Towards new generation of neuro-implantable devices : engineering neuron/carbon nanotubes integrated functional units

2008/2009Le nanotecnologie sono un campo delle scienze che utilizza materiali e dispositivi ingegnerizzati aventi la più piccola organizzazione funzionale a livello di dimensioni nanometriche. Questo implica che nanodispositivi e nanomateriali possano interagire con i sistemi biologici a livello molecolare con un elevato grado di specificità. É largamente accettato che l’applicazione delle nanotecnologie nell’ambito delle neuroscienze abbia un forte potenziale (Silva, 2006). In questo contesto, i nanotubi di carbonio (CNT), un’innovativa forma di carbonio composta da strutture tubulari di grafite dalle dimensioni nanometriche dotate di buone proprietà di conduzione elettrica, si sono dimostrati promettenti candidati per sviluppare la tecnologia di dispositivi impiantabili in ambito biomedico. Diversi studi hanno dimostrato la biocompatibilità dei substrati di CNT per i neuroni in termini di adesione, crescita e differenziamento cellulare (riassunti in Sucapane et al., 2009). Al fine di aumentare la nostra conoscenza riguardo alle interazioni presenti in sistemi ibridi formati da CNT e neuroni, abbiamo caratterizzato l’attività di reti neuronali cresciuti su supporti di CNT attraverso la tecnica del patch clamp. Il nostro gruppo ha riportato che circuti neuronali cresciuti in vitro su substrati di CNT presentano un’aumentata attività sinaptica spontanea rispetto al controllo a fronte di comparabili proprietà base (proprietà passive di membrana, morfologia e densità dei neuroni) delle colture nelle due condizioni di crescita (Lovat et al., 2005). Si è quindi ipotizzato che tale aumentata attività spontanea potesse originare da una modificazione nel modo in cui i singoli neuroni generano il segnale elettrico. A tal fine, si sono monitorate variazioni nelle proprietà elettrogeniche di singoli neuroni, utilizzando un protocollo standard per caratterizzare l’integrazione di potenziali d’azione retropropaganti nei dendriti (Larkum et al., 1999). In configurazione current clamp, attraverso brevi iniezioni di corrente nel soma della cellula, abbiamo indotto una serie di regolari potenziali d’azione (PA) a varie frequenze nel neurone sotto registrazione, quindi abbiamo studiato la presenza di un’addizionale depolarizzazione somatica dopo l’ultimo PA del treno. Abbiamo osservato che neuroni di controllo mostrano nella maggioranza dei casi una iperpolarizzazione (AHP) del potenziale di membrana dopo l’ultimo PA del treno, mentre una depolarizzazione (ADP) è presente solo in una piccola quota di casi. In presenza di CNT, invece, l’ADP risulta essere l’evento predominante. L’ADP è inoltre abolita dall’applicazione di CoCl2, un bloccante non specifico dei canali calcio voltaggio dipendenti. Per di più, l’area dell’ADP può essere diminuita dall’applicazione di nifedipina (10 μM) e l’ulteriore coapplicazione di NiCl2 (50 μM) elimina totalmente l’ADP, suggerendo che sia i canali calcio voltaggio dipendenti ad alta soglia di attivazione, sia quelli a bassa soglia, siano coinvolti in questo processo (Cellot et al., 2009). Attraverso la microscopia elettronica a trasmissione (TEM) e, più recentemente, mediante quella a scansione (SEM) è stata messa in evidenza la presenza di discontinui punti di stretto contatto tra CNT e membrane neuronali: la nostra ipotesi è che tali strutture ibride siano in grado di favorire la retropropagazione dei PA nei dendriti distali. La maggiore eccitabilità a livello del singolo neurone, inoltre, potrebbe essere responsabile dell’incremento di attività spontanea della rete neuronale. Abbiamo quindi ulteriormente caratterizzato l’attività della rete neuronale attraverso registrazioni da coppie di neuroni, dove il neurone presinaptico veniva stimolato ad avere treni di potenziali d’azione a 20 Hz in configurazione current clamp e simultaneamente il neurone postsinaptico era monitorato in configurazione voltage clamp per vedere la presenza o l’assenza di una risposta sinaptica. I nostri esperimenti indicano che la probabilità di trovare connessioni monosinaptiche gabaergiche tra neuroni è aumentata in presenza di CNT (56% vs 40% in controllo). Inoltre, è stato rilevato un ulteriore effetto dei CNT sulla plasticità a breve termine delle sinapsi: nelle condizioni di controllo, treni di potenziali d’azione nella cellula presinaptica evocano nella cellula postsinaptica nel 90% dei casi una chiara depressione nell’ampiezza di consecutivi ePSCs, mentre solo in meno del 10% è possibile rilevare una facilitazione. Al contrario, in presenza di CNT, nel 39% delle coppie, il neurone postsinaptico risponde in modo chiaramente facilitativo. Nelle più recenti serie di esperimenti, abbiamo voluto indagare più approfonditamente l’origine di questa modificazione in termini di plasticità sinaptica; a tal fine, abbiamo trattato neuroni in controllo e su CNT con tetrodotossina 1 µM per 5 giorni, al fine di bloccare completamente l’attività elettrica della rete neuronale, e abbiamo compiuto delle registrazioni da coppie di neuroni. Mentre la risposta prevalentemente di depressione dei controlli non è modificata da tale trattamento, neuroni cresciuti su substrati di cnt in condizioni di blocco dell’attività elettrica non presentano più sinapsi con caratteristiche di facilitazione, ma hanno un comportamento simile ai contolli. Questi risultati indicano che la facilitazione è una proprietà tipica di sinapsi attive sviluppatesi in presenza di CNT.XXII Ciclo198

Thin graphene oxide nanoflakes modulate glutamatergic synapses in the amygdala cultured circuits: exploiting synaptic approaches to anxiety disorders

Anxiety disorders (ADs) are nervous system maladies involving changes in the amygdala synaptic circuitry, such as an upregulation of excitatory neurotransmission at glutamatergic synapses. In the field of nanotechnology, thin graphene oxide flakes with nanoscale lateral size (s-GO) have shown outstanding promise for the manipulation of excitatory neuronal transmission with high temporal and spatial precision, thus they were considered as ideal candidates for modulating amygdalar glutamatergic transmission. Here, we validated an in vitro model of amygdala circuitry as a screening tool to target synapses, towards development of future ADs treatments. After one week in vitro, dissociated amygdalar neurons reconnected forming functional networks, whose development recapitulated that of the tissue of origin. When acutely applied to these cultures, s-GO flakes induced a selective modification of excitatory activity. This type of interaction between s-GO and amygdalar neurons may form the basis for the exploitation of alternative approaches in the treatment of ADs

BDNF impact on synaptic dynamics: extra or intracellular long-term release differently regulates cultured hippocampal synapses

Brain Derived Neurotrophic Factor (BDNF) signalling contributes to the formation, maturation and plasticity of Central Nervous System (CNS) synapses. Acute exposure of cultured brain circuits to BDNF leads to up-regulation of glutamatergic neuro-transmission, by the accurate tuning of pre and post synaptic features, leading to structural and functional synaptic changes. Chronic BDNF treatment has been comparatively less investigated, besides it may represent a therapeutic option to obtain rescue of post-injury alterations of synaptic networks. In this study we used a paradigm of BDNF long-term (4 days) incubation to assess in hippocampal post-natal neurons in culture, the ability of such a treatment to alter synapses. By patch clamp recordings we describe the augmented function of excitatory neurotransmission and we further explore by live imaging the presynaptic changes brought about by long-term BDNF. In our study, exogenous long-term BDNF exposure of post-natal neurons did not affect inhibitory neurotransmission. We further compare, by genetic manipulations of cultured neurons and BDNF release, intracellular overexpression of this neurotrophin at the same developmental age. We describe for the first-time differences in synaptic modulation by BDNF with respect to exogenous or intracellular release paradigms. Such a finding holds the potential of influencing the design of future therapeutic strategies

Premature changes in neuronal excitability account for hippocampal network impairment and autistic-like behavior in neonatal BTBR T+tf/J mice

Coherent network oscillations (GDPs), generated in the immature hippocampus by the synergistic action of GABA and glutamate, both depolarizing and excitatory, play a key role in the construction of neuronal circuits. In particular, GDPs-associated calcium transients act as coincident detectors for enhancing synaptic efficacy at emerging GABAergic and glutamatergic synapses. Here, we show that, immediately after birth, in the CA3 hippocampal region of the BTBR T+tf/J mouse, an animal model of idiopathic autism, GDPs are severely impaired. This effect was associated with an increased GABAergic neurotransmission and a reduced neuronal excitability. In spite its depolarizing action on CA3 pyramidal cells (in single channel experiments EGABA was positive to Em), GABA exerted at the network level an inhibitory effect as demonstrated by isoguvacine-induced reduction of neuronal firing. We implemented a computational model in which experimental findings could be interpreted as the result of two competing effects: a reduction of the intrinsic excitability of CA3 principal cells and a reduction of the shunting activity in GABAergic interneurons projecting to principal cells. It is therefore likely that premature changes in neuronal excitability within selective hippocampal circuits of BTBR mice lead to GDPs dysfunction and behavioral deficits reminiscent of those found in autistic patients

Tuning the Reduction of Graphene Oxide Nanoflakes Differently Affects Neuronal Networks in the Zebrafish

The increasing engineering of biomedical devices and the design of drug-delivery platforms enriched by graphene-based components demand careful investigations of the impact of graphene-related materials (GRMs) on the nervous system. In addition, the enhanced diffusion of GRM-based products and technologies that might favor the dispersion in the environment of GRMs nanoparticles urgently requires the potential neurotoxicity of these compounds to be addressed. One of the challenges in providing definite evidence supporting the harmful or safe use of GRMs is addressing the variety of this family of materials, with GRMs differing for size and chemistry. Such a diversity impairs reaching a unique and predictive picture of the effects of GRMs on the nervous system. Here, by exploiting the thermal reduction of graphene oxide nanoflakes (GO) to generate materials with different oxygen/carbon ratios, we used a high-throughput analysis of early-stage zebrafish locomotor behavior to investigate if modifications of a specific GRM chemical property influenced how these nanomaterials affect vertebrate sensory-motor neurophysiology—exposing zebrafish to GO downregulated their swimming performance. Conversely, reduced GO (rGO) treatments boosted locomotor activity. We concluded that the tuning of single GRM chemical properties is sufficient to produce differential effects on nervous system physiology, likely interfering with different signaling pathways

PEDOT:PSS interfaces support the development of neuronal synaptic networks with reduced neuroglia response in vitro

The design of electrodes based on conductive polymers in brain-machine interface technology offers the opportunity to exploit variably manufactured materials to reduce gliosis, indeed the most common brain response to chronically implanted neural electrodes. In fact, the use of conductive polymers, finely tailored in their physical-chemical properties, might result in electrodes with improved adaptability to the brain tissue and increased charge-transfer efficiency. Here we interfaced poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) doped with different amounts of ethylene glycol (EG) with rat hippocampal primary cultures grown for 3 weeks on these synthetic substrates. We used immunofluorescence and scanning electron microscopy combined to single cell electrophysiology to assess the biocompatibility of PEDOT:PSS in terms of neuronal growth and synapse formation. We investigated neuronal morphology, density and electrical activity. We reported the novel observation that opposite to neurons, glial cell density was progressively reduced, hinting at the ability of this material to down regulate glial reaction. Thus PEDOT:PSS is an attractive candidate for the design of new implantable electrodes, controlling the extent of glial reactivity without affecting neuronal viability and function

Graphene Oxide Nanosheets Hamper Glutamate Mediated Excitotoxicity and Protect Neuronal Survival In An In vitro Stroke Model

Small graphene oxide (s-GO) nanosheets reversibly downregulate central nervous system (CNS) excitatory synapses, with potential developments as future therapeutic tools to treat neuro-disorders characterized by altered glutamatergic transmission. Excitotoxicity, namely cell death triggered by exceeding ambient glutamate fueling over-activation of excitatory synapses, is a pathogenic mechanism shared by several neural diseases, from ischemic stroke to neurodegenerative disorders. In this work, CNS cultures were exposed to oxygen-glucose deprivation (OGD) to mimic ischemic stroke in vitro, and it is show that the delivery of s-GO following OGD, during the endogenous build-up of secondary damage and excitotoxicity, improved neuronal survival. In a different paradigm, excitotoxicity cell damage was reproduced through exogenous glutamate application, and s-GO co-treatment protected neuronal integrity, potentially by directly downregulating the synaptic over-activation brought about by exogenous glutamate. This proof-of-concept study suggests that s-GO may find novel applications in therapeutic developments for treating excitotoxicity-driven neural cell death

Smell Rehabilitation: Recovery of Olfactory Perception and Discrimination in Twelve Cases of Total Laryngectomy

Objective: Total laryngectomy (TL) is a surgical practice widely used in the therapy of advanced laryngeal cancer. Since TL provokes loss of both speech and smell functions, the aim of the present work was to evaluate the effect of a smell rehabilitation cycle in twelve total laryngectomized patients. Methods: Twelve laryngectomized patients were enrolled to undergo a smell rehabilitation cycle in addition to previously performed speech recovery. For this aim the Nasal Airflow-Inducing Maneuver (NAIM) was employed to allow air to reach the nasal cavities again. Both olfactory perception and olfactory discrimination of odorous substances were evaluated by numeric scores to assess the modifications induced by the smell rehabilitative intervention on the recovery of the olfactory functions. Results: Smell capability, as regards the olfactory perception, ameliorated in the group of patients already after the first week of the smell rehabilitation cycle. Subsequently also the olfactory discrimination was evaluated, both at the end of the rehabilitation cycle (day 28) and after a period of twelve months, and we observeda significant amelioration at the end of the rehabilitative intervention that was essentially maintained even after one year although without a constant assistance performed by speech therapists. Conclusions: Smell rehabilitation should be always considered after TL in addition to speech restoration. Recovered smell perception and discrimination could enhance the related taste sensitivity, therefore restored olfactory functions could also ameliorate significantly the quality of life in total laryngectomized patients

Reprogramming fibroblasts to neural-precursor-like cells by structured overexpression of pallial patterning genes

In this study, we assayed the capability of four genes implicated in embryonic specification of the cortico-cerebral field, Foxg1, Pax6, Emx2 and Lhx2, to reprogramme mouse embryonic fibroblasts towards neural identities. Lentivirus-mediated, TetON-dependent overexpression of Pax6 and Foxg1 transgenes specifically activated the neural stem cell (NSC) reporter Sox1-EGFP in a substantial fraction of engineered cells. The efficiency of this process was enhanced up to ten times by simultaneous inactivation of Trp53 and co-administration of a specific drug mix inhibiting HDACs, H3K27-HMTase and H3K4m2-demethylase. Remarkably, a fraction of the reprogrammed population expressed other NSC markers and retained its new identity, even after switching off the reprogramming transgenes. When transferred into a pro-differentiative environment, Pax6/Foxg1-overexpressing cells activated the neuronal marker Tau-EGFP. Frequency of Tau-EGFP positive cells was almost doubled upon delayed delivery of Emx2 and Lhx2 transgenes. A further improvement of the neuron-like cell output was achieved by inhibition of the BMP and TGF\u3b2 pathways. Tau-EGFP positive cells were able to generate action potentials upon injection of depolarizing current pulses, further indicating their neuron-like phenotype

Successful Regrowth of Retinal Neurons When Cultured Interfaced to Carbon Nanotube Platforms

A shared dream of ophthalmologists,neurologists and bioengineers is to recover sight ability in diseased eyes via coupling retinal cells with artificial devices. In the engineering of ophto-prosthetic devices the material directly exposed to the biological milieu is crucial, it has to guarantee tight contacts between retinal neurons and the interface, while assuring cell survival with physiological network development. Carbon nanotubes have been applied in several areas of nerve tissue engineering and are emerging as a promising material for neuro-interfacing applications, given their outstanding physical properties. In the current work we have tested carbon nanotube ability to interface cultured murine and human retinal neurons. We cultured rat retinal neurons on carbon nanotube substrates and described their morphology and synaptic functions via immunofluorescence microscopy and patch-clamp recordings. In a second set of experiments, we explored viability and morphology of human retinal ganglion cells (RGC) when grown on carbon nanotube substrates. We show here carbon nanotube ability to sustain the proper development of rat neurons and, more importantly, of human RGCs. In addition, patch-clamp recordings on rat retinal cells were functional to demonstrate that carbon nanotubes do not perturb the physiological synaptic activity when compared to controls. This result, strengthen by the shown biocompatibility with human cells and the nanotube well described high electrical conductivity, makes these nanomaterials promising candidates to interface, stimulate or record eye nerve cells