The role of P2Y12 in the kinetics of microglial self-renewal and maturation in the adult visual cortex in vivo.

Microglia are the brain's resident immune cells with a tremendous capacity to autonomously self-renew. Because microglial self-renewal has largely been studied using static tools, its mechanisms and kinetics are not well understood. Using chronic in vivo two-photon imaging in awake mice, we confirm that cortical microglia show limited turnover and migration under basal conditions. Following depletion, however, microglial repopulation is remarkably rapid and is sustained by the dynamic division of remaining microglia, in a manner that is largely independent of signaling through the P2Y12 receptor. Mathematical modeling of microglial division demonstrates that the observed division rates can account for the rapid repopulation observed in vivo. Additionally, newly born microglia resemble mature microglia within days of repopulation, although morphological maturation is different in newly born microglia in P2Y12 knock out mice. Our work suggests that microglia rapidly locally and that newly born microglia do not recapitulate the slow maturation seen in development but instead take on mature roles in the CNS.

A Mouse Model for Conditional Secretion of Specific Single-Chain Antibodies Provides Genetic Evidence for Regulation of Cortical Plasticity by a Non-cell Autonomous Homeoprotein Transcription Factor.

During postnatal life the cerebral cortex passes through critical periods of plasticity allowing its physiological adaptation to the environment. In the visual cortex, critical period onset and closure are influenced by the non-cell autonomous activity of the Otx2 homeoprotein transcription factor, which regulates the maturation of parvalbumin-expressing inhibitory interneurons (PV cells). In adult mice, the maintenance of a non-plastic adult state requires continuous Otx2 import by PV cells. An important source of extra-cortical Otx2 is the choroid plexus, which secretes Otx2 into the cerebrospinal fluid. Otx2 secretion and internalization requires two small peptidic domains that are part of the DNA-binding domain. Thus, mutating these "transfer" sequences also modifies cell autonomous transcription, precluding this approach to obtain a cell autonomous-only mouse. Here, we develop a mouse model with inducible secretion of an anti-Otx2 single-chain antibody to trap Otx2 in the extracellular milieu. Postnatal secretion of this single-chain antibody by PV cells delays PV maturation and reduces plasticity gene expression. Induced adult expression of this single-chain antibody in cerebrospinal fluid decreases Otx2 internalization by PV cells, strongly induces plasticity gene expression and reopens physiological plasticity. We provide the first mammalian genetic evidence for a signaling mechanism involving intercellular transfer of a homeoprotein transcription factor. Our single-chain antibody mouse model is a valid strategy for extracellular neutralization that could be applied to other homeoproteins and signaling molecules within and beyond the nervous system.

The expression patterns of MHC class I molecules in the developmental human visual system.

It has been considered that healthy neurons in central nervous system (CNS) do not express major histocompatibility complex (MHC) class I molecules. However, recent studies clearly demonstrated the expression of functional MHC class I in the mammalian embryonic, neonatal and adult brain. Until now, it is still unknown whether MHC I molecules are expressed in the development of human brain. We collected nine human brain tissues from fetuses aged from 21 to 31 gestational weeks (GW), one newborn of postnatal 55 days and one adult. The expression of MHC class I molecules was detected during the development of visual system in human brain by immunohistochemistry and immunofluorescence. MHC class I proteins were located at lateral geniculate nucleus (LGN) and the expression was gradually increased from 21 GW to 31 GW and reached high levels at 30-31 GW when fine-scale refinement phase was mediated by neural electric activity. However, there was no expression of MHC class I molecules in the visual cortical cortex during all the developmental stages examined. We also concluded that MHC class I molecules were mainly expressed in neurons but not in astrocytes at LGN. In the developing visual system, the expression of ß2M protein on neurons was not found in our study.

MHC class I molecules are present both pre- and postsynaptically in the visual cortex during postnatal development and in adulthood.

Immune molecules have been discovered recently to play critical roles in the development, function, and plasticity of the cerebral cortex. MHC class I (MHCI) molecules are expressed in the central nervous system and regulate activity-dependent refinement of visual projections during late postnatal development. They have also been implicated in neurodevelopmental diseases such as schizophrenia and autism. Despite the excitement generated by these unique roles for immune proteins in the brain, little is known about how these molecules regulate cortical connections. The first step toward elucidating the mechanism is to identify the spatial and temporal distribution of MHCI proteins throughout development. Using a pan-specific antibody that recognizes many MHCI variants for biochemistry and immunohistochemistry, we found that MHCI proteins are expressed in the rat visual cortex at all ages examined-during the peak of synaptogenesis, the critical period of synaptic refinement, and adulthood. Their abundance in the cortex peaked during early postnatal development, declining during periods of plasticity and adulthood. In contrast to current assumptions, pre- and postembedding immunogold electron microscopy (EM) revealed that MHCI proteins were present both pre- and postsynaptically at all ages examined. They were often found in the postsynaptic density and were closely associated with synaptic vesicles in the presynaptic terminal. These results suggest a previously undescribed model in which MHCI molecules function on both sides of the synapse to regulate connectivity in the mammalian visual cortex before, during, and after the establishment of connections.

Expression of inflammatory genes in the primary visual cortex of late-stage Alzheimer's disease.

Alzheimer's disease is associated with progressively dysfunctional gene expression in the limbic system of the brain. The thalamus and primary visual cortex are thought to be initially spared of Alzheimer-type changes that ravage the association neocortex. In this study, using DNA arrays and Western immunoassay, gene expression patterns were examined in the thalamus and primary visual cortex of moderate-stage and late-stage Alzheimer's disease and age-matched controls using a set of proinflammatory genes known to be upregulated in the temporal lobe neocortex and hippocampus of moderate-stage Alzheimer's disease. The data indicate that, in late-stage Alzheimer's disease, proinflammatory and proapoptotic gene expression spreads into the primary visual sensory cortex. This upregulation of pathological gene expression could be, in part, responsible for the visual disturbances associated with end-stages of the Alzheimer process.

A possible role for humoral immunity in the pathogenesis of Parkinson's disease.

The pathogenesis of idiopathic Parkinson's disease is unknown, but nigral degeneration and depigmentation are associated with microglial inflammation and anti-inflammatory medications appear to protect against the disease. The possibility that humoral immunity may play a role in initiating or regulating the inflammation has been suggested by experimental studies triggering dopamine cell death using a variety of transfer strategies and the observation of CD8+ T lymphocytes and complement in the nigra in Parkinson's disease. We analysed the association between degeneration and humoral immune markers in brain tissue of patients with idiopathic (n = 13) or genetic (n = 2 with alpha-synuclein and n = 1 with parkin mutations) Parkinson's disease and controls without neurological disease (n = 12) to determine the humoral immune involvement in Parkinson's disease. Formalin-fixed tissue samples from the substantia nigra and primary visual cortex for comparison were stained for alpha-synuclein, major histocompatibility complex II (HLA), immunoglobulin M (IgM), immunoglobulin G (IgG), IgG subclasses 1-4 and IgG receptors FcgammaR I-III. Antigen retrieval and both single immunoperoxidase and double immunofluorescence procedures were employed to determine the cell types involved and their pattern and semiquantitative densities. Significant dopamine neuron loss occurred in all patients with Parkinson's disease, negatively correlating with disease duration (r = -0.76, P = 0.002). Although all patients had increased inflammatory HLA immunopositive microglia, the degree of inflammation was similar throughout the disease (r = 0.08, P = 0.82). All patients with Parkinson's disease had IgG binding on dopamine neurons but not IgM binding. Lewy bodies were strongly immunolabelled with IgG. A mean 30 +/- 12% of dopamine nigral neurons were immunoreactive for IgG in Parkinson's disease with the proportion of IgG immunopositive neurons negatively correlating with the degree of cell loss in the substantia nigra (r = -0.67, P < 0.0001) and positively correlating with the number of HLA immunopositive microglia (r = 0.51, P = 0.01). Most neuronal IgG was the IgG1 subclass with some IgG3 and less IgG2 also found in the damaged substantia nigra. The high affinity activating IgG receptor, FcgammaRI, was expressed on nearby activated microglia. The low affinity activating IgG receptor, FcgammaRIII was expressed on cells morphologically resembling lymphocytes, whereas immunoreactivity for the inhibitory IgG receptor FcgammaRII was absent in all cases. This pattern of humoral immune reactivity is consistent with an immune activation of microglia leading to the targeting of dopamine nigral neurons for destruction in both idiopathic and genetic cases of Parkinson's disease.

c-Fos expression in the rat cerebral cortex during systemic GvH reaction.

OBJECTIVE: It is becoming clear that the CNS receives signals from the peripheral immune system. In order to identify the areas of the brain that receive information about a specific immune response to allogeneic antigens, we studied the expression of c-Fos, a neural activation marker, in the cerebral cortex following the induction of a graft-vs.-host reaction (GvHR) in rats. METHODS: C-Fos expression in the brain was studied by immunohistochemistry. GvHR was induced in (WKY x PVG)F(1) rats by injecting 5 x 10(8) spleen cells from PVG rats. Control rats received syngeneic cells. RESULTS: No c-Fos immunoreactivity (IR) was observed in animals undergoing GvHR in the nucleus tractus solitarii (NTS), the locus coeruleus (LC), the organum vasculosum of lamina terminalis (OVLT), the paraventricular nucleus (PVN) or the central amygdaloid nucleus (Ce). In contrast, 3 days after GvH induction c-Fos IR was observed in the piriform cortex and several other olfactory-related regions indicating the stimulation of the olfactory pathway during GvHR. Strong c-Fos IR was also observed in the occipital visual cortex of animals undergoing a GvHR, suggesting that GvHR can affect visual functions. In addition, GvHR induced c-Fos IR in the prefrontal cortex (Cg3, orbital cortex), a region that has interconnections with most sensory modalities. Double-staining studies indicate that the cells that express the c-Fos signal are neurons. CONCLUSION: We have defined the distribution of brain neurons that are affected during the induction phase of GvHR. Our results also indicate that the integration and processing of information from the immune system at CNS levels involve different areas during different types of immune responses.

The distribution and morphology of calbindin D28K- and calretinin-immunoreactive neurons in the visual cortex of mouse.

We studied the distribution and morphology of calbindin D28K- and calretinin-immunoreactive (IR) neurons in the mouse visual cortex with immunocytochemistry. Most of the calbindin D28K-IR neurons were located in layers II/III and V, while calretinin-IR neurons were predominantly located in layers II/III. The labeled neurons showed variations in morphology. The majority of the calbindin D28K-IR neurons were stellate and round or oval cells with multipolar dendrites. The majority of calretinin-IR neurons were vertical fusiform cells with long processes traveling perpendicular to the pial surface. In the mouse visual cortex, 20.2% of calbindin D28K-IR neurons contained calretinin and 27.2% of calretinin-IR neurons contained calbindin D28K. These results indicate that the calcium-binding proteins, calbindin D28K and calretinin are distributed in specific layers and in selective cell types of the mouse visual cortex.

Regulation of calcium-binding protein immunoreactivity in GABA neurons of macaque primary visual cortex.

Monocular deprivation produces an imbalance in visual drive from the two eyes, which in adult macaque V1 leads to marked changes in the neurochemistry of GABA interneurons. Such changes were further examined by studying immunoreactivity for calbindin, calretinin, and parvalbumin, three calcium-binding proteins that mark distinct subpopulations of GABA neurons, in macaques that had been monocularly deprived by intravitreal injection of tetrodotoxin. Deprivation for 5 d or longer produced a reversal in the normal pattern of calbindin immunostaining in layer III, from one in which intense neuronal immunostaining surrounded the cytochrome oxidase-rich puffs to one in which it occupied the puffs. Over the same period, calbindin immunostaining in other layers was reduced across the entire width of deprived-eye columns or extended into flanking regions of normal-eye columns. In contrast, reduction in parvalbumin immunostaining occurred only in deprived-eye columns and included only terminals with short periods of deprivation (up to 17 d) but both terminals and somata with longer periods. No change in calretinin immunoreactivity was observed. These findings demonstrate that GABA neurons of macaque V1 vary in their response to monocular deprivation according to their neurochemistry and position, suggesting that the weight of inputs from the two eyes and the intrinsic characteristics of each GABA population determine how a neuron responds to a change in visual input.

Transient immunoglobulin-like molecules are present in the subplate zone and cerebral cortex during postnatal development.

A monoclonal antibody (mAb SP-1) labels subplate neurons of the cat visual cortex but does not stain the remnants of the subplate neuronal population that comprise the interstitial cells of adult cortical white matter. mAb SP-1 was shown previously to recognize a cytosolic polypeptide of 56 kDa (Naegele et al., 1991). We have now characterized the distribution of SP-1 immunoreactive neurons in the visual cortex and carried out additional biochemical studies at a range of postnatal ages in various tissues. Brain, liver and serum were found to contain the previously identified 56 kDa polypeptide. This polypeptide was also recognized by a cat immunoglobulin antiserum. The epitope recognized by mAb SP-1 was present on cat IgG Fc fragment but not cat IgG Fab fragment. By 4 weeks postnatal, levels of the 56 kDa antigen decreased in cortex and an additional higher molecular weight SP-1 reactive polypeptide of 75 kDa was detected. In the mature cortex, both polypeptides were absent from cytosolic fractions. Immunocytochemical staining comparing the distributions of SP-1 (SP-1+) and anti-IgG (Ig+) immunoreactive neurons showed complete colocalization in subplate neurons beneath primary visual cortex. By 4 weeks, some pyramidal neurons in cortical areas 17 and 18 were weakly positive for SP-1 but negative for IgG. At subsequent ages, the immunoreactive staining became progressively fainter until it was no longer detectable in white or gray matter of adult cat visual cortex.

An immunohistochemical study of neurotransmitter profiles in developing human visual cortex.

The temporal pattern of development and distribution of gamma aminobutyric acid, serotonin, substance P and neuropeptide Y immunoreactive profiles was studied in the human visual cortex from 16 to 26 weeks of gestation, using an immunohistochemical technique. The immunoreactive profiles showed an increase in number and a change in their morphology and distribution pattern over the time period studied. A large number of neurons, fibers and terminals were stained with GABA antibody at 17-18 weeks and were distributed throughout the five zones of the developing visual cortex. GABA neurons were non-pyramidal and bipolar in form at 17-18 weeks while at 18-19 and 20-21 weeks the cells of subplate and intermediate zones were multipolar. Substance P and serotonin immunopositive fibers were present mainly in the intermediate zone at 16 and 17-18 weeks, where they were oriented in a horizontal manner. At subsequent ages they invaded the other zones also. Substance P positive neurons could be visualized only at 26 weeks of gestation in the intermediate, subventricular and ventricular zones; no cell bodies, however, stained with serotonin antibody. Neuropeptide Y immunoreactive cells and fibers were first seen in the intermediate zone but later were found to be distributed in other zones too. The observations indicate that the intermediate zone of the visual cortex in which the transmitters and peptides appear earlier assumes importance in the normal development as also noted in other mammals.

Human synaptic proteins with a heterogeneous distribution in cerebellum and visual cortex.

Synaptic pathology is likely to be an important feature of a number of neuropsychiatric illnesses. An antibody called EP10 was used previously to demonstrate a regional reduction in a 38 kDa synaptophysin-like protein in Alzheimer's disease. The SP antibodies were developed for further study of this and other synaptic proteins in human brain. Human brain proteins immunoprecipitated with EP10 were used as the immunogen. Hybridoma screening was carried out with a sequential ELISA-immunocytochemical approach. Sixteen antibodies were obtained, the antigens clustered into five groups. Five antibodies were reactive with a 38 kDa synaptophysin-like protein. Another two antibodies were reactive with a 16 kDa antigen which may be synaptobrevin. Immunocytochemical studies indicated these two antigens appeared to be co-localized in human brain. Four antibodies were reactive with a distinct, 34-36 kDa antigen. In the cerebellum, this antigen was restricted to terminals in the molecular layer, putatively in the parallel fibre synapses. Two antibodies were reactive with a 26-27 kDa antigen. In the cerebellum, this antigen localized to a subset of terminals which included the axo-axonal contacts of the Basket and Purkinje cells. The final group of three antibodies detected a complex group of 38 kDa. 40 kDa and higher molecular weight antigens. The results suggest that heterogeneity among synapses can be defined through antibodies directed against distinct proteins. The SP antibodies may be useful probes for studies of human synaptic proteins, and for studies of pathological conditions which disrupt these molecules.

Postnatal development of vasoactive intestinal polypeptide-containing neurons in the visual cortex of normal and dark-reared rats.

The effects of dark rearing on the distribution and density of vasoactive intestinal polypeptide (VIP)-containing neurons in the visual cortical areas (17, 18 and 18a) of rats during postnatal development were examined immunohistochemically. Two groups of Wistar rats, one reared under normal lighting conditions and the other in complete darkness from birth, were used. VIP neurons showed a fairly similar distribution in the three visual areas, being predominantly present in layers II and III. Their pattern of development was found to be similar in the normal and dark-reared animals and was characterized by a marked increase from postnatal day (P) 7 to P21, followed by a gradual diminution to 24-31% of peak densities. Counts of labeled neurons at all ages examined showed that their density was similar in both groups at P7 and P14, but progressively greater in dark-reared animals from P21 and thereafter, so that they only fell to 38-43% of peak densities. Thus, by 60 days of age densities of VIP-labeled neurons in areas 17, 18 and 18a in dark-reared rats were 57%, 49% and 51% higher than in the corresponding areas of the age-matched control rats. These results indicate that the normal decline in the numbers of VIP neurons is not so marked under the conditions of dark rearing.

Calretinin-immunoreactive neurons in the primary visual cortex of dolphin and human brains.

A new class of gamma-aminobutyric acid (GABA)ergic neurons immunoreactive to the calcium-binding protein calretinin (CR) was demonstrated in primary visual cortices of the bottlenose dolphin (Tursiops truncatus) and humans (Homo sapiens). Comparative analysis revealed several differences between dolphin and human visual cortex in the laminar distribution of CR-positive perikarya, although general typology of the immunoreactive CR-positive neurons was similar in both species. Thus, in both human and dolphin primary visual cortex almost all CR-positive neurons are non-pyramidal, either fusiform or bipolar cells, oriented with their long axis along the radial axis of the cortex. Large multipolar stellate cells were also observed in layers I and VI. The CR-positive neurons in the dolphin visual cortex are concentrated almost exclusively in layer I and, to a lesser extent, in layer II. In all other layers (IIIa, b, IIIc/V and VI) of the dolphin visual cortex CR-positive neurons were only rarely seen. In the human primary visual cortex CR-positive neurons are located mainly in layers II, III and IVa, b, c, with considerably lower densities of these cells observed in layers V and VI. CR-positive neurons in layer I of the human visual cortex are represented by Cajal-Retzius horizontal cells, whereas no such cells were seen in layer I of the dolphin neocortex. The numerical density of CR-positive neurons in the dolphin primary visual cortex is significantly lower than in the area of cortex in humans.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative distribution of GABA-immunopositive and -immunonegative neurons and synapses in the monkey striate cortex (area 17).

The number of GABA-immunoreactive [GABA(+)] neurons and synapses was determined in functionally distinct subregions delineated as rich and poor in cytochrome oxidase (CO) in the visual cortex of adult macaque monkeys. The average numerical density (number per unit volume, Nv) of GABA(+) neurons and synapses was not significantly different between the CO-rich and -poor regions. Twenty percent of the total number of cortical neurons and 17% of the synapses were GABA(+). On average, each visual cortical neuron receives 3900 synapses, 660 of them being GABA(+). The latter were distributed on the target cell in a pattern that predicts the site of GABA influences in cortex. The major targets of GABA(+) synapses were dendritic shafts, comprising nearly two-thirds of the postsynaptic elements. About every fourth and every eighth GABA(+) synapse was devoted to dendritic spines and to neuronal somata, respectively. Axon initial segments, although the exclusive targets of GABA(+) cells, comprise less than 0.1% of structures postsynaptic to GABA(+) boutons. From this distribution, we estimate that in each cubic millimeter of striate cortex there were about 20 million GABA(+) synapses on dendritic spines, 47 million on dendritic trunks, 9 million on somata, and fewer than 0.1 million on axon initial segments. The sites of influences of GABA-immunonegative [GABA(-)] synapses were different in that they target mainly dendritic spines and dendritic trunks. About two-thirds of GABA(-) synapses were on dendritic spines, and the remainder were devoted to dendritic trunks. Only a minute fraction innervate somata. We estimate that in 1 mm3 of striate cortex there were about 235 million GABA(-) synapses on spines, 133 million on dendrites, and about 2 million on somata. The proportions of GABA(+) neurons and synapses and their target distribution did not appreciably differ from those of the visual cortex of the cat even though the numerical density of neurons was 2.5 times higher in the monkey.

Morphology and laminar distribution of neuropeptide Y immunoreactive neurons in the human striate cortex.

The morphology, laminar distribution, and distribution relative to cytochrome oxidase patches of neuropeptide-Y immunoreactive (NPY-ir) neurons were studied in the human striate cortex. The density of NPY-ir cells was highest in the white matter. NPY-ir neurons were sparsely distributed within the cortical layers. NPY-ir neurons were located in both cytochrome oxidase dense patch and interpatch regions. However, the paucity of NPY-ir neurons in layer III, where cytochrome oxidase patches are most clearly demonstrated, precluded establishing a clear relationship of NPY-ir neurons to cytochrome oxidase patches. NPY-ir neurons exhibited a variety of nonpyramidal morphologies, and many of them had axons with recurrent or looped trajectories. A dense plexus of NPY-ir axons was located just beneath the pia, and these axons were concentrated at the entry points of pial blood vessels. Other NPY-ir neurons had cell bodies or processes in close proximity to cerebral capillaries. These results suggest a role of NPY in cortical metabolism, control of cerebral circulation, or activity-related changes in local blood flow.

Postnatal development of parvalbumin immunoreactivity in axon terminals of basket and chandelier neurons in monkey neocortex.

1. Two classes of GABAergic inhibitory interneurons, chandelier and basket cells, are known regulators of pyramidal neurons. Parvalbumin (PV) a calcium binding protein, has been shown to be a marker for axon terminals of subpopulations of these interneurons. 2. Immunohistochemical methods were used in this study to examine changes in the distribution of PV-immunoreactive (IR) chandelier and basket axon terminals during postnatal development of monkey neocortex. 3. Our results indicate a differential effect of postnatal development on PV-IR axon terminals of chandelier and basket neurons that is region-specific. 4. The differential regional, laminar and developmental pattern of PV-IR axon terminals of chandelier and basket cells may provide insight into the functional role of these classes of inhibitory neurons in primate neocortex.

Prenatal development of GABA-immunoreactive neurons in the human striate cortex.

The prenatal development of neurons immunoreactive to gamma-aminobutyric acid (GABA) in the striate cortex (area 17) of human foetuses aged from 14 weeks to term was studied immunocytochemically. In the 14 week foetus GABA-immunoreactive cells occurred in all layers of area 17 with the highest density in the marginal zone (MZ), subplate (SP), deep intermediate zone (IZ) and ventricular zone (VZ). The cortical plate (CP), which gives rise to most of the definitive adult cortical layers, had relatively low concentrations of GABAergic cells. By 17 weeks the density in the proliferative VZ had declined. At 20 weeks some of the adult layers were recognisable; the density of GABA-positive neurons was now highest in the definitive cortex, especially the deep layers (layers VI and V), was lower in the superficial cortical plate, and was lowest in IZ, where the white matter would form. The peak of GABA-immunoreactive neuronal density continued to move superficially during development, and was in layer IVc by 30 weeks. The laminar distribution stabilised from 30 weeks with three dense bands: in layer IVc and superficial V, layer IVa, and layers II and superficial III. The tangential distribution of GABAergic neurons was determined in two older brains (32 and 39 weeks) and no unequivocal spatial periodicity was observed in this plane. The mean cross-sectional area of GABAergic neurons in area 17 increased with foetal age, and also increased from superficial to deep layers at each age. Most GABA-immunoreactive neurons in younger brains contained immunonegative or weakly positive nuclei and had few visible processes, while in the older brains most neurons contained positive nuclei and had more visible processes. The proportion of GABA-immunoreactive bipolar cells declined during development while that of multipolar cells increased. GABAergic neurons thus differentiate early in human foetal striate cortex. They are initially most numerous in the proliferative layers deep to the developing definitive cortex; from 20 weeks of gestation, their peak moves superficially into the maturing deep layers (VI and V) and a stable laminar distribution is attained by 30 weeks, with peaks in layers II/IIIm, IVa and IVc/V. There is no obvious horizontal periodic distribution before term.