation, we labelled either layer III- or layer V/VI-pyramidal cells with biocytin in the prefrontal cortex of gerbils (Meriones unguiculatus) from the age of postnatal day (PD) 15 up to adulthood (PD 90). The density of passing fibres and axonal terminals in distinct cortical columns in specific prefrontal projection areas was assessed by digital image analysis. Following layer III injections, fibre densities reached adult values between adolescence (PD 60) and adulthood (PD 90). However, layer V/VI-fibre densities decreased after eye-opening (PD 15), followed by an increase to adult values after weaning (PD 30). These findings are the first to describe dynamic structural changes even beyond adolescence of functionally diverse prefrontal output systems. External interventions might exert adverse influences on the establishment of integrated prefrontal networks especially during the early phase of re-arranging.

Alteration in the GABAergic network of the prefrontal cortex in a potential animal model of psychosis.

The GABAergic input on cortical pyramidal cells has an important influence on the firing activity of the cortex and thus in regulating the behavioural outcome. The aim of the current study was to investigate the long-term neuroplastic adaptation of the GABAergic innervation pattern after an early severe systemic impact. Therefore 40 Mongolian gerbils (Meriones unguiculatus) were either reared under impoverished (IR) or enriched rearing conditions (ER) and received a single early (+)-methamphetamine (MA) challenge (50 mg/kg i.p.) or saline on postnatal day 14. The density of perisomatic immunoreactive GABAergic terminals surrounding layers III and V pyramidal neurons was quantified as well as the overall GABAergic fibre density in layers I/II and V of the medial prefrontal cortex (mPFC) of young adult animals (90 days). We found that IR in combination with an early MA administration led to a significant decrease in GABAergic bouton densities while the overall GABAergic fibre density increased in all investigated layers. The results indicate a shift in inhibition from somatic to dendritic innervation of pyramidal neurons in this potential animal model of psychosis. We conclude that IR combined with early MA trigger changes in the postnatal maturation of the prefrontal cortical GABAergic triggers innervation, which may interfere with proper signal processing within the prefrontal neural network.

Contralateral prefrontal projections in gerbils mature abnormally after early methamphetamine trauma and isolated rearing.

As previously shown, a miswiring of ipsilateral prefrontal projections after methamphetamine (MA) intoxication and/or isolated rearing (IR) may serve as a model of so-called "dysconnection" in human schizophrenia. We here find that deep prefrontal projections to contralateral targets were drastically reduced by both MA and IR alone, but remained equally dense if both impairments cumulated. Projections from superficial layers were not altered by MA and/or IR. These findings confirm that the normal intercortical integration of information is compromised in this animal model of schizophrenia.

A simulation model for compensatory plasticity in the prefrontal cortex inducing a cortico-cortical dysconnection in early brain development.

In the present work, an abstract prefrontal cortex simulation model is used to predict compensatory structural alterations of the cortico-cortical connectivity pattern in the normal and pathologic forebrain maturation. The simulation network shows different representative courses of morphogenesis when developing undisturbed or when suffering from disturbing excitatory afferences. The simulative results could be affirmed by an immuno-histochemical study, revealing a qualitatively comparable development of the glutamatergic projection fibre density in gerbils (Meriones unguiculatus) after juvenile and adult methamphetamine intoxication. The simulation model further allows to consider different rearing conditions (enriched-environment model), and claims contradictory effects of an equal disturbance after enriched or impoverished rearing which are in accordance with the experimental findings.

Isolation rearing or methamphetamine traumatisation induce a "dysconnection" of prefrontal efferents in gerbils: implications for schizophrenia.

A miswiring of prefrontal efferents is generally discussed by the name of "dysconnection" as the anatomical substrate of schizophrenia. Since direct histological confirmation of this hypothesis can hardly be obtained in humans, we used an animal model of schizophrenia to trace prefrontal efferents to distal cortical fields. Mongolian gerbils were intoxicated with a single high dose of methamphetamine on postnatal day 14 and reared in isolation after weaning (day 30). Controls received a saline injection and/or were reared under enriched conditions. Upon reaching adulthood (day 90), biocytin was injected into the medial prefrontal cortex into either deep or superficial laminae. The density of passing fibres and terminal fields in the frontal, parietal and insular cortices was assessed by digital image analysis. Isolation rearing or methamphetamine treatment alone reduced the projections from lamina V/VI to the frontal and from lamina III to the insular cortex, and from both laminae to the parietal cortex. In contrast, isolation rearing of methamphetamine-intoxicated gerbils significantly increased the projections from the deep laminae to the frontal and parietal cortices, compared to isolation-reared controls, with no difference in the efferents from superficial laminae. These results are the first to demonstrate a miswiring of prefrontal efferents in response to adverse systemic influences. They might give a hint at the anatomical basis of "dysconnection" in schizophrenia.

The maturation of the acetylcholine system in the dentate gyrus of gerbils (Meriones unguiculatus) is affected by epigenetic factors.

The current study investigated the influence of impoverished rearing (IR) conditions and a single early methamphetamine challenge (MA; 50 mg/kg i.p.) on day 14 on the postnatal maturation of acetylcholinesterase-positive (AChE+) fibres in the hippocampal dentate gyrus (DG) of gerbils (Meriones unguiculatus). The layer-specific densities of histochemically stained AChE+ fibres were quantified in two planes of the left and right DG in young adults (day 90). Compared to enriched reared (ER) animals, the AChE+ fibre densities turned out to be higher in both the septal and the temporal plane of both hemispheres in saline treated IR and MA treated ER gerbils. The temporal plane was slightly more affected than the septal plane. In IR animals, MA treatment selectively diminished the AChE+ fibre densities in the subgranular layer of both left and right temporal DG. In conclusion, the maturation of AChE+ fibres is vulnerable to both rearing conditions and early MA challenge. The results correlate with our previous studies on the dentate cell proliferation rates and the serotonergic innervation, two parameters which are similarly affected by the experimental design. Thus, disturbances of the ACh system may impair the hippocampal plasticity and hippocampus-related cognitive and emotional function.

Developmentally induced imbalance of dopaminergic fibre densities in limbic brain regions of gerbils ( Meriones unguiculatus).

It is well established that epigenetic factors influence the maturation of neurotransmitter systems. Social isolation as well as an early intervention with methamphetamine (MA) lead to a diminished maturation of dopaminergic (DA) fibres in cortical and striatal areas in the brain of Mongolian gerbils. The aim of this study was to prove whether isolated rearing (IR) and the application of a single dose of MA on postnatal day 14 affect the maturation of DA fibres in caudal limbic areas. Therefore the DA fibre densities were quantified in the dorsolateral and ventrolateral entorhinal cortex (EC), the ventral subiculum (SUB) and in three amygdala nuclei - the basolateral (BLA), the lateral (LA) and the central (CA) nucleus. Our results showed that IR and an early MA application led to an increase of DA fibre densities in various caudal limbic areas. Whereas the BLA was affected by both IR and MA, the LA and the medial left CA were only influenced by MA in IR animals. The DA fibre surplus in the ventrolateral EC was significant in MA treated ER and IR animals in the left and right hemisphere, respectively. The SUB and the dorsolateral EC remained unaffected by both epigenetic factors. Altogether, the BLA seems to be the area which responds most sensitively to IR and MA. Previous studies in our laboratory showed a suppressive maturation of DA fibres in the prefrontal cortex (PFC) and nucleus accumbens (NAC) induced by the same set of epigenetic factors. Thus, due to the close functional connection between the PFC and limbic areas, it could be assumed that the suppressive maturation of prefrontal DA fibres implicates an enhancement of DA innervation densities in caudal limbic areas. Imbalances in the morphology and physiology of the different DA projections are suggested here to be crucial in the aetiology of schizophrenia.

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]

Electromagnetic exposure effects the hippocampal dentate cell proliferation in gerbils (Meriones unguiculatus).

The chronic effect on hippocampal neurogenesis after exposure (30 min/day for 14 days) to a high frequency (35,53 kHz) electromagnetic field, double modulated at extremely low frequencies (ELF; 1, 8, 12, 29 and 50 Hz), was studied in young adult gerbils. Immediately after the last exposure proliferation of dentate granule cells was identified by in vivo labeling with 5-bromo-2-desoxyuridine (BrdU). Exposure to 1, 29 and 50 Hz resulted in a statistically significant reduction of cell proliferation rates, but only the 50 Hz-group manifested the effect highly significantly (-29,3 %). On the other hand, gerbils exposed to 8 and 12 Hz showed no significant change of postmitotic cell proliferation as compared with the sham treated controls. The results suggest that the effects of ELF on the granule cell proliferation are mediated by neurotransmitters and hormones which regulate hippocampal neurogenesis.

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.

Neuronal degeneration and reorganization: a mutual principle in pathological and in healthy interactions of limbic and prefrontal circuits.

Based on developmental principles and insights from animal research about neuroplasticity in cell assemblies, this article is to propose a view of plasticity that promotes a link between hippocampal and prefrontal structure and function. Both the mitotic activity (counting of BrdU-labeled cells) in hippocampal dentatus and the maturation of dopamine fibres (quantitative immunochemistry of mesoprefrontal projection) in the prefrontal cortex proved to be a measurable combination for investigating the complex chain of events that relate activity dependent neuroplasticity to normal as well as to pathological maturational processes. With our animal model we demonstrate that both rearing conditions and neuroactive substances can effectively interfere with developmental plasticity and induce a malfunctional adaptation of prefrontal structures and neurotransmitter systems (dopamine, GABA). In the hippocampal dentatus, where ontogenetic plasticity proved to be preserved by continued neuro- and synaptogenesis, serious damage can be internalized without simultaneous disruption of neural dynamics offering an approach to reverse dysfunctional reorganization in the prefrontal cortex.

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.

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.