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Resected hippocampal tissue from patients with drug-resistant epilepsy presents a unique possibility to test novel treatment strategies directly in target tissue. The post-resection time for testing and analysis however is normally limited. Acute tissue slices allow for electrophysiological recordings typically up to 12 hours. To enable longer time to test novel treatment strategies such as, e.g., gene-therapy, we developed a method for keeping acute human brain slices viable over a longer period. Our protocol keeps neurons viable well up to 48 hours. Using a dual-flow chamber, which allows for microscopic visualisation of individual neurons with a submerged objective for whole-cell patch-clamp recordings, we report stable electrophysiological properties, such as action potential amplitude and threshold during this time. We also demonstrate that epileptiform activity, monitored by individual dentate granule whole-cell recordings, can be consistently induced in these slices, underlying the usefulness of this methodology for testing and/or validating novel treatment strategies for epilepsy.

Epilepsy is a neurological disorder with a prevalence of ≈1% of general population. Available antiepileptic drugs (AEDs) have multiple side effects and are ineffective in 30% of patients. Therefore, development of effective treatment strategies is highly needed, requiring drug-screening models that are relevant and reliable. We investigated novel chemogenetic approach, using DREADDs (designer receptors exclusively activated by designer drugs) as possible inhibitor of epileptiform activity in organotypic hippocampal slice cultures (OHSCs). The OHSCs are characterized by increased overall excitability and closely resemble features of human epileptic tissue. Studies suggest that chemically induced epileptiform activity in rat OHSCs is pharmacoresistant to most of AEDs. However, high-frequency electric stimulus train-induced bursting (STIB) in OHSCs is responsive to carbamazepine and phenytoin. We investigated whether inhibitory DREADD, hM4Di, would be effective in suppressing STIB in OHSC. hM4Di is a mutated muscarinic receptor selectively activated by otherwise inert clozapine-N-oxide, which leads to hyperpolarization in neurons. We demonstrated that this hyperpolarization effectively suppresses STIB in mouse OHSCs. As we also found that STIB in mouse OHSCs is resistant to common AED, valproic acid, collectively our findings suggest that DREADD-based strategy may be effective in suppressing epileptiform activity in a pharamcoresitant epileptic brain tissue.

Downscaling of microfluidic cell culture and detection devices for electrochemical monitoring has mostly focused on miniaturization of the microfluidic chips which are often designed for specific applications and therefore lack functional flexibility. We present a compact microfluidic cell culture and electrochemical analysis platform with in-built fluid handling and detection, enabling complete cell based assays comprising on-line electrode cleaning, sterilization, surface functionalization, cell seeding, cultivation and electrochemical real-time monitoring of cellular dynamics. To demonstrate the versatility and multifunctionality of the platform, we explored amperometric monitoring of intracellular redox activity in yeast (Saccharomyces cerevisiae) and detection of exocytotically released dopamine from rat pheochromocytoma cells (PC12). Electrochemical impedance spectroscopy was used in both applications for monitoring cell sedimentation and adhesion as well as proliferation in the case of PC12 cells. The influence of flow rate on the signal amplitude in the detection of redox metabolism as well as the effect of mechanical stimulation on dopamine release were demonstrated using the programmable fluid handling capability. The here presented platform is aimed at applications utilizing cell based assays, ranging from e.g. monitoring of drug effects in pharmacological studies, characterization of neural stem cell differentiation, and screening of genetically modified microorganisms to environmental monitoring.

Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson's disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.

Controlling activity of defined populations of neurons without affecting other neurons in the brain is now possible by a new gene- and neuroengineering technology termed optogenetics. Derived from microbial organisms, opsin genes encoding light-activated ion channels and pumps (channelrhodopsin-2 [ChR2]; halorhodopsin [NpHR], respectively), engineered for expression in the mammalian brain, can be genetically targeted into specific neural populations using viral vectors. When exposed to light with appropriate wavelength, action potentials can be triggered in ChR2-expressing neurons, whereas inhibition of action potentials can be obtained in NpHR-expressing neurons, thus allowing for powerful control of neural activity. Optogenetics is now intensively used in laboratory animals, both in vitro and in vivo, for exploring functions of complex neural circuits and information processing in the normal brain and during various neurological conditions. The clinical perspectives of adopting optogenetics as a novel treatment strategy for human neurological disorders have generated considerable interest, largely because of the enormous potential demonstrated in recent rodent and nonhuman primate studies. Restoration of dopamine-related movement dysfunction in parkinsonian animals, amelioration of blindness and recovery of breathing after spinal cord injury are a few examples of such perspectives.

A versatile microfluidic, multichamber cell culture and analysis system with an integrated electrode array and potentiostat suitable for electrochemical detection and microscopic imaging is presented in this paper. The system, which allows on-line electrode cleaning and modification, was developed for real-time monitoring of cellular dynamics, exemplified in this work by monitoring of redox metabolism inside living yeast cells and dopamine release from PC12 cells.

One of the most promising and novel strategies to interfere with neurological disease processes is gene therapy using recombinant adeno-associated viral (rAAV) vectors. Such a rAAV-based gene delivery approach is rapidly advancing towards clinical trials. In this regard, neuropeptide Y (NPY) gene transduction into the brain tissue has attracted particular interest due to its potential to regulate and perhaps even ameliorate epileptic conditions. NPY gene transduction by viral vectors in epileptogenic regions of the brain can effectively suppress seizures in animals. The mechanisms underlying the seizure-suppressant effects of an NPY transgene are not well understood; in particular, under which circumstances transgene NPY is released, and whether and how it acts on synaptic transmission within the area of viral vector transduction are not known. These questions are of fundamental importance not only for the implementation of such a gene therapy approach in clinical trials with patients, but also for our general understanding of how transgene neuropeptides may act in the brain.

Udgivelsesdato: 2009-Dec, Both brain-derived neurotrophic factor (BDNF) and the serotonin receptor 2A (5-HT(2A)) have been related to depression pathology. Specific 5-HT(2A) receptor changes seen in BDNF conditional mutant mice suggest that BDNF regulates the 5-HT(2A) receptor level. Here we show a direct effect of BDNF on 5-HT(2A) receptor protein levels in primary hippocampal neuronal and mature hippocampal organotypic cultures exposed to different BDNF concentrations for either 1, 3, 5 or 7 days. In vivo effects of BDNF on hippocampal 5-HT(2A) receptor levels were further corroborated in (BDNF +/-) mice with reduced BDNF levels. In primary neuronal cultures, 7 days exposure to 25 and 50ng/mL BDNF resulted in downregulation of 5-HT(2A), but not of 5-HT(1A), receptor protein levels. The BDNF-associated downregulation of 5-HT(2A) receptor levels was also observed in mature hippocampal organotypic cultures, excluding confounding effects of BDNF on immature tissue. BDNF +/- mice showed significant increased 5-HT(2A) receptor levels in hippocampus confirming the association between 5-HT(2A) receptor and BDNF levels in vivo. In conclusion, our results point to a regulatory role of BDNF on 5-HT2A receptor levels. This interaction may be an important mechanism in the role of BDNF in affective disorders emphasizing the need for further elucidating the specificity and the mechanism behind this regulation.

Gene therapy-based overexpression of endogenous seizure-suppressing molecules represents a promising treatment strategy for epilepsy. Viral vector-based overexpression of the neuropeptide galanin has been shown to effectively suppress generalized seizures in various animal models of epilepsy. However, it has not been explored whether such treatment can also prevent the epileptogenesis. Using a recombinant adeno-associated viral (rAAV) vector, we induced hippocampal galanin overexpression under the neuron specific enolase promoter in rats. Here we report that in animals with galanin overexpression, the duration of electrographic afterdischarges was shortened and initiation of convulsions was delayed at generalized seizure stages. However, the hippocampal kindling development was unchanged. Short-term plasticity of mossy fiber-cornu ammonis (CA) 3 synapses was unaltered, as assessed by paired-pulse and frequency facilitation of field excitatory postsynaptic potentials (fEPSPs) in hippocampal slices, suggesting that despite high transgene galanin expression, overall release probability of glutamate in these synapses was unaffected. These data indicate that hippocampal rAAV-based galanin overexpression is capable of mediating anticonvulsant effects by lowering the seizure susceptibility once generalized seizures are induced, but does not seem to affect kindling development or presynaptic short-term plasticity in mossy fibers.