Introduction: Annually, an estimated 2.5 million traumatic brain injuries (TBI) occur in the United States, of which, over 50,000 result in deaths. Currently, 5.3 million Americans are living with neurological dysfunction secondary to TBIs leading to a $60 billion dollar cost in medical expenses and productivity losses. To date, there are limited treatments available to cure or ease the morbidity of TBI. Despite preventative efforts, traumatic brain injuries (TBI) occur at a staggering rate and it is estimated that 15-20% of survivors develop persistent post-traumatic neurological impairment. The purposed source of neurological dysfunction is a result of circuit reorganization when the brain rebuilds itself. After diffuse TBI, rodents have been shown to develop a late-onset, gain-of-function sensory sensitivity to whisker stimulation; similar to phonophobia and photophobia experienced by human TBI survivors. This morbidity coincides with evidence of post-TBI circuit reorganization, however the etiology of post-traumatic neurological impairment remains largely unknown. Thrombospondin-1 (TSP-1) and thrombospondin-2 (TSP-2) are heavily expressed during pediatric neuronal synapse development. Expression of TSPs, however declines with age. Mechanistically during development, TSP mediates synaptogenesis via bindingα2δ-1 subunit of the voltage-gated calcium channel receptor (α2δ-1). After neurological insult, re-expression of TSPs has been demonstrated and experimental modulation of the TSP/α2δ-1 interaction has led to changes in morbidity. We therefore hypothesize that experimental diffuse TBI will result in re-expression of TSPs, which will be synchronous with increases in synaptic markers in the thalamocortical whisker circuit. Methods: Adult male Sprague-Dawley rats underwent sham or moderate midline fluid percussion brain injury. At multiple time points over 2-months post-injury, expression of TSPs and synaptic markers were quantified from thalamocortical circuit (ventroposterior medial thalamus (VPM), primary somatosensory barrel fields (S1BF)) biopsies using qPCR and automated capillary westerns, respectively. Results: TSP-1 gene expression and protein levels increase in the VPM during the first week after injury. Gene expression of TSP-1 did not significantly change over time in the S1BF, however, there was a significant increase in protein levels in the first and second weeks after injury. No significant changes were demonstrated in synaptic markers in the VPM over the time course. TSP-1 protein levels demonstrated a similar multimodal response to synaptic markers in the S1BF.Conclusion: Re-expression of TSP-1 and synchronous changes in synaptic marker supports a role for TSP-1 mediated synaptogenesis after experimental diffuse TBI in the S1BF. These data positions us for future investigation of pharmacological inhibition of TSP-mediated synaptogenesis after TBI; which may represent a prophylactic strategy against circuit reorganization and neurological dysfunction after TBI.

No description available.

Ketamine has shown promise as a novel treatment for depression and as a means to investigate the biology of depression. The drug effectively and rapidly treats depressed patients with the effects lasting approximately 1 week. However, concerns about ketamine’s efficacy do exist because of the inadequacy of blinding procedures used in existing trials. A dose of 0.5 mg/kg has been found to be most effective. Prolonged ketamine infusions have not extended the antidepressant effect beyond the timeframe of a regular infusion. Repeat infusions may be successful in extending ketamine’s effect, but definite conclusions cannot yet be made in this regard. Combination treatment with escitalopram and cognitive behavioral therapy (CBT) hold promise, as does the development of an intranasal formulation. Ketamine has shown additional efficacy as an acute anti-suicide treatment. Side effects from a single administration usually fade within a few hours and commonly include dissociation, elevations of blood pressure, nausea, and anxiety. Less data is available on the side effects caused by repeated ketamine infusions. Concerns exist regarding genitourinary, hepatic, and cognitive side effects after repeated infusions, as well as a risk of addiction. Research on ketamine’s mechanism of action has focused on the glutamate system in the brain. Ketamine may act by inhibiting release of γ–aminobutyric acid (GABA) from interneurons, activating intrasynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), increasing mammalian target of rapamycin complex 1 (mTORC1) and extracellular signal-regulated kinase (ERK) signaling, enhancing brain-derived neurotrophic factor (BDNF) production, inhibiting glycogen synthase kinase 3 (GSK3), blocking extrasynaptic N-Methyl-D-aspartate receptors (NMDARs), and promoting synaptogenesis and neuroplasticity. The two existing ketamine stereoisomers, (R)- versus (S)-ketamine, have different actions and potentially different efficacies and side effect profiles. Ketamine also produces regional changes in brain activity and connectivity. These include decreased burst firing in the lateral habenula (LHb), increased activity in the prefrontal cortex (PFC) and subgenual anterior cingulate cortex (ACC), and alterations in the amygdala’s response to angry and happy faces. Ketamine has the potential to be developed into a novel and useful clinical tool in the treatment of depression and to advance the understanding of the biology of depression.

No description available.

Dans le cerveau adulte, la signalisation liée à l’adénosine facilite ou inhibe la libération vésiculaire de neurotransmetteurs suite à l’activation des récepteurs de l’adénosine de type 2A ou 1 (A2AR ou A1R), respectivement. Cependant, son rôle dans le développement est mal connu. Au cours de ma thèse, j’ai étudié le rôle de la signalisation adénosine dans la synaptogenèse GABAergiques de l’hippocampe. Nous avons mis en évidence (i) une sécrétion activité-dépendante accrue d’adénosine et d’ATP pendant la période de synaptogenèse, (ii) un pic d’expression de l’enzyme limitant la formation de l’adénosine à partir de l’ATP extracellulaire, l’ecto-5’-nucleotidase, aux synapses pendant cette période critique, et (iii) un pic d’expression péri/post- synaptique du A2AR concomitant de la période de synaptogenèse. Cette expression développementale des molécules clés de la signalisation adénosine dépendante du A2AR corrélait avec un rôle de ce récepteur dans la stabilisation des synapses GABAergiques naissantes, une régulation restreinte à la période de synaptogenèse. De plus, la suppression de A2AR par une approche shRNA dans des neurones isolés conduisait à une perte de synapses GABAergiques équivalente à celle observée après un blocage pharmacologique de l’activité du A2AR, signifiant que la stabilisation synaptique médiée par le A2AR est un processus « cellule autonome » indépendant de l’activité du réseau neuronal et qu’elle requiert l’activation du A2AR dans la cellule post-synaptique.L’ATP et l’adénosine sont secrétés par la glie et les neurones ; cependant, nous avons montré in vitro que la libération neuronale activité-dépendante suffit à stabiliser les synapses GABAergiques naissantes. En utilisant la vidéomicroscopie sur cellules vivantes, nous avons montré que la signalisation adénosine stabilise les synapses actives. Puis, nous avons caractérisé le mécanisme moléculaire sous-jacent. Nous rapportons la contribution de la cascade adénylate cyclase/adénosine monophosphate cyclique/protéine kinase A et nous avons identifié une ciblé clé, la géphrine, la molécule d’ancrage postsynaptique des récepteurs GABAA. Enfin, nous avons mis en évidence que la stabilisation de l’élément présynaptique requiert probablement le complexe trans-synaptique Slitrk3-PTPd.Puisque le GABA exerce une fonction similaire au cours du développement et que le GABA et l’adénosine sont co-libérés à certaines synapses, j’ai étudié l’interaction entre ces deux voies de signalisation. Mes résultats favorisent l’hypothèse que la signalisation GABA, en activant la calcium-calmoduline, converge vers la signalisation adénosine en potentiant les adenylates cyclases sensibles au calcium. Mon travail m’a permis de proposer que, au cours d’une période clé du développement, les A2ARs postsynaptiques agissent comme des senseurs de l’activité des terminaisons présynaptiques GABAergiques pour stabiliser les synapses actives. En absence d’activité et donc de libération d’adénosine/ATP, les synapses seraient éliminées. / In the adult brain, adenosine signaling facilitates or inhibits neurotransmitter vesicular release mainly through activation of type 2A or 1 adenosine receptors (A2AR or A1R), respectively. However, its role in development remains to be elucidated. During my PhD, I addressed the role of A2AR-mediated signalling in GABAergic synaptogenesis in the hippocampus. We found (i) a larger activity-dependent release of ATP and adenosine during the period of synaptogenesis in the hippocampus, (ii) a peak of expression of the ecto-5’-nucleotidase, the rate-limiting enzyme for the formation of adenosine from extracellular ATP in synapses during this critical period, and (iii) a peak of peri/post-synaptic expression of A2AR concomitant with the period of synaptogenesis. This developmental expression of the key molecules of the adenosine A2AR signalling pathway correlated with a role of A2AR in the stabilization of nascent GABA synapses, a regulation restricted to the period of synaptogenesis. Furthermore, suppressing A2AR with a shRNA approach in isolated neurons led to a loss of synapses equivalent to that seen upon A2AR activity blockade, reporting that the A2AR-mediated synapse stabilization is a cell autonomous process that requires A2AR activation in the postsynaptic cell. ATP/adenosine can be secreted by both glia and neurons; however, we found that activity-dependent release of neuronal adenosine is sufficient to stabilize newly formed GABA synapses in vitro. Using live cell imaging, we showed adenosine signalling stabilizes active synapses. We then characterized the molecular mechanism downstream postsynaptic A2AR. We report the contribution of adenylyl cyclase/cyclic adenosine monophosphate/protein kinase A signalling cascade and we identified a key target, the postsynaptic scaffolding molecule gephyrin. We further showed the A2AR-mediated stabilization of the presynaptic compartment most probably requires the trans-synaptic Slitrk3-PTPd complex. Since GABA exerts a similar function during development and GABA and adenosine are co-released at some synapses, I further investigated the interplay between these two pathways. My results support the hypothesis that GABA signalling converge onto the adenosine signalling pathway by potentiating calcium-sensitive adenylyl cyclases through the activation of calmodulin.Altogether these results let us propose that, during a key developmental period, postsynaptic A2ARs act as sensors of the activity of GABAergic presynaptic terminals to stabilize active nascent GABAergic synapses. In absence of activity and therefore secretion of adenosine/ATP, synapses will be eliminated.

No description available.

Synapse development is a stepwise process that requires the recruitment of key synaptic components to active zones, followed by continual maintenance of these structures to maintain connectivity and stability throughout the life of the organisms. Early synapse development requires the recruitment of early scaffolding proteins to establish stable connectivity as well as provide sites of recruitment of other vital synaptic proteins. One of the earliest proteins to be localized to the synapse is the conserved protein Syd-1. Syd-1 proteins contain a Rho GTPase activating protein (GAP)-like domain of unclear significance. Here I show that Drosophila Syd-1 interacts with all six fly Rhos and has GAP activity towards RAC1. I then show that lacks GAP activity localizes normally to presynaptic sites and is sufficient to recruit Nrx-1 but fails to cluster Brp normally and genetically interacts with RAC1 in vivo. I conclude that contrary to previous models, the GAP domain of fly Syd-1 is active and required for presynaptic development. Additionally, I’ve identified a previously uncharacterized protein, Vezl, as being critical for retrograde axonal transport and synaptic maintenance. I found that Vezl required for normal neuronal growth and that vezl loss resulted in decreased neuron size and the formation of swollen neuronal terminals that accumulated membrane markers and axonal transport cargo. I found that vezl mutants specifically retrograde transport of cargo and particularly affected signaling endosomes. The signaling endosomes were unable to initiate retrograde transport in vezl mutants and remained stuck within the distal boutons unable to relay their signaling peptides back to the nucleus. I conclude that Vezl is serving a role in attaching retrograde cargo to dynein and the microtubules specifically at neuron tips so that they can undergo retrograde axonal transport. This dissertation includes previously published and unpublished co-authored material. / 2020-09-06

The environmental neurotoxin trimethyltin (TMT) destroys parts of the hippocampal formation as well as the entorhinal cortex but leaves the septal cholinergic projection to the hippocampus and dentate gyrus intact. In this study we measured choline acetyltransferase (ChAT) activity in micropunch samples of the dentate gyrus, the CA1 region of Ammon's horn, and the caudate-putamen as a measure of density of cholinergic innervation in control rats and rats exposed to 7 mg/kg TMT by means of gastric intubation. Three months after the rats were exposed to a single dose of TMT both the dentate gyrus and CA1 demonstrated significantly higher ChAT activity in TMT-exposed rats than in control rats. No differences were found between groups for the caudate-putamen samples. These results support the hypothesis that exposure to TMT causes reactive synaptogenesis in the cholinergic septohippocampal system.

Synaptogenesis is a critical process in the establishment of neuronal connectivity during brain development. The key step is to transduce external stimuli into the internal signaling cascades. Cell adhesion molecules and scaffold proteins facilitate the transduction to achieve optimal connectivity through PDZ domain mediated interaction. FRMPD2, a product of a human-specific multi-copy gene with three PDZ domains, has been shown to localize to the tight junctions in epithelial cells, suggesting a role in inter-cellular interaction. Although the correlation between neurodevelopmental disorders and gene dosage alteration of FRMPD2 has been observed, its role in the nervous system remains unknown. Therefore, I investigated the role of FRMPD2 in neurodevelopment. I found that FRMPD2 localizes at the excitatory synapses and promotes synaptogenesis in rat neurons. Mechanistically, FERM domain is required for synaptic localization of FRMPD2 through the interaction with F-actin in spines. More importantly, I found that FRMPD2 associates with cell adhesion molecule Neuroligin-1 through PDZ domain mediated interaction, resulting in an increase in Neuroligin-1 surface expression and up-regulation of synaptogenesis. Results from in utero electroporation showed that overexpression of FRMPD2 in mouse brains delayed neuronal migration and increased dendritic arborization and spine formation. Remarkably, viral overexpression of FRMPD2 in mouse brains improved memory retention. Abnormalities in synaptogenesis during neurodevelopment can cause neurodevelopmental disorders, such as Autism Spectrum Disorders (ASDs). Genomic studies from cohorts of ASD patients have revealed the prevalence of dysfunctional genes in the ubiquitin-proteasome pathway, especially the E3 ligases, suggesting the E3 ligase as a key component in ASD pathogenesis. Genomic duplication or deletion of PARK2 gene, a E3 ligase gene, has been identified in ASD patients. Therefore, I explored the autistic phenotypes of the Park2 knockout (KO) mice. Indeed, the KO mice demonstrated features of typical ASD behaviors. Further, Park2 KO mice showed a reduction in spine number, dendritic arborization, and levels of neuronal activity. The alterations in synaptic property in Park2 KO mice may serve as the etiological factor for ASD. These findings provide insights into the role of a novel synaptic organizer scaffold protein for synapse formation during brain development, and a novel ASD model. / 2020-11-12T00:00:00Z

Increased survival of presynaptic-like neuronal profiles was found in cell cultures of rat cerebellum when the non-neuronal cell numbers were reduced with an antimitotic drug. In both treated and untreated cell cultures, neurites grew onto the polylysine-coated surface of sepharose beads and formed a swelling. The neuronal swelling contained an accumulation of synaptic vesicles and a membrane density at the site of contact with the bead and was called an apparent presynaptic element. The apparent presynaptic elements in untreated cultures increased in number from the time the beads were added to the culture to 7 days incubation and then showed a decrease to one half the 7-day value at 14 days incubation. A 75% reduction in cell division of non-neuronal cells was seen in cultures exposed to a 5 × 10-6 M cytosine arabinoside (Ara-C) for 2 days. Adding polylysine-coated beads to cultures treated with Ara-C showed at 14 days incubation a 7-fold increase in the number of apparent presynaptic elements as compared to untreated cultures. Additional experiments examined the numbers of neurites on the beads and found only small differences between treated and untreated cultures. A decrease, however, was shown in the number of glial fibrillary acidic protein staining astrocytes on the surface of the beads in treated cultures. The reduction of astrocytes by Ara-C appeared to enhance the survival of apparent presynaptic elements but did not enhance the growth of neurites. These results suggest that proliferating non-neuronal cells at a site of injury in the central nervous system may inhibit the formation of synaptic contacts and the growth of neurites through the site of injury.