Role of ATP in migraine mechanisms: focus on P2X3 receptors.

Migraine is a major health burden worldwide with complex pathophysiology and multifarious underlying mechanisms. One poorly understood issue concerns the early steps in the generation of migraine pain. To elucidate the basic process of migraine pain further, it seems useful to consider key molecular players that may operate synergistically to evoke headache. While the neuropeptide CGRP is an important contributor, we propose that extracellular ATP (that generally plays a powerful nociceptive role) is also a major component of migraine headache, acting in concert with CGRP to stimulate trigeminal nociceptive neurons. The aim of the present focused review is to highlight the role of ATP activating its P2X3 membrane receptors selectively expressed by sensory neurons including their nerve fiber terminals in the meninges. Specifically, we present data on the homeostasis of ATP and related purines in the trigeminovascular system and in the CNS; the basic properties of ATP signalling at peripheral and central nerve terminals; the characteristics of P2X3 and related receptors in trigeminal neurons; the critical speed and persistence of P2X3 receptor activity; their cohabitation at the so-called meningeal neuro-immune synapse; the identity of certain endogenous agents cooperating with ATP to induce neuronal sensitization in the trigeminal sensory system; the role of P2X3 receptors in familial type migraine; the current state of P2X3 receptor antagonists and their pharmacological perspectives in migraine. It is proposed that the unique kinetic properties of P2X3 receptors activated by ATP offer an interesting translational value to stimulate future studies for innovative treatments of migraine pain.

A study of methylprednisolone neuroprotection against acute injury to the rat spinal cord in vitro.

Methylprednisolone sodium succinate (MPSS) has been proposed as a first-line treatment for acute spinal cord injury (SCI). Its clinical use remains, however, controversial because of the modest benefits and numerous side-effects. We investigated if MPSS could protect spinal neurons and glia using an in vitro model of the rat spinal cord that enables recording reflexes, fictive locomotion and morphological analysis of damage. With this model, a differential lesion affecting mainly either neurons or glia can be produced via kainate-evoked excitotoxicity or application of a pathological medium (lacking O2 and glucose), respectively. MPSS (6-10 µM) applied for 24 h after 1-h pathological medium protected astrocytes and oligodendrocytes especially in the ventrolateral white matter. This effect was accompanied by the return of slow, alternating oscillations (elicited by NMDA and 5-hydroxytryptamine (5-HT)) reminiscent of a sluggish fictive locomotor pattern. MPSS was, however, unable to reverse even a moderate neuronal loss and the concomitant suppression of fictive locomotion evoked by kainate (0.1 mM; 1 h). These results suggest that MPSS could, at least in part, contrast damage to spinal glia induced by a dysmetabolic state (associated to oxygen and glucose deprivation) and facilitate reactivation of spinal networks. Conversely, when even a minority of neurons was damaged by excitotoxicity, MPSS did not protect them nor did it restore network function in the current experimental model.

Modulatory effects by CB1 receptors on rat spinal locomotor networks after sustained application of agonists or antagonists.

Sustained administration of cannabinoid agonists acting on neuronal CB1 receptors (CB1Rs) are proposed for treating spasticity and chronic pain. The impact of CB1Rs on mammalian locomotor networks remains, however, incompletely understood. To clarify how CB1Rs may control synaptic activity and locomotor network function, we used the rat spinal cord in vitro which is an advantageous model to investigate locomotor circuit mechanisms produced by the local central pattern generator. Neither the CB1 agonist anandamide (AEA) nor the CB1R antagonist AM-251 evoked early (<3h) changes in mono or polysynaptic reflexes or in locomotor rhythms. Application of AEA (24h) significantly decreased the ability of dorsal root (DR) afferents to elicit oscillatory cycles, and left synaptic responses unchanged. Similar application of LY 2183240, or JZL 184, inhibitors of endocannabinoid uptake processes, produced analogous results. Application of the antagonist AM-251 (or rimonabant) for >3-24h largely impaired locomotor network activity induced by DR stimuli or neurochemicals, and depressed disinhibited bursting without changing reflex amplitude or inducing neurotoxicity even if CB1R immunoreactivity was lowered in the central region. Since CB1R activation usually inhibits cyclic adenosine monophosphate (cAMP) synthesis, we investigated how a 24-h application of AEA or AM-251 affected basal or forskolin-stimulated cAMP levels. While AEA decreased them in an AM-251-sensitive manner, AM-251 per se did not change resting or stimulated cAMP. Our data suggest that CB1Rs may control the circuit gateway regulating the inflow of sensory afferent inputs into the locomotor circuits, indicating a potential site of action for restricting peripheral signals disruptive for locomotor activity.

The volatile anesthetic methoxyflurane protects motoneurons against excitotoxicity in an in vitro model of rat spinal cord injury.

Neuroprotection of the spinal cord during the early phase of injury is an important goal to determine a favorable outcome by prevention of delayed pathological events, including excitotoxicity, which otherwise extend the primary damage and amplify the often irreversible loss of motor function. While intensive care and neurosurgical intervention are important treatments, effective neuroprotection requires further experimental studies focused to target vulnerable neurons, particularly motoneurons. The present investigation examined whether the volatile general anesthetic methoxyflurane might protect spinal locomotor networks from kainate-evoked excitotoxicity using an in vitro rat spinal cord preparation as a model. The protocols involved 1h excitotoxic stimulation on day 1 followed by electrophysiological and immunohistochemical testing on day 2. A single administration of methoxyflurane applied together with kainate (1h), or 30 or even 60 min later prevented any depression of spinal reflexes, loss of motoneuron excitability, and histological damage. Methoxyflurane per se temporarily decreased synaptic transmission and motoneuron excitability, effects readily reversible on washout. Spinal locomotor activity recorded as alternating electrical discharges from lumbar motor pools was fully preserved on the second day after application of methoxyflurane together with (or after) kainate. These data suggest that a volatile general anesthetic could provide strong electrophysiological and histological neuroprotection that enabled expression of locomotor network activity 1 day after the excitotoxic challenge. It is hypothesized that the benefits of early neurosurgery for acute spinal cord injury (SCI) might be enhanced if, in addition to injury decompression and stabilization, the protective role of general anesthesia is exploited.

A hyperexcitability phenotype in mouse trigeminal sensory neurons expressing the R192Q Cacna1a missense mutation of familial hemiplegic migraine type-1.

Missense mutation R192Q in the CACNA1A gene causes familial hemiplegic migraine type-1 (FHM1), a monogenic subtype of migraine with aura. Using knock-in (KI) gene targeting we introduced this mutation into the mouse gene and generated a transgenic mouse model to investigate basic mechanisms of migraine pathophysiology. While FHM1 R192Q KI trigeminal ganglia were previously shown to exhibit constitutive up-regulation of ATP-gated P2X3 receptors, little is known about the firing properties of trigeminal sensory neurons, which convey nociceptive inputs to higher brain centers. We patch-clamped trigeminal sensory neurons to search for differences in firing properties between wildtype (WT) and KI cells in culture. Although various subclasses of trigeminal neurons were observed with respect to their firing patterns evoked by intracellular current injection, their distribution among WT and KI cells was similar with only small differences in rheobase or input resistance values. However, when neurons were excited by either α,ß-methyl-ATP to stimulate P2X3 receptors or capsaicin to activate transient receptor potential vanilloid (TRPV1) receptors, the firing threshold in KI neurons was significantly lowered and followed by a larger number of spikes. Activation by α,ß-methyl-ATP was associated with a transient cluster of action potentials, while capsaicin elicited more persistent firing. Using α,ß-methyl-ATP or capsaicin, two functional classes of WT or KI neurons were distinguished according to the first spike latency, which suggests that a subgroup of neurons may be indirectly activated, probably via crosstalk between neurons and satellite glial cells. Thus, our results are consistent with reported facilitated trigeminal pain behavior of FHM1 R192Q KI mice.

Excitotoxic cell death induces delayed proliferation of endogenous neuroprogenitor cells in organotypic slice cultures of the rat spinal cord.

The aim of the present report was to investigate whether, in the mammalian spinal cord, cell death induced by transient excitotoxic stress could trigger activation and proliferation of endogenous neuroprogenitor cells as a potential source of a lesion repair process and the underlying time course. Because it is difficult to address these issues in vivo, we used a validated model of spinal injury based on rat organotypic slice cultures that retain the fundamental tissue cytoarchitecture and replicate the main characteristics of experimental damage to the whole spinal cord. Excitotoxicity evoked by 1 h kainate application produced delayed neuronal death (40%) peaking after 1 day without further losses or destruction of white matter cells for up to 2 weeks. After 10 days, cultures released a significantly larger concentration of endogenous glutamate, suggesting functional network plasticity. Indeed, after 1 week the total number of cells had returned to untreated control level, indicating substantial cell proliferation. Activation of progenitor cells started early as they spread outside the central area, and persisted for 2 weeks. Although expression of the neuronal progenitor phenotype was observed at day 3, peaked at 1 week and tapered off at 2 weeks, very few cells matured to neurons. Astroglia precursors started proliferating later and matured at 2 weeks. These data show insult-related proliferation of endogenous spinal neuroprogenitors over a relatively brief time course, and delineate a narrow temporal window for future experimental attempts to drive neuronal maturation and for identifying the factors regulating this process.

Mechanisms underlying cell death in ischemia-like damage to the rat spinal cord in vitro.

New spinal cord injury (SCI) cases are frequently due to non-traumatic causes, including vascular disorders. To develop mechanism-based neuroprotective strategies for acute SCI requires full understanding of the early pathophysiological changes to prevent disability and paralysis. The aim of our study was to identify the molecular and cellular mechanisms of cell death triggered by a pathological medium (PM) mimicking ischemia in the rat spinal cord in vitro. We previously showed that extracellular Mg(2+) (1 mM) worsened PM-induced damage and inhibited locomotor function. The present study indicated that 1 h of PM+Mg(2+) application induced delayed pyknosis chiefly in the spinal white matter via overactivation of poly (ADP-ribose) polymerase 1 (PARP1), suggesting cell death mediated by the process of parthanatos that was largely suppressed by pharmacological block of PARP-1. Gray matter damage was less intense and concentrated in dorsal horn neurons and motoneurons that became immunoreactive for the mitochondrial apoptosis-inducing factor (the intracellular effector of parthanatos) translocated into the nucleus to induce chromatin condensation and DNA fragmentation. Immunoreactivity to TRPM ion channels believed to be involved in ischemic brain damage was also investigated. TRPM2 channel expression was enhanced 24 h later in dorsal horn and motoneurons, whereas TRPM7 channel expression concomitantly decreased. Conversely, TRPM7 expression was found earlier (3 h) in white matter cells, whereas TRPM2 remained undetectable. Simulating acute ischemic-like damage in vitro in the presence of Mg(2+) showed how, during the first 24 h, this divalent cation unveiled differential vulnerability of white matter cells and motoneurons, with distinct changes in their TRPM expression.

A study of the potential neuroprotective effect of riluzole on locomotor networks of the neonatal rat spinal cord in vitro damaged by excitotoxicity.

Excitotoxicity triggered by over-stimulation of glutamatergic receptors is considered to be a major component of damage following acute spinal cord injury (SCI). Using an in vitro model of neonatal rat SCI caused by transient application (1h) of the glutamate agonist kainate (0.05-0.1 mM) to produce limited excitotoxicity, the present study investigated whether riluzole, a drug inhibiting glutamate release and neuronal excitability, could prevent neuronal loss and protect locomotor patterns 24 h later. Immunohistochemical analysis of neuronal and motoneuronal populations was associated with recording of fictive locomotion induced by neurochemicals or dorsal root stimuli. Riluzole (5 µM; 24 h application) per se exerted strong and persistent neurodepressant effects on network synaptic transmission from which recovery was very slow. When continuously applied after kainate, riluzole partially reduced the number of pyknotic cells in the gray matter, although motoneurons remained vulnerable and no fictive locomotion was present. In further experiments, riluzole per se was applied for 3 h (expected to coincide with kainate peak excitotoxicity) and washed out for 24 h with full return of fictive locomotion. When this protocol was implemented after kainate, no efficient histological or functional recovery was observed. No additional benefit was detected even when riluzole was co-applied with kainate and continued for the following 3 h. These results show that modest neuronal losses evoked by excitotoxicity have a severe impact on locomotor network function, and that they cannot be satisfactorily blocked by strong neurodepression with riluzole, suggesting the need for more effective pharmacological approaches.

An innovative spinal cord injury model for the study of locomotor networks.

An acute lesion to the spinal cord triggers complex mechanisms responsible for amplification of the initial damage and its chronicity. In vitro preparations of the rodent spinal cord retain the intrinsic ability to produce locomotor-like discharges from lumbar ventral roots and, thus, offer the opportunity to study the still unclear process of lesion progression in relation to cell number and topography. In addition, these models enable a detailed approach to the molecular mechanisms of damage and to pharmacological tools to counteract them. Using the rat spinal cord in vitro, our laboratory has shown how to reliably produce discrete lesions by applying the glutamate agonist kainate that evokes delayed neuronal loss via a non-apoptotic cell death mechanism termed parthanatos. Parthanatos is believed to be due to mitochondrial damage and exhaustion of cell energy stores caused by hyperactivation of enzymatic systems initially set to repair DNA damage. Locomotor network activity is irreversibly destroyed by kainate in a virtually all-or-none manner, suggesting destruction of a highly-vulnerable cell population crucial for the expression of locomotion. Hypoxic challenge to the spinal cord together with toxic radicals primarily damages white matter cells with deficit (without full suppression) of locomotor network function, while neurons are less vulnerable. Pharmacological agents to inhibit different targets involved in the early pathophysiology of spinal injury provided limited success, indicating that novel approaches based on newly identified steps in the biochemical cascade leading to cell death should be investigated for their potential to improve the outcome of spinal cord injury.

Delayed neuroprotection by riluzole against excitotoxic damage evoked by kainate on rat organotypic spinal cord cultures.

Kainate-mediated excitotoxicity of organotypic spinal cord cultures is an in vitro model advantageous to investigate basic mechanisms of acute spinal injury and its pharmacological neuroprotection. Using such cultures, the putative neuroprotective agent riluzole applied at 5 µM (plasma therapeutic concentration) was studied for its ability to prevent neurotoxicity evoked by 1 h administration of kainate. We monitored real-time release of glutamate, release of lactate dehydrogenase (LDH) (cell damage marker), occurrence of cell pyknosis, the number of surviving neurons and motoneurons, and cell culture metabolic activity. Co-applied riluzole strongly blocked the kainate-evoked early rise in extracellular glutamate (via calcium dependent or independent processes) and suppressed LDH release (limited to <20% of total). Although there were no significant cell losses within the first h after kainate washout, pyknosis, fewer neurons and motoneurons were observed 24 h later. MTT assay demonstrated that surviving cells were metabolically competent. Co-application of kainate and tetrodotoxin also failed to protect spinal cord slices 24 h later. When riluzole application begun at kainate washout and continued for 24 h, significant neuroprotection was observed for neurons in the central and dorsal regions, while ventral horn cells (including motoneurons) were not protected. Our data suggest that riluzole neuroprotection against excitotoxicity was feasible, although it paradoxically required delayed drug administration, and was not extended to the ventral horn. We propose that riluzole was acting on yet-unidentified processes downstream of glutamate release and receptor activation. Deciphering their identity and role in cell death mechanisms may be an important goal to develop neuroprotection.

Extensive glial apoptosis develops early after hypoxic-dysmetabolic insult to the neonatal rat spinal cord in vitro.

The current etiopathogenesis of spinal cord injury comprises a growing number of nontraumatic causes, including ischemia generating hypoxic-dysmetabolic conditions. To mimic the metabolic disruption accompanying nontraumatic acute spinal cord injury and to characterize the type and dynamics of cell death in relation to locomotor network function, we used, as a model, the rat neonatal spinal cord preparation in vitro transiently (1 h) exposed to a "pathological medium" (PM), i.e. hypoxic/aglycemic solution containing toxic radicals. PM induced, in the ventrolateral spinal region, pyknosis already detectable after 2 h and stabilized 24 h later (affecting 55% of white matter cells). Glial cells were much more vulnerable than neurons. The amplitude of fictive locomotor patterns recorded from lumbar ventral roots was decreased and periodicity delayed by PM, in keeping with substantial preservation of neuronal networks. Repeated application of PM intensified such a functional impairment. White matter astrocytes and oligodendrocytes displayed nucleolytic pyknosis mainly dependent on caspase-mediated death processes as shown by active caspase-3 and terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) positivity. Expression of cleaved poly(ADP-ribose) polymerase-1 (PARP-1) (the active caspase-3 executor) also grew with similar time course. The caspase-3 inhibitor II counteracted, in a dose-dependent fashion, white matter pyknosis. Our results suggest the important involvement of apoptotic pathways in early glial cell death during the first 24 h after a hypoxic-dysmetabolic insult, associated with impaired locomotor output. Residual locomotor network activity together with distinctive apoptotic damage to white matter cells suggests that early protection against glial destruction may help to prevent subsequent damage extension responsible for paraplegia.

Kainate-induced delayed onset of excitotoxicity with functional loss unrelated to the extent of neuronal damage in the in vitro spinal cord.

While excitotoxicity is a major contributor to the pathophysiology of acute spinal injury, its time course and the extent of cell damage in relation to locomotor network activity remain unclear. We used two in vitro models, that is, the rat isolated spinal cord and spinal organotypic cultures, to explore the basic characteristics of excitotoxicity caused by transient application of the glutamate analogue kainate followed by washout and analysis 24 h later. Electrophysiological records showed that fictive locomotion was slowed down by 10 microM kainate (with no histological loss) and fully abolished by 50 microM, while disinhibited bursting with unchanged periodicity persisted. Kainate concentrations (> or =50 microM) larger than those necessary to irreversible suppress fictive locomotion could still elicit dose-dependent motoneuron pool depolarization, and dose-dependent neuronal loss in the grey matter, especially evident in central and dorsal areas. Motoneuron numbers were largely decreased. A similar regional pattern was detected in organotypic slices, as extensive cell loss was dose related and affected motoneurons and premotoneurons: the number of dead neurons (already apparent 1 h after kainate) grew faster with the higher kainate concentration. The histological damage was accompanied by decreased MTT formazan production commensurate with the number of surviving cells. Our data suggest locomotor network function was very sensitive to excitotoxicity, even without observing extensive cell death. Excitotoxicity developed gradually leaving a time window in which neuroprotection might be attempted to preserve circuits still capable of expressing basic rhythmogenesis and reconfigure their function in terms of locomotor output.

Neuroprotection of locomotor networks after experimental injury to the neonatal rat spinal cord in vitro.

Treatment to block the pathophysiological processes triggered by acute spinal injury remains unsatisfactory as the underlying mechanisms are incompletely understood. Using as a model the in vitro spinal cord of the neonatal rat, we investigated the feasibility of neuroprotection of lumbar locomotor networks by the glutamate antagonists 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) and aminophosphonovalerate (APV) against acute lesions induced by either a toxic solution (pathological medium (PM) to mimic the spinal injury hypoxic-dysmetabolic perturbation) or excitotoxicity with kainate. The study outcome was presence of fictive locomotion 24 h after the insult and its correlation with network histology. Inhibition of fictive locomotion by PM was contrasted by simultaneous and even delayed (1 h later) co-application of CNQX and APV with increased survival of ventral horn premotoneurons and lateral column white matter. Neither CNQX nor APV alone provided neuroprotection. Kainate-mediated excitotoxicity always led to loss of fictive locomotion and extensive neuronal damage. CNQX and APV co-applied with kainate protected one-third of preparations with improved motoneuron and dorsal horn neuronal counts, although they failed with delayed application. Our data suggest that locomotor network neuroprotection was possible when introduced very early during the pathological process of spinal injury, but also showed how the borderline between presence or loss of locomotor activity was a very narrow one that depended on the survival of a certain number of neurons or white matter elements. The present report provides a model not only for preclinical testing of novel neuroprotective agents, but also for estimating the minimal network membership compatible with functional locomotor output.

Extracellular magnesium enhances the damage to locomotor networks produced by metabolic perturbation mimicking spinal injury in the neonatal rat spinal cord in vitro.

An acute injury to brain or spinal cord produces profound metabolic perturbation that extends and exacerbates tissue damage. Recent clinical interventions to treat this condition with i.v. Mg(2+) to stabilize its extracellular concentration provided disappointing results. The present study used an in vitro spinal cord model from the neonatal rat to investigate the role of extracellular Mg(2+) in the lesion evoked by a pathological medium mimicking the metabolic perturbation (hypoxia, aglycemia, oxidative stress, and acid pH) occurring in vivo. Damage was measured by taking as outcome locomotor network activity for up to 24 h after the primary insult. Pathological medium in 1 mM Mg(2+) solution (1 h) largely depressed spinal reflexes and suppressed fictive locomotion on the same and the following day. Conversely, pathological medium in either Mg(2+)-free or 5 mM Mg(2+) solution evoked temporary network depression and enabled fictive locomotion the day after. While global cell death was similar regardless of extracellular Mg(2+) solution, white matter was particularly affected. In ventral horn the number of surviving neurons was the highest in Mg(2+) free solution and the lowest in 1 mM Mg(2+), while motoneurons were unaffected. Although the excitotoxic damage elicited by kainate was insensitive to extracellular Mg(2+), 1 mM Mg(2+) potentiated the effect of combining pathological medium with kainate at low concentrations. These results indicate that preserving Mg(2+) homeostasis rendered experimental spinal injury more severe. Furthermore, analyzing ventral horn neuron numbers in relation to fictive locomotion expression might provide a first estimate of the minimal size of the functional locomotor network.

Kainate and metabolic perturbation mimicking spinal injury differentially contribute to early damage of locomotor networks in the in vitro neonatal rat spinal cord.

Acute spinal cord injury evolves rapidly to produce secondary damage even to initially spared areas. The result is loss of locomotion, rarely reversible in man. It is, therefore, important to understand the early pathophysiological processes which affect spinal locomotor networks. Regardless of their etiology, spinal lesions are believed to include combinatorial effects of excitotoxicity and severe stroke-like metabolic perturbations. To clarify the relative contribution by excitotoxicity and toxic metabolites to dysfunction of locomotor networks, spinal reflexes and intrinsic network rhythmicity, we used, as a model, the in vitro thoraco-lumbar spinal cord of the neonatal rat treated (1 h) with either kainate or a pathological medium (containing free radicals and hypoxic/aglycemic conditions), or their combination. After washout, electrophysiological responses were monitored for 24 h and cell damage analyzed histologically. Kainate suppressed fictive locomotion irreversibly, while it reversibly blocked neuronal excitability and intrinsic bursting induced by synaptic inhibition block. This result was associated with significant neuronal loss around the central canal. Combining kainate with the pathological medium evoked extensive, irreversible damage to the spinal cord. The pathological medium alone slowed down fictive locomotion and intrinsic bursting: these oscillatory patterns remained throughout without regaining their control properties. This phenomenon was associated with polysynaptic reflex depression and preferential damage to glial cells, while neurons were comparatively spared. Our model suggests distinct roles of excitotoxicity and metabolic dysfunction in the acute damage of locomotor networks, indicating that different strategies might be necessary to treat the various early components of acute spinal cord lesion.

N-methyl-D-aspartate triggers neonatal rat hypoglossal motoneurons in vitro to express rhythmic bursting with unusual Mg2+ sensitivity.

The brainstem nucleus hypoglossus innervates the tongue which must contract rhythmically during respiration, chewing and swallowing. Such rhythmic discharges are due to network bursting mediated by AMPA receptor-dependent glutamatergic transmission. The contribution by hypoglossal motoneurons themselves to rhythmicity remains, however, unclear as they might simply express cyclic patterns produced by premotoneurons or, in analogy to spinal motoneurons, might participate to bursting due to activation of their N-methyl-D-aspartate (NMDA) receptors. Using patch clamp recording from hypoglossal motoneurons in slice preparations of neonatal rat brainstem, we observed that NMDA directly depolarized motoneurons to generate various discharge patterns. Most motoneurons produced transient bursts which were consistently restored by repolarizing membrane potential to rest. Fewer motoneurons generated either sustained bursting or random firing. Rhythmic bursts were recorded from XII nerve rootlets even when single motoneuron bursting required hyperpolarization. NMDA evoked bursts were blocked by the Ca2+ antagonist Cd2+, the gap junction blocker carbenoxolone, or Mg2+ free solution, and partially inhibited by tetrodotoxin or nifedipine. Under voltage clamp, NMDA-induced bursting persisted at negative or positive potentials and was resistant to high extracellular Mg2+ in accordance with the observation of widespread motoneuron expression of NMDA 2D receptor subunits that confer poor Mg2+ sensitivity. It is proposed that NMDA depolarized motoneurons with the contribution of Mg2+ insensitive channels, and triggered bursting via cyclic activation/deactivation of voltage-dependent Na+, Ca2+ and K+ currents spread through gap junctions. The NMDA-evoked bursting pattern was similar to the rhythmic discharges previously recorded from the XII nerve during milk sucking by neonatal rats.

Nicotinic acetylcholine receptors of adrenal chromaffin cells.

In the adrenal medulla, acetylcholine released by the sympathetic splanchnic nerves activates neuronal-type nicotinic acetylcholine receptors (nAChRs) on the membrane of chromaffin cells which liberate catecholamines into the bloodstream in preparation for the fight and flight reactions. On adrenal chromaffin cells the main class of nAChRs is a pentameric assembly of alpha3 and beta4 subunits that forms ion channels which produce membrane depolarization by increasing Na+, K+ and Ca2+ permeability. Homomeric alpha7 nicotinic receptors are expressed in a species-dependent manner and do not contribute to catecholamine secretion. Chromaffin cell nAChRs rapidly activate and desensitize with full recovery on washout. nAChR activity is subjected to various types of dynamic regulation. It is allosterically modulated by the endogenous neuropeptide substance P that stabilizes receptors in their desensitized state, thus depressing their responsiveness. The full-length peptide CGRP acts as a negative allosteric modulator by inhibiting responses without changing desensitization, whereas its N-terminal fragments act as positive allosteric modulators to transiently enhance nAChR function. nAChR expression increases when cells are chronically exposed to either selective antagonists or agonists such as nicotine, a protocol mimicking the condition of chronic heavy smokers. In this case, large upregulation of nAChRs occurs even though most of the extra nAChRs remain inside the cells, creating a mismatch between the increase in total nAChRs and increase in functional nAChRs on the cell surface. These findings highlight the plastic properties of cholinergic neurotransmission in the adrenal medulla to provide robust mechanisms for adapting catecholamine release to acute and chronic changes in sympathetic activity.

Differential modulation by tetraethylammonium of the processes underlying network bursting in the neonatal rat spinal cord in vitro.

In the rat spinal cord in vitro, block of synaptic inhibition evokes persistent, regular disinhibited bursting which is a manifestation of the intrinsic network rhythmicity and is readily recorded from ventral roots. This model is advantageous to explore the network mechanisms controlling burst periodicity, and duration. We questioned the relative contribution of K+ conductances to spontaneous rhythmicity by investigating the effects of the broad K+ channel blocker tetraethylammonium (TEA). In TEA (10 mM) solution, bursts occurred at the same rate but became substantially longer, thus showing an unusual dissociation between mechanisms of burst periodicity and duration. In the presence of TEA, electrical stimulation of a single dorsal root or N-methyl-D-aspartate application (5 microM) could, however, fasten bursting associated with immediate decrease in burst length, thus demonstrating maintenance of short-term plasticity. Either riluzole (1 microM) or surgical sectioning that isolated a single spinal segment strongly depressed bursting which could, however, be revived by TEA. In the presence of TEA, the L-type channel blocker nifedipine (20 microM) made bursting faster and shorter. Our data are best explained by assuming that TEA increased network excitability to generate rhythmic bursting, an effect that was counteracted by intrinsic mechanisms, partly dependent on L-type channel activity, to retain standard periodicity. TEA-sensitive mechanisms were, nevertheless, an important process to regulate burst duration. Our results are consistent with the proposal of a hierarchical structural of the central pattern generator in which the circuits responsible for rhythmicity (the clock) drive the discharges of those creating the motor commands (pattern).

The effects induced by the sulphonylurea glibenclamide on the neonatal rat spinal cord indicate a novel mechanism to control neuronal excitability and inhibitory neurotransmission.

BACKGROUND AND PURPOSE: Using the neonatal rat spinal cord in vitro, we investigated the action of glibenclamide, a drug possessing dual pharmacological effects, namely block of K(ATP) channels and of the cystic fibrosis transmembrane conductance regulator (CFTR). EXPERIMENTAL APPROACH: Intra- and extracellular recordings were performed on motoneurons and interneurons. RT-PCR and western immunoblotting were used to determine gene and protein expression. KEY RESULTS: Glibenclamide (50 microM) facilitated mono- and polysynaptic reflexes, hyperpolarized motoneuron resting potential, increased action potential amplitude, decreased Renshaw cell-mediated recurrent inhibition, and increased network excitability by depressing GABA- and glycine-mediated transmission. The action of glibenclamide was mimicked by tolbutamide (500 microM) or the CFTR blocker diphenylamine-2,2-dicarboxylic acid (500 microM). The action of glibenclamide was independent from pharmacological inhibition of the Na(+)-K(+) pump with strophanthidin (4 microM) and was associated with a negative shift in the extrapolated reversal potential for CI(-) dependent synaptic inhibition. On interneurons, intracellularly-applied 8-bromo-cAMP elicited an inward current and resistance decrease; effects antagonized by the selective CFTR antagonist, CFTR(inh)-172 (5 microM). RT-PCR and western immunoblotting indicated strong expression of the CFTR in neonatal rat spinal cord. CONCLUSIONS AND IMPLICATIONS: These data suggest the CFTR expressed in motoneurons and interneurons of the neonatal spinal cord is involved in the control of Cl(-) homeostasis and neuronal excitability. CFTR appeared to contribute to the relatively depolarized equilibrium potential for synaptic inhibition, an important process to control hyperexcitability and seizure-predisposition in neonates.

Fictive locomotor patterns generated by tetraethylammonium application to the neonatal rat spinal cord in vitro.

Intrinsic spinal networks generate a locomotor rhythm characterized by alternating electrical discharges from flexor and extensor motor pools. Because this process is preserved in the rat isolated spinal cord, this preparation in vitro may be a useful model to explore methods to reactivate locomotor networks damaged by spinal injury. The present electrophysiological investigation examined whether the broad spectrum potassium channel blocker tetraethylammonium could generate locomotor-like patterns. Low (50-500 microM) concentrations of tetraethylammonium induced irregular, synchronous discharges incompatible with locomotion. Higher concentrations (1-10 mM) evoked alternating discharges between flexor and extensor motor pools, plus large depolarization of motoneurons with spike broadening. The alternating discharges were superimposed on slow, shallow waves of synchronous depolarization. Rhythmic alternating patterns were suppressed by blockers of glutamate, GABA(A) and glycine receptors, disclosing a background of depolarizing bursts inhibited by antagonism of group I metabotropic glutamate receptors. Furthermore, tetraethylammonium also evoked irregular discharges on dorsal roots. Rhythmic alternating patterns elicited by tetraethylammonium on ventral roots were relatively stereotypic, had limited synergy with fictive locomotion induced by dorsal root stimuli, and were not accelerated by 4-aminopyridine. Horizontal section of the spinal cord preserved irregular ventral root discharges and dorsal root discharges, demonstrating that the action of tetraethylammonium on spinal networks was fundamentally different from that of 4-aminopyridine. These results show that a potassium channel blocker such as tetraethylammonium could activate fictive locomotion in the rat isolated spinal cord, although the pattern quality lacked certain features like frequency modulation and strong synergy with other inputs to locomotor networks.