Culturing hippocampal neurons

Kaech, Stefanie; Banker, Gary

doi:10.1038/nprot.2006.356

Cited by 1,525 publications

(1,411 citation statements)

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“…6, 7). While astrocytes were traditionally used for nurturing neuronal culture [51], we demonstrated the possibility to directly pattern neuronal adhesion and morphogenesis on astrocytes. As it is widely accepted that astrocytes are functionally indispensable parts of neural microcircuits, the capability to precisely engineer neurons and astrocytes simultaneously gives unprecedented access to study the interaction between neurons and astrocytes.…”

Section: Discussionmentioning

confidence: 99%

“…There is accumulating evidence that astrocyte is a functionally indispensable part of neural circuits [50]. Banker and coworkers successfully evolved a method for co-culturing neurons with astrocytes for improving the viability of neurons [51]. In addition, the viability of astrocytes is more robust in culture, making it more straightforward to pattern astrocytes.…”

Section: Astrocyte As Substrates For Engineering Neuronalmentioning

confidence: 99%

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Surface Coating as a Key Parameter in Engineering Neuronal Network Structures In Vitro

et al. 2012

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By quantitatively comparing a variety of macromolecular surface coating agents, we discovered that surface coating strongly modulates the adhesion and morphogenesis of primary hippocampal neurons and serves as a switch of somata clustering and neurite fasciculation in vitro. The kinetics of neuronal adhesion on poly-lysine-coated surfaces is much faster than that on laminin and Matrigel-coated surfaces, and the distribution of adhesion is more homogenous on poly-lysine. Matrigel and laminin, on the other hand, facilitate neuritogenesis more than poly-lysine does. Eventually, on Matrigel-coated surfaces of self-assembled monolayers, neurons tend to undergo somata clustering and neurite fasciculation. By replacing coating proteins with cerebral astrocytes, and patterning neurons on astrocytes through self-assembled monolayers, microfluidics and micro-contact printing, we found that astrocyte promotes soma adhesion and astrocyte processes guide neurites. There, astrocytes could be a versatile substrate in engineering neuronal networks in vitro. Besides, quantitative measurements of cellular responses on various coatings would be valuable information for the neurobiology community in the choice of the most appropriate coating strategy.

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Section: Discussionmentioning

confidence: 99%

Section: Astrocyte As Substrates For Engineering Neuronalmentioning

confidence: 99%

Surface Coating as a Key Parameter in Engineering Neuronal Network Structures In Vitro

et al. 2012

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“…These tests were scored on a visual scale: 0, no binding; 1, low but specific binding; to 4, strong binding to all transfected cells by two independent observers as previously described3, 5 and in use for routine diagnostics. All positive samples were confirmed, and then tested blind by LW as part of a routine antibody service, with dilutions to assess the titer; the positives samples were also tested for binding to the surface of live hippocampal neurons in culture, prepared from P0 Sprague‐Dawley rat pups as described previously 23, 24…”

Section: Methodsmentioning

confidence: 99%

Neuronal antibodies in pediatric epilepsy: Clinical features and long‐term outcomes of a historical cohort not treated with immunotherapy

et al. 2016

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SummaryObjectiveIn autoimmune encephalitis the etiologic role of neuronal cell‐surface antibodies is clear; patients diagnosed and treated early have better outcomes. Neuronal antibodies have also been described in patients with pediatric epilepsy without encephalitis. The aim was to assess whether antibody presence had any effect on long‐term outcomes in these patients.MethodsPatients (n = 178) were recruited between 1988 and 1992 as part of the prospective Dutch Study of Epilepsy in Childhood; none received immunotherapy. Healthy age‐matched bone‐marrow donors served as controls (n = 112). All sera were tested for serum N‐methyl‐d‐aspartate receptor (NMDAR), alpha amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor, leucine rich glioma inactivated 1, contactin associated protein like 2 (CASPR2), contactin‐2, glutamic acid decarboxylase, and voltage gated potassium channel (VGKC)‐complex antibodies by standard techniques. No cerebrospinal fluid (CSF) samples were available. Results were correlated with clinical data collected over 15 years.ResultsSeventeen patients (9.5%) were positive for VGKC complex (n = 3), NMDAR (n = 7), CASPR2 (n = 4), and contactin‐2 (n = 3), compared to three (3/112; 2.6%) healthy controls (VGKC complex [n = 1], NMDAR [n = 2]; p = 0.03; Fisher's exact test). Titers were relatively low (≤1:100 for cell‐surface antibodies), but 8 (47%) of the 17 positive samples bound to the surface of live hippocampal neurons consistent with a potential pathogenic antibody. Preexisting cognitive impairment was more frequent in antibody‐positive patients (9/17 vs. 33/161; p = 0.01). Fourteen antibody‐positive patients were treated with standard antiepileptic drugs (AEDs); three (17%) became intractable but this was not different from the 16 (10%) of 161 antibody‐negative patients. In 96 patients with available follow‐up samples at 6 and/or 12 months, 6 of 7 positive antibodies had disappeared and, conversely, antibodies had appeared for the first time in a further 7 patients.SignificanceNeuronal antibodies were found at low levels in 9.5% of patients with new‐onset pediatric epilepsy but did not necessarily persist over time, and the development of antibodies de novo in later samples suggests they could be due to a secondary response to neuronal damage or inflammation. Moreover, as the response to standard AEDs and the long‐term outcome did not differ from those of antibody‐negative pediatric patients, these findings suggest that routine neuronal antibody testing is unlikely to be helpful in pediatric epilepsy. However, the higher incidence of preexisting cognitive problems in the antibody‐positive group, the CASPR2 and contactin‐2 antibodies in 7 of 17 patients, and the binding of 8 of 17 of serum samples to live hippocampal neurons suggest that neuronal antibodies, even if secondary, could contribute to the comorbidities of pediatric epilepsy.

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confidence: 99%

“…18 In dissociated hippocampal neurons, the transition for axon formation and maturation involves the following five stages: 19 stage 1 neurons (~2 to 4 h after plating) display abundant lamellipodia and filopodia that develop into several immature short neurites at stage 2 (~12 to 24 h); polarization occurs at stage 3 (~24 to 48 h), in which a single neurite initiates a rapid elongation to become the axon while others acquire dendritic identity; stage 4 (~3-4 days) is characterized by rapid outgrowth of axon and dendrites; and at stage 5 (7 days onwards), the maturation of axon and dendrites is essential for functional synapse formation. 20,21 In the present study, we have identified a novel role for the MT2 receptor in functional axonogenesis and show that activation of the MT2 receptor is crucial for functional axonogenesis and synaptic transmission in central neurons.…”

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confidence: 99%

The MT2 receptor stimulates axonogenesis and enhances synaptic transmission by activating Akt signaling

Man

et al. 2014

Cell Death Differ

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The MT2 receptor is a principal type of G protein-coupled receptor that mainly mediates the effects of melatonin. Deficits of melatonin/MT2 signaling have been found in many neurological disorders, including Alzheimer's disease, the most common cause of dementia in the elderly, suggesting that preservation of the MT2 receptor may be beneficial to these neurological disorders. However, direct evidence linking the MT2 receptor to cognition-related synaptic plasticity remains to be established. Here, we report that the MT2 receptor, but not the MT1 receptor, is essential for axonogenesis both in vitro and in vivo. We find that axon formation is retarded in MT2 receptor knockout mice, MT2-shRNA electroporated brain slices or primary neurons treated with an MT2 receptor selective antagonist. Activation of the MT2 receptor promotes axonogenesis that is associated with an enhancement in excitatory synaptic transmission in central neurons. The signaling components downstream of the MT2 receptor consist of the Akt/GSK-3β/CRMP-2 cascade. The MT2 receptor C-terminal motif binds to Akt directly. Either inhibition of the MT2 receptor or disruption of MT2 receptor-Akt binding reduces axonogenesis and synaptic transmission. Our data suggest that the MT2 receptor activates Akt/GSK-3β/CRMP-2 signaling and is necessary and sufficient to mediate functional axonogenesis and synaptic formation in central neurons. Synaptic circuits are established at the sites of axon-dendritic, axon-somatic or axon-axonal contact, in which functional axonogenesis is a critical step. 1 Axonogenesis can be regulated by many intracellular signals that involve cytoskeletal rearrangements, 2 local protein degradation, 3 as well as diffusional barriers. 4 Additionally, several extracellular neurotrophic factors and hormones have also been shown to have a role in axon guidance and synaptic formation in central neurons. 5,6 To date, the role of melatonin and its receptors in axonogenesis remains unclear. Most of the biological functions of melatonin are mediated by its two receptors, MT1 and MT2 receptors, both of them belong to the G protein-coupled receptor (GPCR) subfamily and are widely expressed throughout the central nervous system (CNS). 7 Activation of the MT2 receptor in response to melatonin is critical for controlling circadian rhythms 7 and regulation of slow wave sleep. 8,9 Early studies have shown that activation of the MT2 receptor in the retina reduces the release of dopamine, while dopamine inhibits growth cone motility and neurite outgrowth during embryonic development, 10,11 suggesting the involvement of the MT2 receptor in functional axonogenesis. In mutant mice with deficient expression of the MT2 gene, the induction of long-term potentiation (LTP) of excitatory synaptic transmission is impaired, and this impairment is closely related to deficits in learning. 12 In the hippocampus, the MT2 receptor inhibits GABA A receptor-mediated current, 13 which is implicated in the synaptic transmission. In Alzheimer's disease, expression of the MT...

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Culturing hippocampal neurons

Cited by 1,525 publications

References 27 publications

Surface Coating as a Key Parameter in Engineering Neuronal Network Structures In Vitro

Surface Coating as a Key Parameter in Engineering Neuronal Network Structures In Vitro

Neuronal antibodies in pediatric epilepsy: Clinical features and long‐term outcomes of a historical cohort not treated with immunotherapy

The MT2 receptor stimulates axonogenesis and enhances synaptic transmission by activating Akt signaling

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