Prenatal infection decreases calbindin, decreases Purkinje cell volume and density and produces long-term motor deficits in Sprague-Dawley rats.

The cerebellum is involved in the control of motor functions with Purkinje cells serving as the only output from the cerebellum. Purkinje cells are important targets for toxic substances and are vulnerable to prenatal insults. Intrauterine infection (IUI) has been shown to selectively target the developing cerebral white matter through lesioning, necrosis and inflammatory cytokine activation. Developmental and cognitive delays have been associated with animal models of IUI. The aim of this study was to determine if IUI leads to damage to Purkinje cells in the developing cerebellum and if any damage is associated with decreases in calbindin and motor behaviors in surviving pups. Pregnant rats were injected with Escherichia coli (1 × 105 colony-forming units) or sterile saline at gestational day 17. Beginning at postnatal day (PND) 2, the pups were subjected to a series of developmental tests to examine developmental milestones. At PND 16, some pups were sacrificed and their brains extracted and processed for histology or protein studies. Hematoxylin and eosin (HE) staining was done to examine the general morphology of the Purkinje cells and to examine Purkinje cell density, area and volume. Calbindin expression was examined in the cerebellum via immunohistochemistry and Western blot techniques. The remaining rat pups were used to examine motor coordination and balance on a rotating rotarod at the prepubertal and adult ages. Prenatal E. coli injection did not significantly change birth weight or delivery time, but did delay surface righting and negative geotaxis in pups. Pups in the E. coli group also had a decrease in the number of Purkinje cells, as well as a decrease in Purkinje cell density and volume. HE staining demonstrated a change in Purkinje cell morphology. Calbindin expression was decreased in rats from the E. coli group as well. Locomotor tests indicated that while there were no significant changes in gross motor activity, motor coordination and balance was impaired in both prepubertal and adult rats from the E. coli group. In this model of IUI, we observed changes in Purkinje cell development which were associated with alterations in cerebellum-dependent motor behaviors. The decreases in calbindin and Purkinje cells were associated with developmental delays. These data further support the importance of IUI in brain development.

Gliogenesis and glial pathology in depression.

Recent research has changed the perception of glia from being no more than silent supportive cells of neurons to being dynamic partners participating in brain metabolism and communication between neurons. This discovery of new glial functions coincides with growing evidence of the involvement of glia in the neuropathology of mood disorders. Unanticipated reductions in the density and number of glial cells are reported in fronto-limbic brain regions in major depression and bipolar illness. Moreover, age-dependent decreases in the density of glial fibrillary acidic protein (GFAP) - immunoreactive astrocytes and levels of GFAP protein are observed in the prefrontal cortex of younger depressed subjects. Since astrocytes participate in the uptake, metabolism and recycling of glutamate, we hypothesize that an astrocytic deficit may account for the alterations in glutamate/GABA neurotransmission in depression. Reductions in the density and ultrastructure of oligodendrocytes are also detected in the prefrontal cortex and amygdala in depression. Pathological changes in oligodendrocytes may be relevant to the disruption of white matter tracts in mood disorders reported by diffusion tensor imaging. Factors such as stress, excess of glucocorticoids, altered gene expression of neurotrophic factors and glial transporters, and changes in extracellular levels of neurotransmitters released by neurons may modify glial cell number and affect the neurophysiology of depression. Therefore, we will explore the role of these events in the possible alteration of glial number and activity, and the capacity of glia as a promising new target for therapeutic medications. Finally, we will consider the temporal relationship between glial and neuronal cell pathology in depression.

Neuroprotection by memantine against neurodegeneration induced by beta-amyloid(1-40).

Progressive neuronal loss and cognitive decline in Alzheimer's disease (AD) might be aggravated by beta-amyloid-enhanced excitotoxicity. Memantine is an uncompetitive NMDA receptor antagonist under clinical development for the treatment of AD. Memantine has neuroprotective actions in several in vitro and in vivo models. In the present study, we determined whether memantine protected against beta-amyloid induced neurotoxicity and learning impairment in rats. Twenty Sprague-Dawley rats received vehicle or vehicle plus memantine (steady-state plasma concentrations of 2.34+/-0.23 microM, n=10) s.c. by osmotic pump for 9 days. After 2 days of treatment, 2 microl of water containing beta-amyloid 1-40 [Abeta(1-40)] were injected into the hippocampal fissure. On the ninth day of treatment, animals were sacrificed, and morphological and immunohistochemical techniques were used to determine the extent of neuronal degeneration and astrocytic and microglial activation in the hippocampus. Psychomotor activity and spatial discrimination were tested on the eighth day of treatment. Abeta(1-40), but not water, injections into hippocampus led to neuronal loss in the CA1 subfield, evidence of widespread apoptosis, and astrocytic and microglial activation and hypertrophy. Memantine treated animals had significant reductions in the amount of neuronal degeneration, pyknotic nuclei, and GFAP immunostaining as compared with vehicle treated animals. These data suggest that memantine, at therapeutically relevant concentrations, can protect against neuronal degeneration induced by beta-amyloid.

SB-271046 (SmithKline Beecham).

SmithKline Beecham is developing the 5-HT6 antagonist, SB-271046, as a potential cognition enhancer. By December 1999, phase I trials had commenced [360354]. This drug was originally being developed primarily for the treatment of shizophrenia [284490], however, cognitive disorders, including but not limited to Alzheimer's disease, have been the main target since 1998 [394309]. SB-271046 is a potent, selective 5-HT6 antagonist with a pKi value of 8.9 [333710]. SB-258585, also known as 4-iodo-N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]benzenesulfonamide is an analog of SB-271046 [322488]. Data recently presented at the Society for Neuroscience annual meeting in November 2000 demonstrated that administration of SB-271046 resulted in a signficant increase in glutamate and aspartate levels in the frontal cortex, without affecting noradrenaline, dopamine or 5-HT levels. This was stated to suggest that 5-HT6 antagonists might therefore be useful for treating cognitive dysfunction [390469]. The drug has also been radiolabeled in order to provide an assay for estimating in vivo 5-HT6 receptor occupancy [390470].

Glial fibrillary acidic protein immunoreactivity in the prefrontal cortex distinguishes younger from older adults in major depressive disorder.

BACKGROUND: Recent postmortem studies in major depressive disorder (MDD) provide evidence for a reduction in the packing density and number of glial cells in different regions of the prefrontal cortex; however, the specific types of glia involved in those morphologic changes are unknown. METHODS: The territory occupied by the astroglial marker glial fibrillary acidic protein (GFAP) was measured as an areal fraction in cortical layers III, IV, and V in sections from the dorsolateral prefrontal cortex (dlPFC) of MDD and control subjects. In addition, the packing density of GFAP-immunoreactive somata was measured by a direct three-dimensional cell counting method. RESULTS: The mean areal fraction and packing density of GFAP-immunoreactive astrocytes in the dlPFC of MDD subjects were not significantly different from those in control subjects; however, in MDD there was a significant strong positive correlation between age and GFAP immunoreactivity. When the MDD group was divided into younger (30-45 years old) and older (46-86) adults, in the five younger MDD adults, areal fraction and packing density were smaller than the smallest values of the control subjects. In contrast, among older MDD subjects these parameters tended to be greater than in the older control subjects. CONCLUSIONS: The present results suggest that the GFAP-immunoreactive astroglia is differentially involved in the pathology of MDD in younger compared with older adults.

Immunohistochemistry of neural markers for the study of the laminar architecture in celloidin sections from the human cerebral cortex.

Morphometric studies of the cerebral cortex in celloidin sections provide reliable quantitative estimates of cytoarchitectural features in individual brain regions. To increase our knowledge about the morphology and distribution of neuronal and glial cell types using specific cellular markers, we compared two methods of celloidin removal/antigen recovery, and subsequent immunohistochemical staining of free floating sections with specific antibodies. The method based on methanol and NaOH for celloidin removal was the most adequate for optimal recovery of immunoreactivity of the neural markers NF200, MAP2, GFAP, calretinin, parvalbumin, calbindin-D28kD, and synaptophysin. The other method, based on a treatment with ethanol/ether and formic acid, gave good results in the immunostaining of NF200, GFAP and MAP2, but not the other markers named above. The immunostained sections were compared with nearby sections stained with cresyl violet in order to assign the immunoreactive structures to individual layers in the prefrontal cortex. Sections from blocks not embedded in celloidin showed a comparable distribution of all the antigens included in the present study. The present paper provides an antigen recovery technique for celloidin sections that can be applied to optimize studies on the cytoarchitecture and distribution of specific neural elements in the human cerebral cortex.

Histone H1(o) expression in the developing cat retina.

Histone H1(o) is a subtype of the non-core H1 histones located in the linker region of DNA between nucleosome cores and postulated to be involved in the regulation of gene expression. Studies in both the mouse retina and rat brain have correlated the terminal differentiation of cell types in these tissues to the expression of H1(o)a The expression of H1(o) in mouse retina occurs after light exposure suggesting that light may trigger the expression of H1(o). The aims of the present research were to: (1) describe the relationship of the appearance of H1(o) protein immunoreactivity to the formation of cell types and layers in the cat retina; and (2) determine whether H1(o) may be dependent on exposure to light or on other postnatal developmental events. We find the nuclei of ganglion, amacrine, and prospective bipolar cells contain H1(o) immunoreactivity before birth, prior to the terminal differentiation of these cells. In the cat retina, expression of H1(o) occurs prior to light exposure. These results show that the expression of H1 degrees protein is not required for the terminal differentiation of retinal cell types in the cat. Additionally, we find no requirement for light exposure prior to H1(o) expression. These findings are at variance with the findings in the mouse retina and are inconsistent with any cross species requirement for the expression of this histone in the terminal differentiation of cell types in the retina.

Morphometric evidence for neuronal and glial prefrontal cell pathology in major depression.

BACKGROUND: This report provides histopathological evidence to support prior neuroimaging findings of decreased volume and altered metabolism in the frontal cortex in major depressive disorder. METHODS: Computer-assisted three-dimensional cell counting was used to reveal abnormal cytoarchitecture in left rostral and caudal orbitofrontal and dorsolateral prefrontal cortical regions in subjects with major depression as compared to psychiatrically normal controls. RESULTS: Depressed subjects had decreases in cortical thickness, neuronal sizes, and neuronal and glial densities in the upper (II-IV) cortical layers of the rostral orbitofrontal region. In the caudal orbitofrontal cortex in depressed subjects, there were prominent reductions in glial densities in the lower (V-VI) cortical layers that were accompanied by small but significant decreases in neuronal sizes. In the dorsolateral prefrontal cortex of depressed subjects marked reductions in the density and size of neurons and glial cells were found in both supra- and infragranular layers. CONCLUSIONS: These results reveal that major depression can be distinguished by specific histopathology of both neurons and glial cells in the prefrontal cortex. Our data will contribute to the interpretation of neuroimaging findings and identification of dysfunctional neuronal circuits in major depression.

Neuroprotective role of S12024 against neurodegeneration in the rat dentate gyrus.

We assessed the neuroprotective capabilities of S12024 (R,S 1-methyl 8-(2-morpholinylmethoxy)-1,2,3,4-tetrahydroquinoleine methane sulphonate) in a model of neuronal degeneration in the dentate gyrus of the rat hippocampus. Specific degeneration of a large part of neurons in the lateral blade of the gyrus dentatus occurred after small intrahippocampal injections of water with or without amyloid-beta 1-28 fragment. S12024 reduced the number of animals with neuronal loss in the hippocampus, diminished the extent of the lesion, and reversed deficits of passive avoidance learning acquisition in animals with deposits of amyloid-beta 1-28. These results suggest that S12024 has neuroprotective effects on hippocampal cells and that the neurodegeneration by fluid injection combined with deposit of amyloid-beta 1-28 may be used to assay the neuroprotective activity of pharmacological compounds.

Plasticity of Congo red staining displayed by subpopulations of neurons within the rat central nervous system.

We document the presence of subpopulations of neurons within the rat central nervous system that are labelled with a new Congo red staining technique. These neurons (CR neurons) show shrunken somata, and smaller and darker nuclei than Congo red-negative cells (non-CR cells). With the Bielschowsky and the cresyl violet Nissl staining methods, two comparable subpopulations of cells can be distinguished by the same morphometrical criteria as those used for CR and non-CR cells. CR neurons are located preferentially in some brain regions while in others they are virtually absent. Their distribution and proportion varied greatly from animal to animal and after particular treatments. Injections of water that damaged the hippocampal dentate gyrus, cortical lesions or eye enucleation decreased the number of CR-cells in the CA1 subfield, reflected in a shift from the CR-staining subclass to the non-CR subclass. Treatment with 200 mg/kg of CDP-choline also significantly reduced the number of CR cells observed in CA1. In the red nucleus, CR neurons showed a characteristic distribution of beta-amyloid precursor protein (APP) immunoreactivity. The population of dendrites immunolabelled for microtubule-associated protein 2 was markedly decreased in the areas of the hippocampus with high numbers of CR cells. Therefore, it is proposed that neurons labelled with the present Congo red technique might be in a reversible degenerative state or represent a particular physiological state in some areas of the central nervous system.

Beta-amyloid(1-40)-induced neurodegeneration in the rat hippocampal neurons of the CA1 subfield.

Small volumes of solutions injected into the hippocampus produce dramatic degeneration in dentate gyrus neurons, but not in neurons of the CA1 subfield. The aim of the present study was to ascertain whether solutions with different fragments of the beta-amyloid protein (Abeta) could produce further degeneration in areas beyond the dentate gyrus. It was found that 5 days after injection of an aqueous solution containing the Abeta 1-40 fragment into the hippocampus, long stretches of the CA1 subfield were either deprived of neurons or most of the neurons were degenerating. By contrast, in animals with deposits containing Abeta 1-28, Abeta 1-42 or water, neuronal degeneration or depletion only occurred in a reduced area around the place where the implant needle penetrated the CA1 subfield. In animals injected with Abeta 1-40, many profiles in the CA1 subfield and dentate gyrus were undergoing apoptosis, as seen using preparations processed by routine histology or the TUNEL technique for detection of fragmented DNA. In addition, there was higher infiltration by ED1-positive, activated microglia-macrophagic cells in Abeta 1-42 deposits than in deposits of Abeta 1-40. The present results suggest that the intrahippocampal injection of toxic Abeta fragments produces neuronal degeneration in the rat CA1 subfield when using the appropriate protocol, and, thus, can provide an in vivo model to investigate the neurotoxic effects of Abeta and for the evaluation of drugs with potential anti-neurodegenerative activity.

Intrahippocampal injections of the beta-amyloid 1-28 fragment induces behavioral deficits in rats.

beta-amyloid (beta A) deposition is a key event in the etiopathogenesis of Alzheimer's disease (AD), contributing to neuronal degeneration and cognitive impairment in AD patients. Both neurotrophic and neurotoxic actions of beta A have been demonstrated in experimental conditions. In order to further characterize the effects of brain beta A deposits on behavioral processes, we evaluated psychomotor activity (PMA), psychomotor coordination (PMC) and learning in a passive avoidance task (PAL) in rats with unilateral or bilateral 2 microliters injections of beta-amyloid (1-28) protein (beta A; 1.5 nmol/microliter) or vehicle (water; W) into the hippocampus, 1 and 4 weeks after neurosurgery. The extent of neuronal loss in the lateral blade of the gyrus dentatus (LBGD) and the area percentage occupied by APP immunoreactivity in neurons of the CA3c subfield of the hippocampus were also measured in animals with unilateral beta A implants. PMA levels were similar in water- and beta A-injected animals 1 and 4 weeks after recovery. As compared to water-injected rats, beta A animals showed reduced PMC values 1 week, but not 4 weeks, after injections. beta A also impaired learning acquisition in a passive avoidance task, reducing the number of avoidances and mean latency per trial at both 1 and 4 weeks postsurgery in rats with unilateral or bilateral beta A implants. The extent of neuronal loss in the LBGD) was not different in rats receiving water or beta A injections. Hippocampal APP expression tended to increase in beta A-implanted rats and showed a negative correlation with cognitive performance at the 4-week period. According to these results it seems that beta A implants into the hippocampus reduce psychomotor coordination performance in a transient manner, with no effect on psychomotor activity, and induce durable learning impairment in rats, and that changes in cognitive performance correlate with histochemical parameters such as APP expression. In conclusion, the present results contribute to a better understanding of beta A-induced behavioral alterations and to the identification of potential molecular mechanisms involved in cognitive dysfunctions in this animal model of neurodegeneration.

Effects of anapsos on the activity of the enzyme Cu-Zn-superoxide dismutase in an animal model of neuronal degeneration.

The enzyme Cu-Zn-SOD is a metalloenzyme that catalyzes the dismutation of the superoxide radical into hydrogen peroxide and molecular oxygen, being a defense system against free radical formation. Free radical reactions are implicated in a variety of physiological and pathological processes as aging, apoptosis and neurodegenerative diseases, and abnormalities associated with SOD have been recently documented in several neurodegenerative processes. In this study, we have evaluated the effect of anapsos on Cu-Zn-SOD activity in rats with injections of beta-amyloid protein or water bilaterally into the hippocampus. These injections caused severe cell depletion in the gyrus dentatus. Anapsos is a biological extract obtained from the fern Polypodium leucotomos with immunomodulatory and anti-neoplastic effects tested in animals and humans. Cu-Zn-SOD activity was measured in the hypothalamus, hippocampus, cerebral cortex, liver and spleen of rats treated i.p. with three doses of anapsos for 7 days (4, 20 and 100 mg/kg/day). Control animals were treated with saline solution under the same conditions. Anapsos significantly modified enzyme activity in all the areas tested. Lower doses of anapsos produced decreased SOD activity in the hypothalamus, hippocampus, liver and spleen, while in the cerebral cortex, a significant dose-dependent increase in SOD activity was observed. These results indicate that anapos was able to modify Cu-Zn-SOD activity in this animal model of neuronal degeneration, which may indicate the participation of anapsos in mechanisms of tissue repair after brain damage.

Distinct temporal patterns of expression of sodium channel-like immunoreactivity during the prenatal development of the monkey and cat retina.

Polyclonal and monoclonal antibodies prepared against the alpha-subunit of the voltage-gated sodium channel (alpha NaCh) were used to examine the distribution of sodium channel-like immunoreactivity during the prenatal development of the cat and rhesus monkey (Macaca mulatta) retina. At all prenatal ages studied, beginning on embryonic day 29 (E29) in the cat and E52 in the monkey, both antibodies labelled optic axons. With the polyclonal antibodies, the appearance of positive cells largely mirrored the onset of their morphological maturation. Immunoreactivity appeared first in the somata of ganglion cells, and subsequently the inner plexiform layer could be distinguished by its intense immunolabelling. A few weeks later horizontal cells displayed immunolabelling that extended to their dendrites in the developing outer plexiform layer. This was followed by immunoreactive cones, with bipolar cells labelled only postnatally. By contrast, with the monoclonal antibody some cells were found to be immunoreactive while their somata were still in the ventricular layer (E33 in cat and E52 in monkey). Many of these cells appeared to migrate to the outer portion of the prospective inner nuclear layer, where they gradually acquired the morphological appearance of bipolar cells. Transient expression of immunolabelling with monoclonal sodium channel antibody was found in the cones of the cat and cones and rods of the monkey. These results indicate that different types of alpha NaCh-like proteins are expressed in the mammalian retina at distinct developmental periods. Their presence at very early stages during development suggests that these proteins could play a specific role in the commitment and/or differentiation of specific retinal cell types.

Voltage-dependent sodium channel alpha subunit immunoreactivity is expressed by distinct cell types of the cat and monkey retina.

Polyclonal (7493 and 7317) and monoclonal (mAb3) antibodies, generated to the alpha subunit of the voltage-gated sodium channel (alpha NaCh), were employed to assess the cell types containing alpha NaCh-like immunoreactivity in the mature cat and monkey retina. Immunoblot analyses of retinal proteins in the cat revealed that the polyclonal and monoclonal antibodies we employed labeled a band in the 260-kDa region which corresponds to the molecular mass of the alpha subunit of the NaCh. In both the cat and monkey, these antibodies immunolabeled several distinct types of retinal cells. With the polyclonal antibodies immunoreactivity was observed in ganglion cells and their intraretinal axons, in horizontal cells, and unexpectedly, in cones. In addition, in both species, a limited number of heavily labeled profiles, presumed to be bipolar cells, were seen in the inner nuclear layer. In cat and monkey the monoclonal antibody labeled axons in the fiber layer, ganglion cell somata, and a continuous band of immunoreactive cell bodies (presumed bipolar cells) situated in the outer half of the inner nuclear layer. By immunolabeling isolated cells dissociated from the cat retina, it was possible to demonstrate unequivocally that a population of bipolar cells was labeled by the monoclonal and the polyclonal antibodies we employed. The differences in the labeling observed with the monoclonal antibody as compared to the polyclonal antibodies were interpreted as reflecting the presence of different alpha-subunit subtypes in the mammalian retina. Collectively, our findings suggest that alpha NaCh-like proteins are expressed by a more diverse population of retinal cells than expected on the basis of previous physiological and immunohistochemical studies.

Projections of tachykinin- and glutaminase-containing rat retinal ganglion cells.

Glutamate (Glu) and the tachykinin substance P (SP) have been proposed as neurotransmitters or neuromodulators of the retinal projection to the brain. In the present study, we demonstrate that tachykinin-like (TK) immunoreactivity (IR) accumulates in rat retinal axons following electrical lesions to the optic tract, indicating that SP is conveyed in the optic nerve to its central targets. In addition, we show that eye enucleation causes a dramatic decrease in TK-IR fibers in the pretectal olivary nucleus (PON), but not in other retinorecipient nuclei of the thalamus and the midbrain, and that Fluorogold injected into the pretectum is retrogradely transported to the somata of TK-IR retinal ganglion cells (RGCs), indicating an important projection of TK-IR RGCs to the PON. We also show that most rat RGCs are labeled with antibodies against phosphate-activated glutaminase, an enzyme considered to generate the transmitter pool of glutamate. Unlike TK-IR fibers, phosphate-activated glutaminase-IR structures disappear in most retinorecipient nuclei following eye enucleation. The present results give neuroanatomical support to the idea that glutamate is a neurotransmitter in the retinal projection and suggest an important role for TK-IR RGCs in the relay of visual information to the PON.

Substance P-immunoreactive innervation from the retina to the suprachiasmatic nucleus in the rat.

The present study examined substance P (SP) innervation in the suprachiasmatic nucleus (SCN) of the rat. In the colchicine-untreated rat, SP-immunoreactive fibers formed a dense oval plexus in the ventral part of the SCN. After bilateral eye enucleation, there was a marked reduction in SP-immunoreactive fibers in the ventral part of the SCNs. The SP-immunoreactive neurons in the retinal ganglion cell layer were retrogradely labeled after injection of Fluoro-gold into the SCN. These findings indicate the presence of the SP innervation from the retina to the SCN in the rat. The role of SP in the retino-hypothalamic tract was discussed from the light-dark cycle.

Ultrastructure and retinal innervation of deafferentation-induced enkephalin-immunoreactive elements in the superficial layers of the rat superior colliculus.

Leu-enkephalin-like immunoreactive (ENK-I) elements appearing in the superficial layers of the rat superior colliculus (SC) after eye-enucleation were examined by means of immunoelectronmicroscopy. ENK-I somata were of a single type and formed symmetric and asymmetric synapses with non-immunoreactive axon terminals. Some degenerating retinal terminals made synaptic contacts only with small ENK-I dendrites, suggesting that deafferentation-induced ENK-I neurons in the rat SC receive retinal input onto the distal portions of their dendrites.