Slices Of Human Neocortex Research Articles

Differential contribution of THIK-1 K+ channels and P2X7 receptors to ATP-mediated neuroinflammation by human microglia

Neuroinflammation is highly influenced by microglia, particularly through activation of the NLRP3 inflammasome and subsequent release of IL-1β. Extracellular ATP is a strong activator of NLRP3 by inducing K+ efflux as a key signaling event, suggesting that K+-permeable ion channels could have high therapeutic potential. In microglia, these include ATP-gated THIK-1 K+ channels and P2X7 receptors, but their interactions and potential therapeutic role in the human brain are unknown. Using a novel specific inhibitor of THIK-1 in combination with patch-clamp electrophysiology in slices of human neocortex, we found that THIK-1 generated the main tonic K+ conductance in microglia that sets the resting membrane potential. Extracellular ATP stimulated K+ efflux in a concentration-dependent manner only via P2X7 and metabotropic potentiation of THIK-1. We further demonstrated that activation of P2X7 was mandatory for ATP-evoked IL-1β release, which was strongly suppressed by blocking THIK-1. Surprisingly, THIK-1 contributed only marginally to the total K+ conductance in the presence of ATP, which was dominated by P2X7. This suggests a previously unknown, K+-independent mechanism of THIK-1 for NLRP3 activation. Nuclear sequencing revealed almost selective expression of THIK-1 in human brain microglia, while P2X7 had a much broader expression. Thus, inhibition of THIK-1 could be an effective and, in contrast to P2X7, microglia-specific therapeutic strategy to contain neuroinflammation.Graphical

δ1-OPIOID receptor-mediated controlofacetylcholine (ACh) release in human neocortex slices

In slices of human neocortex, prelabelled with [3H]-choline, the release of [3H]-acetylcholine reflects the evoked release of endogenous acetylcholine whichwaselicited by the same electrical stimulation paradigm. [3H]-Acetylcholine releasewasdepressed by the δ-opioid receptor agonist d-Pen2-d-Pen5-enkephalin. When the nerve endings were depolarized byelevatingextracellular potassium the evoked [3H]-acetylcholine release was similarlydepressed by d-Pen2-d-Pen5-enkephalin in theabsence, but notin the presence, of tetrodotoxin which blocks action potential propagation.Therefore, the δ-opioid receptor inhibiting [3H]-acetylcholine release should notbe located tocholinergic nerve terminals, but rather to interneurons. The somatostatin2receptorpartial agonist octreotide per se did not influence action potential-evoked [3H]-acetylcholine release, but prevented the inhibition of release of [3H]-acetylcholine byd-Pen2-d-Pen5-enkephalin.Similarly, the δ1-opioid receptor antagonist 7-benzylidenenaltrexon perse did notinfluence [3H]-acetylcholine release, but prevented of the inhibition ofrelease by d-Pen2-d-Pen5-enkephalin.From the presentfindings we conclude : (1) The evoked release of [3H]-acetylcholine from humanneocortex slices reflects the release of endogenous acetylcholine.(2) It is inhibited in an indirectmanner by opioid receptors of the δ1-subtype,which (3) are not localized oncholinergic axon terminals but on soma and dendrites ofsomatostatin-containing interneurons,where they inhibit somatostatin release. (4) Theseinterneurons innervate cholinergic nerveendings in the human neocortex and appear to facilitateacetylcholine release via somatostatin2 receptors.

Opioid receptor-mediated control of acetylcholine release in human neocortex tissue.

The effects of various opioid receptor agonists and antagonists on evoked acetylcholine release were studied in slices of human neocortex prelabelled with [3H]-choline, superfused and depolarized electrically (2 min, 3 Hz, 2 ms, 24 mA) or by K+ (20 mM). The delta-opioid receptor agonist DPDPE and the kappa-opioid receptor agonist U50488 reduced the evoked [3H]-overflow (acetylcholine release) in a concentration-dependent fashion; the delta-opioid receptor antagonist naltrindole and the kappa-opioid receptor antagonist norbinaltorphimine, respectively, antagonized these effects. Application of the mu-opioid receptor agonist DAGO also resulted in an inhibition of acetylcholine release; however, both delta- and kappa-opioid receptor antagonists were able to block this effect. The mu-opioid receptor agonists morphine and (+)-nortilidine had no effect. These results indicate that acetylcholine release in human neocortex is inhibited through delta- and kappa-opioid receptors, but not through mu-opioid receptors. Acetylcholine release was significantly increased by the delta-opioid receptor antagonist naltrindole in the presence of a mixture of peptidase inhibitors providing evidence for a delta-opioid receptor-mediated inhibition of acetylcholine release by endogenous enkephalin. K(+)-evoked acetylcholine release in the presence of TTX was inhibited by U50488, but not by DPDPE, suggesting the presence of kappa-opioid receptors on cholinergic terminals and the localization of delta-receptors on cortical interneurons. Therefore, the potent effect of DPDPE on acetylcholine release is likely to be indirect, by modulation of intrinsic cortical neurons. These interneurons probably do not use GABA as neurotransmitter since both GABAA and GABAB receptor agonists (muscimol and baclofen, respectively) were without effect on acetylcholine release.

Characterization of the 5-hydroxytryptamine receptor modulating the release of 5-[3H]hydroxytryptamine in slices of the human neocortex.

In the rat brain, the presynaptic 5-hydroxytryptamine (5-HT) autoreceptors located on 5-HT terminals correspond to the 5-HT1B subtype. The presence of a 5-HT receptor probably located on 5-HT nerve endings and modulating transmitter release in the human neocortex has been reported, but its detailed pharmacological characterization is not yet available. On the other hand, receptor binding and autoradiographic results indicate that the 5-HT1B receptor subtype is not present in the human brain. We, therefore, studied the modulation of the electrically evoked release of [3H]5-HT by various 5-HT receptor agonists and antagonists in preloaded slices of human neocortex obtained from 18 patients undergoing neurosurgery. The nonselective 5-HT1A/1B/1D receptor agonist 5-carboxamidotryptamine produced a potent inhibition (70% at 0.03 microM) of the electrically evoked release of [3H]5-HT which was blocked by 5-HT receptor antagonists with the following relative order of potency: methiothepin greater than metergoline = methysergide greater than propranolol. The selective 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin at 0.1 microM did not modify the electrically evoked release of [3H]5-HT. The 5-HT1A/1B receptor agonist RU 24969 was 10 times more potent at inhibiting [3H]5-HT overflow in the rat frontal cortex than in the human neocortex. The potent 5-HT1B receptor antagonist cyanopinodolol did not modify the 5-carboxamidotryptamine-induced inhibition of the electrically evoked release of [3H]5-HT in slices of the human neocortex, but produced by itself a small inhibition of [3H]5-HT overflow.(ABSTRACT TRUNCATED AT 250 WORDS)

M-current in human neocortical neurones

Intracellular recordings were made in slices of human neocortex that had been surgically excised from patients in order to remove deep lying brain tumours. In more than half the neurones studied under voltage-clamp (n = 9), a non-inactivating K +-current was detected that was turned on at potentials positive to around −60 mV. This conductance persisted when Ca 2+-flux into neurones was blocked with Cd 2+ and it was suppressed by muscarine (20 μM). The slow kinetics and voltage sensitivity of this K + conductance, together with its muscarinic suppression, identified it as the M-current (I M). In addition to I M, evidence for the existence of Ca 2+ and Ca 2+-activated conductances was obtained in human neurones. These results validate the extrapolation of animal-derived data and identify I M as a target for cholinergic modulation in the human.