Axonal Activity In Vivo: Technical Considerations and Implications for the Exploration of Neural Circuits in Freely Moving Animals

While extracellular somatic action potentials from freely moving rats have been well characterized, axonal activity has not. We have recently reported extracellular tetrode recordings of short duration waveforms (SDWs) with an average peak-trough duration less than 172μs. These waveforms have significantly shorter duration than somatic action potentials and tend to be triphasic. The present review discusses further data that suggests SDWs are representative of axonal activity, how this characterization allows for more accurate classification of somatic activity and could serve as a means of exploring signal integration in neural circuits. The review also discusses how axons may function as more than neural cables and the implications this may have for axonal information processing. While the technical challenges necessary for the exploration of axonal processes in functional neural circuits during behavior are impressive, preliminary evidence suggests that the in vivo study of axons is attainable. The resulting theoretical implications for systems level function make refinement of this approach a necessary goal toward developing a more complete understanding of the processes underlying learning, memory and attention as well as the pathological states underlying mental illness and epilepsy

Potentiation of sensory responses in ventrobasal thalamus in vivo via selective modulation of mGlu1 receptors with a positive allosteric modulator.

Metabotropic glutamate subtype 1 (mGlu1) receptor is thought to play a role in synaptic responses in thalamic relay nuclei. The aim of this study was to evaluate the positive allosteric modulator (PAM) Ro67-4853 as a tool to modulate thalamic mGlu1 receptors on single thalamic neurones in vivo. Ro67-4853, applied by iontophoresis onto ventrobasal thalamus neurones of urethane-anaesthetised rats, selectively enhanced responses to the agonist (S)-3,5-dihydroxy-phenylglycine (DHPG), an effect consistent with mGlu1 potentiation. The PAM was also able to enhance maintained responses to 10 Hz trains of sensory stimulation of the vibrissae, but had little effect on responses to single sensory stimuli. Thus Ro67-4853 appears to be a highly selective tool that can be useful in investigating how mGlu1 receptor potentiation can alter neural processing in vivo. Our results show the importance of mGlu1 in sensory processing and attention mechanisms at the thalamic level and suggest that positive modulation of mGlu1 receptors might be a useful mechanism for enhancing cognitive and attentional processes

Neuronal activity patterns in the mediodorsal thalamus and related cognitive circuits are modulated by metabotropic glutamate receptors.

The mediodorsal thalamus (MD) likely plays an important role in cognition as it receives abundant afferent connections from the amygdala and prefrontal cortex (PFC). Indeed, disturbed activity within the MD is thought to precipitate cognitive deficits associated with schizophrenia. As compounds acting at the Group II metabotropic glutamate (mGlu) receptors (subtypes mGlu2/mGlu3) have efficacy in animal models of schizophrenia, we investigated whether a Group II agonist and an mGlu2 positive allosteric modulator (PAM) could modulate MD activity. Extracellular single-unit recordings were made in vivo from MD neurones in anaesthetised rats. Responses were elicited by electrical stimulation of the PFC and/or amygdala, with Group II compounds locally applied as required. The Group II agonist reduced inhibition evoked in the MD: an effect manifested as an increase in short-latency responses, and a decrease in long-latency burst-firing. This disinhibitory action of the Group II receptors in the MD represents a mechanism of potential therapeutic importance as increased inhibition in the MD has been associated with cognitive deficit-onset. Furthermore, as co-application of the mGlu2 PAM did not potentiate the Group II agonist effects in the MD, we suggest that the Group II disinhibitory effect is majority-mediated via mGlu3. This heterogeneity in Group II receptor thalamic physiology bears consequence, as compounds active exclusively at the mGlu2 subtype are unlikely to perturb maladapted MD firing patterns associated with cognitive deficits, with activity at mGlu3 receptors possibly more appropriate. Indeed, polymorphisms in the mGlu3, but not the mGlu2, gene have been detected in patients with schizophrenia

Characterization of panglial gap junction networks in the thalamus and hippocampus reveals glial heterogeneity

Increasing evidence over the past decades revealed the importance of glia in the brain. In this study, astrocytes in the ventrobasal thalamus were characterized in detail for the first time. Glial heterogeneity was investigated by comparing their properties with those of astrocytes in the hippocampus, a brain region in which glial cells have been widely studied. During development, astrocytes establish gap junction channels with each other. In this study, the networks in the ventrobasal thalamus increased in size until the end of the second postnatal week. Cx30 expression was strongly upregulated during postnatal development and had a predominating role in glial network formation in the mature thalamus. Cx30 and Cx43, but not Cx26, mediated gap junction coupling in the hippocampus and thalamus. Strikingly, some astrocytes were devoid of Cx43. Expression of Cx43 in RG-like cells influences proliferation. The effect of two Cx43 mutations on network sizes were studied to identify the key function, gap junction coupling or adhesion, for proliferation. The function remained elusive. In summary, connexin expression differs among brain regions revealing glial heterogeneity throughout the brain. Tracer diffusion from astrocytes into the myelin sheath was observed in the ventrobasal thalamus. Such functional gap junction channels between astrocytes and oligodendrocytes were so far unknown. In this study, a panglial network formed by oligodendrocytes and astrocytes was discovered in the hippocampus and the thalamus. Employing Cx30/47 DKO mice, a major impact of Cx30 on panglial network formation was observed in the thalamus. Cx30 and Cx47 do not form functional channelsin vitro. This leads to the conclusion that panglial coupling is mainly mediated by Cx30:Cx32 channels. Immunohistochemical analysis in PLP-GFP and Cx43-ECFP mice identified overlapping protein expression of "classical" markers in thalamic astrocytes and oligodendrocytes in contrast to hippocampal glia. S100β was the most reliable marker to distinguish astrocytes and oligodendrocytes in the ventrobasal thalamus. Unexpectedly, an "intermediate" cell-type was identified co-expressing Cx43 and Olig2. It is a mature cell-type which is part of thalamic glial networks and has been described for the first time in this study. The functional role of panglial networks in metabolite supply to neurons was analysed in the present study. Glucose diffusion from astrocytes to oligodendrocytes was demonstrated in the thalamus employing the fluorescent glucose analogue 2-NBDG in PLP-GFP mice with SR101 labelled astrocytes. For further investigation of glia-neuron interactions, a method to analyse neuronal field potentials in the ventrobasal thalamus was established and characterized. Extracellular glucose deprivation abolished neuronal postsynaptic field potentials, thereby confirming the neuronal requirement of glial energy supply. In addition to connexin mediated gap junction channels, the expression of ionotropic AMPA and GABAA receptors was studied in thalamic astrocytes. In the juvenile thalamus, two astrocyte populations were distinguished by the presence or absence of AMPA receptor expression. The GluA2 subunit was abundantly expressed when AMPA receptors were expressed. All astrocytes expressed GABAA receptors. They were devoid of the α3 subunit and rarely expressed the γ2 subunit required for synaptic GABAA receptor localization. Instead, all cells expressed the γ1 subunit. These data indicate that glial heterogeneity occurs even within a given brain regions. The present study describes panglial networks in grey matter and their distinct properties among brain regions for the first time. In addition, glial heterogeneity was observed between and within brain regions and enhanced our understanding of glial specializations. In addition, a new "intermediate" cell-type was discovered which is abundantly present in the ventrobasal thalamus

Abnormal hippocampal melatoninergic system: a potential link between absence epilepsy and depression-like behavior in WAG/Rij rats?

Absence epilepsy and depression are comorbid disorders, but the molecular link between the two disorders is unknown. Here, we examined the role of the melatoninergic system in the pathophysiology of spike and wave discharges (SWDs) and depression-like behaviour in the Wistar Albino Glaxo from Rijswijk (WAG/Rij) rat model of absence epilepsy. In WAG/Rij rats, SWD incidence was higher during the dark period of the light-dark cycle, in agreement with previous findings. However, neither pinealectomy nor melatonin administration had any effect on SWD incidence, suggesting that the melatoninergic system was not involved in the pathophysiology of absence-like seizures. Endogenous melatonin levels were lower in the hippocampus of WAG/Rij rats as compared to non-epileptic control rats, and this was associated with higher levels of melatonin receptors in the hippocampus, but not in the thalamus. In line with the reduced melatonin levels, cell density was lower in the hippocampus ofWAG/Rij rats and was further reduced by pinealectomy. As expected, WAG/Rij rats showed an increased depression-like behaviour in the sucrose preference and forced swim tests, as compared to non-epileptic controls. Pinealectomy abolished the difference between the two strains of rats by enhancing depression-like behaviour in non-epileptic controls. Melatonin replacement displayed a significant antidepressant-like effect in bothWAG/Rij and control rats. These findings suggest that a defect of hippocampal melatoninergic system may be one of the mechanisms underlying the depression-like phenotype inWAG/Rij rats and that activation of melatonin receptors might represent a valuable strategy in the treatment of depression associated with absence epilepsy

Dying neurons in thalamus of asphyxiated term newborns and rats are autophagic

Enjeu: Déterminer si la macroautophagie est activée de façon excessive dans les neurones en souffrance dans l'encéphalopathie anoxique-ischémique du nouveau-né à terme. Contexte de la recherche: L'encéphalopathie anoxique-ischémique suite à une asphyxie néonatale est associée à une morbidité neurologique à long terme. Une diminution de son incidence reste difficile, son primum movens étant soudain, imprévisible voire non identifiable. Le développement d'un traitement pharmacologique neuroprotecteur post-anoxie reste un défi car les mécanismes impliqués dans la dégénérescence neuronale sont multiples, interconnectés et encore insuffisamment compris. En effet, il ressort des études animales que la notion dichotomique de mort cellulaire apoptotique (type 1)/nécrotique (type 3) est insuffisante. Une même cellule peut présenter des caractéristiques morphologiques mixtes non seulement d'apoptose et de nécrose mais aussi parfois de mort autophagique (type 2) plus récemment décrite. L'autophagie est un processus physiologique normal et essentiel de dégradation de matériel intracellulaire par les enzymes lysosomales. La macroautophagie, nommée simplement autophagie par la suite, consiste en la séquestration de parties de cytosol à éliminer (protéines et organelles) dans des compartiments intermédiaires, les autophagosomes, puis en leur fusion avec des lysosomes pour former des autolysosomes. Dans certaines conditions de stress telles que l'hypoxie et l'excitoxicité, une activité autophagique anormalement élevée peut être impliquée dans la mort cellulaire soit comme un mécanisme de mort indépendant (autodigestion excessive correspondante à la mort cellulaire de type 2) soit en activant d'autres voies de mort comme celles de l'apoptose. Description de l'article: Ce travail examine la présence de l'autophagie et son lien avec la mort cellulaire dans les neurones d'une région cérébrale fréquemment atteinte chez le nouveau- né humain décédé après une asphyxie néonatale sévère, le thalamus ventro-latéral. Ces résultats ont été comparés à ceux obtenus dans un modèle d'hypoxie-ischémie cérébrale chez le raton de 7 jours (dont le cerveau serait comparable à celui d'un nouveau-né humain de 34-37 semaines de gestation). Au total 11 nouveau-nés à terme décédés peu après la naissance ont été rétrospectivement sélectionnés, dont 5 présentant une encéphalopathie hypoxique- ischémique sévère et 6 décédés d'une cause autre que l'asphyxie choisis comme cas contrôle. L'autophagie et l'apoptose neuronale ont été évaluées sur la base d'une étude immunohistochimique et d'imagerie confocale de coupes histologiques en utilisant des marqueurs tels que LC3 (protéine dont la forme LC3-II est liée à la membrane des autophagosomes), p62/SQSTM1 (protéine spécifiquement dégradée par autophagie), LAMP1 (protéine membranaire des lysosomes et des autolysosomes), Cathepsin D ou B (enzymes lysosomales), TUNEL (détection de la fragmentation de l'ADN se produisant lors de l'apoptose), CASPASE-3 activée (protéase effectrice de l'apoptose) et PGP9.5 (protéine spécifique aux neurones). Chez le raton l'étude a pu être étendue en utilisant d'autres méthodes complémentaires telles que la microscopie électronique et le Western-blot. Une quantification des différents marqueurs montre une augmentation statistiquement significative de l'autophagie neuronale dans les cas d'asphyxie par rapport aux cas contrôles chez l'humain comme chez le raton. En cas d'asphyxie, les mêmes neurones expriment une densité accrue d'autophagosomes et d'autolysosomes par rapport aux cas contrôles. De plus, les neurones hautement autophagiques présentent des caractéristiques de l'apoptose. Conclusion: Cette étude montre, pour la première fois, que les neurones thalamiques lésés en cas d'encéphalopathie hypoxique-ischémique sévère présentent un niveau anormalement élevé d'activité autophagique comme démontré chez le raton hypoxique-ischémique. Ce travail permet ainsi de mettre en avant l'importance de considérer l'autophagie comme acteur dans la mort neuronale survenant après asphyxie néonatale. Perspectives: Récemment un certain nombre d'études in vitro ou sur des modèles d'ischémie cérébrale chez les rongeurs suggèrent un rôle important de la macroautophagie dans la mort neuronale. Ainsi, l'inhibition spécifique de la macroautophagie devrait donc être envisagée dans le futur développement des stratégies neuroprotectrices visant à protéger le cerveau des nouveau-nés à terme suite à une asphyxie

Alterations in the α2 δ ligand, thrombospondin-1, in a rat model of spontaneous absence epilepsy and in patients with idiopathic/genetic generalized epilepsies

Objectives Thrombospondins, which are known to interact with the α2δ subunit of voltage-sensitive calcium channels to stimulate the formation of excitatory synapses, have recently been implicated in the process of epileptogenesis. No studies have been so far performed on thrombospondins in models of absence epilepsy. We examined whether expression of the gene encoding for thrombospondin-1 was altered in the brain of WAG/Rij rats, which model absence epilepsy in humans. In addition, we examined the frequency of genetic variants of THBS1 in a large cohort of children affected by idiopathic/genetic generalized epilepsies (IGE/GGEs). Methods We measured the transcripts of thrombospondin-1 and α2δ subunit, and protein levels of α2δ, Rab3A, and the vesicular glutamate transporter, VGLUT1, in the somatosensory cortex and ventrobasal thalamus of presymptomatic and symptomatic WAG/Rij rats and in two control strains by real-time polymerase chain reaction (PCR) and immunoblotting. We examined the genetic variants of THBS1 and CACNA2D1 in two independent cohorts of patients affected by IGE/GGE recruited through the Genetic Commission of the Italian League Against Epilepsy (LICE) and the EuroEPINOMICS-CoGIE Consortium. Results Thrombospondin-1 messenger RNA (mRNA) levels were largely reduced in the ventrobasal thalamus of both presymptomatic and symptomatic WAG/Rij rats, whereas levels in the somatosensory cortex were unchanged. VGLUT1 protein levels were also reduced in the ventrobasal thalamus of WAG/Rij rats. Genetic variants of THBS1 were significantly more frequent in patients affected by IGE/GGE than in nonepileptic controls, whereas the frequency of CACNA2D1 was unchanged. Significance These findings suggest that thrombospondin-1 may have a role in the pathogenesis of IGE/GGEs

Astrocytes modulate thalamic sensory processing via mGlu2 receptor activation.

Astrocytes possess many of the same signalling molecules as neurons. However, the role of astrocytes in information processing, if any, is unknown. Using electrophysiological and imaging methods, we report the first evidence that astrocytes modulate neuronal sensory inhibition in the rodent thalamus. We found that mGlu2 receptor activity reduces inhibitory transmission from the thalamic reticular nucleus to the somatosensory ventrobasal thalamus (VB): mIPSC frequencies in VB slices were reduced by the Group II mGlu receptor agonist LY354740, an effect potentiated by mGlu2 positive allosteric modulator (PAM) LY487379 co-application (30 nM LY354740: 10.0 ± 1.6% reduction; 30 nM LY354740 & 30 μM LY487379: 34.6 ± 5.2% reduction). We then showed activation of mGlu2 receptors on astrocytes: astrocytic intracellular calcium levels were elevated by the Group II agonist, which were further potentiated upon mGlu2 PAM co-application (300 nM LY354740: ratio amplitude 0.016 ± 0.002; 300 nM LY354740 & 30 μM LY487379: ratio amplitude 0.035 ± 0.003). We then demonstrated mGlu2-dependent astrocytic disinhibition of VB neurons in vivo: VB neuronal responses to vibrissae stimulation trains were disinhibited by the Group II agonist and the mGlu2 PAM (LY354740: 156 ± 12% of control; LY487379: 144 ± 10% of control). Presence of the glial inhibitor fluorocitrate abolished the mGlu2 PAM effect (91 ± 5% of control), suggesting the mGlu2 component to the Group II effect can be attributed to activation of mGlu2 receptors localised on astrocytic processes within the VB. Gating of thalamocortical function via astrocyte activation represents a novel sensory processing mechanism. As this thalamocortical circuitry is important in discriminative processes, this demonstrates the importance of astrocytes in synaptic processes underlying attention and cognition

Anti-absence activity of mGlu1 and mGlu5 receptor enhancers and their interaction with a GABA reuptake inhibitor: effect of local infusions in the somatosensory cortex and thalamus

OBJECTIVE Glutamate and γ-aminobutyric acid (GABA) are the key neurotransmitter systems in the cortical-thalamocortical network, involved in normal and pathologic oscillations such as spike-wave discharges (SWDs), which characterize different forms of absence epilepsy. Metabotropic glutamate (mGlu) and GABA receptors are widely expressed within this network. Herein, we examined the effects of two selective positive allosteric modulators (PAMs) of mGlu1 and mGlu5 receptors, the GABA reuptake inhibitor, tiagabine, and their interaction in the somatosensory cortex and thalamus on SWDs in WAG/Rij rats. METHODS Male WAG/Rij rats were equipped with bilateral cannulas in the somatosensory cortex (S1po) or the ventrobasal (VB) thalamic nuclei, and with cortical electroencephalography (EEG) electrodes. Rats received a single dose of the mGlu1 receptor PAM, RO0711401, or the mGlu5 receptor PAM, VU0360172, various doses of tiagabine, or VU0360172 combined with tiagabine. RESULTS Both PAMs suppressed SWDs regardless of the site of injection. Tiagabine enhanced SWDs when injected into the thalamus, but, unexpectedly, suppressed SWDs in a dose-dependent manner when injected into the cortex. Intracortical co-injection of VU0360172 and tiagabine produced slightly larger effects as compared to either VU0360172 or tiagabine alone. Intrathalamic co-injections of VU0360172 and subthreshold doses of tiagabine caused an antiabsence effect similar to that exhibited by VU0360172 alone in the first 10 min. At 30 min, however, the antiabsence effect of VU0360172 was prevented by subthreshold doses of tiagabine, and the combination produced a paradoxical proabsence effect at 40 and 50 min. SIGNIFICANCE These data (1) show that mGlu1 and mGlu5 receptor PAMs reduce absence seizures acting at both thalamic and cortical levels; (2) demonstrate for the first time that tiagabine, despite its established absence-enhancing effect, reduces SWDs when injected into the somatosensory cortex; and (3) indicate that the efficacy of VU0360172 in the thalamus may be critically affected by the availability of (extra)synaptic GABA

Neural Substrates of Chronic Pain in the Thalamocortical Circuit

Chronic pain (CP), a pathological condition with a large repertory of signs and symptoms, has no recognizable neural functional common hallmark shared by its diverse expressions. The aim of the present research was to identify potential dynamic markers shared in CP models, by using simultaneous electrophysiological extracellular recordings from the rat ventrobasal thalamus and the primary somatosensory cortex. We have been able to extract a neural signature attributable solely to CP, independent from of the originating conditions. This study showed disrupted functional connectivity and increased redundancy in firing patterns in CP models versus controls, and interpreted these signs as a neural signature of CP. In a clinical perspective, we envisage CP as disconnection syndrome and hypothesize potential novel therapeutic appraisal