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BackgroundWhen the homeostasis of the central nervous system (CNS) is altered, microglial cells become activated displaying a wide range of phenotypes that depend on the specific site, the nature of the activator, and particularly the microenvironment generated by the lesion. Cytokines are important signals involved in the modulation of the molecular microenvironment and hence play a pivotal role in orchestrating microglial activation. Among them, interleukin-6 (IL-6) is a pleiotropic cytokine described in a wide range of pathological conditions as a potent inducer and modulator of microglial activation, but with contradictory results regarding its detrimental or beneficial functions. The objective of the present study was to evaluate the effects of chronic IL-6 production on the immune response associated with CNS-axonal anterograde degeneration.MethodsThe perforant pathway transection (PPT) paradigm was used in transgenic mice with astrocyte-targeted IL6-production (GFAP-IL6Tg). At 2, 3, 7, 14, and 21 days post-lesion, the hippocampal areas were processed for immunohistochemistry, flow cytometry, and protein microarray.ResultsAn increase in the microglia/macrophage density was observed in GFAP-IL6Tg animals in non-lesion conditions and at later time-points after PPT, associated with higher microglial proliferation and a major monocyte/macrophage cell infiltration. Besides, in homeostasis, GFAP-IL6Tg showed an environment usually linked with an innate immune response, with more perivascular CD11b+/CD45high/MHCII+/CD86+macrophages, higher T cell infiltration, and higher IL-10, IL-13, IL-17, and IL-6 production. After PPT, WT animals show a change in microglia phenotype expressing MHCII and co-stimulatory molecules, whereas transgenic mice lack this shift. This lack of response in the GFAP-IL6Tg was associated with lower axonal sprouting.ConclusionsChronic exposure to IL-6 induces a desensitized phenotype of the microglia.

Cortical regions of the brain stand out for their high content in synaptic zinc, which may thus be involved in synaptic function. The relative number, chemical nature and transmitter receptor profile of synapses that sequester vesicular zinc are largely unknown. To address this, we combined pre-embedding zinc histochemistry and post-embedding immunogold electron microscopy in rat hippocampus. All giant mossy fibre (MF) terminals in the CA3 region and approximately 45% of boutons making axospinous synapses in stratum radiatum in CA1 contained synaptic vesicles that stained for zinc. Both types of zinc-positive boutons selectively expressed the vesicular zinc transporter ZnT-3. Zinc-positive boutons further immunoreacted to the vesicular glutamate transporter VGLUT-1, but not to the transmitter gamma-aminobutyric acid. Most dendritic spines in CA1 immunoreacted to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) subunits GluR1-3 (approximately 80%) and to N-methyl-D-aspartate receptor (NMDAR) subunits NR1 + NR2A/B (approximately 90%). Synapses made by zinc-positive boutons contained 40% less AMPAR particles than those made by zinc-negative boutons, whereas NMDAR counts were similar. Further analysis indicated that this was due to the reduced synaptic expression of both GluR1 and GluR2 subunits. Hence, the levels of postsynaptic AMPARs may vary according to the presence of vesicular zinc in excitatory afferents to CA1. Zinc-positive and zinc-negative synapses may represent two glutamatergic subpopulations with distinct synaptic signalling.

The appearance and distribution of zinc-rich terminal fields in the rat forebrain was analyzed at 12 stages of postnatal development using the selenium method. Zinc stain was detected in neonates in piriform, cingulate, and motor cortices, septal area, and hippocampal formation. In the neocortex, a laminar pattern appeared progressively following an inside-out gradient: layer VI at postnatal day 0 (P0), layer V at P1, layers Va and Vb at P5, layer II–III at P9, and layer IV at P12. In the hippocampal formation the layered pattern in the dentate molecular layer appeared at P1–P3, and in the hilus and mossy fibers the stain was observed at P5. Patches in the caudate-putamen were sharply delimited at P1–P3. At these ages, staining was observed in the amygdaloid complex. In the thalamic and hypothalamic nuclei, stain appeared at P5–P7. Thus, a general increase in vesicular zinc over different telencephalic areas was determined until P15–P21, which was followed by a slight decrease thereafter (at P41). The increased stain in zinc-rich terminal fields is consistent with the development of telencephalic circuits. The rise in zinc might be relevant for the establishment and maturation of these circuits. On the other hand, the decrease in staining for zinc at later stages might be due to methodological problems but it might also reflect pruning of supernumerary connections and programmed cell death affecting zinc-rich circuits.

Mitochondrial fusion and fission is a dynamic process critical for the maintenance of mitochondrial function and cell viability. During excitotoxicity neuronal mitochondria are fragmented, but the mechanism underlying this process is poorly understood. Here, we show that Mfn2 is the only member of the mitochondrial fusion/fission machinery whose expression is reduced in in vitro and in vivo models of excitotoxicity. Whereas in cortical primary cultures, Drp1 recruitment to mitochondria plays a primordial role in mitochondrial fragmentation in an early phase that can be reversed once the insult has ceased, Mfn2 downregulation intervenes in a delayed mitochondrial fragmentation phase that progresses even when the insult has ceased. Downregulation of Mfn2 causes mitochondrial dysfunction, altered calcium homeostasis, and enhanced Bax translocation to mitochondria, resulting in delayed neuronal death. We found that transcription factor MEF2 regulates basal Mfn2 expression in neurons and that excitotoxicity-dependent degradation of MEF2 causes Mfn2 downregulation. Thus, Mfn2 reduction is a late event in excitotoxicity and its targeting may help to reduce excitotoxic damage and increase the currently short therapeutic window in stroke.

Infusion of sodium selenite to the occipital cortex of the rat was used for the specific tracing of zinc-rich pathways. Large numbers of labeled somata were found ipsilaterally in the visual, orbital and frontal cortices, and contralaterally in homotopic and heterotopic visual areas. Labeled neurons were also found ipsilaterally in the retrosplenial, parietal, sensory-motor, temporal and perirhinal cortex. In contrast to the cortico-cortical connections, ascending afferents to the visual cortex were not zinc-rich except for a few labeled neurons in the claustrum. Additional injections showed reciprocal zinc-rich connections between the visual cortex and the orbital and frontal cortices. The latter cortices also received ascending zinc-rich afferents from the claustrum. Selenite injections revealed the layered distribution and the morphology of these labeled neurons in the neocortex. Zinc-rich neurons were found in layers II–III, V and VI. However, none was found in layer IV. Zinc-rich somata appeared as pyramidal and inverted neurons. The contrasting chemical properties of cortical and subcortical visual afferents may account for the functional differences between these systems.

Female Zucker lean and obese rats were treated for 14 days with 3.5 μmol/kg oleoyl-estrone (OE) in liposomes (Merlin-2). After 0, 3, 6, 10, and 14 days of treatment, the rats were killed and hypothalamic nuclei (lateral preoptic, median preoptic, paraventricular, ventromedial and arcuate) were used for neuropeptide Y (NPY) radioimmunoassay. In 14 days, OE decreased food intake by 26% in lean and 38% in obese rats and energy expenditure by 6% in lean and 47% in obese rats; the body weight gap between controls and treated rats becoming −17.8% of initial b.wt. in the lean and −13.6% in the obese rats. Obese rats showed higher NPY levels in all the nuclei than the lean rats. Despite a negative energy balance and decreased food intake, there were practically no changes in NPY with OE treatment. The results indicate that oleoyl-estrone does not act through NPY in its control of either food intake or thermogenesis in lean and genetically obese rats.

In this paper we study the septal complex architecture in the lizard Podarcis hispanica (Lacertidae). Histochemical and immunohistochemical techniques were used to define the distribution of zinc (Timm stain), acetyl cholinesterase (AChase), gamma-aminobutyric acid (GABA), tyrosine hydroxylase (TH), dopamine (DA), serotonin (5-HT), and two neuropeptides: leu-enkephalin (L-ENK) and substance P (SP). These reactions delineate a coherent map of nine septal nuclei that are named with a topographical nomenclature: anterior, lateral, ventromedial, medial, dorsolateral, ventrolateral, and dorsal septal nuclei, nucleus septalis impar, and nucleus of the posterior pallial commissure. The anterior septal nucleus is characterized by intense reaction for zinc and the presence of fibers immunoreactive for GABA, 5-HT, and L-ENK, which form pericellular nests. The lateral septal nucleus shows intense reaction for zinc, a high density of GABA-immunoreactive cells, and L-ENK-immunoreactive fibers forming basketlike figures around unstained somata. The ventromedial septal nucleus shows intense AChase reactivity, a dense network of 5-HT-immunoreactive fibers, and virtually no labeling for the other histochemical stains. The medial septal nucleus is defined by heavy reactivity for zinc, dense DA/TH and L-ENK innervations, and the presence of L-ENK-immunoreactive cells. The dorsolateral septal nucleus shows intense AChase staining in the neuropile and a dense network of fibers immunoreactive for 5-HT and DA/TH, but it shows low staining for zinc, The ventrolateral septal nucleus shows L-ENK-immunoreactive cells and a dense L-ENK innervation, but low reactivity for zinc. The dorsal septal nucleus, intermingled with the fimbrial fibers, shows a dense population of GABA-immunoreactive cells and terminals, but it is unreactive for zinc. Two subdivisions can be established in this dorsal septal nucleus: the dorsal part, intensely reactive for AChase and innervated by 5-HT fibers, and the central part, which shows L-ENK-immunoreactive neurons and fibers without reactivity for either AChase or 5-HT. The nucleus septalis impar, traversed by the fibers of the anterior pallial commissure (mildly reactive for zinc), shows reaction for AChase but low (if present) reactivity for the remaining markers. The nucleus of the posterior pallial commissure shows a generally low reactivity for the histochemical reactions employed. The distribution of these markers is similar to that found in other squamate reptiles and allows for a direct comparison with the septal formation of mammals. Such a comparison reintorces the view that the limbic system has undergone a conservative evolution within vertebrates. © 1995 Wiley-Liss, Inc.

Sodium selenide was used as a retrograde tracer to assess the callosal origin of zinc-rich boutons in the neocortex of the rat. Selenide injections were placed in the lateral end of area Oc1 (area 17). Zinc present in synaptic boutons precipitated as zinc selenide and was transported retrogradely to the parent cell bodies. In addition to the ipsilateral labelling, contralateral retrogradely labelled somata were mainly observed along the border between areas Oc1 and Oc2L (area 18a). Labelled neurones were present in layer 2-3 and layer 6. In contrast to tracing studies with peroxidase, no labelled neurone was observed in layers 4 and 5 except at the inner border of layer 5. This study reveals the chemical heterogeneity of callosal projections, which may be divided into zinc-rich and zinc-poor systems.

Staining for zinc in terminal fields of the rat neocortex was studied by applying the sulphide/silver histochemical method. The stain was arranged in a distinct layered pattern. Two bands of heavy reaction were found in deep layer 1 plus layers 2-3 and in upper layer 5. A band of moderate-to-heavy reaction was found in layer 6. Three bands of lighter staining were found in upper layer 1, layer 4 and deep layer 5. The layers of reaction showed variations in width and intensity of staining from area to area. In the frontal and cingulate cortices and in the association cortices, the heavily stained bands were dominant over the narrow, inconspicuous, lightly stained bands. In contrast, in primary sensory regions (Par1, Oc1 and Te1), the lightly stained bands were wide and prominent. The sulphide/silver method gives a straightforward delimitation of many cortical areas and reveals a clear distinction between (A) allocortical and isocortical areas, and (B) primary sensory areas, secondary or association areas, and prefrontal plus motor areas. The presence of vivid bands of staining for zinc appears to be linked to areas with prominent pyramidal layers.

Lipoprotein lipase is a key enzyme in lipoprotein metabolism present primarily in extrahepatic tissues with high turnover of fatty acids. Using immunocytochemistry we have explored where lipoprotein lipase is localized in guinea pig brain. The enzyme was found to be associated with neuronal cells and vascular endothelial surfaces. The distribution was strikingly uneven with intense reaction in some areas, and virtually no reaction in adjacent areas. The highest reactivity was in neocortex, in hippocampus, in Purkinje cells of the cerebellum and in some motor nuclei of the brainstem. The results suggest marked differences between individual brain areas in utilization of plasma lipoproteins.

The distribution of zinc in the forebrain and midbrain of the lizard Gekko gecko was studied with the recently modified Timm method. Areas with a high intensity of staining are almost exclusively found in the telencephalon, although also some structures in the diencephalon display notable staining. Cortical areas that stain heavily are the deep zone of the subcortical layer of the small-celled medial cortex, the longitudinal association bundle that encompasses the large-celled medial cortex, and the dorsal cortex. Of the subcortical areas, particularly the anterior septal nucleus shows a high intensity of staining. Moderate to dense Timm staining is further observed in the ventral part of the anterior lateral cortex, the lateral septal nucleus, the striatum, the amygdaloid complex, and the dorsal ventricular ridge. Staining in the diencephalon is primarily confined to the stria terminalis and the ventromedial hypothalamic nucleus, whereas in the midbrain weak staining is observed in the ventral tegmental area and the periventricular layers of the tectum and the tegmentum. The presence of zinc in the gekkonid brain is discussed in relation to connections and neurotransmitters as studied in same species. Moreover, similarities in pattern of staining for zinc in mammals and reptiles and possible evolutionary implications are mentioned.

The Timm method for the histochemical detection of metals defines accurately many terminal fields in the brain of mammals. This pattern is based on the presence of zinc within the synaptic vesicles of some boutons. The aim of this study was to use the Timm method for the anatomical description of the brain in a reptile. In the telencephalon, zinc staining was observed in the inner layer of the medial cortex, the inner and outer layers of both dorsomedial and dorsal cortices, the inner layer of the lateral cortex pars anterior ventralis, the lateral cortex pars profunda, the intermediate and caudal aspects of the anterior dorsal ventricular ridge, the marginal layer and hilus of the nucleus sphericus, the perifascicular nucleus of the accessory olfactory tract, the striatum pars medialis, the olfactory tubercle, the septum pars anterior, and embedded in the fibres of both pallial and anterior commissures. In the diencephalon, staining was observed in the ventromedial hypothalamic nucleus and among the fibers of the stria terminalis. Stained somata and dendrites were observed in the infundibulum. In the mesencephalon and rhombencephalon, sparse staining was observed in the central gray, torus semicircularis, nucleus interpeduncularis, raphe, reticular formation, Purkinje and granular cell layers in the cerebellum, and nucleus cerebellaris medialis. The present results suggest that the histochemical detection of zinc may be a useful method for the accurate definition of terminal fields in the brain of reptiles also. The presence of zinc-containing terminal fields is discussed in relation to the connections and histochemistry in the reptilian brain. Similarities in the pattern of staining for zinc between mammals and reptiles are mentioned.

Many regions of the basal forebrain are innervated by zinc-containing axonal boutons. In the present work, the lesion/degeneration method, coupled with histochemical staining for zinc-containing boutons, was used to determine the origins and efferent pathways of these zinc-containing projections to the basal forebrain. Knife cuts of the stria terminalis or extensive electrolytic lesions of the amygdala resulted in the bleaching of the staining for zinc (Timm stain) and terminal degeneration (Fink-Heimer method) ipsilaterally in the following areas: granule cell layer of the accessory olfactory bulb, shell of nucleus accumbens, bed nucleus of the stria terminalis, striohypothalamic nucleus, retrochiasmatic area, ventromedial hypothalamic nucleus (in the cell-sparse shell), medial tuberal nucleus, terete hypothalamic nucleus, and ventral premammillary nucleus. Small lesions made with ibotenic acid in the posteromedial part of the amygdalohippocampal area caused bleaching of the stain for zinc in the accessory olfactory bulb, in the medial zone of the bed nucleus of the stria terminalis, and in the ventral premammillary nucleus. Lesions in either the ventral subiculum or the anterolateral part of the amygdalohippocampal area caused bleaching in the ventromedial hypothalamic nucleus. Lesions in the hippocampus or in the neocortex did not produce bleaching of the stain for zinc in the above-mentioned terminal fields. The present results agree with previous studies on amygdaloid efferents and suggest that neurons in the amygdalhippocampal area and, possibly, in the ventral subiculum give origin to zinc-containing boutons.

Background: Zinc concentrates at excitatory synapses, both at the postsynaptic density and in a subset of glutamatergic boutons. Zinc can modulate synaptic plasticity, memory formation and nociception by regulating transmitter receptors and signal transduction pathways. Also, intracellular zinc accumulation is a hallmark of degenerating neurons in several neurological disorders. To date, no single zinc extrusion mechanism has been directly localized to synapses. Based on the presence of a canonical PDZ I motif in the Zinc Transporter-1 protein (ZnT1), we hypothesized that ZnT1 may be targeted to synaptic compartments for local control of cytosolic zinc. Using our previously developed protocol for the co-localization of reactive zinc and synaptic proteins, we further asked if ZnT1 expression correlates with presynaptic zinc content in individual synapses. Findings: Here we demonstrate that ZnT1 is a plasma membrane protein that is enriched in dendritic spines and in biochemically isolated synaptic membranes. Hippocampal CA1 synapses labelled by postembedding immunogold showed over a 5-fold increase in ZnT1 concentration at synaptic junctions compared with extrasynaptic membranes. Subsynaptic analysis revealed a peak ZnT1 density on the postsynaptic side of the synapse, < 10 nm away from the postsynaptic membrane. ZnT1 was found in the vast majority of excitatory synapses regardless of the presence of vesicular zinc in presynaptic boutons. Conclusions: Our study has identified ZnT1 as a novel postsynaptic density protein, and it may help elucidate the role of zinc homeostasis in synaptic function and disease.

The development and maturation of cortical circuits relies on the coordinated actions of long and short range axonal guidance cues. In this regard, the class 3 semaphorins and their receptors have been seen to be involved in the development and maturation of the hippocampal connections. However, although the role of most of their family members have been described, very few data about the participation of Semaphorin 3E (Sema3E) and its receptor PlexinD1 during the development and maturation of the entorhino-hippocampal (EH) connection are available. In the present study, we focused on determining their roles both during development and in adulthood. We determined a relevant role for Sema3E/PlexinD1 in the layer-specific development of the EH connection. Indeed, mice lacking Sema3E/PlexinD1 signalling showed aberrant layering of entorhinal axons in the hippocampus during embryonic and perinatal stages. In addition, absence of Sema3E/PlexinD1 signalling results in further changes in postnatal and adult hippocampal formation, such as numerous misrouted ectopic mossy fibers. More relevantly, we describe how subgranular cells express PlexinD1 and how the absence of Sema3E induces a dysregulation of the proliferation of dentate gyrus progenitors leading to the presence of ectopic cells in the molecular layer. Lastly, Sema3E mutant mice displayed increased network excitability both in the dentate gyrus and the hippocampus proper.