Design and Synthesis of Fluorescent Methylphenidate Analogues for a FRET-Based Assay of Synapsin III Binding.

We previously described synapsin III (Syn III) as a synaptic phosphoprotein that controls dopamine release in cooperation with α-synuclein (aSyn). Moreover, we found that in Parkinson's disease (PD), Syn III also participates in aSyn aggregation and toxicity. Our recent observations point to threo-methylphenidate (MPH), a monoamine re-uptake inhibitor that efficiently counteracts the freezing-gait characteristic of advanced PD, as a ligand for Syn III. We have designed and synthesised two different fluorescently labelled MPH derivatives, one with Rhodamine Red (RHOD) and one with 5-carboxytetramethylrhodamine (TAMRA), to be used for assessing MPH binding to Syn III by FRET. TAMRA-MPH exhibited the ideal characteristics to be used as a FRET acceptor, as it was able to enter into the SK-N-SH cells and could interact specifically with human green fluorescent protein (GFP)-tagged Syn III but not with GFP alone. Moreover, the uptake of TAMRA-MPH and co-localization with Syn III was also observed in primary mesencephalic neurons. These findings support that MPH is a Syn III ligand and that TAMRA-conjugated drug molecules might be valuable tools to study drug-ligand interactions by FRET or to detect Syn III in cytological and histological samples.

[Gangliocytic paragangliomas: a clinicopathologic study].

Objective: To investigate the clinicopathological features of gangliocytic paraganglioma(GP). Methods: Clinical data and pathological diagnosis of the 4 cases of GP were obtained through the medical record inquiry from January 2011 to December 2017 at the First Affiliated Hospital of Zhengzhou University. Routine HE staining and immunohistochemistry of CKpan, Syn, CgA, CD56, NSE and NF were performed. Clinical follow-up of the patients was obtained through telephone communication. Results: All 4 patients, including 2 male and 2 female patients, presented with intermittent abdominal pain and distention. The median age was 56 years. Preoperative CT showed local thickening of the duodenum wall with slight enhancement in all four cases. Endoscopic ultrasonography showed low level echo in the mucous layer and submucosa involved by the tumor in 3 of 4 cases. The maximal diameter of the tumor ranged from 0.6 to 1.8 cm with an average of 1.2 cm. Microscopically, the tumors consisted of epithelioid, spindle and ganglion-like cells, and the proportion of the three cell types was different among cases. Epithelioid cells expressed CKpan, Syn, CgA and CD56. Spindle cells expressed S-100 protein and SOX-10 and ganglion-like cells expressed NF, Syn, CgA and CD56.All tumour cells expressed NSE. All 4 patients had no recurrence a post-surgery follow-up period of 3 to 30 months. Conclusions: GP of the duodenum is a benign tumor with excellent prognosis after endoscopic excision. Although its incidence is very low, its diagnosis should be considered for any mass lesion of the duodenum, especially involving mucosa and submucosa of the second dudenal segment.

Rapid Sequential in Situ Multiplexing with DNA Exchange Imaging in Neuronal Cells and Tissues.

To decipher the molecular mechanisms of biological function, it is critical to map the molecular composition of individual cells or even more importantly tissue samples in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit but are not widely adopted due to the common limitation of requiring multirounds of slow (typically over 2 h at room temperature to overnight at 4 °C in practice) immunostaining. We present here a practical and robust method, which we call DNA Exchange Imaging (DEI), for rapid in situ spectrally unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-round immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 min) buffer exchange of fluorophore-bearing DNA imager strands. The programmability of DEI allows us to apply it to diverse microscopy platforms (with Exchange Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here) at multiple desired resolution scales (from ∼300 nm down to sub-20 nm). We optimized and validated the use of DEI in complex biological samples, including primary neuron cultures and tissue sections. These results collectively suggest DNA exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

Sphingosine-1-Phosphate (S1P) Impacts Presynaptic Functions by Regulating Synapsin I Localization in the Presynaptic Compartment.

Growing evidence indicates that sphingosine-1-P (S1P) upregulates glutamate secretion in hippocampal neurons. However, the molecular mechanisms through which S1P enhances excitatory activity remain largely undefined. The aim of this study was to identify presynaptic targets of S1P action controlling exocytosis. Confocal analysis of rat hippocampal neurons showed that S1P applied at nanomolar concentration alters the distribution of Synapsin I (SynI), a presynaptic phosphoprotein that controls the availability of synaptic vesicles for exocytosis. S1P induced SynI relocation to extrasynaptic regions of mature neurons, as well as SynI dispersion from synaptic vesicle clusters present at axonal growth cones of developing neurons. S1P-induced SynI relocation occurred in a Ca(2+)-independent but ERK-dependent manner, likely through the activation of S1P3 receptors, as it was prevented by the S1P3 receptor selective antagonist CAY1044 and in neurons in which S1P3 receptor was silenced. Our recent evidence indicates that microvesicles (MVs) released by microglia enhance the metabolism of endogenous sphingolipids in neurons and stimulate excitatory transmission. We therefore investigated whether MVs affect SynI distribution and whether endogenous S1P could be involved in the process. Analysis of SynI immunoreactivity showed that exposure to microglial MVs induces SynI mobilization at presynaptic sites and growth cones, whereas the use of inhibitors of sphingolipid cascade identified S1P as the sphingolipid mediating SynI redistribution. Our data represent the first demonstration that S1P induces SynI mobilization from synapses, thereby indicating the phosphoprotein as a novel target through which S1P controls exocytosis. SIGNIFICANCE STATEMENT: Growing evidence indicates that the bioactive lipid sphingosine and its metabolite sphingosine-1-P (S1P) stimulate excitatory transmission. While it has been recently clarified that sphingosine influences directly the exocytotic machinery by activating the synaptic vesicle protein VAMP2 to form SNARE fusion complexes, the molecular mechanism by which S1P promotes neurotransmission remained largely undefined. In this study, we identify Synapsin I, a presynaptic phosphoprotein involved in the control of availability of synaptic vesicles for exocytosis, as the key target of S1P action. In addition, we provide evidence that S1P can be produced at mature axon terminals as well as at immature growth cones in response to microglia-derived signals, which may be important to stabilize nascent synapses and to restore or potentiate transmission.

Changes in Synapsin Levels in the Millipede Gymnostreptus olivaceus Schubart, 1944 Exposed to Different Concentrations of Deltamethrin.

Millipedes are ecologically important soil organisms and may also be an economically threatening species in rural and urban areas when population outbreaks occur. In order to control infestations commercial formulations of deltamethrin have been commonly applied, even though there are few studies about the effects of such insecticide on millipedes. This paper describes the effects of this insecticide on millipedes showing neurotoxic effects assessed by synapsin labeling and confocal microscopy. Deltamethrin concentrations related to the DL50 of the active ingredient and a field concentration were applied topically in the diplopod Gymnostreptus olivaceus to evaluate the behavior, mortality rate, and synapsin levels in the brain 12, 24, and 48h after contact with deltamethin. The insecticide caused mortality at the higher concentrations employed, in which no change was observed in neurotransmission in the survivors. In contrast, at field concentrations, deltamethrin did not cause any deaths, but triggered significant changes in synapsin levels. The results obtained form the synapsin labeling provide several interpretations suggesting that the isolated application of this tool must be associated with additional tools in order to evaluate biologically induced effects of deltamethrin in an accurate way. In addition, the feasibility of chemical control of millipedes with deltamethrin is questioned.

Synapsin IIb as a functional marker of submissive behavior.

Dominance and submissiveness are important functional elements of the social hierarchy. By employing selective breeding based on a social interaction test, we developed mice with strong and stable, inheritable features of dominance and submissiveness. In order to identify candidate genes responsible for dominant and submissive behavior, we applied transcriptomic and proteomic studies supported by molecular, behavioral and pharmacological approaches. We clearly show here that the expression of Synapsin II isoform b (Syn IIb) is constitutively upregulated in the hippocampus and striatum of submissive mice in comparison to their dominant and wild type counterparts. Moreover, the reduction of submissive behavior achieved after mating and delivery was accompanied by a marked reduction of Syn IIb expression. Since submissiveness has been shown to be associated with depressive-like behavior, we applied acute SSRI (Paroxetine) treatment to reduce submissiveness in studied mice. We found that reduction of submissive behavior evoked by Paroxetine was paired with significantly decreased Syn IIb expression. In conclusion, our findings indicate that submissiveness, known to be an important element of depressive-like behavioral abnormalities, is strongly linked with changes in Syn IIb expression.

Increased synapsin I expression in cerebral malaria.

Synapsin I is a neuronal phosphoprotein contained in the synaptic vesicles of mammalian central and peripheral nervous systems. It regulates both neurotransmitter release and synaptic formation. Variations in synapsin I expression in the brain have been reported to cause brain malfunction. In severe malaria, neurological complications, such as convulsion, delirium and coma, suggest abnormalities in the release of neurotransmitters. This study evaluated synapsin I expression in cerebral malaria (CM). An immunohistochemical method was used to study the semi-quantitative and qualitative expression of synapsin I in the brain of CM patients (10 cases) who died with Plasmodium falciparum, compared with non-cerebral malaria (NCM) (4 cases), and control brain tissues (5). Synapsin I was expressed in the gray matter of the cerebral cortex and the molecular layer of the cerebellum, as a diffusely dense precipitate pattern in the neuropil, with no immunoreactivity in the neurons, neuronal dendrites, glial cells, endothelial cells, and Purkinje cells. The findings were similarly demonstrated in CM, NCM, and control brain tissues. However, in the granular layer of the cerebellum, a significant increase in synapsin I expression was observed in the granule cells, and the glomerular synaptic complex, from the CM group, compared with the NCM, and control brain tissues (all P < 0.05). Parasitemia showed a positive correlation with synapsin I expression in the granule cells (on admission: Spearman's ρ = 0.600, P = 0.023) (before death: Spearman's ρ = 0.678, P = 0.008), and glomerular synaptic complex (before death: Spearman's ρ = 0.571, P = 0.033). It was hypothesized that CM causes pre-synaptic excitation and eventually activation of synapsin I, leading to increased neurotransmitter release. Synapsin I inhibitor should be investigated further as a target for a therapeutic intervention to alleviate neurological symptoms in severe malaria.

Neuronal and astroglial TGFß-Smad3 signaling pathways differentially regulate dendrite growth and synaptogenesis.

To address the role of the transforming growth factor beta (TGFß)-Smad3 signaling pathway in dendrite growth and associated synaptogenesis, we used small inhibitory RNA to knockdown the Smad3 gene in either cultured neurons and or primary astrocytes. We found that TGFß1 treatment of primary neurons increased dendrite extensions and the number of synapsin-1-positive synapses. When Smad3 was knockdown in primary neurons, dendrite growth was inhibited and the number of synapsin-1-positive synapses reduced even with TGFß1 treatment. When astrocyte-conditioned medium (ACM), collected from TGFß1-treated astrocytes (TGFß1-stimulated ACM), was added to cultured neurons, dendritic growth was inhibited and the number of synapsin-1-positive puncta reduced. When TGFß1-stimulated ACM was collected from astrocytes with Smad3 knocked down, this conditioned media promoted the growth of dendrites and the number of synapsin-1-positive puncta in cultured neurons. We further found that TGFß1 signaling through Smad3 increased the expression of chondroitin sulfate proteoglycans, neurocan, and phosphacan in ACM. Application of chondroitinase ABC to the TGFß1-stimulated ACM reversed its inhibitory effects on the dendrite growth and the number of synapsin-1-positive puncta. On the other hand, we found that TGFß1 treatment caused a facilitation of Smad3 phosphorylation and translocation to the nucleus induced by status epilepticus (SE) in wild-type (Smad3(+/+)) mice, and this treatment also caused a promotion of γ-aminobutyric acid-ergic synaptogenesis impaired by SE in Smad3(+/+) as well as in Smad3(-/-) mice, but more dramatic promotion in Smad3(+/+) mice. Thus, we provide evidence for the first time that TGFß-Smad3 signaling pathways within neuron and astrocyte differentially regulate dendrite growth and synaptogenesis, and this pathway may be involved in the pathogenesis of some central nervous system diseases, such as epilepsy.

Nicotine prevents synaptic impairment induced by amyloid-ß oligomers through α7-nicotinic acetylcholine receptor activation.

An emerging view on Alzheimer disease's (AD) pathogenesis considers amyloid-ß (Aß) oligomers as a key factor in synaptic impairment and rodent spatial memory decline. Alterations in the α7-nicotinic acetylcholine receptor (α7-nAChR) have been implicated in AD pathology. Herein, we report that nicotine, an unselective α7-nAChR agonist, protects from morphological and synaptic impairments induced by Aß oligomers. Interestingly, nicotine prevents both early postsynaptic impairment and late presynaptic damage induced by Aß oligomers through the α7-nAChR/phosphatidylinositol-3-kinase (PI3K) signaling pathway. On the other hand, a cross-talk between α7-nAChR and the Wnt/ß-catenin signaling pathway was revealed by the following facts: (1) nicotine stabilizes ß-catenin, in a concentration-dependent manner; (2) nicotine prevents Aß-induced loss of ß-catenin through the α7-nAChR; and (3) activation of canonical Wnt/ß-catenin signaling induces α7-nAChR expression. Analysis of the α7-nAChR promoter indicates that this receptor is a new Wnt target gene. Taken together, these results demonstrate that nicotine prevents memory deficits and synaptic impairment induced by Aß oligomers. In addition, nicotine improves memory in young APP/PS1 transgenic mice before extensive amyloid deposition and senile plaque development, and also in old mice where senile plaques have already formed. Activation of the α7-nAChR/PI3K signaling pathway and its cross-talk with the Wnt signaling pathway might well be therapeutic targets for potential AD treatments.

Terminals of the major thalamic input to visual cortex are devoid of synapsin proteins.

Synapsins are nerve-terminal proteins that are linked to synaptic transmission and key factors in several forms of synaptic plasticity. While synapsins are generally assumed to be ubiquitous in synaptic terminals, whether they are excluded from certain types of terminals is of interest. In the visual pathway, synapsins are lacking in photoreceptor and bipolar cell terminals as well as in retinogeniculate synapses. These are the terminals of the first three feedforward synapses in the visual pathway, implying that lack of synapsins may be a common property of terminals that provide the primary driver activity onto their postsynaptic neurons. To further investigate this idea, we studied the fourth driver synapse, thalamocortical synapses in visual cortex, using glutamatergic terminal antibody markers anti-VGluT1 and VGluT2, anti-Synapsin I and II, and confocal microscopy to analyze co-localization of these proteins in terminals. We also used pre-embedding immunocytochemical labeling followed by electron microscopy to investigate morphological similarities or differences between terminals containing synapsins or VGluT2. In visual cortex, synapsin coincided extensively with non-TC-neuron marker, VGluT1, while thalamocortical terminal marker VGluT2 and synapsin overlap was sparse. Morphologically, synapsin-stained terminals were smaller than non-stained, while VGluT2-positive thalamocortical terminals constituted the largest terminals in cortex. The size discrepancy between synapsin- and VGluT2-positive terminals, together with the complementary staining patterns, indicates that thalamocortical synapses are devoid of synapsins, and support the hypothesis that afferent sensory information is consistently transmitted without the involvement of synapsins. Furthermore, VGluT2 and synapsins were colocalized in other brain structures, suggesting that lack of synapsins is not a property of VGluT2-containing terminals, but a property of primary driver terminals in the visual system.

Serotonin immunoreactive interneurons in the brain of the Remipedia: new insights into the phylogenetic affinities of an enigmatic crustacean taxon.

BACKGROUND: Remipedia, a group of homonomously segmented, cave-dwelling, eyeless arthropods have been regarded as basal crustaceans in most early morphological and taxonomic studies. However, molecular sequence information together with the discovery of a highly differentiated brain led to a reconsideration of their phylogenetic position. Various conflicting hypotheses have been proposed including the claim for a basal position of Remipedia up to a close relationship with Malacostraca or Hexapoda. To provide new morphological characters that may allow phylogenetic insights, we have analyzed the architecture of the remipede brain in more detail using immunocytochemistry (serotonin, acetylated α-tubulin, synapsin) combined with confocal laser-scanning microscopy and image reconstruction techniques. This approach allows for a comprehensive neuroanatomical comparison with other crustacean and hexapod taxa. RESULTS: The dominant structures of the brain are the deutocerebral olfactory neuropils, which are linked by the olfactory globular tracts to the protocerebral hemiellipsoid bodies. The olfactory globular tracts form a characteristic chiasm in the center of the brain. In Speleonectes tulumensis, each brain hemisphere contains about 120 serotonin immunoreactive neurons, which are distributed in distinct cell groups supplying fine, profusely branching neurites to 16 neuropilar domains. The olfactory neuropil comprises more than 300 spherical olfactory glomeruli arranged in sublobes. Eight serotonin immunoreactive neurons homogeneously innervate the olfactory glomeruli. In the protocerebrum, serotonin immunoreactivity revealed several structures, which, based on their position and connectivity resemble a central complex comprising a central body, a protocerebral bridge, W-, X-, Y-, Z-tracts, and lateral accessory lobes. CONCLUSIONS: The brain of Remipedia shows several plesiomorphic features shared with other Mandibulata, such as deutocerebral olfactory neuropils with a glomerular organization, innervations by serotonin immunoreactive interneurons, and connections to protocerebral neuropils. Also, we provided tentative evidence for W-, X-, Y-, Z-tracts in the remipedian central complex like in the brain of Malacostraca, and Hexapoda. Furthermore, Remipedia display several synapomorphies with Malacostraca supporting a sister group relationship between both taxa. These homologies include a chiasm of the olfactory globular tract, which connects the olfactory neuropils with the lateral protocerebrum and the presence of hemiellipsoid bodies. Even though a growing number of molecular investigations unites Remipedia and Cephalocarida, our neuroanatomical comparison does not provide support for such a sister group relationship.

Valproic acid improves the tolerance for the stress in learned helplessness rats.

In this study, we investigated whether previously stressed rats with learned helplessness (LH) paradigm could recover from depressive-like behavior four weeks after the exposure, and also whether chronic treatment with valproic acid (VPA) could prevent behavioral despair due to the second stress on days 54 in these animals. Four weeks after induction of LH, we confirmed behavioral remission in the previously stressed rats. Two-way analysis of variance (ANOVA) performed with two factors, pretreatment (LH or Control) and drug (VPA or Saline), revealed a significant main effect of the drug on immobility time in forced swimming test. Post hoc test showed a shorter immobility time in the LH+VPA group than in the LH+Saline group. Immunohistochemical study of synapsin I showed a significant effect of drug by pretreatment interaction on immunoreactivity of synapsin I in the hippocampus: its expression levels in the regions were higher in the LH+VPA group than in the LH+Saline group. These results suggest that VPA could prevent the reappearance of stress-induced depressive-like behaviors in the rats recovering from prior stress, and that the drug-induced presynaptic changes in the expression of synapsin I in the hippocampus of LH animals might be related to improved tolerance toward the stress.

Comparative immunohistochemistry of synaptic markers in the rodent hippocampus in pilocarpine epilepsy.

Pilocarpine-induced epileptic state (Status epilepticus) generates an aberrant sprouting of hippocampal mossy fibers, which alter the intrahippocampal circuits. The mechanisms of the synaptic plasticity remain to be determined. In our studies in mice and rats, pilocarpine-induced seizures were done in order to gain information on the process of synaptogenesis. After a 2-month survival period, changes in the levels of synaptic markers (GAP-43 and Syn-I) were examined in the hippocampus by means of semi-quantitative immunohistochemistry. Mossy fiber sprouting (MFS) was examined in each brain using Timm's sulphide-silver method. Despite the marked behavioral manifestations caused by pilocarpine treatment, only 40% of the rats and 56% of the mice showed MFS. Pilocarpine treatment significantly reduced the GAP-43 immunoreactivity in the inner molecular layer in both species, with some minor differences in the staining pattern. Syn-I immunohistochemistry revealed species differences in the sprouting process. The strong immunoreactive band of the inner molecular layer in rats corresponded to the Timm-positive ectopic mossy fibers. The staining intensity in this layer, representing the ectopic mossy fibers, was weak in the mouse. The Syn-I immunoreactivity decreased significantly in the hilum, where Timm's method also demonstrated enhanced sprouting. This proved that, while sprouted axons displayed strong Syn-I staining in rats, ectopic mossy fibers in mice did not express this synaptic marker. The species variability in the expression of synaptic markers in sprouted axons following pilocarpine treatment indicated different synaptic mechanisms of epileptogenesis.

Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers.

A lack of methods for measuring the protein compositions of individual synapses in situ has so far hindered the exploration and exploitation of synapse molecular diversity. Here, we describe the use of array tomography, a new high-resolution proteomic imaging method, to determine the composition of glutamate and GABA synapses in somatosensory cortex of Line-H-YFP Thy-1 transgenic mice. We find that virtually all synapses are recognized by antibodies to the presynaptic phosphoprotein synapsin I, while antibodies to 16 other synaptic proteins discriminate among 4 subtypes of glutamatergic synapses and GABAergic synapses. Cell-specific YFP expression in the YFP-H mouse line allows synapses to be assigned to specific presynaptic and postsynaptic partners and reveals that a subpopulation of spines on layer 5 pyramidal cells receives both VGluT1-subtype glutamatergic and GABAergic synaptic inputs. These results establish a means for the high-throughput acquisition of proteomic data from individual cortical synapses in situ.

Perinatal development of the mammillothalamic tract and innervation of the anterior thalamic nuclei.

Axonal projections originating from the mammillary bodies represent important pathways that are essential for spatial information processing. Mammillothalamic tract is one of the main efferent projection systems of the mammillary body belonging to the limbic "Papez circuit". This study was aimed to describe the schedule of the mammillothalamic tract development in the rat using carbocyanine dye tracing. It was shown for the first time that fibers of the mammillothalamic tract being the collaterals of the mammillotegmental tract axons start bifurcating from the mammillotegmental tract on E17. The axons of the mammillothalamic tract grow simultaneously and reach the ventral region of the anterior thalamus where they form first terminal arborizations on E20-E21. Ipsilateral projections from the medial mammillary nucleus to the anteromedial and anteroventral thalamic nuclei develop from E20 to P6. Bilateral projections from the lateral mammillary nucleus to the anterodorsal thalamic nuclei develop later, on P3-P6, after the formation of the thalamic decussation of the mammillary body axons. Unique spatial and temporal pattern of the perinatal development of ascending mammillary body projections to the anterior thalamic nuclei may reflect the importance of these connections within the limbic circuitry.

IgLON cell adhesion molecule Kilon is a crucial modulator for synapse number in hippocampal neurons.

Kilon is a member of the IgLON family belonging to the immunoglobulin superfamily of cell adhesion molecules. In the present study, we investigated temporal and spatial changes of Kilon expression and its modulatory functions for synapse number using hippocampal cultured neurons. Kilon was observed to localize chiefly at axons and presynaptic terminals at early culture stage, however, it was seen mainly at dendritic postsynaptic spine of mature neurons at late culture stages. Kilon was solubilized with detergent treatment at early culture stages, while it resisted to extraction of the detergent in mature neurons. The overexpression of Kilon gene using a plasmid vector decreased the number of dendritic synapses at early culture stages, whereas the overexpression increased the number of dendritic synapses at late culture. These results demonstrate the alteration of modulatory function of Kilon for the number of dendritic synapses concomitant with changes in its localization and detergent solubility during neuronal culture development.

Confocal microscopy in large insect brains: zinc-formaldehyde fixation improves synapsin immunostaining and preservation of morphology in whole-mounts.

Confocal microscopy enables the analysis of immunofluorescence in whole-mount brains and is therefore widely used in the functional and comparative neuroanatomy of invertebrates. Three difficulties, however, are commonly encountered. First, poor penetration of antibodies after formaldehyde fixation impedes the immunostaining in central neuropile regions. Second, formaldehyde can cause a loss of antigenicity by epitope masking. Third, large brains must be cleared in hydrophobic media, a procedure that may distort morphology. I present a new methodology that overcomes these three problems by using zinc-formaldehyde (ZnFA) for fixation. The success of this technique is demonstrated in the brain of the desert locust and evaluated by comparison with fixation in formaldehyde and immunostaining against synapsin to reveal the regions of synaptic integration throughout the brain. ZnFA fixation markedly increased antibody penetration, prevented synapsin epitope masking, and in the cleared preparation the morphology of the brain was preserved with great fidelity. Possible mechanisms responsible for these improvements are discussed. Successful double labelling for synapsin and serotonin shows that small-molecule antigens are also retained by ZnFA fixation. The methodology should facilitate a range of applications including whole-mount brain stereology and the generation of digital standard brains. It may furthermore facilitate the detection of other protein antigens in large intact specimens such as vertebrate embryos.

Dendritic projections of different types of octopaminergic unpaired median neurons in the locust metathoracic ganglion.

Octopaminergic dorsal unpaired median (DUM) neurons of locust thoracic ganglia are important components of motor networks and are divided into various sub-populations. We have examined individually stained metathoracic DUM neurons, their dendritic projection patterns, and their relationship to specific architectural features of the metathoracic ganglion, such as longitudinal tracts, transverse commissures, and well-defined sensory neuropils. The detailed branching patterns of individually characterized DUM neurons of various types were analyzed in vibratome sections in which architectural features were revealed by using antibodies against tubulin and synapsin. Whereas DUM3,4,5 and DUM5 neurons (the group innervating leg and "non-wing-power" muscles) had many ventral and dorsal branches, DUM1 and DUM3,4 neurons (innervating "wing-power" muscles) branched extensively only in dorsal areas. The structure of DUM3 neurons differed markedly from that of the other DUM neurons examined in that they sent branches into dorsal areas and had differently structured side branches that mostly extended laterally. The differences between the branching patterns of these neurons were quantified by using currently available new reconstruction algorithms. These structural differences between the various classes of DUM neurons corresponded to differences in their function and biophysical properties.

Reduction in the number of astrocytes and their projections is associated with increased synaptic protein density in the hypothalamus of poorly controlled diabetic rats.

Processes under hypothalamic control, such as thermogenesis, feeding behavior, and pituitary hormone secretion, are disrupted in poorly controlled diabetes, but the underlying mechanisms are poorly understood. Because glial cells regulate neurosecretory neurons through modulation of synaptic inputs and function, we investigated the changes in hypothalamic glia in rats with streptozotocin-induced diabetes mellitus. Hypothalamic glial fibrillary acidic protein (GFAP) levels decreased significantly 6 wk after diabetes onset. This was coincident with decreased GFAP immunoreactive surface area, astrocyte number, and the extension of GFAP immunoreactive processes/astrocyte in the arcuate nucleus. Cell death, analyzed by terminal deoxyuridine 5-triphosphate nick-end labeling and ELISA, increased significantly at 4 wk of diabetes. Proliferation, measured by Western blot for proliferating cell nuclear antigen and immunostaining for phosphorylated histone H-3, decreased in the hypothalamus of diabetic rats throughout the study, becoming significantly reduced by 8 wk. Both proliferation and death affected astroctyes because both phosphorylated histone H-3- and terminal deoxyuridine 5-triphosphate nick-end labeling-labeled cells were GFAP positive. Western blot analysis revealed that postsynaptic density protein 95 and the presynaptic proteins synapsin I and synaptotagmin increased significantly at 8 wk of diabetes, suggesting increased hypothalamic synaptic density. Thus, in poorly controlled diabetic rats, there is a decrease in the number of hypothalamic astrocytes that is correlated with modifications in synaptic proteins and possibly synaptic inputs. These morphological changes in the arcuate nucleus could be involved in neurosecretory and metabolic changes seen in diabetic animals.

A component of BRAF-HDAC complex, BHC80, is required for neonatal survival in mice.

BHC80 is a component of BRAF-HDAC complex (BHC) involved in transcriptional repression of neuron-specific genes in non-neuronal cells. However, BHC80 is present in both neuronal and non-neuronal cells. To explore the physiological importance of BHC80 in vivo, and the precise mechanism underlying neuron-specific gene repression by BHC80, we have produced mutant mice lacking Bhc80. The loss of Bhc80 resulted in neonatal lethality without sucking mother's breast milk sufficiently. Although Bhc80-deficient mice showed no developmental defect in the neuronal and non-neuronal tissues, Bhc80 is indispensable for the survival of neonatal pups.