Bernardo Castellano

Demonstration of poly-N-acetyl lactosamine residues in ameboid and ramified microglial cells in rat brain by tomato lectin binding.

This study was designed to demonstrate the localization of poly-N-acetyl lactosamine residues in postnatal and adult rat brain, visualized by their specific binding to a lectin obtained from Lycopersicon esculentum (tomato). Lectin histochemistry was carried out on cryostat, paraffin, and vibratome sections and was examined by light microscopy. Selected vibratome sections were processed for electron microscopy. Our results showed that tomato lectin histochemistry was found in relation to blood vessels and glial cells in both postnatal and adult rat brain. Since tomato lectin-positive glial cells did not show GFAP immunoreactivity and displayed the same morphological features and overall distribution as nucleoside diphosphatase (NDPase)-positive cells, they were consequently identified as microglial cells. At the electron microscopic level, both ameboid and ramified microglial cells displayed intracytoplasmic and plasma membrane lectin reactivity. In postnatal brain, ameboid microglial cells always showed stronger binding of tomato lectin compared with ramified microglial cells in the adult brain. The putative significance of this decrease in poly-N-acetyl lactosamine from ameboid to ramified microglial cells and the possible role(s) of this sugar residue are discussed.

Neonatal Handling and Environmental Enrichment Effects on Emotionality, Novelty/Reward Seeking, and Age-Related Cognitive and Hippocampal Impairments: Focus on the Roman Rat Lines

Roman high- and low-avoidance (RHA/Verh and RLA/Verh) rats are selected and bred for extreme divergence in two-way active avoidance acquisition. In addition, compared to RLA/Verh rats, RHA/Verh rats are (behaviorally and physiologically) less anxious or reactive to stressors, show increased novelty (sensation)-seeking behavior as well as a higher preference for rewarding substances, and are usually less efficient in learning tasks not involving shock administration. The present article reviews evidence showing that neonatal handling and/or environmental enrichment leads to enduring effects (their magnitude frequently depending upon the rat line) on those behaviors. For example, it has been found that neonatal handling reduces most of the (behavioral and physiological) signs of emotionality/anxiety in RLA/Verh rats, while environmental enrichment increases their novelty seeking (also the case with RHA/Verh rats), saccharin and ethanol intake, and sensitivity to amphetamine. Finally, initial results (currently being further elaborated upon) support a preventive action of both environmental treatments on age-related impairments in learning a spatial, water maze task as well as on hippocampal neuronal atrophy.

Microglial and astroglial reactions to anterograde axonal degeneration: a histochemical and immunocytochemical study of the adult rat fascia dentata after entorhinal perforant path lesions.

The reaction of microglial and a stroglial cells to anterograde axonal degeneration was studied in the fascia dentata of adult rats at various timepoints after removal of the entorhinal perforant path projection. Microglial cells were identified by histochemical staining for nucleoside diphosphatase (NDPase) at light and electron microscopical levels. Astroglial cells were stained immunocytochemically for glial fibrillary acidic protein (GFAP). Activated astroglial cells and some microglial cells also stained immunocytochemically for the intermediate filament protein vimentin. Phagocytotic activity was detected by histochemical staining for acid phosphatase. The postlesional connective reorganization of the cholinergic septohippocampal projection was monitored by histochemical staining for acetyl cholinesterase. Twenty-four hours after entorhinal cortex ablation, microglial cells in the perforant path zones of the fascia dentata and the adjacent neuropil reacted by shortening and coarsening of processes and an increase in NDPase reactivity. These changes occurred prior to a noticeable increase in GFAP immunoreactivity and hypertrophy of astroglial cells (first evident on postlesional day 2) or sprouting of cholinergic septohippocampal fibres (first evident on day 3). There was evidence of an early, local proliferation of microglial cells in the denervated perforant path zones and migration into these zones of microglial cells from adjacent intact areas. The specific accumulation of strongly stained microglial cells within the denervated parts of the dentate molecular layer persisted for at least 4 weeks, while the astroglial reaction subsided at 3 weeks. The results demonstrate an early activation of microglial cells by axonal degeneration, and indicate that these cells may play a pivotal, inductive role in the subsequent glial and neural events.

Neuronal, astroglial and microglial cytokine expression after an excitotoxic lesion in the immature rat brain

Cytokines are important intercellular messengers involved in neuron–glia interactions and in the microglial‐astroglial crosstalk, modulating the glial response to brain injury and the lesion outcome. In this study, excitotoxic lesions were induced by the injection of N‐methyl‐d‐aspartate in postnatal day 9 rats, and the cytokines interleukin‐1 beta (IL‐1β), interleukin‐6 (IL‐6), tumour necrosis factor alpha (TNFα) and transforming growth factor beta 1 (TGF‐β1) analysed by ELISA and/or immunohistochemistry. Moreover, cytokine‐expressing glial cells were identified by means of double labelling with glial fibrillary acidic protein or tomato lectin binding. Our results show that both neurons and glia were capable of cytokine expression following different patterns in the excitotoxically damaged area vs. the nondegenerating surrounding grey matter (SGM). Excitotoxically damaged neurons showed upregulation of IL‐6 and downregulation of TNFα and TGF‐β1 before they degenerated. Moreover, in the SGM, an increased expression of neuronal IL‐6, TNFα and TGF‐β1 was observed. A subpopulation of microglial cells, located in the SGM and showing IL‐1β and TNFα expression, were the earliest glial cells producing cytokines, at 2–10 h postinjection. Later on, cytokine‐positive glial cells were found within the excitotoxically damaged area and the adjacent white matter: some reactive astrocytes expressed TNFα and IL‐6, and microglia/macrophages showed mild IL‐1β and TGF‐β1. Finally, the expression of all cytokines was observed in the glial scar. As discussed, this pattern of cytokine production suggests their implication in the evolution of excitotoxic neuronal damage and the associated glial response.

Ontogeny of sensorimotor gating and immune impairment induced by prenatal immune challenge in rats: implications for the etiopathology of schizophrenia

It has been hypothesized that the maternal immune response to infection may influence fetal brain development and lead to schizophrenia. Animal experimentation has supported this notion by demonstrating altered sensorimotor gating (prepulse inhibition, PPI) in adult rats prenatally exposed to an immune challenge. In the present study, pregnant rats were exposed to the bacterial endotoxin lipopolysaccharide (LPS) throughout gestation and the offspring were examined by evaluating the PPI, dopaminergic function, brain protein expression and cytokine serum levels from weaning to late adulthood. Prenatal LPS exposure induced a deficit in PPI that emerged at ‘puberty’ and that persisted throughout adult life. This prenatal insult caused age-specific changes in accumbal dopamine levels and in synaptophysin expression in the frontal cortex. Moreover, serum cytokine levels were altered in an age- and cytokine-dependent manner. Here we show that prenatal LPS administration throughout pregnancy causes maturation-dependent PPI deficits and age-dependent alterations in dopamine activity, as well as in synaptophysin expression and cytokine levels.

Dynamics of microglia in the developing rat brain

Entrance of mesodermal precursors into the developing CNS is the most well‐accepted origin of microglia. However, the contribution of proliferation and death of recruited microglial precursors to the final microglial cell population remains to be elucidated. To investigate microglial proliferation and apoptosis during development, we combined proliferating cell nuclear antigen (PCNA) immunohistochemistry, in situ detection of nuclear DNA fragmentation (TUNEL), and caspase‐3 immunohistochemistry with tomato lectin histochemistry, a selective microglial marker. The study was carried out in Wistar rats from embryonic day (E) 16 to postnatal day (P) 18 in cerebral cortex, subcortical white matter, and hippocampus. Proliferating microglial cells were found at all ages in the three brain regions and represented a significant fraction of the total microglial cell population. The percentage of microglia expressing PCNA progressively increased from the embryonic period (25–51% at E16) to a maximum at P9, when the great majority of microglia expressed PCNA (92–99%) in all the brain regions analyzed. In spite of the remarkable proliferation and expansion of the microglial population with time, the density of microglia remained quite constant in most brain regions because of the considerable growth of the brain during late prenatal and early postnatal periods. In contrast, apoptosis of microglia was detected only at certain times and was restricted to some ameboid cells in white matter and primitive ramified cells in gray matter, representing a small fraction of the microglial population. Therefore, our results point to proliferation of microglial precursors in the developing brain as a physiological mechanism contributing to the acquisition of the adult microglial cell population. In contrast, microglial apoptosis occurs only locally at certain developmental stages and thus seems less crucial for the establishment of the final density of microglia. J. Comp. Neurol. 458:144–157, 2003.

Development of microglia in the postnatal rat hippocampus

During the prenatal development of the hippocampus, microglial cell precursors progressively occur in all subfields in accordance with known ontogenetic gradients of the region (Dalmau et al., J. Comp. Neurol. 1997a;377:70–84). The present study follows the regional distribution of these microglial cell precursors and their morphological differentiation in the rat hippocampus from birth to postnatal (P) day 18. The results demonstrate that the cellular differentiation and the subregional distribution of microglia follow the specific developmental gradients of the different parts of Ammons horn and the dentate gyrus. Microglial cell distribution in the dentate gyrus is thus delayed compared with that in Ammons horn. The appearance of microglia in the hippocampal subregions and differentiation of cell precursors into adult microglia occur earlier at temporal levels than at septal levels. Distribution of microglial cells follows an outside‐to‐inside pattern from the hippocampal fissure to the main cell layers in either Ammons horn or the dentate gyrus. Meanwhile, the resident microglial cells located in the stratum oriens and dentate hilus at birth also increase in number and gradually disperse throughout the whole tissue of the two layers with age. In Ammons horn, microglial differentiation occurs earlier in CA3 than in CA1. In the dentate gyrus, microglia appear earlier in relation to the external limb than to the internal limb, largely following a lateral‐to‐medial gradient. The differentiation and appearance of microglia in the various hippocampal and dentate subregions often correspond to the developmental stage of intrinsic and extrinsic afferent nerve fiber projections. Finally, in both Ammons horn and the dentate gyrus, cells resembling reactive microglia are also observed and, in particular, in the perforant path projections from P9 to P18, suggesting their participation not only in phagocytosis of dead cells but also in axonal elimination and/or fiber reorganization. Hippocampus 1998;8:458–474.

Neurobehavioral and Immunological Consequences of Prenatal Immune Activation in Rats. Influence of Antipsychotics

Increasing evidence suggests that pre- or perinatal events that influence the immune system contribute to the development of behavioral or neuropsychiatric disorders. For instance, exposure of pregnant rats to the bacterial endotoxin lipopolysaccharide (LPS) disrupts sensorimotor information processing, as assessed by the prepulse inhibition test (PPI), and also the immune function in adult offspring, which might be of particular relevance as regards schizophrenia. However, the consequences of maternal LPS exposure during pregnancy on synaptic functioning in adult offspring and, more importantly, the therapeutic opportunity to re-establish PPI and immune function have still to be demonstrated. In this work, we analyzed the consequences of prenatal LPS exposure on dopaminergic neurotransmission and presynaptic markers in adult brain areas related to PPI circuitry. In addition, we tested whether oral treatment with the typical antipsychotic drug haloperidol (HAL) could reinstate PPI performances and cytokine serum levels in six-month-old male rats with prenatal LPS exposure. Both sensory information processing deficits and immune anomalies induced by prenatal exposure to LPS were accompanied by changes in dopaminergic neurotransmission and synaptophysin expression. It is important to note that PPI disruption and serum increases in cytokines induced by prenatal LPS exposure were both reversed by HAL. Taken together, these results demonstrate the critical influence of prenatal immune events on the functioning of adult nervous and immune systems, in association with the putative role of the immune system in the development of behavior relevant to schizophrenia.

Leukocyte infiltration and glial reactions in xenografts of mouse brain tissue undergoing rejection in the adult rat brain. A light and electron microscopical immunocytochemical study

Neural mouse xenografts undergoing rejection in the adult recipient rat brain were characterized with regard to infiltrating host leukocytes and reactions of graft and host astro- and microglial cells. Rejection occurred within 35 days with infiltration of the grafts by in particular macrophages and T-cells as well as blood-brain barrier (BBB) leakage for IgG. In the surrounding host brain microglial cells showed increased histochemical staining for nucleoside diphosphatase (NDPase) and increased immunocytochemical expression of complement receptor type 3 (CR3), while astroglial cells displayed an increased immunoreactivity for glial fibrillary acidic protein (GFAP). Light microscopic findings of rat major histocompatibility complex (MHC) antigen class I on microglial cells, endothelial cells and leukocytes were confirmed at the ultrastructural level and extended to include a few astrocytes. Rat and mouse MHC antigen class II was only detected on leukocytes and activated microglia. We suggest that host macrophages and activated host and xenograft microglial cells act in situ as immunostimulatory cells on T-helper cells, and that increased levels of donor MHC antigen class I may further enhance the killer activity exerted by host T-cytotoxic cells.

DEVELOPMENT OF MICROGLIA IN THE PRENATAL RAT HIPPOCAMPUS

The distribution and appearance of microglial cell precursors in the prenatal hippocampus were examined in embryonic day 14 (E14) to E21 rats by nucleoside diphosphatase histochemistry. For comparison, the differentiation of astroglial cells was analyzed from E17 by vimentin and glial fibrillary acidic protein immunohistochemistry.