Nicotine Neurotoxicity Involves Low Wnt1 Signaling in Spinal Locomotor Networks of the Postnatal Rodent Spinal Cord.

The postnatal rodent spinal cord in-vitro is a useful model to investigate early pathophysiological changes after injury. While low dose nicotine (1 µM) induces neuroprotection, how higher doses affect spinal networks is unknown. Using spinal preparations of postnatal wild-type Wistar rat and Wnt1Cre2:Rosa26Tom double-transgenic mouse, we studied the effect of nicotine (0.5-10 µM) on locomotor networks in-vitro. Nicotine 10 µM induced motoneuron depolarization, suppressed monosynaptic reflexes, and decreased fictive locomotion in rat spinal cord. Delayed fall in neuronal numbers (including motoneurons) of central and ventral regions emerged without loss of dorsal neurons. Conversely, nicotine (0.5-1 µM) preserved neurons throughout the spinal cord and strongly activated the Wnt1 signaling pathway. High-dose nicotine enhanced expression of S100 and GFAP in astrocytes indicating a stress response. Excitotoxicity induced by kainate was contrasted by nicotine (10 µM) in the dorsal area and persisted in central and ventral regions with no change in basal Wnt signaling. When combining nicotine with kainate, the activation of Wnt1 was reduced compared to kainate/sham. The present results suggest that high dose nicotine was neurotoxic to central and ventral spinal neurons as the neuroprotective role of Wnt signaling became attenuated. This also corroborates the risk of cigarette smoking for the foetus/newborn since tobacco contains nicotine.

Overexpressed Na V 1.7 Channels Confer Hyperexcitability to in vitro Trigeminal Sensory Neurons of Ca V 2.1 Mutant Hemiplegic Migraine Mice.

Trigeminal sensory neurons of transgenic knock-in (KI) mice expressing the R192Q missense mutation in the α1A subunit of neuronal voltage-gated Ca V 2.1 Ca2+ channels, which leads to familial hemiplegic migraine type 1 (FHM1) in patients, exhibit a hyperexcitability phenotype. Here, we show that the expression of Na V 1.7 channels, linked to pain states, is upregulated in KI primary cultures of trigeminal ganglia (TG), as shown by increased expression of its α1 subunit. In the majority of TG neurons, Na V 1.7 channels are co-expressed with ATP-gated P2X3 receptors (P2X3R), which are important nociceptive sensors. Reversing the trigeminal phenotype with selective Ca V 2.1 channel inhibitor ω-agatoxin IVA inhibited Na V 1.7 overexpression. Functionally, KI neurons revealed a TTX-sensitive inward current of larger amplitude that was partially inhibited by selective Na V 1.7 blocker Tp1a. Under current-clamp condition, Tp1a raised the spike threshold of both wild-type (WT) and KI neurons with decreased firing rate in KI cells. Na V 1.7 activator OD1 accelerated firing in WT and KI neurons, a phenomenon blocked by Tp1a. Enhanced expression and function of Na V 1.7 channels in KI TG neurons resulted in higher excitability and facilitated nociceptive signaling. Co-expression of Na V 1.7 channels and P2X3Rs in TGs may explain how hypersensitivity to local stimuli can be relevant to migraine.

GABAergic Mechanisms Can Redress the Tilted Balance between Excitation and Inhibition in Damaged Spinal Networks.

Correct operation of neuronal networks depends on the interplay between synaptic excitation and inhibition processes leading to a dynamic state termed balanced network. In the spinal cord, balanced network activity is fundamental for the expression of locomotor patterns necessary for rhythmic activation of limb extensor and flexor muscles. After spinal cord lesion, paralysis ensues often followed by spasticity. These conditions imply that, below the damaged site, the state of balanced networks has been disrupted and that restoration might be attempted by modulating the excitability of sublesional spinal neurons. Because of the widespread expression of inhibitory GABAergic neurons in the spinal cord, their role in the early and late phases of spinal cord injury deserves full attention. Thus, an early surge in extracellular GABA might be involved in the onset of spinal shock while a relative deficit of GABAergic mechanisms may be a contributor to spasticity. We discuss the role of GABA A receptors at synaptic and extrasynaptic level to modulate network excitability and to offer a pharmacological target for symptom control. In particular, it is proposed that activation of GABA A receptors with synthetic GABA agonists may downregulate motoneuron hyperexcitability (due to enhanced persistent ionic currents) and, therefore, diminish spasticity. This approach might constitute a complementary strategy to regulate network excitability after injury so that reconstruction of damaged spinal networks with new materials or cell transplants might proceed more successfully.

Modeling a Nociceptive Neuro-Immune Synapse Activated by ATP and 5-HT in Meninges: Novel Clues on Transduction of Chemical Signals Into Persistent or Rhythmic Neuronal Firing.

Extracellular ATP and serotonin (5-HT) are powerful triggers of nociceptive firing in the meninges, a process supporting headache and whose cellular mechanisms are incompletely understood. The current study aimed to develop, with the neurosimulator NEURON, a novel approach to explore in silico the molecular determinants of the long-lasting, pulsatile nature of migraine attacks. The present model included ATP and 5-HT release, ATP diffusion and hydrolysis, 5-HT uptake, differential activation of ATP P2X or 5-HT3 receptors, and receptor subtype-specific desensitization. The model also tested the role of branched meningeal fibers with multiple release sites. Spike generation and propagation were simulated using variable contribution by potassium and sodium channels in a multi-compartment fiber environment. Multiple factors appeared important to ensure prolonged nociceptive firing potentially relevant to long-lasting pain. Crucial roles were observed in: (i) co-expression of ATP P2X2 and P2X3 receptor subunits; (ii) intrinsic activation/inactivation properties of sodium Nav1.8 channels; and (iii) temporal and spatial distribution of ATP/5-HT release sites along the branches of trigeminal nerve fibers. Based on these factors we could obtain either persistent activation of nociceptive firing or its periodic bursting mimicking the pulsating nature of pain. In summary, our model proposes a novel tool for the exploration of peripheral nociception to test the contribution of clinically relevant factors to headache including migraine pain.

Modulation of extrasynaptic GABAergic receptor activity influences glutamate release and neuronal survival following excitotoxic damage to mouse spinal cord neurons.

Excitotoxic levels of released glutamate trigger a cascade of deleterious cellular events leading to delayed neuronal death. This phenomenon implies extensive dysregulation in the balance between network excitation and inhibition. Our hypothesis was that enhancing network inhibition should prevent excitotoxicity and provide neuroprotection. To test this notion, we used mouse organotypic spinal slice cultures and explored if excitotoxicity caused by the potent glutamate analogue kainate was blocked by pharmacological increase in GABAA receptor activity. To this end we monitored (with a biosensor) real-time glutamate release following 1 h kainate application and quantified neuronal survival 24 h later. Glutamate release evoked by kainate was strongly decreased by the allosteric GABAA modulator midazolam (10 nM) or the GABA agonist THIP (10 µM), leading to neuroprotection. On the contrary, much higher glutamate release was induced by the GABA antagonist bicuculline (20 µM) that inhibits synaptic and extrasynaptic GABAA receptors. Gabazine (20 µM), an antagonist of synaptic GABAA receptors, had no effect on glutamate release or neuroprotection. No effect was observed with the glycine antagonist strychnine or the glycine agonist L-alanine. These findings indicate that enhancement of GABA receptor activity was an effective tool to counteract excitotoxic death in spinal networks. In view of the potent activity by THIP, preferentially acting on extrasynaptic GABAA receptors, the present data imply a significant role for extrasynaptic GABAA receptors in sparing spinal cord neurons from injury.

Investigation on 2',3'-O-Substituted ATP Derivatives and Analogs as Novel P2X3 Receptor Antagonists.

Antagonists of the purinergic P2X3 receptors represent promising drugs for the treatment of inflammation and pain. The ATP derivative 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) has been described as a potent competitive inhibitor of this receptor. In this work, the design and synthesis of novel TNP-ATP analogues bearing alkyl groups in the 2',3'-position are reported. These compounds were biologically evaluated as P2X3 antagonists using the patch clamp recording technique on mouse trigeminal ganglionic sensory neurons. Some of the compounds showed nanomolar inhibitory potency for the P2X3 receptor. Further modification of these derivatives was made by substitution of the triphosphate chain with different acidic groups. All compounds were additionally tested at five human P2X receptor subtypes stably expressed in 1321N1 astrocytoma cells to evaluate their potency and P2X3 selectivity. Results confirmed the P2X3 antagonist potency for some derivatives.

Long-term application of cannabinoids leads to dissociation between changes in cAMP and modulation of GABAA receptors of mouse trigeminal sensory neurons.

Antinociception caused by cannabinoids may have a partial peripheral origin in addition to its central site of action. In fact, we have observed that anandamide selectively and reversibly inhibits GABAA receptors of putative nociceptive neurons of mouse trigeminal sensory ganglia via CB1 receptor activation to inhibit adenylyl cyclase and decrease cAMP with downstream posttranslational alterations. Since cannabinoids are often used chronically, we studied changes in cAMP levels and GABA-mediated currents of trigeminal neurons following 24 h application of anandamide (0.5 µM) or the synthetic cannabinoid WIN 55,212-2 (5 µM). With this protocol GABA responses were similar to control despite persistent fall in cAMP levels. Inhibition by WIN 55,212-2 of GABA effects recovered after 30 min washout and was not associated with changes in CB1 receptor expression, indicating lack of CB1 receptor inactivation and transient loss of negative coupling between CB1 receptors and GABAA receptors. The phosphodiesterase inhibitor rolipram (100 µM; 24 h) enhanced cAMP levels and GABA-mediated currents, suggesting GABAA receptors were sensitive to persistent upregulation via cAMP. While the adenylyl cyclase activator forskolin (1-20 µM) facilitated cAMP levels and GABA currents following 30 min application, this action was lost after 24 h in line with the drug limited lifespan. The PKA inhibitor PKI 14-22 (10 µM) increased cAMP without changing GABA currents. These data indicate that modulation of GABAA receptors by intracellular cAMP could be lost following persistent application of cannabinoids. Thus, these observations provide an insight into the waning antinociceptive effects of these compounds.

Mechanism of Neuroprotection Against Experimental Spinal Cord Injury by Riluzole or Methylprednisolone.

Any spinal cord injury carries the potential for persistent disability affecting motor, sensory and autonomic functions. To prevent this outcome, it is highly desirable to block a chain of deleterious reactions developing in the spinal areas immediately around the primary lesion. Thus, early timing of pharmacological neuroprotection should be one major strategy whose impact may be first studied with preclinical models. Using a simple in vitro model of the rat spinal cord it is possible to mimic pathological processes like excitotoxicity that damages neurons because of excessive glutamate receptor activation due to injury, or hypoxic/dysmetabolic insult that preferentially affects glia following vascular dysfunction. While ongoing research is exploring the various components of pathways leading to cell death, current treatment principally relies on the off-label use of riluzole (RLZ) or methylprednisolone sodium succinate (MPSS). The mechanism of action of these drugs is diverse as RLZ targets mainly neurons and MPSS targets glia. Even when applied after a transient excitotoxic stimulus, RLZ can provide effective prevention of secondary excitotoxic damage to premotoneurons, although not to motoneurons that remain very vulnerable. This observation indicates persistent inability to express locomotor activity despite pharmacological treatment conferring some histological protection. MPSS can protect glia from dysmetabolic insult, yet it remains poorly effective to prevent neuronal death. In summary, it appears that these pharmacological agents can produce delayed protection for certain cell types only, and that their combined administration does not provide additional benefit. The search should continue for better, mechanism-based neuroprotective agents.

Pharmacological induction of Heat Shock Protein 70 by celastrol protects motoneurons from excitotoxicity in rat spinal cord in vitro.

The secondary phase of spinal cord injury arising after the primary lesion largely extends the damage severity with delayed negative consequences for sensory-motor pathways. It is, therefore, important to find out if enhancing intrinsic mechanisms of neuroprotection can spare motoneurons that are very vulnerable cells. This issue was investigated with an in vitro model of rat spinal cord excitotoxicity monitored for up to 24 hr after the primary injury evoked by kainate. This study sought to pharmacologically boost the expression of heat shock proteins (HSP) to protect spinal motoneurons using celastrol to investigate if the rat spinal cord can upregulate HSP as neuroprotective mechanism. Despite its narrow range of drug safety in vitro, celastrol was not toxic to the rat spinal cord at 0.75 µM concentration and enhanced the expression of HSP70 by motoneurons. When celastrol was applied either before or after kainate, the number of dead motoneurons was significantly decreased and the nuclear localization of the cell death biomarker AIF strongly inhibited. Nevertheless, electrophysiological recording showed that protection of lumbar motor networks by celastrol was rather limited as reflex activity was impaired and fictive locomotion largely depressed, suggesting that functional deficit persisted, though the networks could express slow rhythmic oscillations. While our data do not exclude further recovery at later times beyond the experimental observations, the present results indicate that the upregulated expression of HSP in the aftermath of acute injury may be an interesting avenue for early protection of spinal motoneurons.

Loss of inhibitory synapses causes locomotor network dysfunction of the rat spinal cord during prolonged maintenance in vitro.

The isolated spinal cord of the neonatal rat is widely employed to clarify the basic mechanisms of network development or the early phase of degeneration after injury. Nevertheless, this preparation survives in Krebs solution up to 24 h only, making it desirable to explore approaches to extend its survival for longitudinal studies. The present report shows that culturing the spinal cord in oxygenated enriched Basal Medium Eagle (BME) provided excellent preservation of neurons (including motoneurons), glia and primary afferents (including dorsal root ganglia) for up to 72 h. Using DMEM medium was unsuccessful. Novel characteristics of spinal networks emerged with strong spontaneous activity, and deficit in fictive locomotion patterns with stereotypically slow cycles. Staining with markers for synaptic proteins synapsin 1 and synaptophysin showed thoroughly weaker signal after 3 days in vitro. Immunohistochemical staining of markers for glutamatergic and glycinergic neurons indicated significant reduction of the latter. Likewise, there was lower expression of the GABA-synthesizing enzyme GAD65. Thus, malfunction of locomotor networks appeared related to loss of inhibitory synapses. This phenomenon did not occur in analogous opossum preparations of the spinal cord kept in vitro. In conclusion, despite histological data suggesting that cultured spinal cords were undamaged (except for inhibitory biomarkers), electrophysiological data revealed important functional impairment. Thus, the downregulation of inhibitory synapses may account for the progressive hyperexcitability of rat spinal networks despite apparently normal histological appearance. Our observations may help to understand the basis of certain delayed effects of spinal injury like chronic pain and spasticity.

Nicotine-mediated neuroprotection of rat spinal networks against excitotoxicity.

Activation of neuronal nicotinic acetylcholine receptors (nAChRs) by nicotine is reported to protect brain neurons from glutamate excitotoxicity. We inquired whether a similar phenomenon can occur in the rat isolated spinal cord (or spinal slice culture) challenged by a transient (1 hr) application of kainate (a powerful glutamate receptor agonist) to induce excitotoxicity mimicking spinal injury in vitro. We recorded spinal reflexes and fictive locomotion generated by the locomotor central pattern generator before and 24 hr after applying kainate. We also monitored network activity with Ca2+ imaging and counted neurons and glia with immunohistochemical methods. In control conditions, nicotine (1 µM; 4 hr) depressed reflexes and fictive locomotion with slow recovery and no apparent neurotoxicity at 24 hr although synchronous Ca2+ transients appeared in slice cultures. Kainate nearly halved neuron numbers (while sparing glia), decreased reflexes and Ca2+ transients, and suppressed fictive locomotion. When nicotine was applied (4 hr) after washout of kainate, fictive locomotor cycles appeared 24 hr later though with low periodicity, and significant protection of neurons, including motoneurons, was observed. Nicotine applied together with kainate and maintained for further 4 hr yielded better neuroprotection, improved fictive locomotion expression and reversed the depression of Ca2+ transients. nAChR antagonists did not intensify kainate neurotoxicity and inhibited the neuroprotective effects of nicotine. These data suggest that nicotine was efficacious to limit histological and functional excitotoxic damage probably because it activated and then desensitized nAChRs on excitatory and inhibitory network neurons to prevent triggering intracellular cell death pathways.

Functional up-regulation of the M-current by retigabine contrasts hyperexcitability and excitotoxicity on rat hypoglossal motoneurons.

KEY POINTS: Excessive neuronal excitability characterizes several neuropathological conditions, including neurodegenerative diseases such as amyotrophic lateral sclerosis. Hypoglossal motoneurons (HMs), which control tongue muscles, are extremely vulnerable to this disease and undergo damage and death when exposed to an excessive glutamate extracellular concentration that causes excitotoxicity. Our laboratory devised an in vitro model of excitotoxicity obtained by pharmacological blockade of glutamate transporters. In this paradigm, HMs display hyperexcitability, collective bursting and eventually cell death. The results of the present study show that pharmacological up-regulation of a K+ current (M-current), via application of the anti-convulsant retigabine, prevented all hallmarks of HM excitotoxicity, comprising bursting, generation of reactive oxygen species, expression of toxic markers and cell death. ○Our data may have translational value to develop new treatments against neurological diseases by using positive pharmacological modulators of the M-current. ABSTRACT: Neuronal hyperexcitability is a symptom characterizing several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). In the ALS bulbar form, hypoglossal motoneurons (HMs) are an early target for neurodegeneration because of their high vulnerability to metabolic insults. In recent years, our laboratory has developed an in vitro model of a brainstem slice comprising the hypoglossal nucleus in which HM neurodegeneration is achieved by blocking glutamate clearance with dl-threo-ß-benzyloxyaspartate (TBOA), thus leading to delayed excitotoxicity. During this process, HMs display a set of hallmarks such as hyperexcitability (and network bursting), reactive oxygen species (ROS) generation and, finally, cell death. The present study aimed to investigate whether blocking early hyperexcitability and bursting with the anti-convulsant drug retigabine was sufficient to achieve neuroprotection against excitotoxicity. Retigabine is a selective positive allosteric modulator of the M-current (IM ), an endogenous mechanism that neurons (comprising HMs) express to dampen excitability. Retigabine (10 µm; co-applied with TBOA) contrasted ROS generation, release of endogenous toxic factors into the HM cytoplasm and excitotoxicity-induced HM death. Electrophysiological experiments showed that retigabine readily contrasted and arrested bursting evoked by TBOA administration. Because neuronal IM subunits (Kv7.2, Kv7.3 and Kv7.5) were expressed in the hypoglossal nucleus and in functionally connected medullary nuclei, we suggest that they were responsible for the strong reduction in network excitability, a potent phenomenon for achieving neuroprotection against TBOA-induced excitotoxicity. The results of the present study may have translational value for testing novel positive pharmacological modulators of the IM under pathological conditions (including neurodegenerative disorders) characterized by excessive neuronal excitability.

Differential neuromodulatory role of endocannabinoids in the rodent trigeminal sensory ganglion and cerebral cortex relevant to pain processing.

Endocannabinoids are suggested to control pain, even though their clinical use is not fully validated and the underlying mechanisms are incompletely understood. To clarify the targets of endocannabinoid actions, we studied how activation of the endocannabinoid CB1 receptor (CB1R) affects neuronal responses in two in vitro preparations of rodents, namely the trigeminal sensory ganglion (TG) in culture and a coronal slice of the cerebral cortex. On TG small-medium size neurons, we tested whether submicromolar concentrations of the endogenous CB1R agonist anandamide (AEA) modulated inhibitory GABAA receptors and excitatory ATP-gated P2X3 receptors. AEA reversibly depressed GABA-mediated membrane currents without altering P2X3 receptor responses. The AEA antagonism was non-competitive, prevented by the CB1R antagonist AM-251, mimicked by the other cannabinoids 2-arachidonylglycerol and WIN 55,212-2, and insensitive to TRPV1 blocker capsazepine. AEA inhibited the potentiation of GABAergic responses by the cAMP activator forskolin, in line with the canonical inhibition of cAMP synthesis by CB1Rs. In the cerebral cortex, AEA or WIN 55,212-2 did not affect electrically-evoked local field potentials or characteristics of cortical spreading depolarization (CSD) elicited by high potassium application. The GABAA receptor blocker gabazine, however, strongly enhanced field potentials without affecting CSD properties, suggesting that CSD was not dominantly controlled by GABAergic mechanisms. Our data propose that, despite the widespread expression of CB1Rs peripherally and centrally, the functional effects of AEA are region-specific and depend on CB1R coupling to downstream effectors. Future studies concerned with the mechanisms of AEA analgesia should perhaps be directed to discrete subcortical nuclei processing trigeminal inputs.

Propofol Protects Rat Hypoglossal Motoneurons in an In Vitro Model of Excitotoxicity by Boosting GABAergic Inhibition and Reducing Oxidative Stress.

In brainstem motor networks, hypoglossal motoneurons (HMs) play the physiological role of driving tongue contraction, an activity critical for inspiration, phonation, chewing and swallowing. HMs are an early target of neurodegenerative diseases like amyotrophic lateral sclerosis that, in its bulbar form, is manifested with initial dysphagia and dysarthria. One important pathogenetic component of this disease is the high level of extracellular glutamate due to uptake block that generates excitotoxicity. To understand the earliest phases of this condition we devised a model, the rat brainstem slice, in which block of glutamate uptake is associated with intense bursting of HMs, dysmetabolism and death. Since blocking bursting becomes a goal to prevent cell damage, the present report enquired whether boosting GABAergic inhibition could fulfill this aim and confer beneficial outcome. Propofol (0.5 µM) and midazolam (0.01 µM), two allosteric modulators of GABAA receptors, were used at concentrations yielding analogous potentiation of GABA-mediated currents. Propofol also partly depressed NMDA receptor currents. Both drugs significantly shortened bursting episodes without changing single burst properties, their synchronicity, or their occurrence. Two hours later, propofol prevented the rise in reactive oxygen species (ROS) and, at 4 hours, it inhibited intracellular release of apoptosis-inducing factor (AIF) and prevented concomitant cell loss. Midazolam did not contrast ROS and AIF release. The present work provides experimental evidence for the neuroprotective action of a general anesthetic like propofol, which, in this case, may be achieved through a combination of boosted GABAergic inhibition and reduced ROS production.

In situ imaging reveals properties of purinergic signalling in trigeminal sensory ganglia in vitro.

Chronic pain is supported by sterile inflammation that induces sensitisation of sensory neurons to ambient stimuli including extracellular ATP acting on purinergic P2X receptors. The development of in vitro methods for drug screening would be useful to investigate cell crosstalk and plasticity mechanisms occurring during neuronal sensitisation and sterile neuroinflammation. Thus, we studied, at single-cell level, membrane pore dilation based on the uptake of a fluorescent probe following sustained ATP-gated P2X receptor function in neurons and non-neuronal cells of trigeminal ganglion cultures from wild-type (WT) and R192Q CaV2.1 knock-in (KI) mice, a model of familial hemiplegic migraine type 1 characterised by neuronal sensitisation and higher release of soluble mediators. In WT cultures, pore responses were mainly evoked by ATP rather than benzoyl-ATP (BzATP) and partly inhibited by the P2X antagonist TNP-ATP. P2X7 receptors were expressed in trigeminal ganglia mainly by non-neuronal cells. In contrast, KI cultures showed higher expression of P2X7 receptors, stronger responses to BzATP, an effect largely prevented by prior administration of CaV2.1 blocker ω-agatoxin IVA, small interfering RNA (siRNA)-based silencing of P2X7 receptors or the P2X7 antagonist A-804598. No cell toxicity was detected with the protocols. Calcitonin gene-related peptide (CGRP), a well-known migraine mediator, potentiated BzATP-evoked membrane permeability in WT as well as R192Q KI cultures, demonstrating its modulatory role on trigeminal sensory ganglia. Our results show an advantageous experimental approach to dissect pharmacological properties potentially relevant to chronic pain and suggest that CGRP is a soluble mediator influencing purinergic P2X pore dilation and regulating inflammatory responses.

Neurotoxicity of propofol on rat hypoglossal motoneurons in vitro.

Although propofol is a widely used intravenous general anaesthetic, many studies report its toxic potential, particularly on the developing central nervous system. We investigated its action on hypoglossal motoneurons (HMs) that control two critical functions in neonates, namely tongue muscle activity and airway patency. Thus, clinically relevant concentrations of propofol (1 and 5µM) were applied (4h) to neonatal rat brainstem slices to evaluate the expression of apoptosis-inducing factor (AIF) as biomarker of toxicity. This anaesthetic strongly increased AIF in the cytoplasm and the nucleus, without early loss of HMs. Electrophysiological recordings from HMs showed that propofol (5µM) enhanced GABA- and glycine-evoked current amplitude and lengthened GABAergic current decay time. Propofol also depressed NMDA receptor-mediated responses without affecting AMPA receptors. Since GABA and glycine depolarize neonatal HMs, we propose that the damaging action by propofol on these motoneurons might arise from the facilitated action of these transmitters with subsequent cytoplasmic Ca2+ overload. This phenomenon, in turn, may trigger cell death mechanisms manifested as increased expression of AIF and its translocation into the nucleus. Since propofol is also employed for induction and maintenance of paediatric surgery, caution is needed because its potential neurotoxicity might negatively impact neurodevelopment.

Nicotine protects rat hypoglossal motoneurons from excitotoxic death via downregulation of connexin 36.

Motoneuron disease including amyotrophic lateral sclerosis may be due, at an early stage, to deficit in the extracellular clearance of the excitatory transmitter glutamate. A model of glutamate-mediated excitotoxic cell death based on pharmacological inhibition of its uptake was used to investigate how activation of neuronal nicotinic receptors by nicotine may protect motoneurons. Hypoglossal motoneurons (HMs) in neonatal rat brainstem slices were exposed to the glutamate uptake blocker DL-threo-ß-benzyloxyaspartate (TBOA) that evoked large Ca2+ transients time locked among nearby HMs, whose number fell by about 30% 4 h later. As nicotine or the gap junction blocker carbenoxolone suppressed bursting, we studied connexin 36 (Cx36), which constitutes gap junctions in neurons and found it largely expressed by HMs. Cx36 was downregulated when nicotine or carbenoxolone was co-applied with TBOA. Expression of Cx36 was preferentially observed in cytosolic rather than membrane fractions after nicotine and TBOA, suggesting protein redistribution with no change in synthesis. Nicotine raised the expression of heat shock protein 70 (Hsp70), a protective factor that binds the apoptotic-inducing factor (AIF) whose nuclear translocation is a cause of cell death. TBOA increased intracellular AIF, an effect blocked by nicotine. These results indicate that activation of neuronal nicotinic receptors is an early tool for protecting motoneurons from excitotoxicity and that this process is carried out via the combined decrease in Cx36 activity, overexpression of Hsp70 and fall in AIF translocation. Thus, retarding or inhibiting HM death may be experimentally achieved by targeting one of these processes leading to motoneuron death.

Hyperpolarization-activated current Ih in mouse trigeminal sensory neurons in a transgenic mouse model of familial hemiplegic migraine type-1.

Transgenic knock-in (KI) mice that express CaV2.1 channels containing an R192Q gain-of-function mutation in the α1A subunit known to cause familial hemiplegic migraine type-1 in patients, exhibit key disease characteristics and provide a useful tool to investigate pathophysiological mechanisms of pain transduction. Previously, KI trigeminal sensory neurons were shown to exhibit constitutive hyperexcitability due to up-regulation of ATP-gated P2X3 receptors that trigger spike activity at a more negative threshold. This implies that intrinsic neuronal conductances may shape action potential generation in response to ATP, which could act as a mediator of migraine headache. Here we investigated whether the hyperpolarization-activated conductance Ih, mediated by hyperpolarization activated cyclic nucleotide-gated channels (HCN), contributes to sub-threshold behavior and firing in wild-type (WT) and KI trigeminal ganglia (TG) neurons. Whereas most WT and KI trigeminal neurons expressed Ih current, blocked by the specific inhibitor ZD7288, it was smaller in KI neurons despite similar activation and deactivation kinetics. HCN1 and HCN2 were the most abundantly expressed subunits in TG, both in situ and in culture. In KI TG neurons, HCN2 subunits were predominantly present in the cytoplasm, not at the plasma membrane, likely accounting for the smaller Ih of such cells. ZD7288 hyperpolarized the membrane potential, thereby raising the firing threshold, and prolonging the spike trajectory to generate fewer spikes due to P2X3 receptor activation. The low amplitude of Ih in KI TG neurons suggests that down-regulation of Ih current in sub-threshold behavior acts as a compensatory mechanism to limit sensory hyperexcitability, manifested under certain stressful stimuli.

Ceftriaxone-mediated upregulation of the glutamate transporter GLT-1 contrasts neurotoxicity evoked by kainate in rat organotypic spinal cord cultures.

Excitotoxicity is a major pathological trigger of neurodegenerative diseases like amyotrophic lateral sclerosis. This process is caused by excessive release of the transmitter glutamate that overwhelms the capacity of astroglia transporters to maintain a low extracellular level of this aminoacid and strongly stimulates neurons. Using an in vitro model of rat organotypic spinal slice culture, we explored if the excitotoxicity caused by the potent glutamate analogue kainate, widely employed as a paradigm to evoke neurotoxicity in the central nervous system, was prevented by the antibiotic ceftriaxone known to enhance glutamate transporter expression. We also tested if excitotoxicity was made worse by inhibiting glutamate uptake with dl-threo-ß-benzyloxyaspartate (TBOA). These experiments were aimed at clarifying the relative contribution to neurotoxicity by kainate-activation of glutamate receptors or kainate-mediated release of glutamate. Neither ceftriaxone nor TBOA alone had adverse effects. Ceftriaxone (10µM; 3days) significantly decreased delayed cell death induced by kainate (100µM; 1h) and limited neuronal damage especially to motoneurons. This effect was associated to stronger astrocytic immunostaining of the glutamate transporter GLT-1. Conversely, pharmacological inhibition of glutamate uptake with TBOA was per se unable to induce neurotoxicity, yet it intensified cell death evoked by kainate. These data indicate that kainate-mediated glutamate release was critical to damage neurons, an effect prevented by up regulating glutamate uptake. These data suggest that modulating glutamate uptake is an important strategy to preserve neuronal networks.

Electrophysiological characterization of the M-current in rat hypoglossal motoneurons.

The M-current (IM) is a voltage-dependent, persistent K+ current so termed because it is strongly inhibited by the cholinergic agonist muscarine. The IM main function is to limit neuronal excitability by contrasting action potential firing. Although motoneurons are sensitive to acetylcholine, the role of IM in modulating their excitability is still controversial. The aim of the present report was to examine the presence of IM in hypoglossal motoneurons (HMs) and its role in the modulation of firing properties using an in vitro model of rat brainstem slice. For this purpose, we employed the whole-cell patch-clamp technique to record HM responses upon stimulation with either a standard IM deactivation voltage protocol or depolarizing current steps. Voltage commands from depolarized potential induced inward relaxations with the common characteristics of IM, comprising inhibition by either muscarine (10µM) or the selective IM inhibitor linopirdine (30µM). IM was pharmacologically distinguished from the hyperpolarization-activated inward-rectifying current and, within the -20 to -50mV range, deactivated with >100-ms time constant. Current-clamp experiments demonstrated that IM strongly regulated HM action potential firing, since both muscarine and linopirdine increased spike frequency whereas the M-channel opener retigabine (20µM) reduced it. Conversely, IM seemed uninvolved in the generation of the medium afterhyperpolarizing potential. Our results suggest that HMs possess IM, whose pharmacological modulation is an important tool to up- or down-regulate excitability, to be explored in experimental models of neurodegeneration.