Glutamatergic neurons induce expression of functional glutamatergic synapses in primary myotubes.

The functioning of the nervous system depends upon the specificity of its synaptic contacts. The mechanisms triggering the expression of the appropriate receptors on postsynaptic membrane and the role of the presynaptic partner in the differentiation of postsynaptic structures are little known.To address these questions we cocultured murine primary muscle cells with several glutamatergic neurons, either cortical, cerebellar or hippocampal. Immunofluorescence and electrophysiology analyses revealed that functional excitatory synaptic contacts were formed between glutamatergic neurons and muscle cells. Moreover, immunoprecipitation and immunofluorescence experiments showed that typical anchoring proteins of central excitatory synapses coimmunoprecipitate and colocalize with rapsyn, the acetylcholine receptor anchoring protein at the neuromuscular junction.These results support an important role of the presynaptic partner in the induction and differentiation of the postsynaptic structures

Two-photon microscopy imaging of thy1GFP-M transgenic mice: a novel animal model to investigate brain dendritic cell subsets in vivo

Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for in vivo brain studies with two-photon fluorescence (TPF) microscopy. Mice of the thy1GFP-M line have been engineered for selective expression of green fluorescent protein (GFP) in neuronal populations. Here, we report that TPF microscopy reveals, at the brain surface of these mice, also motile non-neuronal GFP+ cells. We have analyzed the behavior of these cells in vivo and characterized in brain sections their immunophenotype. With TPF imaging, motile GFP+ cells were found in the meninges, subarachnoid space and upper cortical layers. The striking feature of these cells was their ability to move across the brain parenchyma, exhibiting evident shape changes during their scanning-like motion. In brain sections, GFP+ cells were immunonegative to antigens recognizing motile cells such as migratory neuroblasts, neuronal and glial precursors, mast cells, and fibroblasts. GFP+ non-neuronal cells exhibited instead the characteristic features and immunophenotype (CD11c and major histocompatibility complex molecule class II immunopositivity) of dendritic cells (DCs), and were immunonegative to the microglial marker Iba-1. GFP+ cells were also identified in lymph nodes and blood of thy1GFP-M mice, supporting their identity as DCs. Thus, TPF microscopy has here allowed the visualization for the first time of the motile behavior of brain DCs in situ. The results indicate that the thy1GFP-M mouse line provides a novel animal model for the study of subsets of these professional antigen-presenting cells in the brain. Information on brain DCs is still very limited and imaging in thy1GFP-M mice has a great potential for analyses of DC-neuron interaction in normal and pathological conditions

Altered wiring of sleep-wake regulatory hypothalamic neurons in a murine model of chronic neuroinflammation

Peptidergic neurons of the lateral hypothalamus containing orexin (OX) or melanin-concentrating hormone (MCH) are implicated in several physiological functions, including energy homeostasis and sleep regulation. In particular, OX neurons play a role in wakefulness consolidation and MCH neurons are sleep-active. Sleeping sickness (African trypanosomiasis), caused by the extracellular parasites African trypanosomes, is a severe chronic inflammatory condition, fatal if untreated, which causes in humans neuropsychiatric disturbances including characteristic sleep-wake alterations documented also in rodent models of the infection. The pathogenetic mechanisms of these alterations remain to be fully clarified. Using an established approach to the quantitative evaluation of synaptic input to hypothalamic peptidergic neurons (Cristino et al., PNAS 2013), we here analyzed glutamatergic and GABAergic synapses apposed to OX-A and MCH somata in African trypanosome-infected mice, maintained under a 12h/12h light/dark cycle. By triple immunofluorescence (synaptophysin, OX-A or MCH, V-glut or V-GAT), inhibitory and excitatory terminals were analyzed during the encephalitic stage of the infection versus controls, in mice sacrificed during daytime. The total number of synapses apposed to OX-A and MCH neurons was not altered in the infected mice. However, interestingly, inhibitory inputs were significantly increased and excitatory inputs significantly decreased, leading to an overall pattern of synaptic input to OX-A and MCH neurons of infected mice opposite to that found in control mice. The striking alteration of the balance between excitatory and inhibitory inputs to OX-A and MCH neurons opens questions on the susceptibility of the synaptic wiring of these cells to neuroinflammatory signaling and their functional correlates

In vivo visualization of dendritic cells in the mouse brain cortex by multiphoton microscopy in normal conditions and parasitic infection

We are investigating with real-time multiphoton microscopy cells in the brain of GFPM mice, in which about 10% of the neurons express green fluorescent protein (GFP). Through a chronic optical brain window, we have observed in these mice not only fluorescent neuronal subsets but also fluorescent cells which are negative to a variety of tested neuronal antigens, including those of neuronal progenitors, and which exhibit motility. These cells, which are frequently observed to penetrate the brain parenchyma from the meningeal surface, are likely to represent dendritic cells (DCs) on the basis of their immunophenotype (CD68+, CD11b+, F4/80-), the characterization of which is currently being finalized. DCs are professional antigen-presenting cells, which notably play a key role in immuno-tolerogenic and immune responses, and their role in the brain is still largely unexplored. We have investigated these cells in vivo in young adult GFPM mice, both in normal conditions and after infection with the parasite Trypanosoma brucei brucei (T.b.). Infection with T.b. subspecies causes human African trypanosomiasis (also called sleeping sickness), and the non-human pathogenic subspecies T.b. brucei is widely used in rodent models of this disease. In our study, in vivo investigations are complemented by immunolabelling on cryosectioned brains for the study of areas inaccessible in vivo. The findings we have hitherto obtained show that in control non-infected brains DCs are mainly localized in the meninges, they exhibit a round shape and are motionless, and occasionally they move and change their shape. T.b. brucei-infected GFPM mice have been investigated at two time points during the meningoencephalitic phase of the disease, i.e., in the initial phase of parasite invasion of the brain parenchyma and at a more advanced stage of parasite neuroinvasion. At the first of these time points, represented by day post-infection (dpi) 16, we have observed fluorescent DCs rapidly moving (crawling, rolling, etc) within the brain parenchyma, mostly close to blood vessel walls. At a more advanced stage of infection (dpi 22), DCs have been mainly detected within the brain parenchyma, and a large number of fluorescent cell debris (perhaps as a result of autophagy processes) as well as DCs of a relatively small size have also been observed. In vivo experiments with fluorescent T.b. brucei are currently in progress to visualize the eventual occurrence of dynamic interactions of DCs with the parasite

Infezione cerebrale nella tripanosomiasi africana sperimentale: meccanismi patogenetici e biomarcatori

Introduzione. La tripanosomiasi africana umana, denominata anche \u201cmalattia del sonno\u201d, \ue8 una malattia tropicale \u201cnegletta\u201d, tuttora endemica in focolai in zone rurali e remote dell\u2019Africa sub-Sahariana, causata dal parassita extracellulare Trypanosoma brucei (T.b.) trasmesso da punture di mosche del genere Glossina1-3. Nell\u2019ultima decade sono state diagnosticate alcune decine di casi anche in zone non endemiche (in immigrati, espatriati o, raramente, turisti), ponendo un difficile problema diagnostico3. La malattia evolve, nell\u2019uomo e nei modelli sperimentali in roditori, da una prima fase sistemica (emolinfatica) ad una seconda fase meningoencefalitica, con invasione parassitaria del parenchima encefalico e disturbi neuropsichiatrici ingravescenti, che includono caratteristiche alterazioni del sonno e del ciclo sonno/veglia1-4. I meccanismi patogenetici dell\u2019evoluzione della malattia sono solo parzialmente noti1,4. L\u2019infezione \ue8 fatale se non trattata. Sono cruciali a fini terapeutici non solo la diagnosi, ma anche la stadiazione della malattia, poich\ue9 il secondo stadio viene attualmente curato con farmaci di elevata tossicit\ue0 che oltrepassano la barriera emato-encefalica. Il criterio di stadiazione tuttora utilizzato, basato sul numero di globuli bianchi nel liquor, non \ue8 sufficientemente sensibile. I nostri studi pi\uf9 recenti, basati su modelli animali, si sono prefissi un duplice obiettivo di ricerca di A) efficaci biomarcatori dell\u2019infezione cerebrale, B) chiarimento di meccanismi patogenetici neuroimmunitari. Metodi e Risultati. A) Riguardo al primo scopo, abbiamo valutato la correlazione tra le alterazioni del ciclo sonno/veglia (monitorate tramite elettroencefalogramma, EEG, in ratti infetti impiantati con sonde telemetriche) e le concentrazioni di interferone (IFN)-gamma e di chemochine IFN-gamma dipendenti (CXCL-9, CXCL-10, CXCL-11) nel siero e nel liquor (mediante saggi ELISA) e nell\u2019encefalo (mediante qPCR). Le analisi dell\u2019EEG di ratti infetti mostrano, durante la fase meningoencefalitica, una frammentazione del sonno e alterazioni della struttura del sonno (e, in particolare, della sequenza di sonno REM e non-REM, cos\uec come del sonno e della veglia). Lo studio dell\u2019espressione genica ha mostrato un progressivo e significativo incremento nell\u2019encefalo di IFN-gamma e chemochine IFN-gamma dipendenti e, in particolare, di CXCL-10, nella fase avanzata della malattia (4.8 volte rispetto al controllo), concomitante sia con un significativo incremento di parassiti nell\u2019encefalo, che con un picco di concentrazione della CXCL-10 nel liquor. Di particolare interesse risulta la comparsa, nel siero, di un picco di CXCL-10 all\u2019esordio della fase meningoencefalitica. B) Per lo studio della risposta immunitaria dell\u2019encefalo (che, come \ue8 noto, presenta caratteristiche ben diverse dall\u2019immunit\ue0 periferica) abbiamo recentemente indagato il ruolo delle cellule dendritiche (dendritic cells, DCs) cerebrali nella tripanosomiasi sperimentale. Le DCs modulano la risposta immunitaria mediante presentazione dell\u2019antigene alle cellule T. In condizioni fisiologiche la presenza delle DCs all\u2019interno del cranio \ue8 limitata alle regioni meningeee ed alle sedi perivascolari del parenchima cerebrale, suggerendo un ruolo di immunosorveglianza5. Con studi in vivo mediante microscopia multifotonica in topi infetti, abbiamo dimostrato che, nella fase sistemica della malattia, le DCs stabiliscono contatti con il parassita all\u2019interno dei vasi cerebrali. Successivamente, all\u2019esordio della fase meningoencefalitica, le DCs invadono il parenchima cerebrale, esibendo movimenti rapidi ed ampi che indicano un\u2019attivit\ue0 \u201cdi esplorazione\u201d del tessuto. Con il progredire della malattia, le DCs mostrano una significativa diminuzione dei parametri di motilit\ue0, tendendo ad aggregarsi in clusters statici che inglobano il parassita. Al contempo, cellule T citotossiche CD8+ ,extravasate nel parenchima, entrano in contatto con il parassita. Conclusioni. A) I dati puntano a CXCL-10, unitamente al monitoraggio del sonno e della veglia (facilmente realizzabile nell\u2019uomo mediante actigrafia), come possibili biomarcatori di infezione cerebrale nella tripanosomiasi africana. B) I risultati puntano anche ad un ruolo cruciale delle DCs nel presentare antigeni del tripanosoma alle cellule T, e quindi nei meccanismi patogenetici dell\u2019infezione cerebrale, suggerendo la possibilit\ue0 di controllare le funzioni delle DCs per dirigerle, a scopi terapeutici, verso un\u2019attivazione protettiva. Supportato dal Wellcome Trust (WT089992MA). BIBLIOGRAFIA 1. Bentivoglio, M., Mariotti, R., Bertini, G. (2011) Neuroinflammation and brain infections: historical context and current perspectives. Brain Res Rev 66:152-173. 2. Bisoffi, Z., Buonfrate, D., Angheben, A. (2014) Travel, migration and neglected tropical diseases. In: Neglected tropical diseases and conditions of the nervous system (Bentivoglio M, Cavalheiro EA, Kristensson K, Patel N. Eds). Springer, New York, pp. 21-43. 3. Buguet, A., Mpanzou, G., Bentivoglio, M. (2014) Human African trypanosomiasis: a highly neglected neurological disease. In: Neglected tropical diseases and conditions of the nervous system (Bentivoglio M, Cavalheiro EA, Kristensson K, Patel N. Eds). Springer, New York, pp. 165-181. 4. Kristensson K, Nygard M, Bertini G, Bentivoglio M (2010) African trypanosome infections of the nervous system: parasite entry and effects on sleep and synaptic functions. Prog Neurobiol 91:152-171 5. Laperchia C., Allegra Mascaro A. L., Sacconi L., Andrioli A., Grassi-Zucconi G., Bentivoglio M., Buffelli M., Pavone F.S.(2013) Two-photon microscopy imaging of thy1GFP-M transgenic mice: a novel animal model to investigate brain dendritic cell subsets in vivo. PLoS ONE, 8,2, e56144 doi:10.1371/journal.pone.005614

Neural Damage in Experimental Trypanosoma brucei gambiense Infection: Hypothalamic Peptidergic Sleep and Wake-Regulatory Neurons.

Neuron populations of the lateral hypothalamus which synthesize the orexin (OX)/hypocretin or melanin-concentrating hormone (MCH) peptides play crucial, reciprocal roles in regulating wake stability and sleep. The disease human African trypanosomiasis (HAT), also called sleeping sickness, caused by extracellular Trypanosoma brucei (T. b.) parasites, leads to characteristic sleep-wake cycle disruption and narcoleptic-like alterations of the sleep structure. Previous studies have revealed damage of OX and MCH neurons during systemic infection of laboratory rodents with the non-human pathogenic T. b. brucei subspecies. No information is available, however, on these peptidergic neurons after systemic infection with T. b. gambiense, the etiological agent of 97% of HAT cases. The present study was aimed at the investigation of immunohistochemically characterized OX and MCH neurons after T. b. gambiense or T. b. brucei infection of a susceptible rodent, the multimammate mouse, Mastomys natalensis. Cell counts and evaluation of OX fiber density were performed at 4 and 8 weeks post-infection, when parasites had entered the brain parenchyma from the periphery. A significant decrease of OX neurons (about 44% reduction) and MCH neurons (about 54% reduction) was found in the lateral hypothalamus and perifornical area at 8 weeks in T. b. gambiense-infected M. natalensis. A moderate decrease (21% and 24% reduction, respectively), which did not reach statistical significance, was found after T. b. brucei infection. In two key targets of diencephalic orexinergic innervation, the peri-suprachiasmatic nucleus (SCN) region and the thalamic paraventricular nucleus (PVT), densitometric analyses showed a significant progressive decrease in the density of orexinergic fibers in both infection paradigms, and especially during T. b. gambiense infection. Altogether the findings provide novel information showing that OX and MCH neurons are highly vulnerable to chronic neuroinflammatory signaling caused by the infection of human-pathogenic African trypanosomes

The excitatory/inhibitory input to orexin/hypocretin neuron soma undergoes day/night reorganization.

Orexin (OX)/hypocretin-containing neurons are main regulators of wakefulness stability, arousal, and energy homeostasis. Their activity varies in relation to the animal's behavioral state. We here tested whether such variation is subserved by synaptic plasticity phenomena in basal conditions. Mice were sacrificed during day or night, at times when sleep or wake, respectively, predominates, as assessed by electroencephalography in matched mice. Triple immunofluorescence was used to visualize OX-A perikarya and varicosities containing the vesicular glutamate transporter (VGluT)2 or the vesicular GABA transporter (VGAT) combined with synaptophysin (Syn) as a presynaptic marker. Appositions on OX-A <sup>+</sup> somata were quantitatively analyzed in pairs of sections in epifluorescence and confocal microscopy. The combined total number of glutamatergic (Syn <sup>+</sup> /VGluT2 <sup>+</sup> ) and GABAergic (Syn <sup>+</sup> /VGAT <sup>+</sup> ) varicosities apposed to OX-A somata was similar during day and night. However, glutamatergic varicosities were significantly more numerous at night, whereas GABAergic varicosities prevailed in the day. Triple immunofluorescence in confocal microscopy was employed to visualize synapse scaffold proteins as postsynaptic markers and confirmed the nighttime prevalence of VGluT2 <sup>+</sup> together with postsynaptic density protein 95 <sup>+</sup> excitatory contacts, and daytime prevalence of VGAT <sup>+</sup> together with gephyrin <sup>+</sup> inhibitory contacts, while also showing that they formed synapses on OX-A <sup>+</sup> cell bodies. The findings reveal a daily reorganization of axosomatic synapses in orexinergic neurons, with a switch from a prevalence of excitatory innervation at a time corresponding to wakefulness to a prevalence of inhibitory innervations in the antiphase, at a time corresponding to sleep. This reorganization could represent a key mechanism of plasticity of the orexinergic network in basal conditions