Giuliano Taccola

Newborn Analgesia Mediated by Oxytocin during Delivery

The mechanisms controlling pain in newborns during delivery are poorly understood. We explored the hypothesis that oxytocin, an essential hormone for labor and a powerful neuromodulator, exerts analgesic actions on newborns during delivery. Using a thermal tail-flick assay, we report that pain sensitivity is two-fold lower in rat pups immediately after birth than 2 days later. Oxytocin receptor antagonists strongly enhanced pain sensitivity in newborn, but not in 2-day-old rats, whereas oxytocin reduced pain at both ages suggesting an endogenous analgesia by oxytocin during delivery. Similar analgesic effects of oxytocin, measured as attenuation of pain-vocalization induced by electrical whisker pad stimulation, were also observed in decerebrated newborns. Oxytocin reduced GABA-evoked calcium responses and depolarizing GABA driving force in isolated neonatal trigeminal neurons suggesting that oxytocin effects are mediated by alterations of intracellular chloride. Unlike GABA signaling, oxytocin did not affect responses mediated by P2X3 and TRPV1 receptors. In keeping with a GABAergic mechanism, reduction of intracellular chloride by the diuretic NKCC1 chloride co-transporter antagonist bumetanide mimicked the analgesic actions of oxytocin and its effects on GABA responses in nociceptive neurons. Therefore, endogenous oxytocin exerts an analgesic action in newborn pups that involves a reduction of the depolarizing action of GABA on nociceptive neurons. Therefore, the same hormone that triggers delivery also acts as a natural pain killer revealing a novel facet of the protective actions of oxytocin in the fetus at birth.

Tuning and playing a motor rhythm: how metabotropic glutamate receptors orchestrate generation of motor patterns in the mammalian central nervous system

Repeated motor activities like locomotion, mastication and respiration need rhythmic discharges of functionally connected neurons termed central pattern generators (CPGs) that cyclically activate motoneurons even in the absence of descending commands from higher centres. For motor pattern generation, CPGs require integration of multiple processes including activation of ion channels and transmitter receptors at strategic locations within motor networks. One emerging mechanism is activation of glutamate metabotropic receptors (mGluRs) belonging to group I, while group II and III mGluRs appear to play an inhibitory function on sensory inputs. Group I mGluRs generate neuronal membrane depolarization with input resistance increase and rapid fluctuations in intracellular Ca2+, leading to enhanced excitability and rhythmicity. While synchronicity is probably due to modulation of inhibitory synaptic transmission, these oscillations occurring in coincidence with strong afferent stimuli or application of excitatory agents can trigger locomotor‐like patterns. Hence, mGluR‐sensitive spinal oscillators play a role in accessory networks for locomotor CPG activation. In brainstem networks supplying tongue muscle motoneurons, group I receptors facilitate excitatory synaptic inputs and evoke synchronous oscillations which stabilize motoneuron firing at regular, low frequency necessary for rhythmic tongue contractions. In this case, synchronicity depends on the strong electrical coupling amongst motoneurons rather than inhibitory transmission, while cyclic activation of KATP conductances sets its periodicity. Activation of mGluRs is therefore a powerful strategy to trigger and recruit patterned discharges of motoneurons.

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.

ERG conductance expression modulates the excitability of ventral horn GABAergic interneurons that control rhythmic oscillations in the developing mouse spinal cord

During antenatal development, the operation and maturation of mammalian spinal networks strongly depend on the activity of ventral horn GABAergic interneurons that mediate excitation first and inhibition later. Although the functional consequence of GABA actions may depend on maturational processes in target neurons, it is also likely that evolving changes in GABAergic transmission require fine-tuning in GABA release, probably via certain intrinsic mechanisms regulating GABAergic neuron excitability at different embryonic stages. Nevertheless, it has not been possible, to date, to identify certain ionic conductances upregulated or downregulated before birth in such cells. By using an experimental model with either mouse organotypic spinal cultures or isolated spinal cord preparations, the present study examined the role of the ERG current (IK(ERG)), a potassium conductance expressed by developing, GABA-immunoreactive spinal neurons. In organotypic cultures, only ventral interneurons with fast adaptation and GABA immunoreactivity, and only after 1 week in culture, were transformed into high-frequency bursters by E4031, a selective inhibitor of IK(ERG) that also prolonged and made more regular spontaneous bursts. In the isolated spinal cord in which GABA immunoreactivity and m-erg mRNA were colocalized in interneurons, ventral root rhythms evoked by NMDA plus 5-hydroxytryptamine were stabilized and synchronized by E4031. All of these effects were lost after 2 weeks in culture or before birth in coincidence with decreased m-erg expression. These data suggest that, during an early stage of spinal cord development, the excitability of GABAergic ventral interneurons important for circuit maturation depended, at least in part, on the function of IK(ERG).

Modulation of rhythmic patterns and cumulative depolarization by group I metabotropic glutamate receptors in the neonatal rat spinal cord in vitro

The role of group I metabotropic glutamate receptors (mGluRs), and their subtypes 1 or 5, in rhythmic patterns generated by the neonatal rat spinal cord was investigated. Fictive locomotor patterns induced by N‐methyl‐d‐aspartate + serotonin were slowed down by the subtype 1 antagonists (RS)‐1‐aminoindan‐1,5‐dicarboxylic acid (AIDA) or 7‐(hydroxyimino)cyclopropa[b]chromen‐1a‐carboxylate ethyl ester (CPCCOEt) and unaffected by the subtype 5 antagonist 2‐methyl‐6‐(phenylethynyl)pyridine hydrochloride (MPEP). The group I agonist (RS)‐3,5‐dihydroxyphenylglycine (DHPG) depolarized ventral roots and disrupted fictive locomotion, an effect blocked by AIDA (or CPCCOEt) and reversed by increasing the N‐methyl‐d‐aspartate concentration. Cumulative depolarization induced by low frequency trains of dorsal root stimuli was attenuated by DHPG and unchanged by AIDA or MPEP while rhythmic patterns or motoneuron spike wind‐up persisted. Disinhibited bursting induced by strychnine + bicuculline was accelerated by DHPG, slowed down by AIDA (which prevented the action of DHPG), unaffected by MPEP and counteracted by the selective group II agonist (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine. The DHPG transformed regular bursting into arrhythmic bursting, a phenomenon also produced by the group II mGluR antagonist (2S)‐α‐ethylglutamic acid. These results indicate that, during fictive locomotion or disinhibited bursting, endogenous glutamate could activate discrete clusters of subtype 1 mGluRs to facilitate discharges. Diffuse activation by the exogenous agonist DHPG of group I mGluRs throughout spinal networks had an excitatory effect overshadowed by its much stronger depressant action due to concomitant facilitation of glycinergic transmission. Irregular disinhibited bursting caused by activation of subtype 1 receptors or block of group II receptors suggests that mGluRs could control not only the frequency but also the periodicity of bursting patterns, outlining novel mechanisms contributing to burst duration.

Distinct subtypes of group I metabotropic glutamate receptors on rat spinal neurons mediate complex facilitatory and inhibitory effects

While group I glutamate metabotropic (mGlu) receptors show discrete neuronal distribution in the neonatal rat spinal cord, the functional role of their distinct receptor subtypes remains uncertain. Intracellular recording from lumbar motoneurons together with extracellular recording of ventral root (VR) responses was used to investigate the differential contribution by mGlu receptor subtypes to cell excitability and network activity. The group I agonist DHPG evoked motoneuron depolarization (via the AIDA or CPCCOEt‐sensitive mGlu receptor subtype 1) mainly at network level and generated sustained, network‐dependent oscillations (via the MPEP‐sensitive mGlu receptor subtype 5). DHPG also decreased the peak amplitude of synaptic responses induced by dorsal root stimuli, an effect unrelated to depolarization and dependent on glycinergic transmission. Synaptic responses were insensitive to AIDA or MPEP. The present results can be explained by assuming excitation of discrete classes of interneurons by group I mGlu receptor activity. Thus, the cellular distribution of those mGlu receptors at strategic circuit connections may determine the functional outcome of the network in terms of excitation or inhibition. Even if there was insufficient activation by endogenous glutamate of mGlu receptors during synaptic activity evoked by DR stimuli, it is apparent that such receptors are important pharmacological targets for powerful and rapid up‐ or down‐regulation of spinal signal processing at network level, providing a rationale for the proposed use of mGlu receptor agonists in a variety of spinal pathological conditions.

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.

Anoxic persistence of lumbar respiratory bursts and block of lumbar locomotion in newborn rat brainstem–spinal cords

The tolerance of breathing in neonates to oxygen depletion is reflected by persistence of inspiratory‐related motor output during sustained anoxia in newborn rat brainstem preparations. It is not known whether lumbar motor networks innervating expiratory abdominal muscles are, in contrast, inhibited by anoxia similar to locomotor networks in neonatal mouse lumbar cords. To test this, we recorded inspiratory‐related cervical/hypoglossal plus pre/postinspiratory lumbar/facial nerve activities and, sometimes simultaneously, locomotor rhythms in newborn rat brainstem–spinal cords. Chemical anoxia slowed 1 : 1‐coupled cervical and lumbar respiratory rhythms and induced cervical burst doublets associated with depressed preinspiratory and augmented postinspiratory lumbar activities. Similarly, anoxia evoked repetitive hypoglossal bursts and shifted facial activity toward augmented postinspiratory bursting in medullas without spinal cord. Selective lumbar anoxia augmented pre/postinspiratory lumbar bursting without slowing the rhythm. This suggests a medullary origin of both anoxic inspiratory double bursts and preinspiratory depression, but a mixed medullary/lumbar origin of boosted postinspiratory lumbar activity. Lumbar respiratory rhythm is likely to be generated by the parafacial respiratory group expiratory centre as indicated by lack of normoxic and anoxic bursting following brainstem transection between the facial motonucleus and the more caudal pre‐Bötzinger complex inspiratory centre. Opposed to sustained respiratory activities, anoxia reversibly abolished non‐rhythmic spinal discharges and electrically or chemically evoked lumbar locomotor activities, followed by pronounced postanoxic spinal hyperexcitability. We hypothesize that (i) the anoxia tolerance of neonatal breathing includes pFRG‐driven lumbar expiratory networks, (ii) the anoxic respiratory pattern transformation is due to disturbed inspiratory–expiratory centre interactions, and (iii) postanoxic lumbar hyperexcitability contributes to spasticity in cerebral palsy.

Dynamics of early locomotor network dysfunction following a focal lesion in an in vitro model of spinal injury.

It is unclear how a localized spinal cord injury may acutely affect locomotor networks of segments initially spared by the lesion. To investigate the process of secondary damage following spinal injury, we used the in vitro model of the neonatal rat isolated spinal cord with transverse barriers at the low thoracic–upper lumbar region to allow focal application of kainate in hypoxic and aglycemic solution (with reactive oxygen species). The time‐course and nature of changes in spinal locomotor networks downstream of the lesion site were investigated over the first 24 h, with electrophysiological recordings monitoring fictive locomotion (alternating oscillations between flexor and extensor motor pools on either side) and correlating any deficit with histological alterations. The toxic solution irreversibly suppressed synaptic transmission within barriers without blocking spinal reflexes outside. This effect was focally associated with extensive white matter damage and ventral gray neuronal loss. Although cell losses were < 10% outside barriers, microglial activation with neuronal phagocytosis was detected. Downstream motor networks still generated locomotor activity 24 h later when stimulated with N‐methyl‐d‐aspartate (NMDA) and serotonin, but not with repeated dorsal root stimuli. In the latter case, cumulative depolarization was recorded from ventral roots at a slower rate of rise, suggesting failure to recruit network premotoneurons. Our data indicate that, within the first 24 h of injury, locomotor networks below the lesion remained morphologically intact and functional when stimulated by NMDA and serotonin. Nevertheless, microglial activation and inability to produce locomotor patterns by dorsal afferent stimuli suggest important challenges to long‐term network operation.

Low micromolar concentrations of 4-aminopyridine facilitate fictive locomotion expressed by the rat spinal cord in vitro.

Upregulating the operation of spinal locomotor networks is one mechanism to restore, at least partially, lesion-impaired locomotion. We investigated if the K+ channel blocker 4-aminopyridine (4-AP) could facilitate spinal locomotor networks in addition to its well-known effect on motor nerve conduction. Fictive locomotor patterns were recorded from ventral roots (VRs) of the isolated spinal cord of the neonatal rat. 4-AP (0.1-50 microM) produced synchronous VR oscillations which did not develop into fictive locomotion. These oscillations had network origin, required intact glutamatergic transmission and were probably amplified via electrotonic coupling because of their depression by the selective gap junction blocker carbenoxolone. 4-AP (5 microM) slightly increased input resistance of lumbar motoneurons without affecting their action or resting potentials. Dorsal root (DR) evoked synaptic responses were enhanced (217 +/- 65%) by 5 microM 4-AP without changes in axon conduction. 4-AP (5 microM) accelerated fictive locomotion induced by N-methyl-d-aspartate (NMDA) and serotonin (5-HT) without altering cycle amplitude and facilitated the onset of fictive locomotion in the presence of sub-threshold concentrations of NMDA and 5-HT. Furthermore, in the presence of 4-AP, weak DR stimuli, previously insufficient to activate locomotor patterns, generated alternating VR discharges. Thus, although 4-AP per se could not directly activate the locomotor network of the spinal cord, it could strongly facilitate the locomotor program initiated by neurochemicals or electrical stimuli. These data suggest that the reported improvement by 4-AP in locomotor activity of spinal-injury patients may include activation of locomotor networks when low concentrations of this drug are administered in coincidence with appropriate stimuli.