Andrea Nistri

Desensitization of nicotinic ACh receptors: shaping cholinergic signaling

Nicotinic ACh receptors (nAChRs) can undergo desensitization, a reversible reduction in response during sustained agonist application. Although the mechanism of desensitization remains incompletely understood, recent investigations have elucidated new properties underlying desensitization, indicating that it might be important to control synaptic efficacy, responses to cholinergic agents, and certain nAChR-related disease states. Thus, studying how different nAChR subunits contribute to desensitization might help to explain variations in responsiveness to drugs, and might thus improve their therapeutic applications. Agonist-specific desensitization, desensitization arising from resting receptors, natural mutations dramatically altering desensitization, and the possibility that recovery from desensitization is an important process for modulating receptor function, together provide a new framework for considering desensitization as a target to shape cholinergic signaling.

Exocytotic release of ATP from cultured astrocytes

Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca2+]i also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca2+]i attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca2+-dependent. This was confirmed by patch-clamp studies on HEK-293T cells transfected with P2X3 receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca2+. Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared.

Delayed Upregulation of ATP P2X3 Receptors of Trigeminal Sensory Neurons by Calcitonin Gene-Related Peptide

Recent evidence indicates a key role for the neuropeptide calcitonin gene-related peptide (CGRP) in migraine pain, as demonstrated by the strong analgesic action of CGRP receptor antagonists, although the mechanisms of this effect remain unclear. Most trigeminal nociceptive neurons releasing CGRP also express ATP-activated purinergic P2X3 receptors to transduce pain. To understand whether the CGRP action involves P2X3 receptor modulation, the model of trigeminal nociceptive neurons in culture was used to examine the long-term action of this peptide. Although 79% of CGRP-binding neurons expressed P2X3 receptors, acute application of CGRP did not change P2X3 receptor function. Nevertheless, after 1 h of CGRP treatment, strong enhancement of the amplitude of P2X3 receptor currents was observed together with accelerated recovery from desensitization. Receptor upregulation persisted up to 10 h (despite CGRP washout), was accompanied by increased P2X3 gene transcription, and was fully prevented by the CGRP antagonist CGRP8–37. Surface biotinylation showed CGRP augmented P2X3 receptor expression, consistent with confocal microscopy data indicating enhanced P2X3 immunoreactivity beneath the neuronal membrane. These results suggest that CGRP stimulated trafficking of P2X3 receptors to the cell-surface membrane. Using pharmacological tools, we demonstrated that this effect of CGRP was dependent on protein kinase A and PKC activation and was prevented by the trafficking inhibitor brefeldin A. Capsaicin-sensitive TRPV1 vanilloid receptors were not upregulated. The present data demonstrate a new form of selective, slow upregulation of nociceptive P2X3 receptors on trigeminal neurons by CGRP. This mechanism might contribute to pain sensitization and represents a model of neuronal plasticity in response to a migraine mediator.

Riluzole blocks persistent Na+ and Ca2+ currents and modulates release of glutamate via presynaptic NMDA receptors on neonatal rat hypoglossal motoneurons in vitro

The neuroprotective agent riluzole is used for the symptomatic treatment of motoneuron disease, which strongly affects the brainstem nucleus hypoglossus. The mechanism of action of riluzole was investigated using, as a model, patch‐clamp recording from hypoglossal motoneurons of the neonatal rat brainstem slice preparation. In the presence of riluzole (10 µm), theta‐rhythm oscillations evoked by nicotine continued even though the persistent inward current (comprising sodium and calcium components) was halved, but they disappeared when the high frequency of spontaneous glutamatergic currents waned. Riluzole fully inhibited the persistent sodium current and partly depressed a tetrodotoxin (TTX)‐insensitive slow current antagonized by Mn2+ or Cd2+. Repetitive firing was inhibited by riluzole without changing single action potentials. In the presence of TTX, riluzole depressed miniature glutamatergic currents occurring at high rate. Synaptic transmission with low release probability became sensitive to riluzole if release was stimulated by high potassium solution. Miniature current frequency was depressed by the N‐methyl‐d‐aspartic acid (NMDA) receptor antagonist d‐amino‐phosphonovaleriate (50 µm), which fully occluded the action of riluzole. As riluzole is a protein kinase C (PKC) inhibitor, the PKC antagonist chelerythrine (2.5 µm) mimicked the effect of riluzole and prevented it. In summary, riluzole blocked the persistent sodium current fully, and the calcium one partly, plus it decreased glutamatergic transmission probably via inhibition of PKC that regulated presynaptic NMDA receptors having a facilitatory effect on glutamate release. Controlling NMDA receptor function and, thus, excitatory transmitter release via modulation of PKC suggests a novel potential target to contrast glutamate excitotoxicity in this motor nucleus.

Molecular Mechanisms of Sensitization of Pain-transducing P2X3 Receptors by the Migraine Mediators CGRP and NGF

Migraine headache originates from the stimulation of nerve terminals of trigeminal ganglion neurons that innervate meninges. Characteristic features of migraine pain are not only its delayed onset but also its persistent duration. Current theories propose that endogenous substances released during a migraine attack (the neuropeptide calcitonin gene-related peptide [CGRP] and the neurotrophin nerve growth factor [NGF]) sensitize trigeminal neurons to transmit nociceptive signals to the brainstem, though the mechanisms remain poorly understood. Recent studies indicate that acute, long-lasting sensitization of trigeminal nociceptive neurons occurs via distinct processes involving enhanced expression and function of adenosine triphosphate (ATP)-gated P2X3 receptors known to play a role in chronic pain. In particular, on cultured trigeminal neurons, CGRP (via protein kinase A-dependent signaling) induces a slowly developing upregulation of the ionic currents mediated by P2X3 receptors by enhancing receptor trafficking to the neuronal membrane and activating their gene transcription. Such upregulated receptors acquire the ability to respond repeatedly to extracellular ATP, thus enabling long-lasting signaling of painful stimuli. In contrast, NGF induces rapid, reversible upregulation of P2X3 receptor function via protein kinase C phosphorylation, an effect counteracted by anti-NGF antibodies. The diverse intracellular signaling pathways used by CGRP and NGF show that the sensitization of P2X3 receptor function persists if the action of only one of these migraine mediators is blocked. These findings imply that inhibiting a migraine attack might be most efficient by a combinatorial approach. The different time domains of P2X3 receptor modulation by NGF and CGRP suggest that the therapeutic efficacy of novel antimigraine drugs depends on the time of administration.

Expression and dendritic mRNA localization of GABAC receptor ρ1 and ρ2 subunits in developing rat brain and spinal cord

The cellular distribution of GABAC receptor ρ1 and ρ2 subunits in the rat central nervous system remains controversial. We investigated how these subunits were distributed in cerebellum, hippocampus and spinal cord at postnatal day 1, 7 or in adult life. We found that in the adult cerebellum ρ1 and ρ2 mRNAs were expressed in Purkinje cells and basket‐like cells only. In the hippocampus both subunits were expressed throughout the CA1 pyramidal layer, dentate gyrus and scattered interneurons with maximum staining intensity at P7. In the adult hippocampus in situ staining was predominantly found on interneurons. GABAC antibody labelling in P7 and adult hippocampus was largely overlapping with the in situ staining. Western blot analysis showed GABAC receptor in retina, ovary and testis. In the spinal cord the ρ2 signal was consistently stronger than ρ1 with overlapping expression patterns. At P1, the most intensely labelled cells were the motoneurons while on P7 and adult sections, interneurons and motoneurons were likewise labelled. On spinal neurons both ρ1 and ρ2 mRNAs showed somatodendritic localization, extending out for >100 µm with punctate appearance especially in adult cells. A similar spinal distribution pattern was provided with polyclonal antibody labelling, suggesting close correspondence between mRNA and protein compartmentalization. Electrophysiological experiments indicated that P1 spinal motoneurons did possess functional GABAC receptors even though GABAC receptors played little role in evoked synaptic transmission. Our results suggest a pattern of ρ1 and ρ2 subunit distribution more widespread than hitherto suspected with strong developmental regulation of subunit occurrence.

Alternating rhythmic activity induced by dorsal root stimulation in the neonatal rat spinal cord in vitro

1 Electrical stimuli applied to a single dorsal root (DR) of the neonatal rat spinal cord in vitro were used to test the possibility that the central pattern generator responsible for locomotion could be activated by synaptic inputs. 2 Brief pulse trains evoked oscillatory patterns recorded from pairs of lumbar ventral roots. These patterns alternated rhythmically between the left and right sides and between predominantly flexor and extensor motoneuronal pools on the same side, thus displaying properties similar to the fictive locomotor pattern elicited by bath‐applied excitatory transmitter agonists like NMDA and serotonin. 3 Usually pulse trains rather than single pulses were necessary to induce these patterns, the period of which was independent of stimulation frequency (1‐10 Hz) and only moderately dependent on stimulus intensity. 4 Patterns reached a steady rhythm during the stimulus train, lasted for 50 ± 20 s with gradual period lengthening and finally ceased. 5 Since DR stimuli activated the central pattern generator for locomotion in the rat isolated spinal cord, it is suggested that sensory inputs from the periphery can reach the spinal locomotor network and trigger its operation.

Contribution of NMDA and non-NMDA glutamate receptors to locomotor pattern generation in the neonatal rat spinal cord.

The motor programme executed by the spinal cord to generate locomotion involves glutamate–mediated excitatory synaptic transmission. Using the neonatal rat spinal cord as an in vitro model in which the locomotor pattern was evoked by 5–hydroxytryptamine (5–HT), we investigated the role of N–methyl–D–aspartate (NMDA) and non–NMDA glutamate receptors in the generation of locomotor patterns recorded electrophysiologically from pairs of ventral roots. In a control solution, 5–HT (2.5–30 μM) elicited persistent alternating activity in left and right lumbar ventral roots. Increasing 5–HT concentration within this range resulted in increased cycle frequency (on average from 8 to 20 cycles min−1). In the presence of NMDA receptor antagonism, persistent alternating activity was still observed as long as 5–HT doses were increased (range 20–40 μM), even if locomotor pattern frequency was lower than in the control solution. In the presence of non–NMDA receptor antagonism, stable locomotor activity (with lower cycle frequency) was also elicited by 5–HT, albeit with doses larger than in the control solution (15–40 μM). When NMDA and non–NMDA receptors were simultaneously blocked, 5–HT (5–120 μM) always failed to elicit locomotor activity. These data show that the operation of one glutamate receptor class was sufficient to express locomotor activity. As locomotor activity developed at a lower frequency than in the control solution after pharmacological block of either NMDA or non–NMDA receptors, it is suggested that both receptor classes were involved in locomotor pattern generation.

Molecular biology and electrophysiology of neuronal nicotinic receptors of rat chromaffin cells.

Neuronal nicotinic acetylcholine receptors of chromaffin cells in the adrenal medulla are physiologically activated by acetylcholine to mediate catecholamine release into the bloodstream. The present study examined the subunit composition and functional properties of rat chromaffin cell neuronal nicotinic acetylcholine receptors using molecular biology, immunocytochemistry and whole‐cell patch‐clamp. Reverse transcription‐polymerase chain reaction analysis indicated the presence of α2, α3, α4, α5, α7, β2 and β4 transcripts (α6 and β3 could not be detected). Immunocytochemistry revealed most cells positive for α3, β2, β4 and α5 proteins. Few cells were immunoreactive for α2 and α4, while none was for α7. At single‐cell level, colocalization could be demonstrated for α3α5 and α4β2. Western blot analysis confirmed antibody specificity for α3, α4, α5, β2 and β4 subunits. Inward currents elicited by nicotine pulses were insensitive to α‐bungarotoxin and low doses of methyllycaconitine, demonstrating lack of functional α7 receptors. Partial block of nicotine currents was observed with either AuIB α‐conotoxin (selective against α3β4 receptors) or MII α‐conotoxin (selective against α3β2 receptors). With high concentrations of co‐applied toxins, antagonism occlusion developed, suggesting loss of subunit selectivity. Antagonism by dihydro‐β‐erythroidine summated nonlinearly with AuIB and MII inhibition, confirming heterogeneity of neuronal nicotinic acetylcholine receptor block. The present results suggest that the most frequently encountered receptors of rat chromaffin cells should comprise α3β4, α3β2 with the addition of α5 subunits. Because of the prevailing subunit composition, rat chromaffin cell neuronal nicotinic acetylcholine receptors are suitable models, particularly for the α3β4 subclasses of mammalian brain receptors recently demonstrated in discrete cerebral areas.

Neutralization of Nerve Growth Factor Induces Plasticity of ATP-Sensitive P2X3 Receptors of Nociceptive Trigeminal Ganglion Neurons

The molecular mechanisms of migraine pain are incompletely understood, although migraine mediators such as NGF and calcitonin gene-related peptide (CGRP) are believed to play an algogenic role. Although NGF block is proposed as a novel analgesic approach, its consequences on nociceptive purinergic P2X receptors of trigeminal ganglion neurons remain unknown. We investigated whether neutralizing NGF might change the function of P2X3 receptors natively coexpressed with NGF receptors on cultured mouse trigeminal neurons. Treatment with an NGF antibody (24 h) decreased P2X3 receptor-mediated currents and Ca2+ transients, an effect opposite to exogenously applied NGF. Recovery from receptor desensitization was delayed by anti-NGF treatment without changing desensitization onset. NGF neutralization was associated with decreased threonine phosphorylation of P2X3 subunits, presumably accounting for their reduced responses and slower recovery. Anti-NGF treatment could also increase the residual current typical of heteromeric P2X2/3 receptors, consistent with enhanced membrane location of P2X2 subunits. This possibility was confirmed with cross-linking and immunoprecipitation studies. NGF neutralization also led to increased P2X2e splicing variant at mRNA and membrane protein levels. These data suggest that NGF controlled plasticity of P2X3 subunits and their membrane assembly with P2X2 subunits. Despite anti-NGF treatment, CGRP could still enhance P2X3 receptor activity, indicating separate NGF- or CGRP-mediated mechanisms to upregulate P2X3 receptors. In an in vivo model of mouse trigeminal pain, anti-NGF pretreatment suppressed responses evoked by P2X3 receptor activation. Our findings outline the important contribution by NGF signaling to nociception of trigeminal sensory neurons, which could be counteracted by anti-NGF pretreatment.