Biological characterization and optical imaging of brain-derived neurotrophic factor-green fluorescent protein suggest an activity-dependent local release of brain-derived neurotrophic factor in neurites of cultured hippocampal neurons

To visualize the release dynamics of the brain-derived neurotrophic factor (BDNF) involved in neural plasticity, we constructed a plasmid encoding green fluorescent protein (GFP) fused with BDNF. First, several biological studies confirmed that this fusion protein (BDNF-GFP) mimics the biological functions and the release kinetics of unfused (native) BDNF. Second, when BDNF-GFP was expressed in cultured hippocampal neurons, we observed that this protein formed striking clusters in the neurites of mature neurons and colocalized with the PSD-95 immunoreactivity. Such a clustered BDNF-GFP rapidly disappeared in response to depolarization with KCl, as revealed by confocal microscopic studies. These data suggest that BDNF is locally and rapidly released at synaptic sites in an activity-dependent manner. Optical studies using BDNF-GFP may provide important evidence regarding the participation of BDNF in synaptic plasticity.     

Nectin-dependent localization of ZO-1 at puncta adhaerentia junctions between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of adult mouse hippocampus

Nectin and afadin constitute a novel intercellular adhesion system that organizes adherens junctions in cooperation with the cadherin-catenin system in epithelial cells. Nectin is a Ca(2+)-independent immunoglobulin-like adhesion molecule and afadin is an actin filament (F-actin)-binding protein that connects nectin to the actin cytoskeleton. At the puncta adhaerentia junctions (PAs) between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of the adult mouse hippocampus, the nectin-afadin system also colocalizes with the cadherin-catenin system and has a role in the formation of synapses. ZO-1 is another F-actin-binding protein that localizes at tight junctions (TJs) and connects claudin to the actin cytoskeleton in epithelial cells. The nectin-afadin system is able to recruit ZO-1 to the nectin-based cell-cell adhesion sites in nonepithelial cells that have no TJs. In the present study, we investigated the localization of ZO-1 in the mouse hippocampus. Immunofluorescence and immunoelectron microscopy revealed that ZO-1 also localized at the PAs between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of the adult mouse hippocampus, as described for afadin. ZO-1 colocalized with afadin during the development of synaptic junctions and PAs. Microbeads coated with the extracellular fragment of nectin, which interacts with cellular nectin, recruited both afadin and ZO-1 to the bead-cell contact sites in cultured rat hippocampal neurons. These results indicate that ZO-1 colocalizes with nectin and afadin at the PAs and that the nectin-afadin system is involved in the localization of ZO-1.     

Activity-dependent energy budget for neocortical signaling: Effect of short-term synaptic plasticity on the energy expended by spiking and synaptic activity

The available estimate of the energy expended for signaling in rat neocortex is refined to examine the separate contribution of spiking and synaptic activity as a function of average neuronal firing rate. By taking into account a phenomenological model of short‐term synaptic plasticity, we show that the transition from low to high cortical activity is accompanied by a substantial increase in relative energy consumed by action potentials vs. synaptic potentials. This consideration might be important for a deeper understanding of how information is represented in the cortex and which metabolic pathways are upregulated to sustain cortical activity. © 2012 Wiley Periodicals, Inc.     

The interaction between collagens and factor VIII/von Willebrand factor: Investigation of the structural requirements for interaction

The blood protein Factor VIII/von Willebrand factor (FVIII/VWF) has been shown to bind to a variety of collagen polymers including (i), the native-type fibres (of collagens types I and III), (ii), segment-long-spacing (SLS) aggregates (of collagens types I, III, IV and V), (iii), the insoluble polymer obtained by random cross-linking of the type I monomer and (iv), the non-striated fibril (of type I) produced by alcohol precipitation. Relatively little binding of FVIII/VWF to the amorphous, non-fibrillar form of collagen (type I) produced by salt precipitation from acid solution was observed. No significant binding either to elastin or to the insoluble polymer derived by random cross-linking of bovine serum albumin was noted. The absorption of FVIII/VWF to collagens was affected by ionic concentration and FVIII/VWF was only totally bound at relatively low ionic strength. Binding of radiolabelled FVIII/VWF could be largely inhibited by an excess of the unlabelled protein. The interaction of FVIII/VWF with collagen fibres was inhibited in a concentration-dependent manner by monomeric collagen when present at relatively high concentrations. Gelatin did not appear to inhibit binding significantly. The structural requirements of collagen for binding to occur appear to resemble those required for collagen-induced platelet aggregation in which collagen quaternary structure rather than collagen type $\text{per se}$ is the important factor.Loss of secondary or higher orders of structure of FVIII/VWF as a result of heat denaturation or reduction of disulphide bonds decreased or prevented binding. In accord with the association of biological activity with FVIII/VWF aggregates, optimal binding appeared to require the presence of aggregated FVIII/VWF.     

Human immunodeficiency virus type 1 glycoprotein gp120 reduces the levels of brain-derived neurotrophic factor in vivo: potential implication for neuronal cell death

Neuronal loss has been observed in post mortem brains of patients with human immunodeficiency virus type 1 (HIV-1). Experimental evidence has implicated HIV-1-derived envelope glycoprotein 120 (gp120) in the neuronal cell death observed in these patients. However, the intrinsic mechanisms by which gp120 causes neurotoxicity are still unknown. We have recently shown that the neurotoxic effect of gp120 in vitro is reduced by brain-derived neurotrophic factor (BDNF). We therefore tested the hypothesis that low levels of BDNF render neurons more sensitive to gp120. Gp120 was injected acutely into the striatum of BDNF heterozygous mice and wild-type littermates. BDNF heterozygous mice exhibited more apoptotic neurons in the striatum than wild-type mice, suggesting that BDNF is neuroprotective also in vivo. Because several neurodegenerative disorders are characterized by lack of trophic support, we tested the hypothesis that gp120 may cause apoptosis by reducing BDNF expression. Gp120 was injected acutely in the rat striatum and BDNF levels determined by a two-site immunoassay at various times after the injection. Gp120 elicited a dramatic decrease in BDNF protein levels by 24 h. Reduced BDNF levels were still present at 4 days. Cellular localization of BDNF immunoreactivity revealed that gp120 decreases BDNF immunoreactivity mainly in neuronal processes. This effect of gp120 precedes the peak of caspase-3 activation and neuronal cell death. We propose that one of the mechanisms whereby gp120 causes neurotoxicity is a reduction of the neurotrophic factor environment crucial for cell survival.     

A novel neurofibromin (NF1) interaction with the leucine‐rich pentatricopeptide repeat motif‐containing protein links neurofibromatosis type 1 and the french canadian variant of leigh's syndrome in a common molecular complex

Loss‐of‐function mutations and deletions in the neurofibromin tumor suppressor gene (NF1) cause neurofibromatosis type 1 (NF‐1), the most common inherited syndrome of the nervous system in humans, with a birth incidence of 1:3,000. The most visible features of NF‐1 are the neoplastic manifestations caused by the loss of Ras‐GTPase‐activating protein (Ras‐GAP) activity mediated through the GAP‐related domain (GRD) of neurofibromin (NF1), the protein encoded by NF1. However, the syndrome is also characterized by cognitive dysfunction and a number of developmental abnormalities. The molecular etiology of many of these nonneoplastic phenotypes remains unknown. Here we show that the tubulin‐binding domain (TBD) of NF1 is a binding partner of the leucine‐rich pentatricopeptide repeat motif‐containing (LRPPRC) protein. These two proteins complex with Kinesin 5B, hnRNP A2, Staufen1, and Myelin Basic Protein (MBP) mRNA, likely in RNA granules. This interaction is of interest in that it links NF‐1 with Leigh's syndrome, French Canadian variant (LSFC), an autosomal recessive neurodegenerative disorder that arises from mutations in the LRPPRC gene. Our findings provide clues to how loss or mutation of NF1 and LRPPRC may contribute to the manifestations of NF‐1 and LSFC. © 2013 Wiley Periodicals, Inc.     

Neural correlates of the interaction between transient and sustained processes: a mixed blocked/event-related fMRI study

Complete understanding of the neural correlates of cognitive processes requires investigation of both event- and state-related correlates of cognitive performance as well as their interaction. Neuroimaging studies using blocked designs confound these two types of processes and studies using event-related designs focus exclusively on the detection of transient effects. Recent fMRI studies used mixed blocked/event-related designs and found that transient and sustained activity can be dissociated, but it is not yet known how event-related and state-related processing interact. Here we used a phonological categorization paradigm in a mixed blocked/event-related design to investigate where in the brain transient activity interacts with sustained activity. Task difficulty was parametrically manipulated based on individually determined categorization thresholds. We found an interaction effect of transient and sustained activity in the left precuneus. In this cortical structure transient activity increased with increasing task difficulty, while sustained neural activity decreased with increasing task difficulty. Our data suggest that sustained activity is enhanced during processing of an easy task, presumably because of ongoing internally cued endogenous processing, still allowing effortless processing of transient stimuli. During performance of a difficult task, sustained activity in the precuneus is reduced to provide resources for processing incoming stimuli. Processing of stimuli that are expected to be difficult elicits increased transient responses independent of the actual physical properties of the stimuli. In showing an interaction between transient and sustained activity in the precuneus, the present results accommodate seemingly diverging results from previous studies using event-related or blocked designs and expand the knowledge emerging from previous studies using mixed blocked/event-related designs.     

Post‐cocaine changes in regulator of G‐protein signaling (RGS) proteins in the dorsal striatum: Relevance for cocaine‐seeking and protein kinase C‐mediated phosphorylation

Persistent cocaine‐induced neuroadaptations within the cortico‐striatal circuitry might be related to elevated risk of relapse observed in human addicts even after months or years of drug‐free abstinence. Identification of these neuroadaptations may lead development of novel, neurobiologically‐based treatments of relapse. In the current study, 12 adult male Sprague‐Dawley rats self‐administered cocaine (or received yoked‐saline) for two weeks followed by three weeks of home‐cage abstinence. At this point, we analyzed expression of proteins involved in regulation of Gαi‐ and Gαq‐protein signaling in the dorsal striatum (dSTR). Animals abstinent from chronic cocaine showed decreased expression of regulator of G‐protein signaling 2 (RGS2) and RGS4, as well as upregulation of RGS9. These data, together with the increased ratio of Gαq‐to‐Gαi proteins indicated, “sensitized” Gαq signaling in the dSTR of abstinent cocaine animals. To evaluate activation of Gαq signaling during relapse, another group of abstinent cocaine animals (and yoked saline controls, 22 rats together) was reintroduced to the cocaine context and PKC‐mediated phosphorylation in the dSTR was analyzed. Re‐exposure to the cocaine context triggered cocaine seeking and increase in phosphorylation of cellular PKC substrates, including phospho‐ERK and phospho‐CREB. In conclusion, this study demonstrates persistent dysregulation of RGS proteins in the dSTR of abstinent cocaine animals that may produce an imbalance in local Gαq‐to‐Gαi signaling. This imbalance might be related to augmented PKC‐mediated phosphorylation during relapse to cocaine‐seeking. Future studies will address whether selective targeting of RGS proteins in the dSTR can be utilized to suppress PKC‐mediated phosphorylation and relapse to cocaine‐seeking.     

Activation of calcium/calmodulin‐dependent protein kinase IV and peroxisome proliferator‐activated receptor γ coactivator‐1α signaling pathway protects against neuronal injury and promotes mitochondrial biogenesis in the hippocampal CA1 subfield after transient global ischemia

Delayed neuronal cell death occurs in the vulnerable CA1 subfield of the hippocampus after transient global ischemia (TGI). We demonstrated previously, based on an experimental model of TGI, that the significantly increased content of oxidized proteins in hippocampal CA1 neuron was observed as early as 30 min after TGI, followed by augmentation of PGC‐1α expression at 1 hr, as well as up‐regulation of mitochondrial uncoupling protein 2 (UCP2) and superoxide dismutases 2 (SOD2). Using the same animal model, the present study investigated the role of calcium/calmodulin‐dependent protein kinase IV (CaMKIV) and PGC‐1α in delayed neuronal cell death and mitochondrial biogenesis in the hippocampus. In Sprague‐Dawley rats, significantly increased expression of nuclear CaMKIV was noted in the hippocampal CA1 subfield as early as 15 min after TGI. In addition, the index of mitochondrial biogenesis, including a mitochondrial DNA‐encoded polypeptide, cytochrome c oxidase subunit 1 (COX1), and mitochondrial number significantly increased in the hippocampal CA1 subfield 4 hr after TGI. Application bilaterally into the hippocampal CA1 subfield of an inhibitor of CaMKIV, KN‐93, 30 min before TGI attenuated both CaMKIV and PGC‐1α expression, followed by down‐regulation of UCP2 and SOD2, decrease of COX1 expression and mitochondrial number, heightened protein oxidation, and enhanced hippocampal CA1 neuronal damage. This study provides correlative evidence for the neuroprotective cascade of CaMKIV/PGC‐1α which implicates at least in part the mitochondrial antioxidants UCP2 and SOD2 as well as mitochondrial biogenesis in ischemic brain injury. © 2010 Wiley‐Liss, Inc.     

Metastasis-associated S100A4 (Mts1) protein is expressed in subpopulations of sensory and autonomic neurons and in Schwann cells of the adult rat

S100A4 (Mts1) is a member of a family of calcium-binding proteins of the EF-hand type, which are widely expressed in the nervous system, where they appear to be involved in the regulation of neuron survival, plasticity, and response to injury or disease. S100A4 has previously been demonstrated in astrocytes of the white matter and rostral migratory stream of the adult rat. After injury, S100A4 is markedly up-regulated in affected central nervous white matter areas as well as in the periventricular area and rostral migratory stream. Here, we show that S100A4 is expressed in a subpopulation of dorsal root, trigeminal, geniculate, and nodose ganglion cells; in a subpopulation of postganglionic sympathetic and parasympathetic neurons; in chromaffin cells of the adrenal medulla; and in satellite and Schwann cells. In dorsal root ganglia, S100A4-positive cells appear to constitute a subpopulation of small ganglion neurons, a few of which coexpressed calcitonin gene-related peptide (CGRP) and Griffonia simplicifolia agglutinin (GSA) isolectin B4 (B4). S100A4 protein appears to be transported from dorsal root ganglia to the spinal cord, where it is deposited in the tract of Lissauer. After peripheral nerve or dorsal root injury, a few S100A4-positive cells coexpress CGRP, GSA, or galanin. Peripheral nerve or dorsal root injury induces a marked up-regulation of S100A4 expression in satellite cells in the ganglion and in Schwann cells at the injury site and in the distal stump. This pattern of distribution partially overlaps that of the previously studied S100B and S100A6 proteins, indicating a possible functional cooperation between these proteins. The presence of S100A4 in sensory neurons, including their processes in the central nervous system, suggests that S100A4 is involved in propagation of sensory impulses in specific fiber types.     

Anatomical and functional implications of corticotrophin‐releasing hormone neurones in a septal nucleus of the avian brain: an emphasis on glial‐neuronal interaction via V1a receptors in vitro

Previously, we showed that corticotrophin‐releasing hormone immunoreactive (CRH‐IR) neurones in a septal structure are associated with stress and the hypothalamic‐pituitary‐adrenal axis in birds. In the present study, we focused upon CRH‐IR neurones located within the septal structure called the nucleus of the hippocampal commissure (NHpC). Immunocytochemical and gene expression analyses were used to identify the anatomical and functional characteristics of cells within the NHpC. A comparative morphometry analysis showed that CRH‐IR neurones in the NHpC were significantly larger than CRH‐IR parvocellular neurones in the paraventricular nucleus of the hypothalamus (PVN) and lateral bed nucleus of the stria terminalis. Furthermore, these large neurones in the NHpC usually have more than two processes, showing characteristics of multipolar neurones. Utilisation of an organotypic slice culture method enabled testing of how CRH‐IR neurones could be regulated within the NHpC. Similar to the PVN, CRH mRNA levels in the NHpC were increased following forskolin treatment. However, dexamethasone decreased forskolin‐induced CRH gene expression only in the PVN and not in the NHpC, indicating differential inhibitory mechanisms in the PVN and the NHpC of the avian brain. Moreover, immunocytochemical evidence also showed that CRH‐IR neurones reside in the NHpC along with the vasotocinergic system, comprising arginine vasotocin (AVT) nerve terminals and immunoreactive vasotocin V1a receptors (V1aR) in glia. Hence, we hypothesised that AVT acts as a neuromodulator within the NHpC to modulate activity of CRH neurones via glial V1aR. Gene expression analysis of cultured slices revealed that AVT treatment increased CRH mRNA levels, whereas a combination of AVT and a V1aR antagonist treatment decreased CRH mRNA expression. Furthermore, an attempt to identify an intercellular mechanism in glial‐neuronal communication in the NHpC revealed that brain‐derived neurotrophic factor (BDNF) and its receptor (TrkB) could be involved in the signalling mechanism. Immunocytochemical results further showed that both BDNF and TrkB receptors were found in glia of the NHpC. Interestingly, in cultured brain slices containing the NHpC, the use of a selective TrkB antagonist decreased the AVT‐induced increase in CRH gene expression levels. The results from the present study collectively suggest that CRH neuronal activity is modulated by AVT via V1aR involving BDNF and TrkB glia in the NHpC.     

Electrophysiological evidence for a functional interaction between 5-HT1A and 5-HT2A receptors in the rat medial prefrontal cortex: An lontophoretic study

In this study, we examined the interaction of 5-HT_1A and 5-HT_2A receptors in the rat medial prefrontal cortex (mPFc) using the techniques of extracellular single unit recording and microiontophoresis. The iontophoresis of the selective 5-HT_1A receptor agonist (±)-8-hydroxy-2-(di-n-propylamino) tetralin (8-OHDPAT) produced a current-dependent suppression (2.5-20 nA) of the basal firing rate of spontaneously active mPFc cells. The iontophoretic (5-10 nA) and systemic administration (0.1-0.5 mg/kg, i.v. ) of the 5-HT_2A/5-HT_2C receptor antagonist ritanserin and the selective 5 HT_2A receptor antagonist MDL 28727 significantly potentiated and prolonged 8-OHDPATs suppressant action. In addition, the systemic administration of another selective 5-HT_2A antagonist MDL 100907, but not its less active enantiomer MDL 100009, also potentiated and prolonged 8-OHDPATs action. The potentiating effect of the 5-HT_2A receptor antagonists on the action of 8-OHDPAT is specific in that neither the iontophoresis of ritanserin nor MDL 28727 altered the suppressant action produced by the iontophoresis of the 5-HT₃ receptor agonist 2-methylserotonin onto mPFc cells. Moreover, the suppressant action of 8-OHDPAT was not altered by the systemic administration of the selective 5-HT₃ receptor antagonist granisetron (0.1-0.5 mg/kg, i.v.). On the other hand, the iontophoresis of a low current (0.5 nA) of the 5-HT_2A,2C receptor agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) potentiated the excitation induced by the iontophoresis of 1-glutamate on quiescent mPFc cells. The iontophoresis of 8-OHD-PAT at a current that had no effect on the firing rate of 1-glutamate activated when administered alone significantly attenuated the excitatory action produced by the iontophoresis of DOI. Overall these results confirm and extend the hypothesis that there is an interaction between 5-HT_1A and 5-HT_2A receptors in the mPFc at the neuronal level. © 1994 Wiley-Liss, Inc.     

Dopamine and Cu+/2+ can induce oligomerization of α‐synuclein in the absence of oxygen: Two types of oligomerization mechanisms for α‐synuclein and related cell toxicity studies

α‐Synuclein oligomers can induce neurotoxicity and are implicated in Parkinson's disease etiology and disease progression. Many studies have reported α‐synuclein oligomerization by dopamine (DA) and transition metal ions, but few studies provide insight into joint influences of DA and Cu²⁺. In this study, DA and Cu²⁺ were coadministered aerobically to measure α‐synuclein oligomerization under these conditions. In the presence of oxygen, DA induced α‐synuclein oligomerization in a dose‐dependent manner. Cu^+/2+ did not effect oligomerization in such a manner in the presence of DA. By electrophoresis, Cu²⁺ was found easily to induce oligomerization with DA. This implies that oligomerization invoked by DA is reversible in the presence of Cu²⁺, which appears to be mediated by noncovalent bond interactions. In the absence of oxygen, DA induced less oligomerization of α‐synuclein, whereas DA/Cu²⁺ induced aerobic‐level amounts of oligomers, suggesting that DA/Cu²⁺ induces oligomerization independent of oxygen concentration. Radical species were detected through electron paramagnetic resonance (EPR) spectroscopic analysis arising from coincubation of DA/Cu²⁺ with α‐synuclein. Redox reactions induced by DA/Cu²⁺ were observed in multimer regions of α‐synuclein oligomers through NBT assay. Cellular toxicity results confirm that, for normal and hypoxic conditions, copper or DA/Cu²⁺ can induce cell death, which may arise from copper redox chemistry. From these results, we propose that DA and DA/Cu²⁺ induce different mechanisms of α‐synuclein oligomerization, cross‐linking with noncovalent (or reversible covalent) bonding vs. likely radical‐mediated covalent modification. © 2013 Wiley Periodicals, Inc.     

Expression of m1‐type muscarinic acetylcholine receptors by parvalbumin‐immunoreactive neurons in the primary visual cortex: A comparative study of rat,guinea pig,ferret, macaque,and human

Cholinergic neuromodulation is a candidate mechanism for aspects of arousal and attention in mammals. We have reported previously that cholinergic modulation in the primary visual cortex (V1) of the macaque monkey is strongly targeted toward GABAergic interneurons, and in particular that the vast majority of parvalbumin‐immunoreactive (PV) neurons in macaque V1 express the m1‐type (pirenzepine‐sensitive, Gq‐coupled) muscarinic ACh receptor (m1AChR). In contrast, previous physiological data indicates that PV neurons in rats rarely express pirenzepine‐sensitive muscarinic AChRs. To examine further this apparent species difference in the cholinergic effectors for the primary visual cortex, we have conducted a comparative study of the expression of m1AChRs by PV neurons in V1 of rats, guinea pigs, ferrets, macaques, and humans. We visualize PV‐ and mAChR‐immunoreactive somata by dual‐immunofluorescence confocal microscopy and find that the species differences are profound; the vast majority (>75%) of PV‐ir neurons in macaques, humans, and guinea pigs express m1AChRs. In contrast, in rats only ～25% of the PV population is immunoreactive for m1AChRs. Our data reveal that while they do so much less frequently than in primates, PV neurons in rats do express Gq‐coupled muscarinic AChRs, which appear to have gone undetected in the previous in vitro studies. Data such as these are critical in determining the species that represent adequate models for the capacity of the cholinergic system to modulate inhibition in the primate cortex. J. Comp. Neurol. 522:986–1003, 2014. © 2013 Wiley Periodicals, Inc.     

Evidence for neuronal regulation of oligodendrocyte development: Cellular localization of platelet-derived growth factor α receptor and A-chain mRNA during cerebral cortex development in the rat

Oligodendrocyte responses in vitro to platelet-derived growth factor (PDGF) include proliferation, survival, migration, and changes in cell morphology and molecular expression. Studies of mixed glial cultures established that astrocytes secrete PDGF; thus astrocytes are considered to be key regulators of oligodendrocyte development in vitro. We previously demonstrated PDGF α receptor mRNA expression by oligodendrocyte progenitors and preoligodendrocytes during postnatal development of rat cerebral cortex. In the present study, we have mapped the spatial and temporal expression of PDGF A-chain ligand mRNA and α receptor mRNA to determine if the cell-cell interactions that form the basis for PDGF regulation of oligodendrocyte development in vitro are also present in vivo. By in situ hybridization (ISH) we demonstrate that at embryonic day 17 (E17) cells expressing receptor mRNA (PDGFRα+) are initially in the subventricular zone, at a distance from cells expressing ligand mRNA (PDGF+) in the cortical plate. By E20 PDGFRα+ cells are found throughout the corpus callosum and cortical gray matter. PDGF+ cells are restricted to the cortical plate prenatally and only appeared in the corpus callosum postnatally. Combined immunocytochemistry and ISH demonstrated the PDGF+ cells colocalized with neurofilament, but not with GFAP. These data establish that PDGF is expressed by neurons during PDGFRα+ oligodendrocyte progenitor migration from the subventricular zone to the corpus callosum and gray matter. Furthermore, neurons continue to express PDGF during the generation and differentiation of appropriate numbers of oligodendrocytes needed to myelinate axons as the nervous system matures. © 1996 Wiley-Liss, Inc.