Specialized astrocytes mediate glutamatergic gliotransmission in the CNS.

Multimodal astrocyte-neuron communications govern brain circuitry assembly and function1. For example, through rapid glutamate release, astrocytes can control excitability, plasticity and synchronous activity2,3 of synaptic networks, while also contributing to their dysregulation in neuropsychiatric conditions4-7. For astrocytes to communicate through fast focal glutamate release, they should possess an apparatus for Ca2+-dependent exocytosis similar to neurons8-10. However, the existence of this mechanism has been questioned11-13 owing to inconsistent data14-17 and a lack of direct supporting evidence. Here we revisited the astrocyte glutamate exocytosis hypothesis by considering the emerging molecular heterogeneity of astrocytes18-21 and using molecular, bioinformatic and imaging approaches, together with cell-specific genetic tools that interfere with glutamate exocytosis in vivo. By analysing existing single-cell RNA-sequencing databases and our patch-seq data, we identified nine molecularly distinct clusters of hippocampal astrocytes, among which we found a notable subpopulation that selectively expressed synaptic-like glutamate-release machinery and localized to discrete hippocampal sites. Using GluSnFR-based glutamate imaging22 in situ and in vivo, we identified a corresponding astrocyte subgroup that responds reliably to astrocyte-selective stimulations with subsecond glutamate release events at spatially precise hotspots, which were suppressed by astrocyte-targeted deletion of vesicular glutamate transporter 1 (VGLUT1). Furthermore, deletion of this transporter or its isoform VGLUT2 revealed specific contributions of glutamatergic astrocytes in cortico-hippocampal and nigrostriatal circuits during normal behaviour and pathological processes. By uncovering this atypical subpopulation of specialized astrocytes in the adult brain, we provide insights into the complex roles of astrocytes in central nervous system (CNS) physiology and diseases, and identify a potential therapeutic target.

Gender differences in health expenditure determinants: A follow-up study.

Researchers' aim was to investigate if patients/physicians characteristics could differently affect males/females health care expenditure. In 2009/2010, a health-related-quality-of-life (HRQL) measure was distributed to 887 general practitioners' (GP) patients in Siena's province-Italy. Severity of diseases was calculated through Cumulative Illness Rating Scale Severity Index (CIRS-SI). Information about GPs' gender and age and patients' gender, age, and socio-economic variables were recorded. 2012 data about pharmaceutical, outpatient and hospital expenditure were obtained. Multivariate regression was carried out. In males, hospital expenditure increased with higher CIRS-SI and female GP whilst in females it was not influenced by any of the variables. Outpatient and pharmaceutical expenditure increased with aging, higher CIRS-SI, and lower HRQL and education, both in males and females. Gender differences in health expenditure determinants emerged for hospital expenditure.

Role of oral nystatin prophylaxis in cardiac surgery with prolongedextracorporeal circulation.

Duration >120 minutes of extracorporeal circulation (ECC) during cardiopulmonary bypass procedure was associated to an increased risk of candidemia in the intensive care unit (ICU). To evaluate oral nystatin prophylaxis in cardiac surgery considering its exclusive effect on Candida, in the absence of systemic effects and selection of resistant strains to polyene. We conducted an observational study in the postcardiac surgery ICU of Policlinico "Umberto I" of Rome. From January 2014, all patients with a prolonged ECC >120 minutes were systematically treated with oral nystatin (Prophylaxis group). This group was compared with all patients hospitalised in the same ICU, who have not received oral nystatin after ECC >120 minutes (No prophylaxis group). Overall, 672 consecutive patients were analyzed: 318 (47.3%) patients belonged to the no prophylaxis group, and 354 (52.7%) patients to the prophylaxis group. Diagnosis of candidemia was confirmed in 7 (2.2%) patients, all belonged to the no prophylaxis group. At multivariate analysis, oral nystatin prophylaxis showed a protective effect for development of candidemia after cardiac surgery. Oral nystatin prophylaxis, in patients who underwent a ECC >120 minutes, seems to reduce development of candidemia; however, the real efficacy of such prophylaxis approach requires further investigation.

Variable electrobehavioral patterns during focal nonconvulsive status epilepticus induced by unilateral intrahippocampal injection of kainic acid.

OBJECTIVE: Nonconvulsive status epilepticus (ncSE) is a severe condition that may result in neurologic sequelae and epilepsy resistant to pharmacologic treatment. We analyze here seizure and electroencephalography (EEG) patterns and their correlation to the development of a chronic epileptic condition in a guinea pig model of focal ncSE induced by intrahippocampal injection of kainic acid (KA). METHODS: Electrobehavioral patterns during ncSE induced by unilateral injection of 1 µg of KA in the CA1 region of the hippocampus were characterized by continuous video-EEG monitoring in 13 guinea pigs bilaterally implanted with recording electrodes in the hippocampus and neocortex. RESULTS: Video-EEG analysis demonstrates a high variability of seizure type and duration during KA-induced ncSE. Seizures showed focal signs correlated with diverse epileptiform EEG discharge distributions, either diffuse or localized. Nonfocal (bilateral motor) signs during seizures most likely correlated with a diffuse EEG pattern. The evolution into a chronic epileptic condition correlated neither with the severity of seizure pattern nor with the diffusion of the EEG discharges observed during the ncSE. SIGNIFICANCE: Video-EEG monitoring in a guinea pig model of ncSE induced by unilateral hippocampal injection of KA demonstrates a high variability of electrobehavioral patterns. We demonstrate that the seizure severity score during focal ncSE is not a predictor of the evolution into a chronic epileptic condition of mesial temporal lobe epilepsy.

Seizure-like discharges induced by 4-aminopyridine in the olfactory system of the in vitro isolated guinea pig brain.

PURPOSE: The study of the interactions leading to network- or region-specific propagation of seizures is crucial to understand ictogenesis. We have recently found that systemic (arterial) application of the potassium channel blocker, 4-aminopyridine (4AP), induces different and independent seizure activities in olfactory and in limbic structures. Here, we have characterized the network and cellular features that support 4AP-induced seizure-like events in the olfactory cortex. METHODS: Simultaneous extracellular recordings were performed from the piriform cortex, the entorhinal cortex, the olfactory tubercle, and the amygdala of the in vitro isolated guinea pig brain preparation. Intracellular, sharp electrode recordings were obtained from neurons of different layers of the region of ictal onset, the piriform cortex. Seizure-like discharges were induced by both arterial perfusion and local intracortical injections of 4AP. KEY FINDINGS: Arterial application of 4AP induces independent seizure activities in limbic and olfactory cortices. Both local applications of 4AP and cortico-cortical disconnections demonstrated that region-specific seizure-like events initiated in the primary olfactory cortex and propagate to anatomically related areas. Seizures induced by arterial administration of 4-AP are preceded by runs of fast activity at circa 30-40 Hz and are independently generated in the hemispheres. Simultaneous extracellular and intracellular recordings in the piriform cortex revealed that the onset of seizure correlates with (1) a gradual amplitude increase of fast activity runs, (2) a large intracellular depolarization with action potential firing of superficial layer neurons, and (3) no firing in a subpopulation of deep layers neurons. During the ictal event, neuronal firing was abolished for 10-30 s in all neurons and gradually restored and synchronized before seizure termination. SIGNIFICANCE: Our data show that olfactory neuronal networks sustain the generation of seizure-like activities that are independent from those observed in adjacent and connected limbic cortex regions. The data support the concept that functionally and anatomically hard-wired networks generate region-specific seizure patterns that could be substrates for system epilepsy.

Hippocampal hyperexcitability and specific epileptiform activity in a mouse model of Dravet syndrome.

PURPOSE: Dravet syndrome (DS) is caused by dominant mutations of the SCN1A gene, encoding the NaV 1.1 sodium channel α subunit. Gene targeted mouse models of DS mutations replicate patients' phenotype and show reduced γ-aminobutyric acid (GABA)ergic inhibition. However, little is known on the properties of network hyperexcitability and on properties of seizure generation in these models. In fact, seizures have been studied thus far with surface electroencephalography (EEG), which did not show if specific brain regions are particularly involved. We have investigated hyperexcitability and epileptiform activities generated in neuronal networks of a mouse model of DS. METHODS: We have studied heterozygous NaV 1.1 knock-out mice performing field potential recordings in combined hippocampal/cortical slices in vitro and video/depth electrode intracerebral recordings in vivo during hyperthermia-induced seizures. KEY FINDINGS: In slices, we have disclosed specific signs of hyperexcitability of hippocampal circuits in both the pre-epileptic and epileptic periods, and a specific epileptiform activity was generated in the hippocampus upon application of the convulsant 4-aminopyridine in the epileptic period. During in vivo hyperthermia-induced seizures, we have observed selective hippocampal activity in early preictal phases and pronounced hippocampal activity in the ictal phase. SIGNIFICANCE: We have identified specific epileptiform activities and signs of network hyperexcitability, and disclosed the important role of the hippocampus in seizure generation in this model. These activities may be potentially used as targets for screenings of antiepileptic approaches.

A guinea pig model of mesial temporal lobe epilepsy following nonconvulsive status epilepticus induced by unilateral intrahippocampal injection of kainic acid.

PURPOSE: Models of temporal lobe epilepsy are commonly utilized to study focal epileptogenesis and ictogenesis. The criteria that define animal models representative of human mesial temporal lobe may vary in different laboratories. We describe herein a focal epilepsy model of mesial temporal (hippocampal) origin that relies on the analysis of interictal and ictal electroencephalography (EEG) patterns and on their correlation with seizure symptoms and neuropathologic findings. The study is based on guinea pigs, a species seldom utilized to develop chronic epilepsy models. METHODS: Young adult guinea pigs were bilaterally implanted under isoflurane anesthesia with epidural electrodes over somatosensory cortex and depth electrodes in CA1 hippocampal region. A stainless steel guide cannula was positioned unilaterally in the right dorsal hippocampus to inject 1 µl of 0.9% NaCl solution containing 1 µg kainic acid (KA). One week after surgery, continuous 24 h/day video-EEG monitoring was performed 48 h before and every other week after KA injection, for no <1 month. EEG data were recorded wide-band at 2 kHz. After video-EEG monitoring, brains were analyzed for thionine and Timm staining and glial fibrillary acid protein (GFAP) immunostaining. KEY FINDINGS: Unilateral injection of KA in dorsal hippocampus of guinea pigs induces an acute nonconvulsive status epilepticus (SE) that terminates within 24 h (n = 22). Chronic seizures with very mild motor signs (undetectable without EEG monitoring) and highly variable recurrence patterns appear in 45.5% (10 of 22) KA-treated animals, with variable delays from the initial SE. In these animals interictal events, CA1 cell loss, gliosis, and altered Timm staining pattern were observed. The induction of a chronic condition did not correlate with the duration of the nonconvulsive acute SE, but correlated with the extension and quality of neuropathologic damage. SIGNIFICANCE: We demonstrate that a model of hippocampal (mesial temporal lobe) epilepsy can be developed in the guinea pig by intrahippocampal injection of KA. Seizure events in this model show little behavioral signs and may be overlooked without extensive video-EEG monitoring. The establishment of a chronic epileptic condition correlates with the extension of the hippocampal damage (mainly cell loss and gliosis) and not with the intensity of the initial SE.

Status epilepticus-induced pathologic plasticity in a rat model of focal cortical dysplasia.

We have generated an experimental 'double-hit' model of chronic epilepsy to recapitulate the co-existence of abnormal cortical structure and frequently recurrent seizures as observed in human focal cortical dysplasia. We induced cortical malformations by exposing rats prenatally to methylazoxymethanol acetate and triggered status epilepticus and recurrent seizures in adult methylazoxymethanol acetate rats with pilocarpine. We studied the course of epilepsy and the long-term morphologic and molecular changes induced by the occurrence of status epilepticus and subsequent chronic epilepsy in the malformed methylazoxymethanol acetate exposed brain. Behavioural and electroencephalographic analyses showed that methylazoxymethanol acetate pilocarpine rats develop more severe epilepsy than naïve rats. Morphologic and molecular analyses demonstrated that status epilepticus and subsequent seizures, but not pilocarpine treatment per se, was capable of affecting both cortical architectural and N-methyl-D-aspartate receptor abnormalities induced by methylazoxymethanol acetate. In particular, cortical thickness was further decreased and N-methyl-D-aspartate regulatory subunits were recruited at the postsynaptic membrane. In addition, methylazoxymethanol acetate pilocarpine rats showed abnormally large cortical pyramidal neurons with neurofilament over-expression. These neurons bear similarities to the hypertrophic/dysmorphic pyramidal neurons observed in acquired human focal cortical dysplasia. These data show that status epilepticus sets in motion a pathological process capable of significantly changing the cellular and molecular features of pre-existing experimental cortical malformations. They suggest that seizure recurrence in human focal cortical dysplasia might be an additional factor in establishing a pathological circuitry that favours chronic neuronal hyperexcitability.

Independent epileptiform discharge patterns in the olfactory and limbic areas of the in vitro isolated Guinea pig brain during 4-aminopyridine treatment.

In vitro studies performed on brain slices demonstrate that the potassium channel blocker 4-aminopyridine (4AP, 50 microM) discloses electrographic seizure activity and interictal discharges. These epileptiform patterns have been further analyzed here in a isolated whole guinea pig brain in vitro by using field potential recordings in olfactory and limbic structures. In 8 of 13 experiments runs of fast oscillatory activity (fast runs, FRs) in the piriform cortex (PC) propagated to the lateral entorhinal cortex (EC), hippocampus and occasionally to the medial EC. Early and late FRs were asynchronous in the hemispheres showed different duration [1.78 +/- 0.51 and 27.95 +/- 4.55 (SD) s, respectively], frequency of occurrence (1.82 +/- 0.49 and 34.16 +/- 6.03 s) and frequency content (20-40 vs. 40-60 Hz). Preictal spikes independent from the FRs appeared in the hippocampus/EC and developed into ictal-like discharges that did not propagate to the PC. Ictal-like activity consisted of fast activity with onset either in the hippocampus (n = 6) or in the mEC (n = 2), followed by irregular spiking and sequences of diffusely synchronous bursts. Perfusion of the N-methyl-d-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid (100 microM) did not prevent FRs, increased the duration of limbic ictal-like discharges and favored their propagation to olfactory structures. The AMPA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (50 microM) blocked ictal-like events and reduced FRs. In conclusion, 4AP-induced epileptiform activities are asynchronous and independent in olfactory and hippocampal-entorhinal regions. Epileptiform discharges in the isolated guinea pig brain show different pharmacological properties compared with rodent in vitro slices.

Studying Axon-Astrocyte Functional Interactions by 3D Two-Photon Ca2+ Imaging: A Practical Guide to Experiments and "Big Data" Analysis.

Recent advances in fast volumetric imaging have enabled rapid generation of large amounts of multi-dimensional functional data. While many computer frameworks exist for data storage and analysis of the multi-gigabyte Ca2+ imaging experiments in neurons, they are less useful for analyzing Ca2+ dynamics in astrocytes, where transients do not follow a predictable spatio-temporal distribution pattern. In this manuscript, we provide a detailed protocol and commentary for recording and analyzing three-dimensional (3D) Ca2+ transients through time in GCaMP6f-expressing astrocytes of adult brain slices in response to axonal stimulation, using our recently developed tools to perform interactive exploration, filtering, and time-correlation analysis of the transients. In addition to the protocol, we release our in-house software tools and discuss parameters pertinent to conducting axonal stimulation/response experiments across various brain regions and conditions. Our software tools are available from the Volterra Lab webpage at https://wwwfbm.unil.ch/dnf/group/glia-an-active-synaptic-partner/member/volterra-andrea-volterra in the form of software plugins for Image J (NIH)-a de facto standard in scientific image analysis. Three programs are available: MultiROI_TZ_profiler for interactive graphing of several movable ROIs simultaneously, Gaussian_Filter5D for Gaussian filtering in several dimensions, and Correlation_Calculator for computing various cross-correlation parameters on voxel collections through time.

Three-dimensional Ca2+ imaging advances understanding of astrocyte biology.

Astrocyte communication is typically studied by two-dimensional calcium ion (Ca2+) imaging, but this method has not yielded conclusive data on the role of astrocytes in synaptic and vascular function. We developed a three-dimensional two-photon imaging approach and studied Ca2+ dynamics in entire astrocyte volumes, including during axon-astrocyte interactions. In both awake mice and brain slices, we found that Ca2+ activity in an individual astrocyte is scattered throughout the cell, largely compartmented between regions, preponderantly local within regions, and heterogeneously distributed regionally and locally. Processes and endfeet displayed frequent fast activity, whereas the soma was infrequently active. In awake mice, activity was higher than in brain slices, particularly in endfeet and processes, and displayed occasional multifocal cellwide events. Astrocytes responded locally to minimal axonal firing with time-correlated Ca2+ spots.

Distribution of the olfactory fiber input into the olfactory tubercle of the in vitro isolated guinea pig brain.

The olfactory tubercle (OT) is a cortical component of the olfactory system involved in reward mechanisms of drug abuse. This region covers an extensive part of the rostral ventral cerebrum and is relatively poorly studied. The intrinsic network interactions evoked by olfactory input are analyzed in the OT of the in vitro isolated guinea pig brain by means of field potential analysis and optical imaging of voltage-sensitive signals. Stimulation of the lateral olfactory tract induces a monosynaptic response that progressively decreases in amplitude from lateral to medial. The monosynaptic input induces a disynaptic response that is proportionally larger in the medial portion of the OT. Direct stimulation of the piriform cortex and subsequent lesion of this pathway showed the existence of an associative disynaptic projection from the anterior part of the piriform cortex to the lateral part of the OT that integrates with the component mediated by the local intra-OT collaterals. Optical and electrophysiological recordings of the signals evoked by stimulation of the olfactory tract during arterial perfusion with the voltage-sensitive dye di-2-ANEPEQ confirmed the pattern of distribution of the mono and disynaptic responses in the OT. Finally, current source density analysis of laminar profiles recorded with 16-channel silicon probes confirmed that the monosynaptic and disynaptic potentials localize in the most superficial and the deep portions of the plexiform layer I, as suggested by previous reports. This study sets the standard for further analysis of the modulation of network properties in this largely unexplored brain region.

Importance of sociodemographic and morbidity aspects in measuring health-related quality of life: performances of three tools: comparison of three questionnaire scores.

BACKGROUND: Since health-related quality of life (HRQL) measures are numerous, comparisons have been suggested. AIM: To compare three HRQL measures: SF6D, HUI3 and EQ5D. METHODS: Three questionnaires (SF36, HUI3, EQ5D) were administered to 1,011 patients attending 16 general practices in two Italian cities. Information about patients' gender, age, education, marital status, smoking, body mass index (BMI) and chronic diseases (hypertension, diabetes, cardiovascular and musculoskeletal diseases) were also collected. Questionnaires scores were calculated using the appropriate algorithms; in particular SF6D scores were obtained from SF36 items. Agreement and correlation between questionnaires scores were investigated using Bland and Altman method and Spearman coefficient. The influence of socio-demographic and morbidity indicators on scores was analysed using the nonparametric quantile regression. RESULTS: The Spearman coefficient was about 0.6 for all questionnaires. The 95% limits of agreement of the scores were approximately from -0.5 to 0.3 except for SF6D and EQ5D when they were from -0.4 to 0.2. The measures were influenced by socio-demographic and clinical variables in a similar way, especially SF6D (the index obtained from SF36) and EQ5D, which appeared to be influenced by the same pattern of factors, including gender, chronic diseases, smoking and BMI. CONCLUSIONS: Overall, the agreement between questionnaires scores was quite low, whilst the correlation level was good. Questionnaire scores were influenced by socio-demographic and clinical variables in a similar way, especially SF6D and EQ5D. Therefore, the descriptive capacity of SF6D and EQ5D was found to be similar.