Effects of environmental complexity on neurogenesis in the hippocampal dentate gyrus

Introduction. Postnatal neurogenesis in the hippocampal dentate gyrus, can be modulated by numerous determinants, such as hormones, transmitters and stress. Among the factors positively interfering with neurogenesis, the complexity of the environment appears to play a particularly striking role. Adult mice reared in an enriched environment produce more neurons and exhibit better performance in hippocampus-specific learning tasks. While the effects of complex environments on hippocampal neurogenesis are well documented, there is a lack of information on the effects of living under socio-sensory deprivation conditions. Due to the immaturity of rats and mice at birth, studies dealing with the effects of environmental enrichment on hippocampal neurogenesis were carried out in adult animals, i.e. during a period of relatively low rate of neurogenesis. The impact of environment is likely to be more dramatic during the first postnatal weeks, because at this time granule cell production is remarkably higher than at later phases of development. The aim of the present research was to clarify whether and to what extent isolated or enriched rearing conditions affect hippocampal neurogenesis during the early postnatal period, a time window characterized by a high rate of precursor proliferation and to elucidate the mechanisms underlying these effects. The experimental model chosen for this research was the guinea pig, a precocious rodent, which, at 4-5 days of age can be independent from maternal care. Experimental design. Animals were assigned to a standard (control), an isolated, or an enriched environment a few days after birth (P5-P6). On P14-P17 animals received one daily bromodeoxyuridine (BrdU) injection, to label dividing cells, and were sacrificed either on P18, to evaluate cell proliferation or on P45, to evaluate cell survival and differentiation. Methods. Brain sections were processed for BrdU immunhistochemistry, to quantify the new born and surviving cells. The phenotype of the surviving cells was examined by means of confocal microscopy and immunofluorescent double-labeling for BrdU and either a marker of neurons (NeuN) or a marker of astrocytes (GFAP). Apoptotic cell death was examined with the TUNEL method. Serial sections were processed for immunohistochemistry for i) vimentin, a marker of radial glial cells, ii) BDNF (brain-derived neurotrofic factor), a neurotrophin involved in neuron proliferation/survival, iii) PSA-NCAM (the polysialylated form of the neural cell adhesion molecule), a molecule associated with neuronal migration. Total granule cell number in the dentate gyrus was evaluated by stereological methods, in Nissl-stained sections. Results. Effects of isolation. In P18 isolated animals we found a reduced cell proliferation (-35%) compared to controls and a lower expression of BDNF. Though in absolute terms P45 isolated animals had less surviving cells than controls, they showed no differences in survival rate and phenotype percent distribution compared to controls. Evaluation of the absolute number of surviving cells of each phenotype showed that isolated animals had a reduced number of cells with neuronal phenotype than controls. Looking at the location of the new neurons, we found that while in control animals 76% of them had migrated to the granule cell layer, in isolated animals only 55% of the new neurons had reached this layer. Examination of radial glia cells of P18 and P45 animals by vimentin immunohistochemistry showed that in isolated animals radial glia cells were reduced in density and had less and shorter processes. Granule cell count revealed that isolated animals had less granule cells than controls (-32% at P18 and -42% at P45). Effects of enrichment. In P18 enriched animals there was an increase in cell proliferation (+26%) compared to controls and a higher expression of BDNF. Though in both groups there was a decline in the number of BrdU-positive cells by P45, enriched animals had more surviving cells (+63) and a higher survival rate than controls. No differences were found between control and enriched animals in phenotype percent distribution. Evaluation of the absolute number of cells of each phenotype showed that enriched animals had a larger number of cells of each phenotype than controls. Looking at the location of cells of each phenotype we found that enriched animals had more new neurons in the granule cell layer and more astrocytes and cells with undetermined phenotype in the hilus. Enriched animals had a higher expression of PSA-NCAM in the granule cell layer and hilus Vimentin immunohistochemistry showed that in enriched animals radial glia cells were more numerous and had more processes.. Granule cell count revealed that enriched animals had more granule cells than controls (+37% at P18 and +31% at P45). Discussion. Results show that isolation rearing reduces hippocampal cell proliferation but does not affect cell survival, while enriched rearing increases both cell proliferation and cell survival. Changes in the expression of BDNF are likely to contribute to he effects of environment on precursor cell proliferation. The reduction and increase in final number of granule neurons in isolated and enriched animals, respectively, are attributable to the effects of environment on cell proliferation and survival and not to changes in the differentiation program. As radial glia cells play a pivotal role in neuron guidance to the granule cell layer, the reduced number of radial glia cells in isolated animals and the increased number in enriched animals suggests that the size of radial glia population may change dynamically, in order to match changes in neuron production. The high PSA-NCAM expression in enriched animals may concur to favor the survival of the new neurons by facilitating their migration to the granule cell layer. Conclusions. By using a precocious rodent we could demonstrate that isolated/enriched rearing conditions, at a time window during which intense granule cell proliferation takes place, lead to a notable decrease/increase of total granule cell number. The time-course and magnitude of postnatal granule cell production in guinea pigs are more similar to the human and non-human primate condition than in rats and mice. Translation of current data to humans would imply that exposure of children to environments poor/rich of stimuli may have a notably large impact on dentate neurogenesis and, very likely, on hippocampus dependent memory functions.

Influenza delle caratteristiche quali-quantitative della luce da fonti artificiali sulla fisio-morfologia di piante verdi per interior landscaping

La conoscenza delle esigenze luminose (intensità, spettro, durata minima, massima ed ottimale del fotoperiodo di illuminazione) e della tolleranza alle condizioni degli interni delle piante ad uso decorativo, è di fondamentale importanza per una giusta tecnica di progettazione (dimensionamento e dislocazione dei punti luce) dell’indoor plantscaping. Il lungo periodo di condizionamento al quale queste piante vengono sottoposte, caratterizzato principalmente dalla scarsa disponibilità di luce naturale e dagli alti livelli di concentrazione di CO2 determina una forte influenza sui processi morfo-fisiologici. Il presente studio analizza il fattore luminoso ed è articolato su più punti quali; • caratterizzazione della riposta fotosintetica all’intensità luminosa di 21 delle principali specie a fogliame decorativo comunemente utilizzate nella realizzazione degli spazi verdi indoor, per stabilire quali siano i minimi ed ottimali livelli di PAR tali da garantire una fotosintesi netta positiva e nel complesso le condizioni di maggior benessere per le piante; • quantificazione dell’incremento fotosintetico netto dovuto ad una maggior concentrazione di CO2 negli interni rispetto alla concentrazione CO2 atmosferica esterna, all’aumentare dell’ intensità luminosa artificiale sulle precedenti specie; • monitoraggio dell’andamento delle attività fotosintetiche durante il periodo di illuminazione di 8 ore comunemente utilizzato in un interno ad uso lavorativo, a PAR costante e variabile in Ficus elastica e Dieffenbachia picta, al fine di stabilire quali possano essere le durate e le modalità di somministrazione della luce per rendere massima la fotosintesi netta riducendo al minimo i consumi energetici dovuti all’accensione delle lampade; • valutazione della risposta morfo-fisiologica e fotosintetica a modificazioni dello spettro luminoso mediante l’uso di LED monocromatici colorati ad emissione nel bianco, blu e rosso in Ficus benjamina e Iresine herbistii al fine di stabilire se questo tipo di lampade possano essere utilizzate come fonte integrativa e/o sostitutiva nella realizzazione degli spazi verdi interni. Vengono analizzati il punto si compensazione alla luce (g), il punto di saturazione alla luce (s), l’efficienza quantica (AQE), il punto di respirazione al buio (Rd) e la fotosintesi netta massima (A max) per (Aglaonema commutatum, Asplenium nidus, Anthurium andreanum, Begonia rex, Calathea luoise, Calathea veitchiana, Calathea rufibarba, Calathea zebrina, Codiaeum variegatum, Cthenanthe oppenheimiana, Dieffenbakia picta, Ficus benjamina, Ficus elatica, Ficus longifolia, Fittonia verschaffeltii, Iresine herbistii, Philodendron erubescens, Philodendron pertusum, Potos aureus, Spathiphillum wallisi, Syngonium podophillum ) e classificate le specie in funzione di Amax in quattro categorie; A max < 2 µmol CO2 m-2 s-1, A max compresa tra 2 e 4 µmol CO2 m-2 s-1, Amax cpmpresa tra 4 e 6 µmol CO2 m-2 s-1, Amax > 6 µmol CO2 m-2 s-1, al fine di mettere in risalto la potenzialità fotosintetiche di ogni singola specie. I valori di PAR compresi tra (g) ed (s) forniscono le indicazioni sulle quali basarsi per scegliere una giusta lampada o dimensionare un punto luce per ogni singola specie e/o composizione. È stimata l’influenza di due livelli di concentrazione di CO2 ambientale (400 e 800 ppm) all’incrementare dell’intensità luminosa sul processo fotosintetico delle specie precedenti. Per quasi tutte le specie 800 ppm di CO2 non favoriscono nessun incremento all’attività fotosintetica ad eccezione di Ficus benjamina, Ficus elatica e Syngonium podophillum se non accompagnati da una disponibilità luminosa superiore alle 10 µmol m-2 s-1. Viene monitorato l’andamento dell’attività fotosintetica a PAR costante e variabile (intervallando periodi di 8 minuti a PAR 40 e 80) durante 8 ore di illuminazione su Ficus elastica e Dieffenbachia picta al fine di stabilire la miglior modalità di somministrazione della luce. La fotosintesi netta cumulativa per l’intera durata di illuminazione a PAR costante mostra un calo dopo alcune ore dall’attivazione in Dieffenbackia, e un andamento oscillatorio in Ficus. L’illuminazione alternata consente di raggiungere i quantitativi di CO2 organicata a 80 µmol m-2 s-1 di PAR, dopo 5 ore e mezza sia in Ficus che Dieffenbackia sebbene le potenzialità fotosintetiche delle due piante siano molto differenti. È stato valutato l’effetto dell’illuminazione artificiale mediante LED (15W) a luce bianca, blu e rossa monocromatica in rapporto alla luce neon(36W) bianca tradizionale (con differenti abbinamenti tra le lampade) sui principali parametri morfologici e fisiologici, in Ficus benjamin ‘Variegata’ e Iresine herbistii per verificare se tali fonti possono rappresentare una valida alternativa nella sostituzione o integrazione di altre lampade per gli spazi verdi indoor. Tutte le combinazioni LED indagate possono rappresentare un’alternativa di sostituzione alla coltivazione con neon ed un risparmio energetico di oltre il 50%. Una PAR di 20,6 µmol m-2 s-1 della singola lampada LED bianco è sufficiente per mantenere la pianta in condizioni di sopravvivenza con un consumo di 15W a fronte dei 36W necessari per il funzionamento di ogni neon. La combinazione LED bianco + LED blu monocromatico favorisce il contenimento della taglia della pianta, caratteristica gradita nella fase di utilizzo indoor, una maggior produzione di sostanza secca e un’attività fotosintetica più elevata.

Wavelet analysis of heart rate variability related to nocturnal frontal lobe epilepsy seizures

Introduction: Nocturnal frontal lobe epilepsy (NFLE) is a distinct syndrome of partial epilepsy whose clinical features comprise a spectrum of paroxysmal motor manifestations of variable duration and complexity, arising from sleep. Cardiovascular changes during NFLE seizures have previously been observed, however the extent of these modifications and their relationship with seizure onset has not been analyzed in detail. Objective: Aim of present study is to evaluate NFLE seizure related changes in heart rate (HR) and in sympathetic/parasympathetic balance through wavelet analysis of HR variability (HRV). Methods: We evaluated the whole night digitally recorded video-polysomnography (VPSG) of 9 patients diagnosed with NFLE with no history of cardiac disorders and normal cardiac examinations. Events with features of NFLE seizures were selected independently by three examiners and included in the study only if a consensus was reached. Heart rate was evaluated by measuring the interval between two consecutive R-waves of QRS complexes (RRi). RRi series were digitally calculated for a period of 20 minutes, including the seizures and resampled at 10 Hz using cubic spline interpolation. A multiresolution analysis was performed (Daubechies-16 form), and the squared level specific amplitude coefficients were summed across appropriate decomposition levels in order to compute total band powers in bands of interest (LF: 0.039062 - 0.156248, HF: 0.156248 - 0.624992). A general linear model was then applied to estimate changes in RRi, LF and HF powers during three different period (Basal) (30 sec, at least 30 sec before seizure onset, during which no movements occurred and autonomic conditions resulted stationary); pre-seizure period (preSP) (10 sec preceding seizure onset) and seizure period (SP) corresponding to the clinical manifestations. For one of the patients (patient 9) three seizures associated with ictal asystole were recorded, hence he was treated separately. Results: Group analysis performed on 8 patients (41 seizures) showed that RRi remained unchanged during the preSP, while a significant tachycardia was observed in the SP. A significant increase in the LF component was instead observed during both the preSP and the SP (p<0.001) while HF component decreased only in the SP (p<0.001). For patient 9 during the preSP and in the first part of SP a significant tachycardia was observed associated with an increased sympathetic activity (increased LF absolute values and LF%). In the second part of the SP a progressive decrease in HR that gradually exceeded basal values occurred before IA. Bradycardia was associated with an increase in parasympathetic activity (increased HF absolute values and HF%) contrasted by a further increase in LF until the occurrence of IA. Conclusions: These data suggest that changes in autonomic balance toward a sympathetic prevalence always preceded clinical seizure onset in NFLE, even when HR changes were not yet evident, confirming that wavelet analysis is a sensitive technique to detect sudden variations of autonomic balance occurring during transient phenomena. Finally we demonstrated that epileptic asystole is associated with a parasympathetic hypertonus counteracted by a marked sympathetic activation.

Studio neuroradiologico (morfologico e funzionale) nei pazienti con Epilessia Frontale Notturna

L'epilessia frontale notturna (EFN) è caratterizzata da crisi motorie che insorgono durante il sonno. Scopo del progetto è studiare le cause fisiopatologiche e morfo-funzionali che sottendono ai fenomeni motori nei pazienti con EFN e identificare alterazioni strutturali e/o metaboliche mediante tecniche avanzate di Risonanza Magnetica (RM). Abbiamo raccolto una casistica di pazienti con EFN afferenti al Centro Epilessia e dei Disturbi del Sonno del Dipartimento di Scienze Neurologiche, Università di Bologna. Ad ogni paziente è stato associato un controllo sano di età (± 5 anni) e sesso corrispondente. Tutti sono stati studiati mediante tecniche avanzate di RM comprendenti Spettroscopia del protone (1H-MRS), Tensore di diffusione ed imaging 3D ad alta risoluzione per analisi morfometriche. In particolare, la 1H-MRS è stata effettuata su due volumi di interesse localizzati nei talami e nel giro del cingolo anteriore. Sono stati inclusi nell’analisi finale 19 pazienti (7 M), età media 34 anni (range 19-50) e 14 controlli (6 M) età media 30 anni (range 19-40). A livello del cingolo anteriore il rapporto della concentrazione di N-Acetil-Aspartato rispetto alla Creatina (NAA/Cr) è risultato significativamente ridotto nei pazienti rispetto ai controlli (p=0,021). Relativamente all’analisi di correlazione, l'analisi tramite modelli di regressione multipla ha evidenziato che il rapporto NAA/Cr nel cingolo anteriore nei pazienti correlava con la frequenza delle crisi (p=0,048), essendo minore nei pazienti con crisi plurisettimanali/plurigiornaliere. Per interpretare il dato ottenuto è possibile solo fare delle ipotesi. L’NAA è un marker di integrità, densità e funzionalità neuronale. E’ possibile che alla base della EFN ci siano alterazioni metaboliche tessutali in precise strutture come il giro del cingolo anteriore. Questo apre nuove possibilità sull’utilizzo di strumenti di indagine basati sull’analisi di biosegnali, per caratterizzare aree coinvolte nella genesi della EFN ancora largamente sconosciute e chiarire ulteriormente l’eziologia di questo tipo di epilessia.

Rationale for an adjunctive therapy with fenofibrate in pharmacoresistant nocturnal frontal lobe epilepsy (NFLE).

Nocturnal Frontal Lobe Epilepsy (NFLE) is characterized by onset during infancy or childhood with persistence in adulthood, family history of similar nocturnal episodes simulating non-REM parasomnias (sleep terrors or sleepwalking), general absence of morphological substrates, often by normal interictal electroencephalographical recordings (EEGs) during wakefulness. A family history of epilepsy may be present with Mendelian autosomal dominant inheritance has been described in some families. Recent studies indicate the involvement of neuronal nicotinic acetylcholine receptors (nAChRs) in the molecular mechanisms of NFLE. Mutations in the genes encoding for the α4 (CHRNA4) and ß2 (CHRNB2) subunits of the nAChR induce changes in the biophysical properties of nAChR, resulting generally in a “gain of function”. Preclinical studies report that activation of a nuclear receptor called type peroxisome proliferator-activated receptor (PPAR-α) by endogenous molecules or by medications (e.g. fenofibrate) reduces the activity of the nAChR and, therefore, may decrease the frequency of seizures. Thus, we hypothesize that negative modulation of nAChRs might represent a therapeutic strategy to be explored for pharmacological treatment of this form of epilepsy, which only partially responds to conventional antiepileptic drugs. In fact, carbamazepine, the current medication for NFLE, abolishes the seizures only in one third of the patients. The aim of the project is: 1)_to verify the clinical efficacy of adjunctive therapy with fenofibrate in pharmacoresistant NFLE and ADNFLE patients; focousing on the analysis of the polysomnographic action of the PPAR- agonist (fenofibrate). 2)_to demonstrate the subtended mechanism of efficacy by means of electrophysiological and behavioral experiments in an animal model of the disease: particularly, transgenic mice carrying the mutation in the nAChR 4 subunit (Chrna4S252F) homologous to that found in the humans. Given that a PPAR-α agonist, FENOFIBRATE, already clinically utilized for lipid metabolism disorders, provides a promising therapeutic avenue in the treatment of NFLE\ADNFLE.