A fourth type of opioid receptor, termed ORL1, has been cloned and nociceptin (also known as orphanin FQ) has been identified as an endogenous ligand at this receptor. We examined whether nociceptin affects the release of noradrenaline in the brain. For this purpose, cerebral cortex slices from the mouse, rat or guinea-pig were preincubated with [3H]noradrenaline and then superfused with medium containing desipramine and rauwolscine. Tritium overflow was evoked electrically (0.3 Hz) or by introduction of Ca2+ 1.3 mM into Ca2+-free K+-rich (15 mM) medium. Nociceptin 1 microM reduced the electrically evoked tritium overflow from mouse, rat and guinea-pig brain cortex slices by 80, 71 and 36%, respectively. Naloxone 10 microM did not change the effect of nociceptin. All subsequent experiments were performed on mouse brain cortex slices and in the presence of naloxone 10 microM. The concentration-response curve of nociceptin (maximum inhibition by 80%, pEC50 7.5) was shifted to the right by the non-selective ORL1 receptor antagonist naloxone benzoylhydrazone and the selective ORL1 receptor antagonist [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)NH2 (pA2 6.6 and 7.2, respectively). Naloxone benzoylhydrazone did not affect the evoked overflow by itself whereas [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 caused an inhibition by maximally 35% (pEC50 7.0; intrinsic activity alpha 0.45). The inhibitory effect of [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)NH2 was counteracted by naloxone benzoylhydrazone. Nociceptin also reduced the Ca2+-evoked tritium overflow in mouse brain cortex slices superfused in the presence of tetrodotoxin. This effect was also antagonized by naloxone benzoylhydrazone, which, by itself, did not affect the evoked tritium overflow. In conclusion, nociceptin inhibits noradrenaline release more markedly in the mouse than in the rat or guinea-pig brain cortex. The effect of nociceptin in the mouse brain cortex involves ORL1 receptors, which are located presynaptically on noradrenergic neurones.

Neural network formation is a complex process involving axon outgrowth and guidance. Axon guidance is facilitated by structural and molecular cues from the surrounding microenvironment. Micro-fabrication techniques can be employed to produce microfluidic chips with a highly controlled microenvironment for neural cells enabling longitudinal studies of complex processes associated with network formation. In this work, we demonstrate a novel open microfluidic chip design that encompasses a freely variable number of nodes interconnected by axon-permissible tunnels, enabling structuring of multi-nodal neural networks in vitro. The chip employs a partially open design to allow high level of control and reproducibility of cell seeding, while reducing shear stress on the cells. We show that by culturing dorsal root ganglion cells (DRGs) in our microfluidic chip, we were able to structure a neural network in vitro. These neurons were compartmentalized within six nodes interconnected through axon growth tunnels. Furthermore, we demonstrate the additional benefit of open top design by establishing a 3D neural culture in matrigel and a neuronal aggregate 3D culture within the chips. In conclusion, our results demonstrate a novel microfluidic chip design applicable to structuring complex neural networks in vitro, thus providing a versatile, highly relevant platform for the study of neural network dynamics applicable to developmental and regenerative neuroscience.

Guinea-pig hippocampal slices preincubated with [3H]noradrenaline were superfused with medium containing desipramine and rauwolscine and rat striatal slices preincubated with [3H]dopamine were superfused with medium containing nomifensine; the effect of cannabinoid receptor ligands on tritium overflow stimulated by NMDA or kainate was examined. Furthermore, the affinity of the drugs for cannabinoid CB1 receptors was determined in rat brain cortex membranes using [3H]SR 141716. In guinea-pig hippocampal slices preincubated with [3H]noradrenaline, tritium overflow stimulated by NMDA 100 microM and 1000 microM and by kainate 1000 microM was inhibited by the cannabinoid receptor agonists CP-55,940 and/or WIN 55,212-2. The CB1 receptor antagonist SR 141716 increased the NMDA (1000 microM)-stimulated tritium overflow but did not affect tritium overflow stimulated by NMDA 100 microM or kainate 1000 microM. The inhibitory effect of WIN 55,212-2 on the NMDA (100 microM)- and kainate (1000 microM)-evoked tritium overflow was antagonized by SR 141716. In rat striatal slices preincubated with [3H]dopamine, WIN 55,212-2 inhibited the NMDA (1000 microM)-stimulated tritium overflow. SR 141716, which, by itself, did not affect tritium overflow, counteracted the inhibitory effect of WIN 55,212-2. [3H]SR 141716 binding to rat cortical membranes was inhibited by SR 141716, CP-55,940 and WIN 55,212-2 (pKi 8.53, 7.34 and 5.93, respectively) but not affected by desipramine, rauwolscine and nomifensine (pKi < 5). In conclusion, activation of CB1 receptors inhibits the NMDA- and kainate-stimulated noradrenaline release in guinea-pig hippocampus and the NMDA-stimulated dopamine release in rat striatum. The explanation for the facilitatory effect of SR 141716 might be that it acts as an inverse agonist at CB1 receptors or that these receptors are activated by endogenous cannabinoids.

In conclusion, nociceptin inhibits noradrenaline release in the mouse cortex via ORL 1 receptors, which interact with presynaptic a 2 -autoreceptors on noradrenergic neurones. The e ect of nociceptin does not desensitize nor does it involve NO, prostanoids or adenosine. Nociceptin also attenuates noradrenaline release from several subcortical regions and serotonin release from cortical slices by a naloxone benzoylhydrazone-sensitive mechanism.

The human brain is a remarkable computing machine, i.e. vastly parallel, self-organizing, robust, and energy efficient. To gain a better understanding into how the brain works, a cyborg (cybernetic organism, a combination of machine and living tissue) is currently being made in an interdisciplinary effort, known as the Cyborg project. In this paper we describe how living cultures of neurons (biological neural networks) are successfully grown in-vitro over Micro-Electrode Arrays (MEAs), which allow them to be interfaced to a robotic body through electrical stimulation and neural recordings. Furthermore, we describe the bio-and nano-technological procedures utilized for the culture of such dissociated neural networks and the interface software and hardware framework used for creating a closed-loop hybrid neuro-system. A Reservoir Computing (RC) approach is used to harness the computational power of the neuronal culture.