Mario Raggenbass

A Novel Positive Allosteric Modulator of the α7 Neuronal Nicotinic Acetylcholine Receptor: In Vitro and In Vivo Characterization

Several lines of evidence suggest a link between the α7 neuronal nicotinic acetylcholine receptor (nAChR) and brain disorders including schizophrenia, Alzheimers disease, and traumatic brain injury. The present work describes a novel molecule, 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596), which acts as a powerful positive allosteric modulator of the α7 nAChR. Discovered in a high-throughput screen, PNU-120596 increased agonist-evoked calcium flux mediated by an engineered variant of the human α7 nAChR. Electrophysiology studies confirmed that PNU-120596 increased peak agonist-evoked currents mediated by wild-type receptors and also demonstrated a pronounced prolongation of the evoked response in the continued presence of agonist. In contrast, PNU-120596 produced no detectable change in currents mediated by α4β2, α3β4, and α9α10 nAChRs. PNU-120596 increased the channel mean open time of α7 nAChRs but had no effect on ion selectivity and relatively little, if any, effect on unitary conductance. When applied to acute hippocampal slices, PNU-120596 increased the frequency of ACh-evoked GABAergic postsynaptic currents measured in pyramidal neurons; this effect was suppressed by TTX, suggesting that PNU-120596 modulated the function of α7 nAChRs located on the somatodendritic membrane of hippocampal interneurons. Accordingly, PNU-120596 greatly enhanced the ACh-evoked inward currents in these interneurons. Systemic administration of PNU-120596 to rats improved the auditory gating deficit caused by amphetamine, a model proposed to reflect a circuit level disturbance associated with schizophrenia. Together, these results suggest that PNU-120596 represents a new class of molecule that enhances α7 nAChR function and thus has the potential to treat psychiatric and neurological disorders.

A three-dimensional multi-electrode array for multi-site stimulation and recording in acute brain slices

Several multi-electrode array devices integrating planar metal electrodes were designed in the past 30 years for extracellular stimulation and recording from cultured neuronal cells and organotypic brain slices. However, these devices are not well suited for recordings from acute brain slice preparations due to a dead cell layer at the tissue slice border that appears during the cutting procedure. To overcome this problem, we propose the use of protruding 3D electrodes, i.e. tip-shaped electrodes, allowing tissue penetration in order to get closer to living neurons in the tissue slice. In this paper, we describe the design and fabrication of planar and 3D protruding multi-electrode arrays. The electrical differences between planar and 3D protruding electrode configuration were simulated and verified experimentally. Finally, a comparison between the planar and 3D protruding electrode configuration was realized by stimulation and recording from acute rat hippocampus slices. The results show that larger signal amplitudes in the millivolt range can be obtained with the 3D electrode devices. Spikes corresponding to single cell activity could be monitored in the hippocampus CA3 and CA1 region using 3D electrodes.

Comparative distribution of nicotinic receptor subtypes during development, adulthood and aging: an autoradiographic study in the rat brain

The distribution in the rat brain of high affinity nicotinic heteromeric acetylcholine receptors and of low affinity nicotinic, alpha7-containing, homomeric receptors was studied using in vitro light microscopic autoradiography. As ligands, we used [3H]epibatidine, or [125I]epibatidine, and [125I]alpha-bungarotoxin, respectively. In adult animals, the two types of binding sites were widely distributed in many different brain structures, including the brainstem, cerebellum, mesencephalic structures, limbic system and cortex, but their anatomical distribution differed markedly. Only in rare instances could a co-localization be observed, for example in the superficial layer of the superior colliculus. In developing animals, both types of labeling were strongly expressed during embryonic and postnatal phases. Their distributions were qualitatively similar to those observed in adult animals, with a few noticeable exceptions in the cerebral cortex, hippocampus and brain stem. In aging animals, neither the distribution nor the density of nicotinic binding sites was significantly altered. Our conclusions are the following. (a) There is little overlap in the distribution of heteromeric and alpha7-containing homomeric nicotinic receptors in the rat brain. (b) The abundance of neuronal nicotinic receptors during embryonic and postnatal development suggests that they may play a role in the establishment of neuronal connectivity. (c) The expression of neuronal nicotinic receptors is unaltered in middle aged animals, suggesting that in the rat these receptors do not play any major role in aging process.

Alpha7 neuronal nicotinic acetylcholine receptors are negatively regulated by tyrosine phosphorylation and Src-family kinases.

Nicotine, a component of tobacco, is highly addictive but possesses beneficial properties such as cognitive improvements and memory maintenance. Involved in these processes is the neuronal nicotinic acetylcholine receptor (nAChR) α7, whose activation triggers depolarization, intracellular signaling cascades, and synaptic plasticity underlying addiction and cognition. It is therefore important to investigate intracellular mechanisms by which a cell regulates α7 nAChR activity. We have examined the role of phosphorylation by combining molecular biology, biochemistry, and electrophysiology in SH-SY5Y neuroblastoma cells, Xenopus oocytes, rat hippocampal interneurons, and neurons from the supraoptic nucleus, and we found tyrosine phosphorylation of α7 nAChRs. Tyrosine kinase inhibition by genistein decreased α7 nAChR phosphorylation but strongly increased acetylcholine-evoked currents, whereas tyrosine phosphatase inhibition by pervanadate produced opposite effects. Src-family kinases (SFKs) directly interacted with the cytoplasmic loop of α7 nAChRs and phosphorylated the receptors at the plasma membrane. SFK inhibition by PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or SU6656 (2,3-dihydro-N,N-dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1H-indole-5-sulfonamide) increased α7 nAChR-mediated responses, whereas expression of active Src reduced α7 nAChR activity. Mutant α7 nAChRs lacking cytoplasmic loop tyrosine residues because of alanine replacement of Tyr-386 and Tyr-442 were more active than wild-type receptors and insensitive to kinase or phosphatase inhibition. Because the amount of surface α7 receptors was not affected by kinase or phosphatase inhibitors, these data show that functional properties of α7 nAChRs depend on the tyrosine phosphorylation status of the receptor and are the result of a balance between SFKs and tyrosine phosphatases. These findings reveal novel regulatory mechanisms that may help to understand nicotinic receptor-dependent plasticity, addiction, and pathology.

Early appearance and transient expression of vasopressin receptors in the brain of rat fetus and infant. An autoradiographical and electrophysiological study

The development of vasopressin (AVP) receptors in the rat brain, spinal cord and pituitary gland was studied by in vitro light microscopic autoradiography. AVP binding sites were labeled using [3H]AVP in tissue sections from animals aged between embryonic day 12 (E12) and postnatal day 90 (PN90); the binding of [3H]AVP to oxytocin receptors was prevented by adding in the incubation medium a highly selective oxytocin agonist. Specific binding was first detected at E16 in the ventral pontine reticular formation. Many other brain areas were progressively labeled between E18 and PN5. The distribution of binding sites observed at PN5 remained unchanged until the beginning of the third postnatal week. Thereafter binding was markedly reduced or even disappeared in several areas, in particular in the facial nucleus. The adult distribution of AVP binding sites was established at the time of weaning. The properties of transient AVP binding sites in the facial nucleus were studied both by autoradiography and by electrophysiology. Non-radioactive AVP displaced [3H]AVP binding in this nucleus as efficiently as it did in the lateral septum of the adult. Single-unit extracellular recordings showed that AVP can excite facial motoneurones by interacting with receptors which are pharmacologically indistinguishable from V1 (vasopressor) type. Thus, AVP binding sites transiently expressed in the brain of fetal and infant rat probably represent functional neuronal receptors, having the same ligand selectivity and affinity than AVP binding sites present in the adult. This suggests that AVP acts not only as a neuropeptide in the adult brain but may play a significant role during maturation of the central nervous system.

A role of central oxytocin in autonomic functions: its action in the motor nucleus of the vagus nerve

Neurones located in the dorsal motor nucleus of the vagus nerve were shown, in slices from the rat brainstem, to respond to oxytocin by a concentration-dependent increase in rate of firing. A newly available oxytocin antagonist suppressed the excitatory effect of oxytocin on single neurones; this antagonism was partially reversible. Further evidence that neurones located in the dorsal motor nucleus of the vagus nerve possess oxytocin receptors was obtained from in vitro light microscopical autoradiography using [125I]-labelled oxytocin antagonist. In conjunction with data by others which showed that oxytocin antagonist microinjected into the dorsal motor nucleus of the vagus nerve blocks gastric and cardiac effects caused by stimulation of the hypothalamic paraventricular nucleus, our results suggest a role for central oxytocin in autonomic efferent activity.

Presence of functional neuronal nicotinic acetylcholine receptors in brainstem motoneurons of the rat

In mammals, nicotinic acetylcholine receptors (nAChRs) play a crucial role in motor control. Muscle‐type nAChRs mediate synaptic excitation of skeletal muscle by motoneurons, and nAChRs are present on Renshaw cells, where they produce recurrent inhibition of spinal motoneurons. We asked whether nAChRs are also present in motoneurons. Whole‐cell recordings were performed on various motor nuclei in brainstem slices of young rats. Neurons were visualized using infrared (IR) videomicroscopy. Acetylcholine (ACh) or the nicotinic agonist, epibatidine, were delivered by pressure microinjection. Facial (VII), hypoglossal (XII) and vagal (X) motoneurons responded to ACh by generating a fast inward current. In VII motoneurons, the ACh effect was mimicked by epibatidine, and nicotine induced a slow inward current and desensitized the ACh‐evoked current. In VII and XII motoneurons, the ACh‐evoked current was blocked by the nicotinic antagonist dihydro‐β‐erythroidine (DHβE), but was unaffected by methyllycaconitine (MLA), an α7‐specific antagonist. By contrast, the ACh‐induced current in X motoneurons was sensitive to MLA. Current–voltage relationships indicated that the currents mediated by either α7‐containing (X) or non‐α7‐containing (VII, XII) nAChRs displayed inward rectification. In accordance with the electrophysiological data, autoradiography revealed that VII, X and XII nuclei of young rats contained binding sites for [3H]epibatidine; binding sites for [125I]α‐bungarotoxin, a selective ligand of α7‐containing nAChRs, were present in X nucleus but were almost undetectable in VII and XII nuclei. Thus, brainstem motoneurons of young rats possess functional nAChRs. They could promote fast synaptic coupling between motoneurons, and thus play a role in somatic and visceral motor functions.

Direct excitatory action of vasopressin in the lateral septum of the rat brain

The electrophysiological action of arginine vasopressin on neurones in the lateral septum of the rat brain was studied using extracellular recordings and the in vitro brain slice technique. Of 177 neurones tested in the presence of vasopressin at 1-1000 nM, 77 (about 44%) responded by a reversible increase in firing rate, 12 (about 7%) were inhibited and the remaining were not affected. The lowest peptide concentration effective in exciting septal neurones ranged between 1 and 50 nM, and the magnitude of the excitatory effect was concentration dependent. At high vasopressin concentrations, the peptide-induced excitation was often followed by a transient pause in firing; this was probably due to action potential inactivation, brought about by the vasopressin-induced neuronal membrane depolarization. The excitatory effect of vasopressin was postsynaptic, since it was not abolished following synaptic blockade in a low calcium-high magnesium perifusion solution. A comparison of the effects of vasopressin and oxytocin suggested that most of the septal vasopressin-sensitive neurones are endowed with vasopressin receptors, whereas a minority of them bear oxytocin receptors.

Oxytocin receptor agonists enhance inhibitory synaptic transmission in the rat hippocampus by activating interneurons in stratum pyramidale

Oxytocin probably plays a role as a neurotransmitter/neuromodulator in the hippocampus of the rat. Oxytocin binding sites are present in the subiculum and CA1 region and oxytocin can excite a class of CA1 nonpyramidal neurons. In the present work we characterized the effect of oxytocin on hippocampal synaptic transmission. Whole‐cell recordings were obtained from pyramidal neurons, in conditions of nearly symmetrical chloride concentrations. The selective oxytocin receptor agonist, [Thr4,Gly7]‐oxytocin (TGOT), caused an increase in the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) in virtually all neurons. These peptide‐enhanced IPSCs were blocked by bicuculline, but not by strychnine, and reversed near 0 mV, indicating that they were mediated by γ‐aminobutyric acid (GABA)A receptors. On average, TGOT caused a nearly threefold increase in the frequency and almost a doubling in the amplitude of spontaneous IPSCs. TGOT did not influence the frequency and the amplitude of miniature IPSCs or spontaneous excitatory postsynaptic currents (EPSCs), and had no effect on evoked IPSCs. The peptide did not affect the basic membrane properties of pyramidal neurons or their GABA sensitivity. Thus, TGOT facilitated inhibitory transmission by exerting an excitatory action on the soma and/or dendrites of GABAergic interneurons. Extracellular recordings were performed in interneurons located in various hippocampal strata. Their sensitivity to TGOT was compared to that of substance P (SP). Interneurons in stratum pyramidale were excited both by TGOT and by SP. By contrast, stratum radiatum interneurons responded to SP but not to TGOT. In stratum oriens, half of the interneurons responded to SP, but only a minority to TGOT. Thus, oxytocin‐responsive interneurons appear to be preferentially located in close vicinity of pyramidal neurons.

Mechanism of action of oxytocin in rat vagal neurones: induction of a sustained sodium-dependent current.

1. The mechanism of action of oxytocin on vagal neurones of the rat was studied using single‐electrode voltage‐clamp recordings from brainstem slices. The ionic basis of the oxytocin‐induced current was examined by changing the composition of the perfusion solution and by making use of channel blockers. 2. In neurones clamped at or near their resting potential, oxytocin generated a sustained, TTX‐insensitive inward current whose peak amplitude was concentration related. This current was detectable at 10 nM, was half‐maximal at about 100 nM and was maximal at micromolar concentrations of peptide. 3. The oxytocin current was inward over membrane potentials ranging from ‐110 to ‐20 mV and was voltage dependent, since it increased in magnitude as the membrane was depolarized from the resting potential toward less negative potentials. 4. Partial replacement of extracellular sodium by equimolar N‐methyl‐D‐glucamine reversibly attenuated or suppressed the oxytocin current. By contrast, substituting part of extracellular chloride or blocking calcium currents did not modify it. Increasing the transmembrane potassium gradient was also without effect and none of the potassium channel blockers TEA, 4‐amino pyridine (4‐AP), apamin, caesium or barium affected the oxytocin current. This current is thus at least in part carried by sodium. 5. The activation of the oxytocin current as a function of the membrane potential could be quantitatively simulated using a Boltzmann equation, suggesting that oxytocin acts by inducing the opening of a voltage‐dependent channel which can exist in either of two states, open or closed. 6. Lowering the extracellular calcium concentration from 2 to 0.1 mM, while keeping the magnesium concentration constant at 1 mM, enhanced the response to oxytocin. This low calcium‐induced potentiation of the oxytocin current was 1.4‐3‐fold and was reversible. 7. We conclude that oxytocin increases the excitability of vagal neurones by generating a persistent, voltage‐gated current which is sodium dependent, is insensitive to TTX and is modulated by divalent cations.