Pascal Fossat

Glial D-Serine Gates NMDA Receptors at Excitatory Synapses in Prefrontal Cortex

N-methyl-D-aspartate receptors (NMDARs) subserve numerous neurophysiological and neuropathological processes in the cerebral cortex. Their activation requires the binding of glutamate and also of a coagonist. Whereas glycine and D-serine (D-ser) are candidates for such a role at central synapses, the nature of the coagonist in cerebral cortex remains unknown. We first show that the glycine-binding site of NMDARs is not saturated in acute slices preparations of medial prefrontal cortex (mPFC). Using enzymes that selectively degrade either D-ser or glycine, we demonstrate that under the present conditions, D-ser is the principle endogenous coagonist of synaptic NMDARs at mature excitatory synapses in layers V/VI of mPFC where it is essential for long-term potentiation (LTP) induction. Furthermore, blocking the activity of glia with the metabolic inhibitor, fluoroacetate, impairs NMDAR-mediated synaptic transmission and prevents LTP induction by reducing the extracellular levels of D-serine. Such deficits can be restored by exogenous D-ser, indicating that the D-amino acid mainly originates from glia in the mPFC, as further confirmed by double-immunostaining studies for D-ser and anti-glial fibrillary acidic protein. Our findings suggest that D-ser modulates neuronal networks in the cerebral cortex by gating the activity of NMDARs and that altering its levels is relevant to the induction and potentially treatment of psychiatric and neurological disorders.

Anxiety-like behavior in crayfish is controlled by serotonin

The crayfish that was afraid of the dark We tend to assume that complex emotions, such as anxiety, only occur in mammals or other cognitively complex vertebrates. But a heightened sense of awareness and the avoidance of novel or dangerous environments could be helpful for any animal species. Fossat et al. show that crayfish exposed to a stressful electric field refuse to enter dark arms in a light/dark maze, even after the electric field has been removed. The animals calmed down when they were injected with an anxiolytic drug used to treat anxiety in humans, and they entered the dark as normal. The stressed animals had increased levels of the neurotransmitter serotonin in the brain, and injections of serotonin induced anxiety-like behavior in control animals. Thus, these invertebrates display a primitive form of anxiety that shares a mechanism with the more complex emotions displayed by vertebrates. Science, this issue p. 1293 Crayfish respond to stress with an apparent fear of the dark that can be abolished with an anxiolytic drug. Anxiety, a behavioral consequence of stress, has been characterized in humans and some vertebrates, but not invertebrates. Here, we demonstrate that after exposure to stress, crayfish sustainably avoided the aversive illuminated arms of an aquatic plus-maze. This behavior was correlated with an increase in brain serotonin and was abolished by the injection of the benzodiazepine anxiolytic chlordiazepoxide. Serotonin injection into unstressed crayfish induced avoidance; again, this effect was reversed by injection with chlordiazepoxide. Our results demonstrate that crayfish exhibit a form of anxiety similar to that described in vertebrates, suggesting the conservation of several underlying mechanisms during evolution. Analyses of this ancestral behavior in a simple model reveal a new route to understanding anxiety and may alter our conceptions of the emotional status of invertebrates.

Knockdown of L Calcium Channel Subtypes: Differential Effects in Neuropathic Pain

The maintenance of chronic pain states requires the regulation of gene expression, which relies on an influx of calcium. Calcium influx through neuronal L-type voltage-gated calcium channels (LTCs) plays a pivotal role in excitation–transcription coupling, but the involvement of LTCs in chronic pain remains unclear. We used a peptide nucleic acid (transportan 10-PNA conjugates)-based antisense strategy to investigate the role of the LTC subtypes CaV1.2 and CaV1.3 in long-term pain sensitization in a rat model of neuropathy (spinal nerve ligation). Our results demonstrate that specific knockdown of CaV1.2 in the spinal dorsal horn reversed the neuropathy-associated mechanical hypersensitivity and the hyperexcitability and increased responsiveness of dorsal horn neurons. Intrathecal application of anti-CaV1.2 siRNAs confirmed the preceding results. We also demonstrated an upregulation of CaV1.2 mRNA and protein in neuropathic animals concomitant to specific CaV1.2-dependent phosphorylation of the cAMP response element (CRE)-binding protein (CREB) transcription factor. Moreover, spinal nerve ligation animals showed enhanced transcription of the CREB/CRE-dependent gene COX-2 (cyclooxygenase 2), which also depends strictly on CaV1.2 activation. We propose that L-type calcium channels in the spinal dorsal horn play an important role in pain processing, and that the maintenance of chronic neuropathic pain depends specifically on channels comprising CaV1.2.

L‐type calcium channels and NMDA receptors: a determinant duo for short‐term nociceptive plasticity

In the dorsal horn of the spinal cord, pain‐transmitting neurons exhibit action potential windup, a form of short‐term plasticity, which consists of a progressive increase in neuronal response during repetitive stimulation of nociceptive input fibers. Windup depends on N‐methyl‐d‐aspartate (NMDA) receptor activation, but previous in vitro studies indicated that windup also relies on intrinsic plateau properties of spinal neurons. In the present study, we considered the possible involvement of these properties in windup in vivo. For this purpose, we first studied a nociceptive flexion reflex in the rat. We showed that windup of the reflex is actually suppressed by blockers of L‐type calcium current and Ca2+‐activated non‐specific cationic current (Ican), the two main depolarizing conductances of plateau potentials. We further showed that, during windup, NMDA receptors provide a critical excitatory component in a dynamic balance of excitatory and inhibitory inputs which ultimately activates L‐type calcium channels. The nociceptive reflex involves at least two neuronal groups, which may express intrinsic amplification properties, motor neurons and dorsal horn neurons. By means of extracellular recordings in the dorsal horn, we showed that windup of dorsal horn neuron discharge was sensitive to the modulators of L‐type calcium current. Altogether, our results suggest that, in vivo, windup also depends on the amplification properties of spinal neurons, the triggering of which requires previous activation of NMDA receptors.

Impairment of GABAB receptor dimer by endogenous 14‐3‐3ζ in chronic pain conditions

In the central nervous system, the inhibitory GABAB receptor is the archetype of heterodimeric G protein‐coupled receptors (GPCRs). However, the regulation of GABAB dimerization, and more generally of GPCR oligomerization, remains largely unknown. We propose a novel mechanism for inhibition of GPCR activity through de‐dimerization in pathological conditions. We show here that 14‐3‐3ζ, a GABAB1‐binding protein, dissociates the GABAB heterodimer, resulting in the impairment of GABAB signalling in spinal neurons. In the dorsal spinal cord of neuropathic rats, 14‐3‐3ζ is overexpressed and weakens GABAB inhibition. Using anti‐14‐3‐3ζ siRNA or competing peptides disrupts 14‐3‐3ζ/GABAB1 interaction and restores functional GABAB heterodimers in the dorsal horn. Importantly, both strategies greatly enhance the anti‐nociceptive effect of intrathecal Baclofen in neuropathic rats. Taken together, our data provide the first example of endogenous regulation of a GPCR oligomeric state and demonstrate its functional impact on the pathophysiological process of neuropathic pain sensitization.

Glia-Dependent Switch of Kainate Receptor Presynaptic Action

Presynaptic kainate receptors (KARs) exert a modulatory action on transmitter release. This effect can be switched from facilitation to inhibition by an increased concentration of KAR agonists. We here report that activation of presynaptic GluK1-containing KARs facilitates GABA release on oxytocin and vasopressin neurons in the supraoptic nucleus of the hypothalamus. Increase in ambient levels of glutamate associated with the physiological reduction of astrocytic coverage of oxytocin neurons in lactating rats switches this KAR-mediated facilitation to inhibition of GABAergic transmission. This effect was reproduced in both oxytocin and vasopressin neurons of virgin rats when glutamate transporters were blocked pharmacologically, thereby establishing that enhanced levels of extracellular glutamate induce the switch in KAR-mediated action. The facilitation of GABA release was inhibited with philanthotoxin, a Ca2+-permeable KAR antagonist, suggesting that this effect was associated with an ionotropic mode of action. Conversely, KAR-mediated inhibition was compromised in the presence of U73122, a phospholipase C inhibitor, in agreement with the involvement of a metabotropic pathway. We thus reveal that physiological astrocytic plasticity modifies the mode of action of presynaptic KARs, thereby inversing their coupling with GABA release.

Serotonin, but not dopamine, controls the stress response and anxiety-like behavior in the crayfish Procambarus clarkii

ABSTRACT In the animal kingdom, biogenic amines are widespread modulators of the nervous system that frequently interact to control mood. Our previous investigations in crayfish (Procambarus clarkii) have established that stress induces changes in brain serotonin (5-HT) concentrations that are responsible for the appearance of anxiety-like behavior (ALB). Here, we further analyze the roles of 5-HT and another biogenic amine, dopamine (DA), on the crayfish response to stress. We show that the intensity of crayfish ALB depends on the intensity of stressful stimulation and is associated with increased concentrations of 5-HT in the brain. These 5-HT levels were significantly correlated, before, as well as after stress, with those of DA, which were approximately 3- to 5-times less abundant. However, whereas the degree of ALB was clearly correlated with brain 5-HT concentrations, it was not significantly correlated with DA. Moreover, in contrast to injections of 5-HT, DA injections were not able to elicit a stress response or ALB. In addition, 5-HT and DA levels were not modified by treatment with the anxiolytic chlordiazepoxide, confirming that suppression of ALB by this GABA-A receptor ligand acts downstream and is independent of changes in crayfish bioamine levels. Our study also provides evidence that the anxiogenic effect of 5-HT injections can be prevented by a preliminary injection of 5-HT antagonists. Altogether, our results emphasize that the rises in brain concentrations of 5-HT, but not DA, play a role in controlling the induction and the intensity of crayfish ALB. Summary: After stress, crayfish brain 5-HT levels increase, which induces anxiety-like behavior (ALB) but the level of dopamine does not change, and in contrast to 5-HT, injection of dopamine does not trigger a metabolic response or ALB.

Intrinsic membrane properties of spinal dorsal horn neurones modulate nociceptive information processing in vivo

Non‐technical summary  The dorsal horn of the spinal cord is the first site in the central nervous system where painful sensory information is processed before transmission to the brain. In vitro recordings in spinal slices established that this processing relies on both plasticity of synaptic connections and intrinsic electrical properties of dorsal horn neurones (DHNs). DHNs may generate plateau potentials, which underlie intense discharges and long‐lasting after‐discharges in response to a brief stimulation, and represent a putative endogenous mechanism for amplification of painful sensory inputs. Using patch‐clamp recordings in the anaesthetized adult rat, we show that DHNs do generate plateau potentials in vivo, which shape their responses to natural sensory stimulation. Moreover, we give direct evidence for the involvement of these amplification properties in both short‐term (windup) and long‐term sensitisation associated with neuropathic pain, raising the possibility that plateau potentials could be putative therapeutic targets to control spinal component of neuropathic pain.

Cav1.2 and Cav1.3 L‐type calcium channels independently control short‐ and long‐term sensitization to pain

L‐type calcium channels in the CNS exist as two subunit forming channels, Cav1.2 and Cav1.3, which are involved in short‐ and long‐term plasticity. We demonstrate that Cav1.3 but not Cav1.2 is essential for wind‐up. These results identify Cav1.3 as a key conductance responsible for short‐term sensitization in physiological pain transmission. We confirm the role of Cav1.2 in a model of long‐term plasticity associated with neuropathic pain. Up‐regulation of Cav1.2 and down‐regultation of Cav1.3 in neuropathic pain underlies the switch from physiology to pathology. Finally, the results of the present study reveal that therapeutic targeting molecular pathways involved in wind‐up may be not relevant in the treatment of neuropathy.

Extracellular signal‐regulated kinase phosphorylation in forebrain neurones contributes to osmoregulatory mechanisms

The mechanisms of osmotically induced vasopressin secretion from the hypothalamic magnocellular neurosecretory cells, which is crucial for body fluid homeostasis, are not yet fully understood. Extracellular signal‐regulated protein kinases (ERK) are mitogen‐activated protein kinases that transduce extracellular stimuli into intracellular post‐translational and transcriptional responses and might be involved in the regulation of vasopressin release in response to changes in osmolality. We found that ERK was dose‐dependently activated (phosphorylated) in the rat osmosensitive forebrain regions, including magnocellular neurosecretory cells, by increases in osmolality induced by hypertonic solutions. Inhibition of ERK phosphorylation reduced hypertonically induced activation of osmosensitive forebrain neurones and vasopressin release. Our results identify ERK activation as a new element contributing to the osmoregulatory mechanisms of vasopressin release.