The metabolite p-cresol impairs dendritic development, synaptogenesis, and synapse function in hippocampal neurons: Implications for autism spectrum disorder.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopment disorder resulting from different etiological factors, both genetic and/or environmental. These factors can lead to abnormal neuronal development on dendrite and synaptic function at the central nervous system. Recent studies have shown that a subset of ASD patients display increased circulation levels of the tyrosine metabolite, p-cresol, related to chronic intestinal disorders because of dysbiosis of the intestinal microbiota. In particular, abnormal presence of intestinal Clostridium sp. has been linked to high levels of p-cresol in ASD children younger than 8 years. However, the role of p-cresol during development of the central nervous system is unknown. Here, we evaluated in vitro the effect of p-cresol on neurite outgrowth in N2a and PC12 cell lines and dendritic morphology, synaptic density, neuronal activity, and calcium responses in primary rat hippocampal neurons. p-cresol inhibits neural differentiation and neurites outgrowth in N2a and PC12 neuronal cell lines. In hippocampal neuronal cultures, Sholl's analysis shows a decrease in the dendritic arborization of neurons treated with p-cresol. Synaptic density analyzed with the synaptic markers Piccolo and Shank2 is diminished in hippocampal neurons treated with p-cresol. Electrically evoked intracellular calcium rise was drastically, but reversely, blocked by p-cresol, whereas that spontaneous neuronal activity was severely affected by early addition of the metabolite. These findings show that p-cresol alters dendrite development, synaptogenesis, and synapse function of neurons in culture, therefore, neuronal alterations occurring in ASD children may be related to this metabolite and dysbiosis of the intestinal microbiota.

The dendritic ERGIC: Microtubule and actin cytoskeletons participate in stop-and-go movement of mobile carriers between stable structures.

The endoplasmic reticulum (ER)-to-Golgi intermediate compartment (ERGIC) is a membranous organelle that mediates protein transport between the ER and the Golgi apparatus. In neurons, clusters of these vesiculotubular structures are situated throughout the cell in proximity to the ER, passing cargo to the cis-Golgi cisternae, located mainly in the perinuclear region. Although ERGIC markers have been identified in neurons, the distribution and dynamics of neuronal ERGIC structures have not been characterized yet. Here, we show that long-distance ERGIC transport occurs via an intermittent mechanism in dendrites, with mobile elements moving between stationary structures. Slow and fast live-cell imaging have captured stable ERGIC structures remaining in place over long periods of time, as well as mobile ERGIC structures advancing very short distances along dendrites. These short distances have been consistent with the lengths between the stationary ERGIC structures. Kymography revealed ERGIC elements that moved intermittently, emerging from and fusing with stationary ERGIC structures. Interestingly, this movement apparently depends not only on the integrity of the microtubule cytoskeleton, as previously reported, but on the actin cytoskeleton as well. Our results indicate that the dendritic ERGIC has a dual nature, with both stationary and mobile structures. The neural ERGIC network transports proteins via a stop-and-go movement in which both the microtubule and the actin cytoskeletons participate.

Leucine-rich repeats and immunoglobulin-like domains 1 deficiency affects hippocampal dendrite complexity and impairs cognitive function.

Leucine-rich repeat (LRR) transmembrane proteins have been directly linked to neurodevelopmental and cognitive disorders. We have previously shown that the LRR transmembrane protein, leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1), is a physiological regulator of dendrite complexity of hippocampal pyramidal neurons and social behavior. In this study, we performed a battery of behavioral tests to evaluate spatial memory and cognitive capabilities in Lrig1 mutant mice. The cognitive assessment demonstrated deficits in recognition and spatial memory, evaluated by novel object recognition and object location tests. Moreover, we found that Lrig1-deficient mice present specific impairments in the processing of similar but not dissimilar locations in a spatial pattern separation task, which was correlated with an enhanced dendritic growth and branching of Doublecortin-positive immature granule cells of the dentate gyrus. Altogether, these findings indicate that Lrig1 plays an essential role in controlling morphological and functional plasticity in the hippocampus.

Ketamine, but not fluoxetine, rapidly rescues corticosterone-induced impairments on glucocorticoid receptor and dendritic branching in the hippocampus of mice.

Although numerous studies have investigated the mechanisms underlying the fast and sustained antidepressant-like effects of ketamine, the contribution of the glucocorticoid receptor (GR) and dendritic branching remodeling to its responses remain to be fully established. This study investigated the ability of a single administration of ketamine to modulate the GR and dendritic branching remodeling and complexity in the hippocampus of mice subjected to chronic corticosterone (CORT) administration. CORT was administered for 21 days, followed by a single administration of ketamine (1 mg ∕kg, i.p.) or fluoxetine (10 mg ∕kg, p.o., conventional antidepressant) in mice. On 22nd, 24 h after the treatments, GR immunocontent in the hippocampus was analyzed by western blotting, while the dendritic arborization and dendrite length in the ventral and dorsal dentate gyrus (DG) of the hippocampus was analyzed by Sholl analysis. Chronic CORT administration downregulated hippocampal GR immunocontent, but this alteration was completely reversed by a single administration of ketamine, but not fluoxetine. Moreover, CORT administration significantly decreased dendritic branching in the dorsal and ventral DG areas and caused a mild decrease in dendrite length in both regions. Ketamine, but not fluoxetine, reversed CORT-induced dendritic branching loss in the ventral and dorsal DG areas, regions associated with mood regulation and cognitive functions, respectively. This study provides novel evidence that a single administration of ketamine, but not fluoxetine, rescued the impairments on GR and dendritic branching in the hippocampus of mice subjected to chronic CORT administration, effects that may be associated with its rapid antidepressant response.

A single administration of ascorbic acid rapidly reverses depressive-like behavior and hippocampal synaptic dysfunction induced by corticosterone in mice.

Ketamine is the prototype for glutamate-based fast-acting antidepressants. The establishment of ketamine-like drugs is still a challenge and ascorbic acid has emerged as a candidate. This study investigated the ascorbic acid's ability to induce a fast antidepressant-like response and to improve hippocampal synaptic markers in mice subjected to chronic corticosterone (CORT) administration. CORT was administered for 21 days, followed by a single administration of ascorbic acid (1 mg ∕Kg, p.o.), ketamine (1 mg ∕Kg, i.p.) or fluoxetine (10 mg ∕Kg, p.o.) in mice. Depressive-like behavior, hippocampal synaptic proteins immunocontent, dendrite spines density in the dentate gyrus (DG) were analyzed 24 h following treatments. The administration of ascorbic acid or ketamine, but not fluoxetine, counteracted CORT-induced depressive-like behavior in the tail suspension test (TST). CORT administration reduced PSD-95, GluA1, and synapsin (synaptic markers) immunocontent, and these alterations were reversed by ascorbic acid or ketamine, but only ketamine reversed the CORT-induced reduction on GluA1 immunocontent. In the ventral and dorsal DG, CORT decreased filopodia-, thin- and stubby-shaped spines, while ascorbic acid and ketamine abolished this alteration only in filopodia spines. Ascorbic acid and ketamine increased mushroom-shaped spines density in ventral and dorsal DG. Therefore, the results show that a single administration of ascorbic acid, in a way similar to ketamine, rapidly elicits an antidepressant-like response and reverses hippocampal synaptic deficits caused by CORT, an effect associated with increased levels of synaptic proteins and dendritic remodeling.

Melatonin Rescues the Dendrite Collapse Induced by the Pro-Oxidant Toxin Okadaic Acid in Organotypic Cultures of Rat Hilar Hippocampus.

The pro-oxidant compound okadaic acid (OKA) mimics alterations found in Alzheimer's disease (AD) as oxidative stress and tau hyperphosphorylation, leading to neurodegeneration and cognitive decline. Although loss of dendrite complexity occurs in AD, the study of this post-synaptic domain in chemical-induced models remains unexplored. Moreover, there is a growing expectation for therapeutic adjuvants to counteract these brain dysfunctions. Melatonin, a free-radical scavenger, inhibits tau hyperphosphorylation, modulates phosphatases, and strengthens dendritic arbors. Thus, we determined if OKA alters the dendritic arbors of hilar hippocampal neurons and whether melatonin prevents, counteracts, or reverses these damages. Rat organotypic cultures were incubated with vehicle, OKA, melatonin, and combined treatments with melatonin either before, simultaneously, or after OKA. DNA breaks were assessed by TUNEL assay and nuclei were counterstained with DAPI. Additionally, MAP2 was immunostained to assess the dendritic arbor properties by the Sholl method. In hippocampal hilus, OKA increased DNA fragmentation and reduced the number of MAP2(+) cells, whereas melatonin protected against oxidation and apoptosis. Additionally, OKA decreased the dendritic arbor complexity and melatonin not only counteracted, but also prevented and reversed the dendritic arbor retraction, highlighting its role in post-synaptic domain integrity preservation against neurodegenerative events in hippocampal neurons.

Oral Administration of Incense Resin (Styrax benzoin) Extract Enhances Spatial Learning, Memory, and Dendrite Complexity of Mice

Abstract Hippocampus is a part of the brain that has a major role in spatial learning and memory which can be affected by herbal extracts. Incense resin (Styrax benzoin) has been used by local communities to improve intelligence. However, there is no scientific evidence of the functions of Styrax benzoin for regulating hippocampal function. The aim of this study was intended to analyze and investigate the effect of incense resin on learning, memory, and dendrite complexity of mice. Three months old male Deutch Democratic Yokohama (DDY) mice were injected orally with graded doses of 100, 150, and 200 mg/kg of incense resin aqueous extract daily for 30 days. Spatial learning and memory performance levels were tested with Y-maze alternation, novel object recognition, and Morris water maze. The branches and maximum dendritic span in the dentate gyrus were observed by the Golgi-Cox staining. Overall, our results showed that incense resin extract increased learning and memory ability, and the number of dendrite branching in the dentate gyrus.

Jan Evangelista Purkinje, a brilliant, multifaceted Czech biologist: nerve tissue / Jan Evangelista Purkinje, um brilhante multifacetado biólogo checo: tecido nervoso

Jan Evangelista Purkinje was a Czech physician with an exceptional capacity for innovative thinking, and he was one of the fathers of experimental physiology, experimental pharmacology, experimental psychology, histology, embryology, and physical anthropology. Several achievements are named after him, from his prodigious productivity. Of special interest of this paper was his pioneering role in the rise of experimental physiology, microscopical anatomy, and histological methods by the 1830´s that allowed him define more accurate data concerning the structure of nerve tissue of animals and humans such as the now known "Purkinje's cells" and others cells of the brain. He investigated the structure of neuronal processes, including the dendrites. Purkinje recognized possible functional differences between a variety of types of neurons and speculated about their interrelations. He was one of the great geniuses of science.

Jan Evangelista Purkinje foi um médico checo com excepcional capacidade de pensamento inovador e um dos pais da fisiologia experimental, farmacologia experimental, psicologia experimental, histologia, embriologia e antropologia física. Várias conquistas receberam o nome dele, de sua produtividade prodigiosa. De interesse especial deste trabalho enaltece-se o seu papel pioneiro no surgimento da fisiologia experimental, anatomia microscópica e métodos histológicos na década de 1830. Isso permitiu que ele definisse dados mais precisos sobre a estrutura do tecido nervoso de animais e humanos, como as agora conhecidas "células de Purkinje" e outras células do cérebro. Ele investigou a estrutura dos processos neuronais, incluindo os dendritos. Purkinje reconheceu possíveis diferenças funcionais entre uma variedade de tipos de neurônios e especulou sobre suas inter-relações. Ele foi um dos grandes gênios da ciência.

The Exocyst Component Exo70 Modulates Dendrite Arbor Formation, Synapse Density, and Spine Maturation in Primary Hippocampal Neurons.

Neurons are highly polarized cells displaying an elaborate architectural morphology. The design of their dendritic arborization and the distribution of their synapses contribute importantly to information processing in the brain. The growth and complexity of dendritic arbors are driven by the formation of synapses along their lengths. Synaptogenesis is augmented by the secretion of factors, like BDNF, Reelin, BMPs, or Wnts. Exo70 is a component of the exocyst complex, a protein complex that guides membrane addition and polarized exocytosis. While it has been linked to cytokinesis and the establishment of cell polarity, its role in synaptogenesis is poorly understood. In this report, we show that Exo70 plays a role in the arborization of dendrites and the development of synaptic connections between cultured hippocampal neurons. Specifically, while the overexpression of Exo70 increases dendritic arborization, synapse number, and spine density, the inhibition of Exo70 expression reduces secondary and tertiary dendrite formation and lowers synapse density. Moreover, increasing Exo70 expression augmented synaptic vesicle recycling as evaluated by FM4-64 dye uptake and the inverse was observed with downregulation of endogenous Exo70. Monitoring the formation of dendritic spines by super-resolution microscopy, we also observed that mRFP-Exo70 accumulates at the tip of EGFP-ß-actin-positive filopodia. Together, these results suggest that Exo70 is essentially involved in the formation of synapses and neuronal dendritic morphology.

Wnt7b signalling through Frizzled-7 receptor promotes dendrite development by coactivating CaMKII and JNK.

The formation of complex dendritic arbors is crucial for the assembly of functional networks as abnormal dendrite formation underlies several neurodevelopmental and psychiatric disorders. Many extracellular factors have been postulated as regulators of dendritic growth. Wnt proteins play a critical role in neuronal development and circuit formation. We previously demonstrated that Wnt7b acts through the scaffold protein dishevelled 1 (Dvl1) to modulate dendrite arborisation by activating a non-canonical Wnt signalling pathway. Here, we identify the seven-transmembrane frizzled-7 (Fz7, also known as FZD7) as the receptor for Wnt7b-mediated dendrite growth and complexity. Importantly, Fz7 is developmentally regulated in the intact hippocampus, and is localised along neurites and at dendritic growth cones, suggesting a role in dendrite formation and maturation. Fz7 loss-of-function studies demonstrated that Wnt7b requires Fz7 to promote dendritic arborisation. Moreover, in vivo Fz7 loss of function results in dendritic defects in the intact mouse hippocampus. Furthermore, our findings reveal that Wnt7b and Fz7 induce the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and JNK proteins, which are required for dendritic development. Here, we demonstrate that Wnt7b-Fz7 signals through two non-canonical Wnt pathways to modulate dendritic growth and complexity.

Overexpression of MAP2 and NF-H Associated with Dendritic Pathology in the Spinal Cord of Mice Infected with Rabies Virus.

Rabies is a viral infection that targets the nervous system, specifically neurons. The clinical manifestations of the disease are dramatic and their outcome fatal; paradoxically, conventional histopathological descriptions reveal only subtle changes in the affected nervous tissue. Some researchers have considered that the pathophysiology of rabies is based more on biochemical changes than on structural alterations, as is the case with some psychiatric diseases. However, we believe that it has been necessary to resort to other methods that allow us to analyze the effect of the infection on neurons. The Golgi technique is the gold standard for studying the morphology of all the components of a neuron and the cytoskeletal proteins are the structural support of dendrites and axons. We have previously shown, in the mouse cerebral cortex and now with this work in spinal cord, that rabies virus generates remarkable alterations in the morphological pattern of the neurons and that this effect is associated with the increase in the expression of two cytoskeletal proteins (MAP2 and NF-H). It is necessary to deepen the investigation of the pathogenesis of rabies in order to find therapeutic alternatives to a disease to which the World Health Organization classifies as a neglected disease.

Pea3 Transcription Factors, Etv4 and Etv5, Are Required for Proper Hippocampal Dendrite Development and Plasticity.

The proper formation and morphogenesis of dendrites is essential to the establishment of neuronal connectivity. We report that 2 members of the Pea3 family of transcription factors, Etv4 and Etv5, are expressed in hippocampal neurons during the main period of dendritogenesis, suggesting that they have a function in dendrite development. Here, we show that these transcription factors are physiological regulators of growth and arborization of pyramidal cell dendrites in the developing hippocampus. Gain and loss of function assays indicate that Etv4 and Etv5 are required for proper development of hippocampal dendritic arbors and spines. We have found that in vivo deletion of either Etv4 or Etv5 in hippocampal neurons causes deficits in dendrite size and complexity, which are associated with impaired cognitive function. Additionally, our data support the idea that Etv4 and Etv5 are part of a brain-derived neurotrophic factor-mediated transcriptional program required for proper hippocampal dendrite connectivity and plasticity.

Dual effect of serotonin on the dendritic growth of cultured hippocampal neurons: Involvement of 5-HT1A and 5-HT7 receptors.

Serotonin acts through its receptors (5-HTRs) to shape brain networks during development and modulates essential functions in mature brain. The 5-HT1AR is mainly located at soma of hippocampal neurons early during brain development and its expression gradually shifts to dendrites during postnatal development. The 5-HT7R expressed early during hippocampus development, shows a progressive reduction in its expression postnatally. Considering these changes during development, we evaluated in cultured hippocampal neurons whether the 5-HT1AR and 5-HT7R change their expression, modulate dendritic growth, and activate signaling pathways such as ERK1/2, AKT/GSK3ß and LIMK/cofilin, which may sustain dendrite outgrowth by controlling cytoskeleton dynamics. We show that mRNA levels of both receptors increase between 2 and 7 DIV; however only protein levels of 5-HT7R increase significantly at 7 DIV. The 5-HT1AR is preferentially distributed in the soma, while 5-HT7R displays a somato-dendritic localization at 7 DIV. Through stimulation with 5-HT at 7 DIV during 24h and using specific antagonists, we determined that 5-HT1AR decreases the number of primary and secondary dendrites and restricts the growth of primary dendrites. The activation of 5-HT1AR and 5-HT7R promotes the growth of short secondary dendrites and triggers ERK1/2 and AKT phosphorylation through MEK and PI3K activation respectively; without changes in the phosphorylation of LIMK and cofilin. We conclude that 5-HT1AR restricts dendritogenesis and outgrowth of primary dendrites, but that both 5-HT1AR and 5-HT7R promote secondary dendrite outgrowth. These data support the role of 5-HT in neuronal outgrowth during development and provide insight into cellular basis of neurodevelopmental disorders.

Mechanisms regulating dendritic arbor patterning.

The nervous system is populated by diverse types of neurons, each of which has dendritic trees with strikingly different morphologies. These neuron-specific morphologies determine how dendritic trees integrate thousands of synaptic inputs to generate different firing properties. To ensure proper neuronal function and connectivity, it is necessary that dendrite patterns are precisely controlled and coordinated with synaptic activity. Here, we summarize the molecular and cellular mechanisms that regulate the formation of cell type-specific dendrite patterns during development. We focus on different aspects of vertebrate dendrite patterning that are particularly important in determining the neuronal function; such as the shape, branching, orientation and size of the arbors as well as the development of dendritic spine protrusions that receive excitatory inputs and compartmentalize postsynaptic responses. Additionally, we briefly comment on the implications of aberrant dendritic morphology for nervous system disease.

The GDNF-GFRα1 complex promotes the development of hippocampal dendritic arbors and spines via NCAM.

The formation of synaptic connections during nervous system development requires the precise control of dendrite growth and synapse formation. Although glial cell line-derived neurotrophic factor (GDNF) and its receptor GFRα1 are expressed in the forebrain, the role of this system in the hippocampus remains unclear. Here, we investigated the consequences of GFRα1 deficiency for the development of hippocampal connections. Analysis of conditional Gfra1 knockout mice shows a reduction in dendritic length and complexity, as well as a decrease in postsynaptic density specializations and in the synaptic localization of postsynaptic proteins in hippocampal neurons. Gain- and loss-of-function assays demonstrate that the GDNF-GFRα1 complex promotes dendritic growth and postsynaptic differentiation in cultured hippocampal neurons. Finally, in vitro assays revealed that GDNF-GFRα1-induced dendrite growth and spine formation are mediated by NCAM signaling. Taken together, our results indicate that the GDNF-GFRα1 complex is essential for proper hippocampal circuit development.

Assembly of Neuronal Connectivity by Neurotrophic Factors and Leucine-Rich Repeat Proteins.

Proper function of the nervous system critically relies on sophisticated neuronal networks interconnected in a highly specific pattern. The architecture of these connections arises from sequential developmental steps such as axonal growth and guidance, dendrite development, target determination, synapse formation and plasticity. Leucine-rich repeat (LRR) transmembrane proteins have been involved in cell-type specific signaling pathways that underlie these developmental processes. The members of this superfamily of proteins execute their functions acting as trans-synaptic cell adhesion molecules involved in target specificity and synapse formation or working in cis as cell-intrinsic modulators of neurotrophic factor receptor trafficking and signaling. In this review, we will focus on novel physiological mechanisms through which LRR proteins regulate neurotrophic factor receptor signaling, highlighting the importance of these modulatory events for proper axonal extension and guidance, tissue innervation and dendrite morphogenesis. Additionally, we discuss few examples linking this set of LRR proteins to neurodevelopmental and psychiatric disorders.

Lrig1 is a cell-intrinsic modulator of hippocampal dendrite complexity and BDNF signaling.

Even though many extracellular factors have been identified as promoters of general dendritic growth and branching, little is known about the cell-intrinsic modulators that allow neurons to sculpt distinctive patterns of dendrite arborization. Here, we identify Lrig1, a nervous system-enriched LRR protein, as a key physiological regulator of dendrite complexity of hippocampal pyramidal neurons. Lrig1-deficient mice display morphological changes in proximal dendrite arborization and defects in social interaction. Specifically, knockdown of Lrig1 enhances both primary dendrite formation and proximal dendritic branching of hippocampal neurons, two phenotypes that resemble the effect of BDNF on these neurons. In addition, we show that Lrig1 physically interacts with TrkB and attenuates BDNF signaling. Gain and loss of function assays indicate that Lrig1 restricts BDNF-induced dendrite morphology. Together, our findings reveal a novel and essential role of Lrig1 in regulating morphogenic events that shape the hippocampal circuits and establish that the assembly of TrkB with Lrig1 represents a key mechanism for understanding how specific neuronal populations expand the repertoire of responses to BDNF during brain development.

Transport along the dendritic endoplasmic reticulum mediates the trafficking of GABAB receptors.

In neurons, secretory organelles within the cell body are complemented by the dendritic endoplasmic reticulum (ER) and Golgi outposts (GOPs), whose role in neurotransmitter receptor trafficking is poorly understood. γ-aminobutyric acid (GABA) type B metabotropic receptors (GABABRs) regulate the efficacy of synaptic transmission throughout the brain. Their plasma membrane availability is controlled by mechanisms involving an ER retention motif and assembly-dependent ER export. Thus, they constitute an ideal molecular model to study ER trafficking, but the extent to which the dendritic ER participates in GABABR biosynthesis has not been thoroughly explored. Here, we show that GABAB1 localizes preferentially to the ER in dendrites and moves long distances within this compartment. Not only diffusion but also microtubule and dynein-dependent mechanisms control dendritic ER transport. GABABRs insert throughout the somatodendritic plasma membrane but dendritic post-ER carriers containing GABABRs do not fuse selectively with GOPs. This study furthers our understanding of the spatial selectivity of neurotransmitter receptors for dendritic organelles.

WNT signaling in neuronal maturation and synaptogenesis.

The Wnt signaling pathway plays a role in the development of the central nervous system and growing evidence indicates that Wnts also regulates the structure and function of the adult nervous system. Wnt components are key regulators of a variety of developmental processes, including embryonic patterning, cell specification, and cell polarity. In the nervous system, Wnt signaling also regulates the formation and function of neuronal circuits by controlling neuronal differentiation, axon outgrowth and guidance, dendrite development, synaptic function, and neuronal plasticity. Wnt factors can signal through three very well characterized cascades: canonical or ß-catenin pathway, planar cell polarity pathway and calcium pathway that control different processes. However, divergent downstream cascades have been identified to control neuronal morphogenesis. In the nervous system, the expression of Wnt proteins is a highly controlled process. In addition, deregulation of Wnt signaling has been associated with neurodegenerative diseases. Here, we will review different aspects of neuronal and dendrite maturation, including spinogenesis and synaptogenesis. Finally, the role of Wnt pathway components on Alzheimer's disease will be revised.