Physical exercise modulates peripheral levels of brain-derived neurotrophic factor (BDNF): A systematic review of experimental studies in the elderly

The objective of this study was to conduct a systematic review of studies that analyzed the effect of physical exercise on the peripheral levels of BDNF in elderly individuals. Method: We conducted a search in PsycINFO, Biological Abstracts, Pubmed, Web of Science, and Science Direct from 1990 to 2011, using the following keywords: physical exercise , physical activity , physical therapy , training , BDNF , neuroplasticity , neurotrophins , neuroplasticity proteins , aged , older , elderly The articles were considered for inclusion in the review if they were studies with elderly, assessed peripheral (serum and/or plasma) BDNF and evaluated an acute exercise or chronic exercise (training). Results: Five randomized controlled trial and one randomized non-controlled trial studies were analyzed. Five out of six studies reported a significantly higher BDNF response to aerobic acute exercise and to aerobic or strength training program in healthy elderly and elderly with different pathologies. Conclusion: It was not possible to establish a recommendation protocol for the type and intensity of physical exercise required to produce an increase in levels BDNF. However, physical exercise, particularly, moderate-intensity exercises seem to be more effective to promote increase the peripheral levels of BDNF in the elderly. © 2012 Elsevier B.V.

Motor Recovery and Synaptic Preservation after Ventral Root Avulsion and Repair with a Fibrin Sealant Derived from Snake Venom

Background:Ventral root avulsion is an experimental model of proximal axonal injury at the central/peripheral nervous system interface that results in paralysis and poor clinical outcome after restorative surgery. Root reimplantation may decrease neuronal degeneration in such cases. We describe the use of a snake venom-derived fibrin sealant during surgical reconnection of avulsed roots at the spinal cord surface. The present work investigates the effects of this fibrin sealant on functional recovery, neuronal survival, synaptic plasticity, and glial reaction in the spinal motoneuron microenvironment after ventral root reimplantation.Methodology/Principal Findings:Female Lewis rats (7 weeks old) were subjected to VRA and root replantation. The animals were divided into two groups: 1) avulsion only and 2) replanted roots with fibrin sealant derived from snake venom. Post-surgical motor performance was evaluated using the CatWalk system twice a week for 12 weeks. The rats were sacrificed 12 weeks after surgery, and their lumbar intumescences were processed for motoneuron counting and immunohistochemistry (GFAP, Iba-1 and synaptophysin antisera). Array based qRT-PCR was used to evaluate gene regulation of several neurotrophic factors and receptors as well as inflammatory related molecules. The results indicated that the root reimplantation with fibrin sealant enhanced motor recovery, preserved the synaptic covering of the motoneurons and improved neuronal survival. The replanted group did not show significant changes in microglial response compared to VRA-only. However, the astroglial reaction was significantly reduced in this group.Conclusions/Significance:In conclusion, the present data suggest that the repair of avulsed roots with snake venom fibrin glue at the exact point of detachment results in neuroprotection and preservation of the synaptic network at the microenvironment of the lesioned motoneurons. Also such procedure reduced the astroglial reaction and increased mRNA levels to neurotrophins and anti-inflammatory cytokines that may in turn, contribute to improving recovery of motor function. © 2013 Barbizan et al.

Altered Neurotrophin, Neuropeptide, Cytokines and Nitric Oxide Levels in Autism

Introduction: Modifications in neurotrophins, neuropeptides, cytokines and nitric oxide (NO) levels in autism may represent different biological aspects of the disease. In the present study we investigate simultaneously all these variables as an attempt to clarify their interrelationships in autism. Methods: Plasma levels of vasoactive intestinal peptide (VIP), neurotrophin-3 (NT-3), cytokines and nitric oxide (NO) were determined in children with DSM-IV autistic disorder (n = 24) and in age- and gender-matched healthy controls (n = 24). VIP, NT-3, IFN-gamma and IL-1 beta levels were measured by ELISA, TNF-alpha, IL-10, IL-6, IL-4, IL-2 were evaluated by flow cytometry, and NO by Griess reaction. Results: Plasma levels of VIP, IFN-gamma and NO were significantly higher and NT-3 plasma levels were significantly lower in children with autism, compared to the healthy subjects. In children with autism there was a positive correlation between plasma levels of NO and IFN-gamma. Discussion: Our results indicate the presence of altered levels of neurotrophin and neuropeptide in infantile autism and provide additional evidence that higher levels of IFN-gamma may be associated with increased oxidative stress in autism.

Motor recovery and synaptic preservation after ventral root avulsion and repair with a fibrin sealant derived from snake venom

Background: Ventral root avulsion is an experimental model of proximal axonal injury at the central/peripheral nervous system interface that results in paralysis and poor clinical outcome after restorative surgery. Root reimplantation may decrease neuronal degeneration in such cases. We describe the use of a snake venom-derived fibrin sealant during surgical reconnection of avulsed roots at the spinal cord surface. The present work investigates the effects of this fibrin sealant on functional recovery, neuronal survival, synaptic plasticity, and glial reaction in the spinal motoneuron microenvironment after ventral root reimplantation. Methodology/Principal Findings: Female Lewis rats (7 weeks old) were subjected to VRA and root replantation. The animals were divided into two groups: 1) avulsion only and 2) replanted roots with fibrin sealant derived from snake venom. Post-surgical motor performance was evaluated using the CatWalk system twice a week for 12 weeks. The rats were sacrificed 12 weeks after surgery, and their lumbar intumescences were processed for motoneuron counting and immunohistochemistry (GFAP, Iba-1 and synaptophysin antisera). Array based qRT-PCR was used to evaluate gene regulation of several neurotrophic factors and receptors as well as inflammatory related molecules. The results indicated that the root reimplantation with fibrin sealant enhanced motor recovery, preserved the synaptic covering of the motoneurons and improved neuronal survival. The replanted group did not show significant changes in microglial response compared to VRA-only. However, the astroglial reaction was significantly reduced in this group. Conclusions/Significance: In conclusion, the present data suggest that the repair of avulsed roots with snake venom fibrin glue at the exact point of detachment results in neuroprotection and preservation of the synaptic network at the microenvironment of the lesioned motoneurons. Also such procedure reduced the astroglial reaction and increased mRNA levels to neurotrophins and anti-inflammatory cytokines that may in turn, contribute to improving recovery of motor function.

Serum levels of brain-derived neurotrophicfactor correlate with the number of T2 MRI lesions in multiple sclerosis

The objective of the present study was to determine if there is a relationship between serum levels of brain-derived neurotrophic factor (BDNF) and the number of T2/fluid-attenuated inversion recovery (T2/FLAIR) lesions in multiple sclerosis (MS). The use of magnetic resonance imaging (MRI) has revolutionized the study of MS. However, MRI has limitations and the use of other biomarkers such as BDNF may be useful for the clinical assessment and the study of the disease. Serum was obtained from 28 MS patients, 18-50 years old (median 38), 21 women, 0.5-10 years (median 5) of disease duration, EDSS 1-4 (median 1.5) and 28 healthy controls, 19-49 years old (median 33), 19 women. BDNF levels were measured by ELISA. T1, T2/FLAIR and gadolinium-enhanced lesions were measured by a trained radiologist. BDNF was reduced in MS patients (median [range] pg/mL; 1160 [352.6-2640]) compared to healthy controls (1640 [632.4-4268]; P = 0.03, Mann-Whitney test) and was negatively correlated (Spearman correlation test, r = -0.41; P = 0.02) with T2/FLAIR (11-81 lesions, median 42). We found that serum BDNF levels were inversely correlated with the number of T2/FLAIR lesions in patients with MS. BDNF may be a promising biomarker of MS.

Role of BDNF secretion and signaling in the activity-dependent refinement of synaptic connections

Neuronal networks exhibit diverse types of plasticity, including the activity-dependent regulation of synaptic functions and refinement of synaptic connections. In addition, continuous generation of new neurons in the “adult” brain (adult neurogenesis) represents a powerful form of structural plasticity establishing new connections and possibly implementing pre-existing neuronal circuits (Kempermann et al, 2000; Ming and Song, 2005). Neurotrophins, a family of neuronal growth factors, are crucially involved in the modulation of activity-dependent neuronal plasticity. The first evidence for the physiological importance of this role evolved from the observations that the local administration of neurotrophins has dramatic effects on the activity-dependent refinement of synaptic connections in the visual cortex (McAllister et al, 1999; Berardi et al, 2000; Thoenen, 1995). Moreover, the local availability of critical amounts of neurotrophins appears to be relevant for the ability of hippocampal neurons to undergo long-term potentiation (LTP) of the synaptic transmission (Lu, 2004; Aicardi et al, 2004). To achieve a comprehensive understanding of the modulatory role of neurotrophins in integrated neuronal systems, informations on the mechanisms about local neurotrophins synthesis and secretion as well as ditribution of their cognate receptors are of crucial importance. In the first part of this doctoral thesis I have used electrophysiological approaches and real-time imaging tecniques to investigate additional features about the regulation of neurotrophins secretion, namely the capability of the neurotrophin brain-derived neurotrophic factor (BDNF) to undergo synaptic recycling. In cortical and hippocampal slices as well as in dissociated cell cultures, neuronal activity rapidly enhances the neuronal expression and secretion of BDNF which is subsequently taken up by neurons themselves but also by perineuronal astrocytes, through the selective activation of BDNF receptors. Moreover, internalized BDNF becomes part of the releasable source of the neurotrophin, which is promptly recruited for activity-dependent recycling. Thus, we described for the first time that neurons and astrocytes contain an endocytic compartment competent for BDNF recycling, suggesting a specialized form of bidirectional communication between neurons and glia. The mechanism of BDNF recycling is reminiscent of that for neurotransmitters and identifies BDNF as a new modulator implicated in neuro- and glio-transmission. In the second part of this doctoral thesis I addressed the role of BDNF signaling in adult hippocampal neurogenesis. I have generated a transgenic mouse model to specifically investigate the influence of BDNF signaling on the generation, differentiation, survival and connectivity of newborn neurons into the adult hippocampal network. I demonstrated that the survival of newborn neurons critically depends on the activation of the BDNF receptor TrkB. The TrkB-dependent decision regarding life or death in these newborn neurons takes place right at the transition point of their morphological and functional maturation Before newborn neurons start to die, they exhibit a drastic reduction in dendritic complexity and spine density compared to wild-type newborn neurons, indicating that this receptor is required for the connectivity of newborn neurons. Both the failure to become integrated and subsequent dying lead to impaired LTP. Finally, mice lacking a functional TrkB in the restricted population of newborn neurons show behavioral deficits, namely increased anxiety-like behavior. These data suggest that the integration and establishment of proper connections by newly generated neurons into the pre-existing network are relevant features for regulating the emotional state of the animal.

Intrazelluläre Lokalisation und synaptische Ausschüttung der Neurotrophine in hippokampalen Neuronen und die Bedeutung von BDNF bei hippokampaler synaptischer Plastizität

Die Mitglieder der Neurotrophin-Familie (NGF, BDNF, NT-3 und NT-4) sind sekretierte Neuropeptide, die eine bedeutende Rolle bei der Entwicklung von Nervenzellen und bei der Modulation der synaptischen Transmission spielen. Wenngleich eine aktivitätsabhängige Sekretion von BDNF bereits gezeigt werden konnte, wurden die subzelluläre Expression und die Ausschüttung der anderen Neurotrophine bislang nur unzureichend charakterisiert. Um die Expression und die Ausschüttung aller Neurotrophine unter identischen Bedingungen untersuchen zu können, wurde in der vorliegenden Arbeit das Expressionsmuster und die synaptische Ausschüttung GFP-markierter Neurotrophine in dissoziierten hippokampalen Neuronen mit Hilfe der konfokalen Fluoreszenz-Videomikroskopie zeitaufgelöst untersucht. Zwei Phänotypen konnten unterschieden werden: der distale vesikuläre Expressionstyp mit Neurotrophin-beinhaltenden Vesikeln in distalen Neuriten, und der proximale Expressionstyp mit einer diffusen Neurotrophin-Verteilung in den Neuriten und Neurotrophin-beinhaltenden Vesikeln im Soma des Neurons und in den proximalen Dendriten. Der distale vesikuläre Phänotyp entsprach einer Verteilung des entsprechenden Neurotrophins in die sekretorischen Granula des aktivitätsabhängigen Sekretionsweges, während der proximale Phänotyp den Transport eines Neurotrophins in den konstitutiven Sekretionsweg widerspiegelte. Alle Neurotrophine erreichten in hippokampalen Neuronen prinzipiell beide Sekretionswege. Jedoch gelangten BDNF und NT-3 mit einer größeren Effizienz in den regulierten Sekretionsweg als NT-4 und NGF (BDNF: in 98% aller Zellen, NT-3: 85%, NT-4: 23% und NGF: 46%). Neurotrophine besitzen, wie es für sekretorische Peptide üblich ist, eine Vorläufersequenz, die während der Reifung des Proteins proteolytisch abgespalten wird. Die Fusion dieser Präpro-Sequenz von BDNF mit der Sequenz des maturen NT-4 bewirkte einen effizienteren Transport von NT-4 in die sekretorischen Granula des regulierten Sekretionsweges, und zeigte die große Bedeutung der Präpro-Sequenz für das zelluläre Verteilungsmuster von Neurotrophinen. In Neuronen, in denen die Neurotrophine in den regulierten Sekretionsweg transportiert wurden, konnte eine aktivitätsabhängige Sekretion der Neurotrophine an postsynaptische Strukturen glutamaterger Synapsen beobachtet werden. Die aktivitätsabhängige postsynaptische Ausschüttung der Neurotrophine zeigte eine Heterogenität in der Kinetik der Sekretion (exponentieller Abfall des Neurotrophin-Signals mit Zeitkonstanten von tau = 121 bis 307s). Die Präinkubtion mit dem Protonen-Ionophor Monensin, welcher die Neutralisation des intragranulären pH-Wertes und somit die Solubilisierung der dicht gepackten Proteinstrukturen in den Vesikeln erzwingt, erhöhte die Geschwindigkeit der Neurotrophin-Ausschüttung auf den Wert des unter physiologischen Bedingungen schnellsten Neurotrophins NT-4. Dennoch blieb die Geschwindigkeit der Neurotrophin-Ausschüttung im Vergleich zur Neurotransmitter-Ausschüttung langsam (tau = 13 ± 2 s). Diese Daten belegen eindeutig, dass die Neutralisation der sekretorischen Granula die Geschwindigkeit der Neurotrophin-Ausschüttung kritisch determiniert und die Geschwindigkeit der Neurotrophin-Ausschüttung im Vergleich zur konventionellen Neurotransmitter-Ausschüttung langsam erfolgt. Des Weiteren konnte gezeigt werden, dass das Neurotrophin BDNF effizient in distale vesikuläre Strukturen von CA1 Pyramidenzellen organotypischer Schnittkulturen des Hippokampus sortiert wird. Die basalen elektrischen Eigenschaften von CA1 Pyramidenzellen BDNF-defizienter Mäuse sind vergleichbar zu den Eigenschaften von Wildtyp Mäusen. Sowohl das Eigenpotential der CA1 Pyramidenzellen, die Form der Aktionspotentiale als auch die evozierten Antworten der CA1 Pyramdenzellen auf eine gepaarte präsynaptische Stimulation der Schaffer-Kollateralen zeigten bei BDNF-/- -, BDNF+/- - und BDNF+/+ -Mäusen keine signifikanten Unterschiede. Die Fähigkeit der CA1 Pyramidenzellen auf eine hochfrequente Reizung mit einer Langzeitpotenzierung (LTP) der postsynaptischen Ströme zu reagieren ist jedoch bei den BDNF-defizienten Mäusen beinträchtigt. Eine verminderte Induktion von LTP war in den BDNF-defizienten Mäusen nach tetanischer Stimulation der präsynaptischen Schaffer-Kollateralen und simultaner postsynaptischer Depolarisation der CA1 Pyramidenzelle zu beobachten.

Aktivitätsabhängige Kontrolle der Apoptose in neonatalen kortikalen Neuronen und Etablierung eines Biosensors zur Echtzeitanalyse der Caspase-3-Aktivierung

Die Apoptose spielt eine entscheidende Rolle während der normalen Entwicklung des zentralen Nervensystems. Elektrische Aktivität und die Versorgung mit trophischen Faktoren sind ausschlaggebend für das Überleben von Neuronen. Um zu untersuchen, welche zellulären Prozesse die aktivitätsabhängige Apoptose in organotypischen Schnittkulturen des neugeborenen Neokortex beeinflussen, wurde in der vorliegenden Arbeit immunzytochemisch das Auftreten aktivierter Caspase-3, nach pharmakologischer Beeinflussung von Ionenkanälen und membranständigen Rezeptoren analysiert. Die Unterdrückung neuronaler Aktivität durch den Natriumionenkanalblocker TTX führte zu einem signifikanten Verlust kortikaler Neuronen. Ein ähnlicher Anstieg der Zahl apoptotischer Neurone konnte durch Applikation von Antagonisten ionotroper Glutamatrezeptoren, GABAA-Rezeptoren oder neuronaler Gap Junctions induziert werden. Jedoch konnte bei einigen Antagonisten die apoptosefördernde Wirkung erst nach längerer Einwirkung beobachtet werden. Im Weiteren wurde eine Methode etabliert, mit deren Hilfe eine Echtzeitanalyse der Apoptose kortikaler Neurone unter dem Entzug trophischer Faktoren in Gegenwart unterschiedlicher extrazellulärer Kaliumkonzentrationen ermöglicht wurde. Dazu wurden dissoziierte kortikale Kulturen mit dem pCaspase3-sensor Vektor transfiziert. Das durch dieses Plasmid codierte fluoreszente Protein wird Caspase-3 abhängig gespalten. In der vorliegenden Arbeit konnte gezeigt werden, dass der Caspase3-sensor spezifisch für die Aktivierung der Caspase-3 ist, und dass die Überlebensfähigkeit der transfizierten Neurone durch das Transfektionsprotokoll nicht beeinflusst wird.

Signalling mechanisms affecting regulated neurotrophin secretion in hippocampal neurons

Die Neurotrophine aus Säugetiere BDNF und NT-3 sind von Neuronen sekretierte Wachstumsfaktoren. Ferner sind Neurotrophine in verschiedene Formen der aktivitätsabhängigen synaptische Plastizität involviert. Obwohl die Ausschüttung von Neurotrophine aus Synapsen beschrieben worden ist, sind die intrazellulären Signalkaskaden, die die synaptische Ausschüttung von Neurotrophine regulieren, bei weitem nicht verstanden. Deswegen ist die Analyse der Sekretion von Neurotrophine auf subzellulärer Ebene erforderlich, um die genaue Rolle von präsynaptische und postsynaptische NT-Sekretion in der synaptischen Plastizität aufzudecken. In der vorliegenden Arbeit wurden die Kulturen von dissoziierten hippocampalen Neuronen aus Ratten mit grün fluoreszierenden Protein-markierten Konstrukten von BDNF und NT-3 transfiziert und Neurotrophine-enthaltenden Vesikeln durch die Colokalisierung mit dem cotransfizierten postsynaptischen Marker PSD-95-DsRed an glutamatergen Synapsen identifiziert. Depolarisationsinduzierte Sekretion von BDNF und NT-3 wurde per Direktaufnahme am Fluoreszenzmikroskop beobachtet. Die unvermittelte postsynaptische Depolarisation mit erhöhtem Kalium, in Gegenwart von Inhibitoren der synaptischen Transmission, erlaubte die Untersuchung der Signalwege, die am postsynaptischen Sekretionsprozess der Neurotrophinvesikel beteiligt sind. Es konnte gezeigt werden, dass die depolarisationsinduzierte postsynaptische Ausschüttung der Neurotrophine durch Calcium-Einstrom ausgelöst wird, entweder über L-Typ-spannungsabhängige Calcium-Kanäle oder über NMDA-Rezeptoren. Eine anschließende Freisetzung von Calcium aus intrazellulären Speichern über Ryanodin-Rezeptoren ist für den Sekretionsprozess erforderlich. Die postsynaptische Neurotrophinausschüttung wird durch KN-62 und KN-93 gehemmt, was auf eine unmittelbare Abhängigkeit von aktiver alpha-Calcium-Calmodulin-abhängige Proteinkinase II (CaMKII) hinweist. Der Inhibitor der cAMP/Proteinkinase A (PKA), Rp-cAMP-S, sowie der NO-Donor, SNP, minderten die Neurotrophinausschüttung. Hingegen blieben die Erhöhung des intrazellulären cAMP und der NO-Synthase-Inhibitor L-NMMA ohne Wirkung. Mit dem Trk-Inhibitor K252a konnte gezeigt werden, dass autokrine Neurotrophin-induzierte Neurotrophinausschüttung nicht an der synaptischen Freisetzung der Neurotrophine beiträgt und, dass BDNF seine eigene postsynaptische Sekretion nicht auslöst. Freisetzungsexperimente mit dem Fluoreszenz-Quencher Bromphenolblau konnten den Nachweis erbringen, dass asynchrone und anhaltende Fusionsporenöffnung von Neurotrophinvesikeln während der Sekretion stattfindet. Wegen der im Vergleich zum komplexen Sekretionsprozess schnellen Fusionsporenöffnung, scheint die Freisetzungsgeschwindigkeit von Neurotrophine durch ihre Diffusion aus dem Vesikel begrenzt. Zusammenfassend zeigen diese Ergebnisse eine starke Abhängigkeit der aktivitätsabhängigen postsynaptischen Neurotrophinausschüttung vom Calcium-Einstrom, von der Freisetzung von Calcium aus internen Speichern, von der Aktivierung der CaMKII und einem intakten Funktion der PKA, während der Trk-Signalweg, die Aktivierung von Natrium-Kanäle und NO-Signale nicht erforderlich sind.

LIF promotes neurogenesis and maintains neural precursors in cell populations derived from spiral ganglion stem cells

BACKGROUND: Stem cells with the ability to form clonal floating colonies (spheres) were recently isolated from the neonatal murine spiral ganglion. To further examine the features of inner ear-derived neural stem cells and their derivatives, we investigated the effects of leukemia inhibitory factor (LIF), a neurokine that has been shown to promote self-renewal of other neural stem cells and to affect neural and glial cell differentiation. RESULTS: LIF-treatment led to a dose-dependent increase of the number of neurons and glial cells in cultures of sphere-derived cells. Based on the detection of developmental and progenitor cell markers that are maintained in LIF-treated cultures and the increase of cycling nestin-positive progenitors, we propose that LIF maintains a pool of neural progenitor cells. We further provide evidence that LIF increases the number of nestin-positive progenitor cells directly in a cell cycle-independent fashion, which we interpret as an acceleration of neurogenesis in sphere-derived progenitors. This effect is further enhanced by an anti-apoptotic action of LIF. Finally, LIF and the neurotrophins BDNF and NT3 additively promote survival of stem cell-derived neurons. CONCLUSION: Our results implicate LIF as a powerful tool to control neural differentiation and maintenance of stem cell-derived murine spiral ganglion neuron precursors. This finding could be relevant in cell replacement studies with animal models featuring spiral ganglion neuron degeneration. The additive effect of the combination of LIF and BDNF/NT3 on stem cell-derived neuronal survival is similar to their effect on primary spiral ganglion neurons, which puts forward spiral ganglion-derived neurospheres as an in vitro model system to study aspects of auditory neuron development.

Fine control of drug delivery for cochlear implant applications

Cochlear implants are neuroprostheses that are inserted into the inner ear to directly electrically stimulate the auditory nerve, thus replacing lost cochlear receptors, the hair cells. The reduction of the gap between electrodes and nerve cells will contribute to technological solutions simultaneously increasing the frequency resolution, the sound quality and the amplification of the signal. Recent findings indicate that neurotrophins (NTs) such as brain derived neurotrophic factor (BDNF) stimulate the neurite outgrowth of auditory nerve cells by activating Trk receptors on the cellular surface (1–3). Furthermore, small-size TrkB receptor agonists such as di-hydroxyflavone (DHF) are now available, which activate the TrkB receptor with similar efficiency as BDNF, but are much more stable (4). Experimentally, such molecules are currently used to attract nerve cells towards, for example, the electrodes of cochlear implants. This paper analyses the scenarios of low dose aspects of controlled release of small-size Trk receptor agonists from the coated CI electrode array into the inner ear. The control must first ensure a sufficient dose for the onset of neurite growth. Secondly, a gradient in concentration needs to be maintained to allow directive growth of neurites through the perilymph-filled gap towards the electrodes of the implant. We used fluorescein as a test molecule for its molecular size similarity to DHF and investigated two different transport mechanisms of drug dispensing, which both have the potential to fulfil controlled low-throughput drug-deliverable requirements. The first is based on the release of aqueous fluorescein into water through well-defined 60-μm size holes arrays in a membrane by pure osmosis. The release was both simulated using the software COMSOL and observed experimentally. In the second approach, solid fluorescein crystals were encapsulated in a thin layer of parylene (PPX), hence creating random nanometer-sized pinholes. In this approach, the release occurred due to subsequent water diffusion through the pinholes, dissolution of the fluorescein and then release by out-diffusion. Surprisingly, the release rate of solid fluorescein through the nanoscopic scale holes was found to be in the same order of magnitude as for liquid fluorescein release through microscopic holes.

Chronic stress promotes tumor growth through increased BDNF production and neo-innervation

Background: Activation of the sympathetic nervous system (SNS) in response to chronic biobehavioral stress results in high levels of catecholamines and persistent activation of adrenergic signaling, which promotes tumor growth and progression. However it is unknown how catecholamine levels within the tumor exceed systemic levels in circulation. I hypothesized that neo-innervation of tumors is required for stress-mediated effects on tumor growth. Results: First, I examined whether sympathetic nerves are present in human ovarian cancer samples as well as orthotopic ovarian cancer models. Immunohistochemical (IHC) staining for neurofilament revealed that catecholaminergic neurons are present within tumor tissue. In order to determine whether chronic stress affects the density of nerves in the tumor, I utilized an orthotopic mouse model of ovarian cancer that was exposed to daily restraint stress. IHC analysis revealed that nerve density in tumors increased by more than three-fold in stressed animals versus non-stressed controls. IHC analysis suggested that this results from both recruitment of existing neurons (axonogenesis) as well as new neuron formation (neurogenesis) within the tumor. To determine how tumors are recruiting nerve growth, I utilized a PCR array analysis of 84 nerve growth related genes and their receptors, which showed that stimulation of the SKOV3 ovarian cancer cell line with norepinephrine (NE) leads to increased expression of several neurotrophins, including brain-derived neurotrophic factor (BDNF). Neurite extension assays showed that media conditioned by ovarian cancer cell lines is capable of inducing neurite outgrowth in differentiated neuron-like PC12 cells, and NE treatment of cancer cells potentiates this effect. Norepinephrine-induced neurite extension was abolished after BDNF silencing by siRNA, suggesting that BDNF is critical to tumor cell-induced nerve growth. in vivo BDNF inhibition resulted in complete abrogation of stress-induced increases in tumor weight and nerve density, as well as downstream markers of stress. Discussion: These studies indicate that adrenergic signalling induced by chronic stress promotes neo-innervation in the tumor microenvironment. This results in a mutually beneficial relationship between the tumor cells and neurons. This work is crucial for providing a link between chronic stress and its effects on the tumor and its microenvironment. The data shown here aims to open new venues that can be used in development of therapies designed to block the stress effects on tumor growth.

Long-range signaling within growing neurites mediated by neurotrophin-3

In addition to well established trophic functions, neurotrophins acutely affect neurotransmitter secretion from the presynaptic nerve terminal, influence synaptic development, and may serve as selective retrograde messengers that regulate synaptic efficacy. The crucial question related to the mechanisms of neurotrophin-mediated signaling is whether acute effects of neurotrophins are spatially restricted to the activated synapses. Here we have used a local perfusion technique for local delivery of neurotrophin-3 (NT-3) to various regions of developing Xenopus embryo neurons in culture. Within minutes after a focal exposure of a soma or a small (≈30 μm in length) axonal segment to NT-3, we observed an increase in the spontaneous neurotransmitter secretion from the presynaptic nerve terminals located ≈300–400 μm away from the site of NT-3 application. Secretory activity along the axonal shaft was not affected. Our findings suggest that the NT-3-mediated signal may rapidly travel through neuronal cytoplasm over unexpectedly long distances and modulate neurotransmitter release specifically at the presynaptic nerve terminals.

Subplate neuron ablation alters neurotrophin expression and ocular dominance column formation

Ocular dominance column formation in visual cortex depends on both the presence of subplate neurons and the endogenous expression of neurotrophins. Here we show that deletion of subplate neurons, which supply glutamatergic inputs to visual cortex, leads to a paradoxical increase in brain-derived neurotrophic factor mRNA in the same region of visual cortex in which ocular dominance columns are absent. Subplate neuron ablation also increases glutamic acid decarboxylase-67 levels, indicating an alteration in cortical inhibition. These observations imply a role for this special class of neurons in modulating activity-dependent competition by regulating levels of neurotrophins and excitability within a developing cortical circuit.

Identification of genes differentially expressed by nerve growth factor- and neurotrophin-3-dependent sensory neurons

The neurotrophins nerve growth factor (NGF) and neurotrophin-3 (NT3) support the survival of subpopulations of primary sensory neurons with defined and distinct physiological characteristics. Only a few genes have been identified as being differentially expressed in these subpopulations, and not much is known about the nature of the molecules involved in the processing of sensory information in NGF-dependent nociceptive neurons or NT3-dependent proprioceptive neurons. We devised a simple dorsal root ganglion (DRG) explant culture system, allowing the selection of neuronal populations preferentially responsive to NGF or NT3. The reliability of this assay was first monitored by the differential expression of the NGF and NT3 receptors trkA and trkC, as well as that of neuropeptides and calcium-binding proteins. We then identified four differentially expressed sodium channels, two enriched in the NGF population and two others in the NT3 population. Finally, using an optimized RNA fingerprinting protocol, we identified 20 additional genes, all differentially expressed in DRG explants cultured with NGF or NT3. This approach thus allows the identification of large number of genes expressed in subpopulations of primary sensory neurons and opens the possibility of studying the molecular mechanisms of nociception and proprioception.