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The L1 cell adhesion molecule NrCAM (Neuron-glia related cell adhesion molecule) functions as a co-receptor for secreted class 3 Semaphorins to prune subpopulations of dendritic spines on apical dendrites of pyramidal neurons in the developing mouse neocortex. The developing spine cytoskeleton is enriched in actin filaments, but a small number of microtubules have been shown to enter the spine apparently trafficking vesicles to the membrane. Doublecortin-like kinase 1 (DCLK1) is a member of the Doublecortin (DCX) family of microtubule-binding proteins with serine/threonine kinase activity. To determine if DCLK1 plays a role in spine remodeling, we generated a tamoxifen-inducible mouse line (Nex1Cre-ERT2: DCLK1 flox/flox: RCE) to delete microtubule binding isoforms of DCLK1 from pyramidal neurons during postnatal stages of spine development. Homozygous DCLK1 conditional mutant mice exhibited decreased spine density on apical dendrites of pyramidal neurons in the prefrontal cortex (layer 2/3). Mature mushroom spines were selectively decreased upon DCLK1 deletion but dendritic arborization was unaltered. Mutagenesis and binding studies revealed that DCLK1 bound NrCAM at the conserved FIGQY 1231 motif in the NrCAM cytoplasmic domain, a known interaction site for the actin-spectrin adaptor Ankyrin. These findings demonstrate in a novel mouse model that DCLK1 facilitates spine growth and maturation on cortical pyramidal neurons in the mouse prefrontal cortex., (Copyright © 2022. Published by Elsevier Ltd.)

Adhesion G protein-coupled receptor A1 (ADGRA1) belongs to the G protein-coupled receptor (GPCR) family, and its physiological function remains largely unknown. We found that Adgra1 is highly and exclusively expressed in the brain, suggesting that Adgra1 may be involved in the regulation of neurological behaviors including anxiety, depression, learning and memory. To this end, we comprehensively analyzed the potential role of ADGRA1 in the neurobehaviors of mice by comparing Adgra1 -/- and their wild-type (wt) littermates. We found that Adgra1 -/- male but not female mice exhibited elevated anxiety levels in the open field, elevated plus maze, and light-dark box tests, with normal depression levels in the tail-suspension and forced-swim tests, and comparable learning and memory abilities in the Morris water maze, Y maze, fear condition, and step-down avoidance tests. Further studies showed that ADGRA1 deficiency resulted in higher dendritic branching complexity and spine density as evidenced by elevated expression levels of SYN and PSD95 in amygdalae of male mice. Finally, we found that PI3K/AKT/GSK-3β and MEK/ERK in amygdalae of Adgra1-deficient male mice were aberrantly activated when compared to wt male mice. Together, our findings reveal an important suppressive role of ADGRA1 in anxiety control and synaptic function by regulating the PI3K/AKT/GSK-3β and MEK/ERK pathways in amygdalae of male mice, implicating a potential, therapeutic application in novel anti-anxiety drug development., Competing Interests: Conflict of interest The authors declare no conflict of interests., (Copyright © 2022 IBRO. Published by Elsevier Ltd. All rights reserved.)

N-methyl-d-aspartate receptor-mediated ( spikes can be causally linked to the induction of synaptic long-term potentiation (LTP) in hippocampal and cortical pyramidal cells. However, it is unclear if they regulate plasticity at a local or global scale in the dendritic tree. Here, we used dendritic patch-clamp recordings and calcium imaging to investigate the integrative properties of single dendrites of hippocampal CA3 cells. We show that local hyperpolarization of a single dendritic segment prevents NMDA spikes, their associated calcium transients, as well as LTP in a branch-specific manner. This result provides direct, causal evidence that the single dendritic branch can operate as a functional unit in regulating CA3 pyramidal cell plasticity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 IBRO. Published by Elsevier Ltd.. All rights reserved.)

Fibroblast Growth Factor Receptors (FGFRs) play crucial roles in promoting dendrite growth and branching during development. In mice, three FGFR genes, Fgfr1, Fgfr2, and Fgfr3, remain expressed in the adult neurogenic niche of the hippocampal dentate gyrus. However, the function of FGFRs in the dendritic maturation of adult-born neurons remains largely unexplored. Here, using conditional alleles of Fgfr1, Fgfr2, and Fgfr3 as well as Fgfr1 alleles lacking binding sites for Phospholipase-Cγ (PLCγ) and FGF Receptor Substrate (FRS) proteins, we test the requirement for FGFRs in dendritogenesis of adult-born granule cells. We find that deleting all three receptors results in a small decrease in proximal dendrite elaboration. In contrast, specifically mutating Tyr766 in FGFR1 (a PLCγ binding site) in an Fgfr2;Fgfr3 deficient background results in a dramatic increase of overall dendrite elaboration, while blocking FGFR1-FRS signaling causes proximal dendrite deficits and, to a lesser extent than Tyr766 mutants, increases distal dendrite elaboration. These findings reveal unexpectedly complex roles for FGFRs and their intracellular mediators in regulating proximal and distal dendrite elaboration, with the most notable role in suppressing distal elaboration through the PLCγbinding site., (Copyright © 2020 IBRO. Published by Elsevier Ltd. All rights reserved.)

Agrin is a multi-domain protein best known for its essential function during formation of the neuromuscular junction. Alternative mRNA splicing at sites named y and z in the C-terminal part of agrin regulates its interaction with a receptor complex consisting of the agrin-binding low-density lipoprotein receptor-related protein 4 (Lrp4) and the muscle-specific kinase (MuSK). Isoforms with inserts at both splice sites bind to Lrp4, activate MuSK and are synaptogenic at the neuromuscular junction. Agrin is also expressed as a transmembrane protein in the central nervous system (CNS) but its function during interneuronal synapse formation is unclear. Recently we demonstrated that transfection of a full-length cDNA coding for transmembrane agrin (TM-agrin) in cultured embryonic cortical neurons induced an Lrp4-dependent but MuSK-independent increase in dendritic glutamatergic synapses and an Lrp4- and MuSK-independent reduction of inhibitory synapses. Here we show that presynaptic specializations were similarly affected by TM-agrin overexpression. In addition, we mapped the regions within TM-agrin responsible for TM-agrin's effects on dendritic aggregates of synapse-associated proteins. We show that the presence of a four amino acid insert at splice site y is essential for the increase in the density of puncta containing the postsynaptic density protein 95 kDa. This effect was independent of splice site z. The reduction of the gephyrin puncta density was independent of the entire extracellular part of TM-agrin but required a highly conserved serine residue in the intracellular domain of TM-agrin. These results provide further evidence for a function of TM-agrin during CNS synaptogenesis and demonstrate that different domains and alternative splicing of TM-agrin differentially affect excitatory and inhibitory synapse formation in cultured embryonic CNS neurons., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Synaptic pruning during adolescence is critical for optimal cognition. The CA3 hippocampus contains unique spine types and plays a pivotal role in pattern separation and seizure generation, where sex differences exist, but adolescent pruning has only been studied in the male. Thus, for the present study we assessed pruning of specific spine types in the CA3 hippocampus during adolescence and investigated a possible mechanism in the female mouse. To this end, we used Golgi-impregnated brains from pubertal (∼PND 35, assessed by vaginal opening) and post-pubertal (PND 56) mice. Spine density was assessed from z-stack (0.1-μm steps) images taken using a Nikon DS-U3 camera through a Nikon Eclipse Ci-L microscope and analyzed with NIS Elements. Spine density decreased significantly (P < 0.05) during adolescence, with 50-60% decreases in mushroom and stubby spine-types (P < 0.05, ∼PND35 vs. PND56) in non-proestrous mice. This was associated with decreases in kalirin-7, a spine protein which stabilizes the cytoskeleton and is required for spine maintenance. Because our previous findings suggest that pubertal increases in α4βδ GABA A receptors (GABARs) trigger pruning in CA1, we investigated their role in CA3. α4 expression in CA3 hippocampus increased 4-fold at puberty (P < 0.05), assessed by immunostaining and verified electrophysiologically by an increased response to gaboxadol (100 nM), which is selective for α4βδ. Knock-out of α4 prevented the pubertal decrease in kalirin-7 and synaptic pruning and also increased the dendritic length, demonstrating a functional link. These data suggest that pubertal α4βδ GABARs alter dendritic morphology and trigger pruning in female CA3 hippocampus., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Retinal horizontal cells (HCs) are inhibitory neurons, which modulate the transmission of light-elicited signals from photoreceptors to bipolar cells in the outer retina. HCs of the same physiological type are extensively coupled via gap junctions. In the zebrafish retina, the population of HCs comprises up to four morphologically distinct subtypes. Four different connexins (Cx52.6, Cx52.7, Cx52.9 and Cx55.5) were detected in these cells with overlapping expression patterns. In this study, we show that Cx52.6 is alternatively spliced in the retina, resulting in an additional isoform, designated as Cx53.4, which differs from the originally described Cx52.6 only by the final C-terminal peptide (12 vs. 4 aa). Further protein sequence alignments revealed that Cx53.4 represents the counterpart of alternatively spliced mouse Cx57 and human Cx62. RT-PCR analyses of mRNA expression in different adult zebrafish tissues showed that Cx53.4 is expressed exclusively in the retina. The localization of Cx53.4 protein within the retina was analyzed using a specific antibody. Immunofluorescence analyses demonstrated that the expression of Cx53.4 is restricted to HCs of all four subtypes. Further, immunoelectron microscopy confirmed the presence of Cx53.4 in gap junctions between HC dendrites and between their axon terminals., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

The brain is capable of improving from a chronically stressed state. The hippocampus in particular appears to "recover" from chronic stress-induced morphological and functional deficits following a post-stress rest period of several weeks. We previously found that hippocampal brain-derived neurotrophic factor (BDNF) was necessary for spatial ability to improve following a post-stress rest period. The following studies are the first to investigate the involvement of BDNF and its TrkB receptor on the recovery process following the end of chronic stress, as it pertains to hippocampal dendritic retraction and spatial memory deficits. In the first study, hippocampal BDNF was downregulated via RNA interference and then hippocampal CA3 and CA1 dendritic complexity were evaluated following chronic stress and a post-stress rest period in male Sprague-Dawley rats. Downregulating hippocampal BDNF prevented the enhancement of CA3 apical dendritic complexity following the rest period. Moreover, chronic stress and downregulated BDNF in the post-stress rest group led to regionally specific enhancements in CA1 dendritic complexity. In the second study, we tested whether the TrkB receptor was involved by administering daily systemic injections of ANA-12, a TrkB receptor antagonist, during the three-week post-stress rest period. ANA-12 prevented the improvement in spatial ability and CA3 apical dendritic complexity following the post-stress rest period. These data demonstrate that hippocampal BDNF acting via its TrkB receptor is necessary during the post-stress rest period in order to improve the impaired hippocampal structural and cognitive outcomes that occur in response to chronic stress., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Morphological screening of mouse brains with known behavioral deficits can give great insight into the relationship between brain regions and their behavior. Oxytocin- and CD38-deficient mice have previously been shown to have behavioral phenotypes, such as restrictions in social memory, social interactions, and maternal behavior. CD38 is reported as an autism spectrum disorder (ASD) candidate gene and its behavioral phenotypes may be linked to ASD. To address whether these behavioral phenotypes relate to brain pathology and neuronal morphology, here we investigate the morphological changes in the CD38-deficient mice brains, with focus on the pathology and neuronal morphology of the cortex and hippocampus, using Nissl staining, immunohistochemistry, and Golgi staining. No difference was found in terms of cortical layer thickness. However, we found abnormalities in the number of neurons and neuronal morphology in the visual cortex and dentate gyrus (DG). In particular, there were arborisation differences between CD38 -/- and CD38 +/+ mice in the apical dendrites of the visual cortex and hippocampal CA1 pyramidal neurons. The data suggest that CD38 is implicated in appropriate development of brain regions important for social behavior., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

The basolateral amygdala receives a very dense cholinergic innervation from the basal forebrain that is important for memory consolidation. Although behavioral studies have shown that both M 1 and M 2 muscarinic receptors are critical for these mnemonic functions, there have been very few neuroanatomical and electrophysiological investigations of the localization and function of different types of muscarinic receptors in the amygdala. In the present study we investigated the subcellular localization of M 2 muscarinic receptors (M 2 Rs) in the anterior basolateral nucleus (BLa) of the mouse, including the localization of M 2 Rs in parvalbumin (PV) immunoreactive interneurons, using double-labeling immunoelectron microscopy. Little if any M 2 R-immunoreactivity (M 2 R-ir) was observed in neuronal somata, but the neuropil was densely labeled. Ultrastructural analysis using a pre-embedding immunogold-silver technique (IGS) demonstrated M 2 R-ir in dendritic shafts, spines, and axon terminals forming asymmetrical (excitatory) or symmetrical (mostly inhibitory) synapses. In addition, about one-quarter of PV+ axon terminals and half of PV+ dendrites, localized using immunoperoxidase, were M 2 R+ when observed in single thin sections. In all M 2 R+ neuropilar structures, including those that were PV+, about one-quarter to two-thirds of M 2 R+ immunoparticles were plasma-membrane-associated, depending on the structure. The expression of M 2 Rs in PV+ and PV-negative terminals forming symmetrical synapses indicates M 2 R modulation of inhibitory transmission. Electrophysiological studies in mouse and rat brain slices, including paired recordings from interneurons and pyramidal projection neurons, demonstrated M 2 R-mediated suppression of GABA release. These findings suggest cell-type-specific functions of M 2 Rs and shed light on organizing principles of cholinergic modulation in the BLa., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

As the predominant mediator of the delayed rectifier current, K V 2.1 is an important regulator of neuronal excitability. K V 2.1, however, also plays a well-established role in apoptotic cell death. Apoptogenic stimuli induce syntaxin-dependent trafficking of K V 2.1, resulting in an augmented delayed rectifier current that acts as a conduit for K + efflux required for pro-apoptotic protease/nuclease activation. Recent evidence suggests that K V 2.1 somato-dendritic clusters regulate the formation of endoplasmic reticulum-plasma membrane junctions that function as scaffolding sites for plasma membrane trafficking of ion channels, including K V 2.1. However, it is unknown whether K V 2.1 somato-dendritic clusters are required for apoptogenic trafficking of K V 2.1. By overexpression of a protein derived from the C-terminus of the cognate channel K V 2.2 (K V 2.2CT), we induced calcineurin-independent disruption of K V 2.1 somato-dendritic clusters in rat cortical neurons, without altering the electrophysiological properties of the channel. We observed that K V 2.2CT-expressing neurons are less susceptible to oxidative stress-induced cell death. Critically, expression of K V 2.2CT effectively blocked the increased current density of the delayed rectifier current associated with oxidative injury, supporting a vital role of K V 2.1-somato-dendritic clusters in apoptogenic increases in K V 2.1-mediated currents., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Mutations in the PI3K/Akt/mTOR signaling pathway or in the upstream negative regulator Pten cause human brain overgrowth disorders, such as focal cortical dysplasia and megalencephaly, and are characterized by the presence of hypertrophic neurons. These disorders often have a pediatric onset and a high comorbidity with drug-resistant epilepsy; however, effective pharmacological treatments are lacking. We established forebrain excitatory neuron-specific Pten-deficient cultures as an in vitro model of brain overgrowth disorders, and investigated the effects of this Pten mutation on PI3K/Akt/mTOR signaling and neuronal growth. Mutant neurons exhibit excessive PI3K/Akt/mTOR signaling activity, enlarged somas and increased dendritic arborization. To understand the contributions of Akt and mTORC1 kinases to the hypertrophy phenotype, we evaluated the effects of short-term treatment with the Akt inhibitor MK-2206, and the mTORC1 inhibitor RAD001, which have shown safety and efficacy in human cancer clinical trials. We found that RAD001 treatment only partially reversed the morphological abnormalities of Pten mutant neurons, whereas MK-2206 treatment completely rescued the phenotype. Interestingly, neither treatment altered the size or morphology of normal neurons. Our results suggest that Akt is a major determinant of neuronal growth, and that Akt inhibition may be an effective strategy for pharmacological intervention in brain overgrowth disorders., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

In the vertebrate retina, horizontal cells (HCs) reveal homologous coupling by gap junctions (gj), which are thought to consist of different connexins (Cx). However, recent studies in mouse, rabbit and zebrafish retina indicate that individual HCs express more than one connexin. To provide further insights into the composition of gj connecting HCs and to determine whether HCs express multiple connexins, we examined the molecular identity and distribution of gj between HCs of the carp retina. We have cloned four carp connexins designated Cx49.5, Cx55.5, Cx52.6 and Cx53.8 with a close relationship to connexins previously reported in HCs of mouse, rabbit and zebrafish, respectively. Using in situ hybridization, Cx49.5 expression was detected in different subpopulations of retinal neurons including HCs, whereas the Cx52.6 transcript was localized exclusively in HCs. Using specific antibodies, Cx55.5 and Cx53.8 were detected on dendrites of all four HC subtypes and axon terminals. Immunoelectron microscopy confirmed the presence of Cx55.5 and Cx53.8 in gap junctions between these processes and Cx55.5 was additionally observed in HC dendrites invaginating cone pedicles, suggesting its participation in the modulation of photoreceptor output in the carp retina. Furthermore, using single-cell RT-PCR, all four connexins were detected in different subtypes of HCs, suggesting overlapping expression patterns. Thus, the composition of gj mediating homologous coupling between subtypes of carp HCs appears to be more complex than expected. Moreover, BLAST searches of the preliminary carp genome, using novel sequences as query, suggest that most of the analyzed connexin genes are duplicated in carp., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

Neuronal gap junctions are ubiquitous in the brain, but their precise positions in actual neuronal circuits have been uncertain, and their functional roles remain unclear. In this study, I visualized connexin36-immunoreactive gap junctions and examined the structural features of the interconnected dendrites arising from parvalbumin (PV)-positive interneurons in layer 4 of the feline visual cortex. I observed evidence for net-like dense linkages of dendrites among virtually all PV neurons (56/58 cells, 96.6%) in the tissue. This dendritic reticulum established connections among clustered cells and further among remote cells. The latter connectivity exhibited a preference for vertical direction, including translaminar linkages, but was also characterized by lateral continuity. Measurement of the distances from each dendritic gap junction back to the two connected somata revealed that at least one of two somata was within 50μm from the junction in 77.5% of the cases and within 75μm in 91.2% of the cases. Thus, distal gap junctions mediated morphologically asymmetrical connection where one soma was close to, but the other soma was far from the connecting junction. This connectivity was typically observed between neurons located apart in the same columnar space, where a long vertical dendrite bridged two neurons through an asymmetrically positioned gap junction. In contrast, gap junctions formed between nearby cells were close to both somata. Thalamocortical afferents established synapses densely on somata of layer 4 PV neurons, indicating the possible involvement of proximal gap junctions in visual stimulus-driven feedforward regulation. These findings provide a new structural basis for cortical investigations., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The subiculum is the output component of the hippocampal formation and holds a key position in the neural circuitry of memory. Previous studies have demonstrated the subiculum's connectivity to other brain areas in detail; however, little is known regarding its internal structure. We investigated the cytoarchitecture of the temporal and mid-septotemporal parts of the subiculum using immunohistochemistry. The border between the CA1 region and subiculum was determined by both cytoarchitecture and zinc transporter 3 (ZnT3)-immunoreactivity (IR), whereas the border between the subiculum and presubiculum (PreS) was partially indicated by glutamate receptor 1 (GluR1)-IR. The subiculum was divided into proximal and distal subfields based on cytoarchitecture and immunohistochemistry for calbindin (CB), nitric oxide synthase (NOS) and Purkinje cell protein 4 (PCP4). The proximal subiculum (defined here as subiculum 2) was composed of five layers: the molecular layer (layer 1), the medium-sized pyramidal cell layer (layer 2) that contained NOS- and PCP4-positive neurons, the large pyramidal cell layer (layer 3) characterized by the accumulation of ZnT3- (more proximally) and vesicular glutamate transporter 2-positive (more distally) boutons, layer 4 containing polymorphic cells, and the deepest layer 5 composed of PCP4-positive cells with long apical dendrites that reached layer 1. The distal subiculum (subiculum 1) consisting of smaller neurons did not show these features. Quantitative analyses of the size and numerical density of somata substantiated this delineation. Both the proximal-distal division and five-layered structure in the subiculum 2 were confirmed throughout the temporal two-thirds of the subiculum. These findings will provide a new structural basis for hippocampal investigations., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Iron-overload can cause cognitive impairment due to blood-brain barrier (BBB) breakdown and brain mitochondrial dysfunction. Although deferiprone (DFP) has been shown to exert neuroprotection, the head-to-head comparison among iron chelators used clinically on brain iron-overload has not been investigated. Moreover, since antioxidant has been shown to be beneficial in iron-overload condition, its combined effect with iron chelator has not been tested. Therefore, the hypothesis is that all chelators provide neuroprotection under iron-overload condition, and that a combination of an iron chelator with an antioxidant has greater efficacy than monotherapy. Male Wistar rats (n=42) were assigned to receive a normal diet (ND) or a high-iron diet (HFe) for 4months. At the 2nd month, HFe-fed rats were treated with a vehicle, deferoxamine (DFO), DFP, deferasirox (DFX), n-acetyl cysteine (NAC) or a combination of DFP with NAC, while ND-fed rats received vehicle. At the end of the experiment, rats were decapitated and brains were removed to determine brain iron level and deposition, brain mitochondrial function, BBB protein expression, brain mitochondrial dynamic, brain apoptosis, tau-hyperphosphorylation, amyloid-β (Aβ) accumulation and dendritic spine density. The results showed that iron-overload induced BBB breakdown, brain iron accumulation, brain mitochondrial dysfunction, impaired brain mitochondrial dynamics, tau-hyperphosphorylation, Aβ accumulation and dendritic spine reduction. All treatments, except DFX, attenuated these impairments. Moreover, combined therapy provided a greater efficacy than monotherapy. These findings suggested that iron-overload induced brain iron toxicity and a combination of an iron chelator with an antioxidant provided a greatest efficacy for neuroprotection than monotherapy., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

The organization of the inhibitory intercalated cell masses (IM) of the primate amygdala is largely unknown despite their key role in emotional processes. We studied the structural, topographic, neurochemical and intrinsic connectional features of IM neurons in the rhesus monkey brain. We found that the intercalated neurons are not confined to discrete cell clusters, but form a neuronal net that is interposed between the basal nuclei and extends to the dorsally located anterior, central, and medial nuclei of the amygdala. Unlike the IM in rodents, which are prominent in the anterior half of the amygdala, the primate inhibitory net stretched throughout the antero-posterior axis of the amygdala, and was most prominent in the central and posterior extent of the amygdala. There were two morphologic types of intercalated neurons: spiny and aspiny. Spiny neurons were the most abundant; their somata were small or medium size, round or elongated, and their dendritic trees were round or bipolar, depending on location. The aspiny neurons were on average slightly larger and had varicose dendrites with no spines. There were three non-overlapping neurochemical populations of IM neurons, in descending order of abundance: (1) Spiny neurons that were positive for the striatal associated dopamine- and cAMP-regulated phosphoprotein (DARPP-32+); (2) Aspiny neurons that expressed the calcium-binding protein calbindin (CB+); and (3) Aspiny neurons that expressed nitric oxide synthase (NOS+). The unique combinations of structural and neurochemical features of the three classes of IM neurons suggest different physiological properties and function. The three types of IM neurons were intermingled and likely interconnected in distinct ways, and were innervated by intrinsic neurons within the amygdala, or by external sources, in pathways that underlie fear conditioning and anxiety., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

The K(+)-dependent Na(+)/Ca(2)-exchanger (NCKX) family is encoded by five related genes, of which NCKX2 (solute carrier family 24, member 2) is the most abundant member present in the brain. Nckx2 knockout mice display profound loss of hippocampal long-term potentiation, and selective deficits in motor learning and spatial working memory. However, the molecular mechanisms underlying these changes have not been established. Thus, the overall goal of this project was to identify the exact subcellular localization of NCKX2 in the hippocampus, as an important step toward understanding the physiological role for NCKX2 in neuronal plasticity. To achieve this goal, we used dual immunofluorescent confocal microscopy and immunoelectron microscopy. Our data demonstrate that the majority of NCKX2 is co-localized with the dendritic marker, microtubule associated protein 2. A smaller fraction is co-localized with the presynaptic marker, synapsin 1, and the smallest amount is co-localized with the glutamatergic spine marker, N-methyl-d-aspartate receptor 1. The data from immunoelectron microscopy are consistent with the observations from dual immunofluorescence, and show that the highest fraction of NCKX2 is located on the plasma membrane of small oblique dendrites, particularly in CA1 neurons of the stratum radiatum. In the molecular layer, a greater fraction of NCKX2 is associated with axon terminals and, in addition, a fraction of NCKX2 is found not associated with the plasma membrane but located in the cytoplasm. These studies describe for the first time the exact location of NCKX2 in the hippocampus of adult mice and suggest that the function of NCKX2 in neuronal plasticity in hippocampal CA1 neurons may be mediated by its kinetic effect on the local Ca(2+) concentration that influences dendritic integration. At other hippocampal locations NCKX2 has a somewhat different spatial distribution, consistent with published reports of NCKX2 expression in other brain regions, suggesting that NCKX2 contributes to Ca(2+) homeostasis in distinct ways in different brain neurons., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Allopregnanolone (APα; 5α-pregnan-3α-ol-20-one) is synthesized in both the periphery and central nervous system and is known to be a potent positive allosteric modulator of the GABAA receptor. Because APα was suggested to improve the symptoms of depression and Alzheimer's disease (AD), which involve synaptic dysfunction and loss, we examined whether APα affects excitatory synapses. Drebrin, which is an actin-binding protein, forms a unique stable actin structure in dendritic spines, and drebrin levels correlate positively with cognitive levels in AD and mild cognitive impairment. We investigated whether APα increases excitatory synapse density along dendrites of mature hippocampal neurons using drebrin-imaging-based evaluation of mature synapses. We prepared primary cultures of hippocampal neurons and either transfected them with GFP or immunostained them against drebrin. Morphological analysis of GFP-transfected neurons revealed that a 24-h exposure to 0.3 or 1 μM APα significantly increased dendritic spine density without any morphological changes to spines. Drebrin cluster density was also increased by 0.3 and 1 μM APα. The protein kinase A (PKA) inhibitor H-89 inhibited the APα-induced increase in drebrin cluster density. These data demonstrate that APα increases mature excitatory synapses via activation of PKA. Therefore, the PKA-cAMP response element-binding protein (CREB) signaling pathway is likely to be involved in the APα-induced increase of mature excitatory synapses. Another possibility is that the PKA-dependent increase in AMPA receptors at dendritic spines mediates the APα function. In conclusion, our study indicates that APα may improve neuropsychiatric disorder outcomes via increasing the numbers of mature excitatory synapses., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Endocannabinoids (eCBs) are involved in a myriad of physiological processes that are mediated through the activation of cannabinoid receptors, which are ubiquitously distributed within the nervous system. One neurochemical target at which cannabinoids interact to have global effects on behavior is brain noradrenergic circuitry. We, and others, have previously shown that CB type 1 receptors (CB1r) are positioned to pre-synaptically modulate norepinephrine (NE) release in the rat frontal cortex (FC). Diacylglycerol lipase (DGL) is a key enzyme in the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). While DGL-α is expressed in the FC in the rat brain, it is not known whether noradrenergic afferents target neurons expressing synthesizing enzymes for the endocannabinoid, 2-AG. In the present study, we employed high-resolution neuroanatomical approaches to better define cellular sites for interactions between noradrenergic afferents and FC neurons expressing DGL-α. Immunofluorescence microscopy showed close appositions between processes containing the norepinephrine transporter (NET) or dopamine-β-hydroxylase (DβH) and cortical neurons expressing DGL-α-immunoreactivity. Ultrastructural analysis using immunogold-silver labeling for DGL-α and immunoperoxidase labeling for NET or DβH confirmed that NET-labeled axon terminals were directly apposed to FC somata and dendritic processes that exhibited DGL-α-immunoreactivity. Finally, tissue sections were processed for immunohistochemical detection of DGL-α, CB1r and DβH. Triple label immunofluorescence revealed that CB1r and DβH were co-localized in common cellular profiles and these were in close association with DGL-α. Taken together, these data provide anatomical evidence for direct synaptic associations between noradrenergic afferents and cortical neurons exhibiting endocannabinoid synthesizing machinery., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Since the discovery that long-term memory is dependent on protein synthesis, several transcription factors have been found to participate in the transcriptional activity needed for its consolidation. Among them, NF-kappa B is a constitutive transcription factor whose nuclear activity has proven to be necessary for the consolidation of inhibitory avoidance in mice. This transcription factor has a wide distribution in the nervous system, with a well-reported presence in dendrites and synaptic terminals. Here we report changes in synaptosomal NF-kappa B localization and activity, during long-term memory consolidation. Activity comparison of synaptosomal and nuclear NF-kappa B, indicates different dynamics for both localizations. In this study we identify two pools of synaptosomal NF-kappa B, one obtained with the synaptoplasm (free fraction) and the second bound to the synaptosomal membranes. During the early steps of consolidation the first pool is activated, as the membrane associated transcription factor fraction increases and concomitantly the free fraction decreases. These results suggest that the activation of synaptic NF-kappa B and its translocation to membranes are part of the consolidation of long-term memory in mice., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Gap junctions provide for direct intercellular electrical and metabolic coupling. The abundance of gap junctions at "large myelinated club ending (LMCE)" synapses on Mauthner cells (M-cells) of the teleost brain provided a convenient model to correlate anatomical and physiological properties of electrical synapses. There, presynaptic action potentials were found to evoke short-latency electrical "pre-potentials" immediately preceding their accompanying glutamate-induced depolarizations, making these the first unambiguously identified "mixed" (i.e., chemical plus electrical) synapses in the vertebrate CNS. We recently showed that gap junctions at these synapses exhibit asymmetric electrical resistance (i.e., electrical rectification), which we correlated with total molecular asymmetry of connexin composition in their apposing gap junction hemiplaques, with connexin35 (Cx35) restricted to axon terminal hemiplaques and connexin34.7 (Cx34.7) restricted to apposing M-cell plasma membranes. We now show that similarly heterotypic neuronal gap junctions are abundant throughout goldfish brain, with labeling exclusively for Cx35 in presynaptic hemiplaques and exclusively for Cx34.7 in postsynaptic hemiplaques. Moreover, the vast majority of these asymmetric gap junctions occur at glutamatergic axon terminals. The widespread distribution of heterotypic gap junctions at glutamatergic mixed synapses throughout goldfish brain and spinal cord implies that pre- vs. postsynaptic asymmetry at electrical synapses evolved early in the chordate lineage. We propose that the advantages of the molecular and functional asymmetry of connexins at electrical synapses that are so prominently expressed in the teleost CNS are unlikely to have been abandoned in higher vertebrates. However, to create asymmetric coupling in mammals, where most gap junctions are composed of connexin36 (Cx36) on both sides, would require some other mechanism, such as differential phosphorylation of connexins on opposite sides of the same gap junction or on asymmetric differences in the complement of their scaffolding and regulatory proteins., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Bone morphogenetic protein-5 (BMP5), a member of the transforming growth factor-β (TGF-β) superfamily, has many effects in several biological events. Although BMP5 expression has been well reported in the early development of the central nervous system (CNS), there is little information about its expression in the adult CNS. Thus, we analyzed BMP5 expression in the adult rat CNS by immunohistochemistry. Abundant BMP5 expression was observed in most neurons, and their dendrites and axons. Furthermore, strong BMP5 expression was also detected in the neuropil of the gray matters with high plasticity, such as the molecular layer of the cerebellum, locus coeruleus, and nucleus of the solitary tract. In addition, we showed BMP5 expression also in astrocytes, ependymal cells and meninges. Our data suggest that BMP5 is widely expressed throughout the adult CNS, and this abundant expression in the adult brain strongly supports the idea that BMP5 plays important roles not only in the developing brain but also in the adult brain., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Extracellular matrix (ECM) accumulates around different neuronal compartments of the central nervous system (CNS) or appears in diffuse reticular form throughout the neuropil. In the adult CNS, the perineuronal net (PNN) surrounds the perikarya and dendrites of various neuron types, whereas the axonal coats are aggregations of ECM around the individual synapses, and the nodal ECM is localized at the nodes of Ranvier. Previous studies in our laboratory demonstrated on rats that the heterogeneous distribution and molecular composition of ECM is associated with the variable cytoarchitecture and hodological organization of the vestibular nuclei and may also be related to their specific functions in gaze and posture control as well as in the compensatory mechanisms following vestibular lesion. Here, we investigated the ECM expression pattern in the climbing fiber-generating inferior olive (IO), which is functionally related to the vestibular nuclei. By using histochemical and immunohistochemical methods, the most characteristic finding was the lack of PNNs, presumably due to the absence of synapses on the perikarya and proximal dendrites of IO neurons. On the other hand, the darkly stained dots or ring-like structures in the neuropil might represent the periaxonal coats around the axon terminals of olivary synaptic glomeruli. We have observed positive ECM reaction for the hyaluronan, tenascin-R, hyaluronan and proteoglycan link protein 1 (HAPLN1) and various chondroitin sulfate proteoglycans. The staining intensity and distribution of ECM molecules revealed a number of differences between the functionally different subnuclei of IO. We hypothesized that the different molecular composition and intensity differences of ECM reaction is associated with different control mechanisms of gaze and posture control executed by the visuomotor-vestibular, somatosensory and integrative subnuclei of the IO., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Fragile X mental retardation protein (FMRP) binds select mRNAs, functions in intracellular transport of these mRNAs and represses their translation. FMRP is highly expressed in neurons and lack of FMRP has been shown to result in dendritic dysmorphology and altered synaptic function. FMRP is known to interact with mRNAs for the Kv3.1b potassium channel which is required for neurons to fire action potentials at high rates with remarkable temporal precision. Auditory brainstem neurons are known for remarkably high spike rates and expression of Kv3.1b potassium channels. Fragile X syndrome (FXS) is a genetic disorder caused by a mutation in the fragile X mental retardation 1 gene (Fmr1) resulting in decreased expression of FMRP and subsequent intellectual disability, seizures, attention deficit and hypersensitivity to auditory and other sensory stimuli. We therefore hypothesize that the auditory difficulties in FXS result, at least in part, from dysfunction of auditory brainstem neurons. To examine this hypothesis, we have studied normal human brainstem tissue with immunohistochemical techniques and confocal microscopy. Our results demonstrate that FMRP is widely expressed in cell bodies and dendritic arbors of neurons in the human cochlear nucleus and superior olivary complex and also that coincidence detector neurons of the medial superior olive colocalization of FMRP and Kv3.1b. We interpret these observations to suggest that the lower auditory brainstem is a potential site of dysfunction in FXS., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Tyrosine-hydroxylase-positive (TH(+)) amacrine cells release dopamine in a paracrine manner and also form GABA-ergic contact sites with inner retinal neurons. The best known sites are formed by TH(+) fibrous rings and AII amacrine cell somata in stratum 1 of the inner plexiform layer (IPL). An AII amacrine cell is a highly compartmentalized neuron with relatively large soma, a stout dendritic stalk and two sets of processes, one showing lobular appearance and extending horizontally in stratum 1 and a second transversally elongated group of fibers in strata 4 and 5. Although, all of these compartments have been reported as tic sites, it is uncertain if TH(+) amacrine cell inputs are homogeneously distributed or they rather target specific AII cell compartments. In this study we investigated the TH(+)/AII cell system by immunohistochemistry to map the potential synaptic contacts in the rabbit retina. We found numerous intimate contacts between the two amacrine cell populations throughout the IPL. However, TH(+) fibers favored the soma/main stalk region of AII amacrine cells and only contacted lobular appendages and transversal processes sporadically. In addition to the well-studied contacts between AII cell somata and TH(+) rings in stratum 1 we found that the main stalk region in stratum 3 serves as a secondary major target for TH(+) axons. These data thus clearly show that TH(+) contacts to AII amacrine cells are highly compartment specific., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Bone morphogenetic proteins (BMPs) exert its biological functions by interacting with membrane bound receptors. However, functions of BMPs are also regulated in the extracellular space by secreted antagonistic regulators. Chordin is an extracellular BMP antagonist that binds BMP-2, 4, and 7 with high affinity and thus interferes with binding to BMP receptors. Although chordin expression has been well described in the early development of the CNS, little information is available for its expression in the adult CNS. We, thus, investigated chordin expression in the adult rat CNS using immunohistochemistry. Chordin was intensely expressed in most neurons, and their dendrites and axons. In addition, abundant chordin expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. Furthermore, we found that astrocytes and ependymal cells also express chordin protein. These data indicate that chordin is more widely expressed throughout the adult CNS than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that chordin plays pivotal roles also in the adult brain., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Social isolation during the vulnerable period of adolescence produces emotional dysregulation manifested by abnormalities in adult behaviors that require emotional processing. The affected brain regions may include the basolateral amygdala (BLA), where plasticity of glutamatergic synapses in principal neurons plays a role in conditioned emotional responses. This plasticity is dependent on NMDA receptor trafficking denoted by intracellular mobilization of the obligatory NR1 NMDA subunit. We tested the hypothesis that the psychosocial stress of adolescent social isolation (ASI) produces a lasting change in NMDA receptor distribution in principal neurons in the BLA of adults that express maladaptive emotional responses to sensory cues. For this, we used behavioral testing and dual electron microscopic immunolabeling of NR1 and calcium calmodulin-dependent protein kinase II (CaMKII), a protein predominantly expressed in principal neurons of the BLA in adult C57Bl/6 mice housed in isolation or in social groups from post-weaning day 22 until adulthood (∼3 months of age). The isolates showed persistent deficits in sensorimotor gating evidenced by altered prepulse inhibition (PPI) of acoustic startle and hyperlocomotor activity in a novel environment. Immunogold-silver labeling for NR1 alone or together with CaMKII was seen in many somatodendritic profiles in the BLA of all mice irrespective of rearing conditions. However, isolates compared with group-reared mice had a significantly lower cytoplasmic (4.72 ± 0.517 vs 6.31 ± 0.517) and higher plasmalemmal (0.397 ± 0.0779 vs 0.216 ± 0.026) density of NR1 immunogold particles in CaMKII-containing dendritic spines. There was no rearing-dependent difference in the size or number of these spines or those of other dendritic profiles within the neuropil, which also failed to show an impact of ASI on NR1 immunogold labeling. These results provide the first evidence that ASI enhances the surface trafficking of NMDA receptors in dendritic spines of principal neurons in the BLA of adult mice showing maladaptive behaviors that are consistent with emotional dysregulation., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Functional interactions between dopaminergic and noradrenergic systems occur in many brain areas, including the prefrontal cortex (PFC). Biochemical, electrophysiological and behavioral data indicate crosstalk between D1 dopamine receptor (D1R) and α1-adrenergic receptor (α1AR) signaling in the PFC. However, it is unknown whether these interactions occur within the same neurons, or between neurons expressing either receptor. In this study, we used electron microscopy immunocytochemistry to demonstrate that D1Rs and α1ARs co-localize in rat PFC neuronal elements, most prominently in dendrites (60-70%), but also significantly in axon terminals, unmyelinated axons and spines (∼20-30%). Our data also showed that the ratio of plasma membrane-bound to intracellular α1ARs is significantly reduced in D1R-expressing dendrites. Similar results were obtained using either a pan-α1AR or a selective α1bAR antibody to label noradrenergic receptors. Thus, these results demonstrate that D1Rs and α1ARs co-localize in PFC dendrites, thereby suggesting that the catecholaminergic effects on PFC function may be driven, at least in part, by cell-autonomous D1R-α1AR interactions., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

TatCN21 is a membrane permeable calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor derived from the inhibitor protein CaMKIIN. TatCN21 has been used to demonstrate the involvement of CaMKII in a variety of physiological and pathological phenomena, and it also limits excitotoxic damage in neurons. Here we use preembedding immunogold electron microscopy to examine the effect of tatCN21 on the redistribution of CaMKII in cultured hippocampal neurons. Incubation of cultures with tatCN21 (20 μM for 20 min) prior to exposure to N-methyl-d-asparic acid (NMDA) (50 μM for 2 min) inhibited both the accumulation of CaMKII at postsynaptic densities (PSDs) and CaMKII clustering in the dendrites. Under these conditions, CaMKII also formed morphologically distinct aggregates with polyribosomes near the PSD and in dendrites. Formation of these CaMKII-polyribosome aggregates requires the presence of both tatCN21 and calcium, and was augmented upon exposure to high K(+) or NMDA. CaMKII-polyribosome aggregates formed consistently with 20 μM tatCN21, but minimally or not at all with 5 μM. However, these aggregates are not induced by another CaMKII inhibitor, KN93. Formation of CaMKII-polyribosome aggregates was completely reversible within 1h after washout of tatCN21. Effects of tatCN21 were largely restricted to dendrites, with minimal effect in the soma. The effects of tatCN21 on CaMKII distribution can be used to dissect the mechanism of CaMKII involvement in cellular events., (Published by Elsevier Ltd.)

Parallel representations of the visual world are already established at the very first synapse of the visual system. Cone photoreceptors, which hyperpolarize in response to light, forward the visual signal onto distinct types of ON and OFF cone bipolar cells (BCs). In the case of OFF BCs, the glutamatergic cone input is integrated by ionotropic glutamate receptors, giving rise to a sign-preserving mode of synaptic transmission. The combination of glutamate receptor (GluR) subunits, i.e. AMPA or kainate subunits, importantly contributes to shaping the OFF bipolar cells' distinct response properties. The mouse is one of the few mammals in which the (most likely) complete set of (five) retinal OFF BC types is identified. However, it is not clear which GluR subtypes are expressed by the different mouse OFF BC types. We addressed this question by combining immunolabeling, electrical whole-cell recordings and pharmacology, and present evidence that the different types of OFF BCs express distinct types of glutamate receptors: Type 1 BCs exclusively expressed AMPA receptors, whereas type 2 and type 3a BCs expressed kainate receptors of different subunit compositions. Additionally, we found that two OFF BC types (3b and 4) very likely express both AMPA and kainate receptors but, interestingly, the two receptor subunits were not co-localized at the same dendritic site. The complex, BC type-specific expression pattern of GluRs we describe here supports their essential role in establishing parallel pathways at the first synapse of the mouse visual system., (Copyright © 2013 IBRO. All rights reserved.)

A state of low dopaminergic activity has been implicated in attention-deficit/hyperactivity disorder (ADHD). The clinical symptoms of ADHD include inattention, impulsivity and hyperactivity, as well as impaired learning; dopaminergic modulation of the functions in the hippocampus is important to both learning and memory. To determine dopamine receptor (DR) density in a well-established animal model for ADHD, we quantified the dopamine D5 receptors in the hippocampus in the spontaneously hypertensive rat. We used immunofluorescence microscopy and immunogold electron microscopy to quantify the dopamine D5 receptor density on CA1 pyramidal cell somas and dendrites and dendritic spines in the stratum radiatum and stratum oriens. The density of the dopamine D5 receptors was significantly lower in the cytoplasm of pyramidal cell somas in the spontaneously hypertensive rat compared to the control, indicating a reduced reservoir for insertion of receptors into the plasma membrane. DRs are important for long-term potentiation and long-term depression, hence the deficit may contribute to the learning difficulties in individuals with the diagnosis of ADHD., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Adolescent experiences of social deprivation result in profound and enduring perturbations in adult behavior, including impaired sensorimotor gating. The behavioral deficits induced by adolescent social isolation in rats can be ameliorated by antipsychotic drugs blocking dopamine D2 receptors in the prefrontal cortex (PFC) or by chronic administration of a cannabinoid CB1 receptor antagonist. The patterning and abundance of D2 receptors in the PFC evolves concurrently with CB1 receptors through the period of adolescence. This evidence suggests that mature expression and/or surface distribution of D2 and CB1 receptors may be influenced by the adolescent social environment. We tested this hypothesis using electron microscopic immunolabeling to compare the distribution of CB1 and D2 receptors in the PFC of adult male Sprague-Dawley rats that were isolated or socially reared throughout the adolescent transition period. Prepulse inhibition (PPI) of acoustic startle was assessed as a measure of sensorimotor gating. Social isolation reduced PPI and selectively decreased dendritic D2 immunogold labeling in the PFC. However, the decrease was only evident in dendrites that were not contacted by axon terminals containing CB1. There was no apparent change in the expression of CB1 or D2 receptors in presynaptic terminals. The D2 deficit therefore may be tempered by local CB1-mediated retrograde signaling. This suggests a biological mechanism whereby the adolescent social environment can persistently influence cortical dopaminergic activity and resultant behavior., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Synaptic activity is a spatially limited process that requires a precise, yet dynamic, complement of proteins within the synaptic micro-domain. The maintenance and regulation of these synaptic proteins is regulated, in part, by local mRNA translation in dendrites. Protein synthesis within the postsynaptic compartment allows neurons tight spatial and temporal control of synaptic protein expression, which is critical for proper functioning of synapses and neural circuits. In this review, we discuss the identity of proteins synthesized within dendrites, the receptor-mediated mechanisms regulating their synthesis, and the possible roles for these locally synthesized proteins. We also explore how our current understanding of dendritic protein synthesis in the hippocampus can be applied to new brain regions and to understanding the pathological mechanisms underlying varied neurological diseases., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

The cerebellum contains more neurons than all other brain regions combined and these cells exhibit complex circuit development and dendritic elaboration during the postnatal period. Neural development, cellular morphogenesis, and synaptic plasticity are dependent on the dynamic regulation of the actin cytoskeleton by actin-binding proteins. The identification of the actin filament interactome, including proteins developmentally regulated in the postnatal cerebellum, could help define important regulators of actin cytoskeletal dynamics in developing cerebellar neurons. Affinity purification of cerebellar proteins on F-actin columns, combined with mass spectrometry, in total, 434 actin filament-associated proteins in postnatal rat cerebellum (P7) were identified. Furthermore, semi-quantitative RT-PCR was performed to screening postnatal developmentally regulated genes involved in actin dynamics and membrane trafficking in rat cerebellum (P0-P56). As the result, nine genes encoding members of the cerebellar F-actin interactome were developmentally regulated in the transcriptional level and at least five of them exhibited a similar pattern at the protein expression level by Western blot analysis. Further fluorescent immunohistochemical observations demonstrated that the actin-associated proteins Lethal(2) giant larvae protein homolog 1 (LLGL1) and metastasis suppressor 1 (MTSS1) were specifically upregulated in granule neurons and Purkinje cells during morphogenesis of axons and dendrites. This work defines a provisional actin filament interactome in rat postnatal cerebellum and identifies several candidate proteins that may be involved in the postnatal development of the cerebellum., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

The olfactory bulb (OB) of mammals contains the major endogenous dopamine-producing system in the forebrain. The vast majority of dopaminergic neurons consists of juxtaglomerular cells, which innervate the olfactory glomeruli and modulate the entrance of sensory information to the OB. Although dopaminergic juxtaglomerular cells have been widely investigated, the presence of dopaminergic interneurons other than juxtaglomerular cells has been largely unexplored. In this study, we analyze a population of tyrosine hydroxylase (TH)-containing interneurons located in the external plexiform layer (EPL) of the rat OB. These interneurons are GABAergic and morphologically heterogeneous. They have an axon and two to four dendrites running throughout the EPL. Frequently, they have appendages similar to spines in the dendrites and, sometimes, the distal portions of the dendritic branches show enlargements or swellings similar to varicosities. Contrary to other interneurons of the EPL, the TH-containing ones do not form dendro-dendritic synapses on principal cells and do not receive dendro-dendritic synapses from them. In fact, no synapses were found from the dendrites of these interneurons. When their dendrites are involved in synaptic contacts, they are always the postsynaptic element. They receive symmetrical and asymmetrical synapses from GABAergic and non-GABAergic axons of unidentified origin. Our data indicate that the local circuits of the EPL are more complex than previously thought. Although most of the interneurons of this layer establish dendro-dendritic synaptic relationships with principal cells, the TH-containing interneurons constitute an exception to this rule, resembling interneurons from other cortical areas., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Cardiorespiratory control neurons in the brainstem nucleus tractus solitarius (NTS) undergo dramatic expansion of dendritic arbors during the early postnatal period, when functional remodeling takes place within the NTS circuitry. However, the underlying molecular mechanisms of morphological maturation of NTS neurons are largely unknown. Our previous studies point to the neurotrophin brain-derived neurotrophic factor (BDNF), which is abundantly expressed by NTS-projecting primary sensory neurons, as a candidate mediator of NTS dendritogenesis. In the current study, we used neonatal rat NTS neurons in vitro to examine the role of BDNF in the dendritic development of neurochemically identified subpopulations of NTS neurons. In the presence of abundant glia, BDNF promoted NTS dendritic outgrowth and complexity, with the magnitude of the BDNF effect dependent on neuronal phenotype. Surprisingly, BDNF switched from promoting to inhibiting NTS dendritogenesis upon glia depletion. Moreover, glia depletion alone led to a significant increase in NTS dendritic outgrowth. Consistent with this result, astrocyte-conditioned medium (ACM), which promoted hippocampal dendritogenesis, inhibited dendritic growth of NTS neurons. The latter effect was abolished by heat-inactivation of ACM, pointing to a diffusible astrocyte-derived negative regulator of NTS dendritic growth. Together, these data demonstrate a role for BDNF in the postnatal development of NTS neurons, and reveal novel effects of glia on this process. Moreover, previously documented dramatic increases in NTS glial proliferation in victims of sudden infant death syndrome (SIDS) underscore the importance of our findings and the need to better understand the role of glia and their interactions with BDNF during NTS circuit maturation. Furthermore, while it has previously been demonstrated that the specific effects of BDNF on dendritic growth are context-dependent, the role of glia in this process is unknown. Thus, our data carry important implications for mechanisms of dendritogenesis likely beyond the NTS., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Mammalian neurokinin-3 (NK(3)) receptors of the tachykinin family of neuropeptides have been shown to activate dopaminergic neurons of the ventral tegmental area (VTA), a midbrain area displaying dopaminergic dysfunctional activity in schizophrenia. The recent finding of NK(3) receptors in VTA neuronal nucleus highlights a new level of neuromodulation, in addition to the traditional tachykinin-induced NK(3) receptor internalization and activation of second messenger signaling pathways. The function of nuclear NK(3) receptors is still unknown. It is also unclear how dopaminergic activation is affecting the NK(3) receptor distribution in the VTA. In the present study, trafficking of the NK(3) receptor in somatodendritic profiles of dopaminergic and non-dopaminergic neurons of the rat VTA was investigated following acute systemic administration of the dopamine D(1)/D(2) receptor agonist apomorphine. VTA sections were dual immunolabeled for the NK(3) receptor (immunogold) and the dopamine synthesizing enzyme tyrosine hydroxylase (TH, immunoperoxidase). Electron microscopic quantifications of somatic and dendritic densities of NK(3) immunogold particles with or without TH immunolabeling were compared in vehicle-injected or apomorphine-injected rats. In dopaminergic (TH) neurons, apomorphine evoked a significant increase in NK(3) receptor densities in cytoplasmic and nuclear portions of the soma. These changes were accompanied by a respective decrease and increase in plasmalemmal and cytoplamic NK(3) receptor densities in dopaminergic dendrites. In non-TH neurons, presumably GABAergic neurons of the VTA, the NK(3) receptor densities in somata and dendrites were not significantly altered by apomorphine. The results suggest that dopaminergic receptor activation is inducing a rapid mobilization of NK(3) receptors in VTA dopaminergic neurons. The apomorphine-evoked NK(3) receptors plasticity might reflect dendritic internalization and translocation of NK(3) receptors toward the soma and nucleus. This trafficking is not observed in non-dopaminergic neurons of the VTA. The selective apomorphine-evoked redistribution of VTA NK(3) receptors might have important implications in normal or pathological conditions such as schizophrenia., (Published by Elsevier Ltd.)

Intrinsically-photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and function as irradiance detectors, responsible for crucial non-image forming visual functions. In addition to their intrinsic photosensitivity, ipRGCs are also activated by synaptic inputs originating at the classical photoreceptors, rods and cones. Little is known about inhibition through these retinal pathways, despite ipRGCs receiving massive synaptic inputs from inhibitory amacrine interneurons. We performed a wide anatomical screening for neurotransmitter receptors possibly involved in the inhibitory modulation of ipRGCs in the macaque retina. We investigated both subtypes of primate ipRGCs described so far and report that outer-stratifying (M1) cells possess mainly GlyR α2 and GABA(A)R α3 subunits, while inner-stratifying (M2) cells are overall subject to less inhibitory modulation. Our results suggest that M1 and M2 ipRGC subtypes are modulated via distinct inhibitory intraretinal circuits., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neuronal morphogenesis plays an important role in neuronal development. TC10β/CDC42 GTPase-activating protein (TCGAP) is known to be a brain-enriched multiple domain protein, but its role in neuronal development process remains poorly understood. In the present study, we showed that TCGAP positively regulated dendritic outgrowth and spine formation in developing cortical neurons. Knocking down TCGAP by RNA interference led to a decrease in the overall length of dendrite arbors and the number of dendrite branches both in vitro and in vivo. Overexpressing TCGAP in cultured cortical neurons increased dendritic outgrowth and branching. Moreover, overexpressing TCGAP lead to an increase of spine density while knocking-down TCGAP decreased spine density in vivo. The defect by downregulating TCGAP could be rescued by expressing a knock-down resistant form of TCGAP both in vivo and in vitro. In contrast, neither downregulating nor overexpressing TCGAP had any effect on axonal morphogenesis in primary cortical neuron cultures. Together, our findings suggest that TCGAP regulates neuronal morphogenesis in developing cortical neurons at both early and late stage., (Copyright © 2011. Published by Elsevier Ltd.)

The dyslexia-associated gene DCDC2 is a member of the DCX family of genes known to play roles in neurogenesis, neuronal migration, and differentiation. Here we report the first phenotypic analysis of a Dcdc2 knockout mouse. Comparisons between Dcdc2 knockout mice and wild-type (wt) littermates revealed no significant differences in neuronal migration, neocortical lamination, neuronal cilliogenesis or dendritic differentiation. Considering previous studies showing genetic interactions and potential functional redundancy among members of the DCX family, we tested whether decreasing Dcx expression by RNAi would differentially impair neurodevelopment in Dcdc2 knockouts and wild-type mice. Consistent with this hypothesis, we found that deficits in neuronal migration, and dendritic growth caused by RNAi of Dcx were more severe in Dcdc2 knockouts than in wild-type mice with the same transfection. These results indicate that Dcdc2 is not required for neurogenesis, neuronal migration or differentiation in mice, but may have partial functional redundancy with Dcx., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Pctaire1, a Cdk-related protein kinase, is prominently expressed in terminally differentiated tissues, including the brain and the testis. We have previously shown that Pctaire1 regulates neurotransmitter release through phosphorylation of NSF, and its kinase activity is regulated by the Cdk5-dependent phosphorylation at Serine-95 (Ser95). Nonetheless, the functional roles of Pctaire1 in neurons during development remained poorly understood. In this study, we found that Pctaire1 is expressed along neurites and is concentrated at the growth cones of early differentiating hippocampal neurons. Upon maturation of these neurons, Pctiare1 is expressed as puncta and co-localized with synaptic marker in dendrites. Phosphorylation of Pctaire1 at Ser95 increases upon neuronal differentiation, concurrent with the elevation in Cdk5 activity. Knockdown of Pctaire1 abolishes dendrite development, and more importantly, expression of Ser95 phosphorylation-deficient mutant of Pctaire1 also reduces dendrite complexity, suggesting that Cdk5 regulates Pctaire1 functions in differentiating neurons. Together, our findings demonstrate that Cdk5-dependent phosphorylation of Pctaire1 at Ser95 plays an important role in dendrite development., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Calmodulin (CaM) and neurogranin (Ng) are two abundant neuronal proteins in the forebrain whose interactions are implicated in the enhancement of synaptic plasticity. To gain further insight into the actions of these two proteins we investigated whether they co-localize in principle neurons and whether they respond to high frequency stimulation in a coordinated fashion. Immunohistochemical staining of CaM and Ng in mouse hippocampal slices revealed that CaM was highly concentrated in the nucleus of CA1 pyramidal neurons, whereas Ng was more broadly localized throughout the soma and dendrites. The asymmetrical localization of CaM in the nucleus of pyramidal neurons was in sharp contrast to the distribution observed in pyramidal cells of the neighboring subiculum, where CaM was uniformly localized throughout the soma and dendrites. The somatic concentrations of CaM and Ng in CA1 pyramidal neurons were approximately 10- and two-fold greater than observed in the dendrites, respectively. High frequency stimulation (HFS) of hippocampal slices promoted mobilization of CaM and Ng from soma to dendrites. These responses were spatially restricted to the area close to the site of stimulation and were inhibited by the N-methyl-D-asparate receptor antagonist 2-amino-5-phosphonopentanoic acid. Furthermore, HFS failed to promote translocation of CaM from soma to dendrites of slices from Ng knockout mice, which also exhibited deficits in HFS-induced long-term potentiation. Translocated CaM and Ng exhibited distinct puncta decorating the apical dendrites of pyramidal neurons and appeared to be concentrated in dendritic spines. These findings suggest that mobilization of CaM and Ng to stimulated dendritic spines may enhance synaptic efficacy by increasing and prolonging the Ca2+ transients and activation of Ca2+/CaM-dependent enzymes., (Published by Elsevier Ltd.)

Activated extracellular signal-regulated kinase (ERK) signaling mediated plasticity-related gene transcription has been proposed for one possible mechanism by which 17β-estradiol (E2) enhances synaptic plasticity and memory. Because activated ERK also enhances plasticity-related mRNA translation in the dendrites of neurons, we sought to determine the effects of E2 on activation of ERK, phosphorylation of translation initiation factors, and dendritic mRNA translation in hippocampal neurons. Acute E2 application resulted in a rapid, transient increase in phosphorylation of translation initiation factors, ribosomal protein (S6) and eIF4E binding protein1 (4EBP1), in an activated ERK-dependent manner. Since phosphorylation of these translation factors enhance mRNA translation, we tested E2's effect on dendritic mRNA translation. Using a green fluorescent protein (GFP)-based dendritic mRNA translation reporter (reporter plasmid construct consisted of a GFP gene fused to the 3' untranslated region (UTR) from CAMKIIα, which contains dendritic resident mRNA targeting and mRNA translational regulatory elements) we showed that E2 treatment resulted in increased somatic and dendritic GFP mRNA translation in GFP-reporter transfected hippocampal neurons. Translation inhibitor anisomycin and ERK inhibitor U0126 blocked E2 effects. Taken together, our results provide a novel mechanism by which E2 may trigger local protein synthesis of α-CaMKII in the dendrites, which is necessary for modulation of synaptic plasticity., (Published by Elsevier Ltd.)

Cocaine administration increases AMPA GluR1 expression and receptor-mediated activation of the ventral tegmental area (VTA). Functionality is determined, however, by surface availability of these receptors in transmitter- and VTA-region-specific neurons, which may also be affected by cocaine. To test this hypothesis, we used electron microscopic immunolabeling of AMPA GluR1 subunits and tyrosine hydroxylase (TH), the enzyme needed for dopamine synthesis, in the cortical-associated parabrachial (PB) and in the limbic-associated paranigral (PN) VTA of adult male C57BL/6 mice receiving either a single injection (acute) or repeated escalating-doses for 14 days (chronic) of cocaine. Acute cocaine resulted in opposing VTA-region-specific changes in TH-containing dopaminergic dendrites. TH-labeled dendrites within the PB VTA showed increased cytoplasmic GluR1 immunogold particle density consistent with decreased AMPA receptor-mediated glutamatergic transmission. Conversely, TH-labeled dendrites within the PN VTA showed greater surface expression of GluR1 with increases in both synaptic and plasmalemmal GluR1 immunogold density after a single injection of cocaine. These changes diminished in both VTA subregions after chronic cocaine administration. In contrast, non-TH-containing, presumably GABAergic dendrites showed VTA-region-specific changes only after repeated cocaine administration such that synaptic GluR1 decreased in the PB, but increased in the PN VTA. Taken together, these findings provide ultrastructural evidence suggesting that chronic cocaine not only reverses the respective depression and facilitation of mesocortical (PB) and mesolimbic (PN) dopaminergic neurons elicited by acute cocaine, but also differentially affects synaptic availability of these receptors in non-dopaminergic neurons of each region. These adaptations may contribute to increased cocaine seeking/relapse and decreased reward that is reported with chronic cocaine use., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

NMDA receptors are found in neurons both at synapses and in extrasynaptic locations. Extrasynaptic locations are poorly characterized. Here we used preembedding immunoperoxidase and postembedding immunogold electron microscopy and fluorescence light microscopy to characterize extrasynaptic NMDA receptor locations in dissociated hippocampal neurons in vitro and in the adult and postnatal hippocampus in vivo. We found that extrasynaptic NMDA receptors on neurons in vivo and in vitro were usually concentrated at points of contact with adjacent processes, which were mainly axons, axon terminals, or glia. Many of these contacts were shown to contain adhesion factors such as cadherin and catenin. We also found associations of extrasynaptic NMDA receptors with the membrane associated guanylate kinase (MAGUKs), postsynaptic density (PSD)-95 and SAP102. Developmental differences were also observed. At postnatal day 2 in vivo, extrasynaptic NMDA receptors could often be found at sites with distinct densities whereas dense material was seen only rarely at sites of extrasynaptic NMDA receptors in the adult hippocampus in vivo. This difference probably indicates that many sites of extrasynaptic NMDA receptors in early postnatal ages represent synapse formation or possibly sites for synapse elimination. At all ages, as suggested in both in vivo and in vitro studies, extrasynaptic NMDA receptors on dendrites or the sides of spines may form complexes with other proteins, in many cases, at stable associations with adjacent cell processes. These associations may facilitate unique functions for extrasynaptic NMDA receptors., (Published by Elsevier Ltd.)

The dystrophin defective mdx mouse, acknowledged model of Duchenne Muscular Dystrophy (DMD), bears outstanding alterations of the cortical architecture, that could be responsible for the cognitive impairment often accompanying this pathological condition. Using a retrograde tract tracing technique to label neurons in Golgi-like fashion, we investigated the fine anatomical organization of associative cortico-cortical projections in mdx mice. While the absolute number of associative pyramidal neurons was significantly higher in mdx than in control animals, the ratio between the number of supra- and infragranular cortico-cortical cells was substantially unmodified. Basal dendrites of layer 2/3 pyramidal neurons displayed longer terminal branches in mdx compared to controls. Finally, the density of dendritic spines was significantly lower in mdx animals. The anomalies of associative cortico-cortical projections provide potential groundwork on the neurobiological bases of cognitive involvement in DMD and value the role of cortical microcircuitry alterations as possible source of interference with peripheral motor impairment., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

We have previously demonstrated that cocaine- and amphetamine-regulated transcript (CART) peptide colocalizes with GABA, dynorphin, D1 receptors, and substance P in some neurons in the nucleus accumbens (NAcc). One of the main nuclei that receive accumbal efferents is the ventral pallidum (VP), and both dynorphin and substance P have been shown to be present in the cell bodies and terminals of this projection. Thus, we investigated whether CART peptide is also present in the VP in terminals that originate in the accumbens. The anterograde tracer Phaseolus vulgaris leukoagglutinin (PHA-L) colocalized with CART in neuronal processes in the VP when injected into the NAcc. Also, CART colocalized with the retrograde tracer r-BDA in accumbens cell bodies after the tracer was injected into the VP. Using electron microscopic immunocytochemistry, we examined CART terminals in the VP and found that CART-immunoreactive terminals formed symmetric synapses consistent with inhibitory GABAergic synapses. These synapses closely resemble GABAergic synapses in the substantia nigra pars reticulata (SNr), another nucleus that receives some CART-containing accumbal efferents. Lastly, we found that intra-pallidal injection of CART 55-102 inhibited cocaine-induced locomotion, indicating that CART peptide in the VP can have functional effects.

The ventral part of the oral pontine reticular nucleus (vRPO) is involved in the generation and maintenance of rapid eye movement (REM) sleep. Both GABAergic and serotonergic neurotransmission have been implicated in the control of the sleep-wakefulness cycle. Nevertheless, the synaptic organization of serotonergic terminals in the vRPO has not yet been characterized. We performed an electron microscope study of serotonin-immunoreactive (5-HT-IR) terminals using immunoperoxidase or immunogold-silver methods. In a second set of experiments, combining GABA immunoperoxidase and 5-HT immunogold-silver techniques, we examined inputs from GABA-immunoreactive (GABA-IR) terminals to serotonergic neurons. 5-HT-IR terminals were located primarily on dendrites and occasionally on somata of unlabeled and 5-HT-IR neurons. The majority of the synapses formed by 5-HT-IR terminals were of the symmetrical type, making contacts primarily with unlabeled dendritic profiles. Moreover, 5-HT-IR terminals contacted unlabeled axon terminals that formed asymmetric synapses on dendrites. Double immunolabeling experiments showed 5-HT-IR and GABA-IR afferents, in apposition to each other, making synapses with the same dendrites. Finally, GABA-IR terminals innervated 5-HT-IR and GABA-IR dendrites. Our findings indicate that serotonin would modulate the neuronal activity through inhibitory or excitatory influences, although the action of serotonin on the vRPO would predominantly be inhibitory. Moreover, the present results suggest that the serotonin modulation of vRPO neurons might involve indirect connections. In addition, GABA might contribute to the induction and maintenance of REM sleep by inhibiting serotonergic and GABAergic neurons in the vRPO.

Connexin 35/36 (Cx35/36) gap junction protein is expressed in various regions of the brain, including the retina. In this work, the expression of Cx35/36 in the outer retina of carp was studied by immunocytochemistry. By light microscopy, strong punctate Cx35/36-immunoreactivity was observed in the outer plexiform layer. Double labeling experiments on vertical retinal sections showed that Cx35/36 puncta were localized beneath cone pedicles, stained by recoverin, but not on them. In addition, few of the dendrites of rod-dominant ON type bipolar cells (rod-ON-BCs), stained by PKCalpha, were labelled with Cx35/36 in the retinal sections. In isolated cell preparations, Cx35/36 was clearly expressed on the dendrites of cone-dominant ON type bipolar cells (cone-ON-BCs), but the expression was much less on rod-ON-BCs. Moreover, Cx35/36 puncta were found in the dendrites of isolated horizontal cells (labelled by GAD 65/67) driven by cones, including H1 and H2 cells, but not in those of cells driven by rods (H4 cells). At the ultrastructural level, reaction product was found in H1 and H2 cell dendrites invaginating cone terminals, but not in H4 cell dendrites invaginating rod terminals. Moreover, dendrites of cone-ON-BCs, were also labebed. These results suggest that Cx35/36 could be specifically involved in modulation of the cone signal pathway in the outer retina of carp.