Giuseppe Bertini

African trypanosome infections of the nervous system: Parasite entry and effects on sleep and synaptic functions.

The extracellular parasite Trypanosoma brucei causes human African trypanosomiasis (HAT), also known as sleeping sickness. Trypanosomes are transmitted by tsetse flies and HAT occurs in foci in sub-Saharan Africa. The disease, which is invariably lethal if untreated, evolves in a first hemo-lymphatic stage, progressing to a second meningo-encephalitic stage when the parasites cross the blood-brain barrier. At first, trypanosomes are restricted to circumventricular organs and choroid plexus in the brain outside the blood-brain barrier, and to dorsal root ganglia. Later, parasites cross the blood-brain barrier at post-capillary venules, through a multi-step process similar to that of lymphocytes. Accumulation of parasites in the brain is regulated by cytokines and chemokines. Trypanosomes can alter neuronal function and the most prominent manifestation is represented by sleep alterations. These are characterized, in HAT and experimental rodent infections, by disruption of the sleep-wake 24h cycle and internal sleep structure. Trypanosome infections alter also some, but not all, other endogenous biological rhythms. A number of neural pathways and molecules may be involved in such effects. Trypanosomes secrete prostaglandins including the somnogenic PGD2, and they interact with the hosts immune system to cause release of pro-inflammatory cytokines. From the sites of early localization of parasites in the brain and meninges, such molecules could affect adjacent brain areas implicated in sleep-wakefulness regulation, including the suprachiasmatic nucleus and its downstream targets, to cause the changes characteristic of the disease. This raises challenging issues on the effects of cytokines on synaptic functions potentially involved in sleep-wakefulness alterations.

Decline of the presynaptic network, including GABAergic terminals, in the aging suprachiasmatic nucleus of the mouse.

Biological rhythms, and especially the sleep/wake cycle, are frequently disrupted during senescence. This draws attention to the study of aging-related changes in the hypothalamic suprachiasmatic nucleus (SCN), the master circadian pacemaker. The authors here compared the SCN of young and old mice, analyzing presynaptic terminals, including the gamma-aminobutyric acid (GABA)ergic network, and molecules related to the regulation of GABA, the main neurotransmitter of SCN neurons. Transcripts of the α3 subunit of the GABAA receptor and the GABA-synthesizing enzyme glutamic acid decarboxylase isoform 67 (GAD67) were analyzed with real-time RT-PCR and GAD67 protein with Western blotting. These parameters did not show significant changes between the 2 age groups. Presynaptic terminals were identified in confocal microscopy with synaptophysin immunofluorescence, and the GABAergic subset of those terminals was revealed by the colocalization of GAD67 and synaptophysin. Quantitative analysis of labeled synaptic endings performed in 2 SCN subregions, where retinal afferents are known to be, respectively, very dense or very sparse, revealed marked aging-related changes. In both subregions, the evaluated parameters (the number of and the area covered by presynaptic terminals and by their GABAergic subset) were significantly decreased in old versus young mice. No significant differences were found between SCN tissue samples from animals sacrificed at different times of day, in either age group. Altogether, the data point out marked reduction in the synaptic network of the aging biological clock, which also affects GABAergic terminals. Such alterations could underlie aging-related SCN dysfunction, including low-amplitude output during senescence.

Cytokine-induced activation of glial cells in the mouse brain is enhanced at an advanced age.

Numerous neurological diseases which include neuroinflammatory components exhibit an age-related prevalence. The aging process is characterized by an increase of inflammatory mediators both systemically and in the brain, which may prime glial cells. However, little information is available on age-related changes in the glial response of the healthy aging brain to an inflammatory challenge. This problem was here examined using a mixture of the proinflammatory cytokines interferon-gamma and tumor necrosis factor-alpha, which was injected intracerebroventricularly in young (2-3.5 months), middle-aged (10-11 months) and aged (18-21 months) mice. Vehicle (phosphate-buffered saline) was used as control. After a survival of 1 or 2 days (all age groups) or 4 days (young and middle-aged animals), immunohistochemically labeled astrocytes and microglia were investigated both qualitatively and quantitatively. In all age groups, astrocytes were markedly activated in periventricular as well as in deeper brain regions 2 days following cytokine treatment, whereas microglia activation was already evident at 24 h. Interestingly, cytokine-induced activation of both astrocytes and microglia was significantly more marked in the brain of aged animals, in which it included numerous ameboid microglia, than of younger age groups. Moderate astrocytic activation was also seen in the hippocampal CA1 field of vehicle-treated aged mice. FluoroJade B histochemistry and the terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling technique, performed at 2 days after cytokine administration, did not reveal ongoing cell death phenomena in young or aged animals. This indicated that glial cell changes were not secondary to neuronal death. Altogether, the findings demonstrate for the first time enhanced activation of glial cells in the old brain, compared with young and middle-aged subjects, in response to cytokine exposure. Interestingly, the results also suggest that such enhancement does not develop gradually since youth, but appears characterized by relatively late onset.

Nitric oxide synthase in the adult and developing thalamus: Histochemical and immunohistochemical study in the rat

The distribution of neuronal elements that express nitric oxide synthase (NOS), the synthetic enzyme of the free radical nitric oxide, was investigated in the adult and developing rat thalamus by means of NADPH‐diaphorase (NADPH‐d) histochemistry, which is a marker of NOS. Immunocytochemistry was also used to confirm the equivalence between the histochemical pattern of staining and the distribution of the expression of the neuronal NOS isoform.

Late-onset Parkinsonism in NFκB/c-Rel-deficient mice

Activation of the nuclear factor κB/c-Rel can increase neuronal resilience to pathological noxae by regulating the expression of pro-survival manganese superoxide dismutase (MnSOD, now known as SOD2) and Bcl-xL genes. We show here that c-Rel-deficient (c-rel−/−) mice developed a Parkinson’s disease-like neuropathology with ageing. At 18 months of age, c-rel−/− mice exhibited a significant loss of dopaminergic neurons in the substantia nigra pars compacta, as assessed by tyrosine hydroxylase-immunoreactivity and Nissl staining. Nigral degeneration was accompanied by a significant loss of dopaminergic terminals and a significant reduction of dopamine and homovanillic acid levels in the striatum. Mice deficient of the c-Rel factor exhibited a marked immunoreactivity for fibrillary α-synuclein in the substantia nigra pars compacta as well as increased expression of divalent metal transporter 1 (DMT1) and iron staining in both the substantia nigra pars compacta and striatum. Aged c-rel−/− mouse brain were characterized by increased microglial reactivity in the basal ganglia, but no astrocytic reaction. In addition, c-rel−/− mice showed age-dependent deficits in locomotor and total activity and various gait-related deficits during a catwalk analysis that were reminiscent of bradykinesia and muscle rigidity. Both locomotor and gait-related deficits recovered in c-rel−/− mice treated with l-3,4-dihydroxyphenylalanine. These data suggest that c-Rel may act as a regulator of the substantia nigra pars compacta resilience to ageing and that aged c-rel−/− mice may be a suitable model of Parkinson’s disease.

Experimental sleep deprivation as a tool to test memory deficits in rodents

Paradigms of sleep deprivation (SD) and memory testing in rodents (laboratory rats and mice) are here reviewed. The vast majority of these studies have been aimed at understanding the contribution of sleep to cognition, and in particular to memory. Relatively little attention, instead, has been devoted to SD as a challenge to induce a transient memory impairment, and therefore as a tool to test cognitive enhancers in drug discovery. Studies that have accurately described methodological aspects of the SD protocol are first reviewed, followed by procedures to investigate SD-induced impairment of learning and memory consolidation in order to propose SD protocols that could be employed as cognitive challenge. Thus, a platform of knowledge is provided for laboratory protocols that could be used to assess the efficacy of drugs designed to improve memory performance in rodents, including rodent models of neurodegenerative diseases that cause cognitive deficits, and Alzheimers disease in particular. Issues in the interpretation of such preclinical data and their predictive value for clinical translation are also discussed.

Cerebral perfusion alterations in epileptic patients during peri-ictal and post-ictal phase: PASL vs DSC-MRI

Non-invasive pulsed arterial spin labeling (PASL) MRI is a method to study brain perfusion that does not require the administration of a contrast agent, which makes it a valuable diagnostic tool as it reduces cost and side effects. The purpose of the present study was to establish the viability of PASL as an alternative to dynamic susceptibility contrast (DSC-MRI) and other perfusion imaging methods in characterizing changes in perfusion patterns caused by seizures in epileptic patients. We evaluated 19 patients with PASL. Of these, the 9 affected by high-frequency seizures were observed during the peri-ictal period (within 5hours since the last seizure), while the 10 patients affected by low-frequency seizures were observed in the post-ictal period. For comparison, 17/19 patients were also evaluated with DSC-MRI and CBF/CBV. PASL imaging showed focal vascular changes, which allowed the classification of patients in three categories: 8 patients characterized by increased perfusion, 4 patients with normal perfusion and 7 patients with decreased perfusion. PASL perfusion imaging findings were comparable to those obtained by DSC-MRI. Since PASL is a) sensitive to vascular alterations induced by epileptic seizures, b) comparable to DSC-MRI for detecting perfusion asymmetries, c) potentially capable of detecting time-related perfusion changes, it can be recommended for repeated evaluations, to identify the epileptic focus, and in follow-up and/or therapy-response assessment.

Neuroinflammation and brain infections: historical context and current perspectives.

An overview of current concepts on neuroinflammation and on the dialogue between neurons and non-neuronal cells in three important infections of the central nervous systems (rabies, cerebral malaria, and human African trypanosomiasis or sleeping sickness) is here presented. Large numbers of cases affected by these diseases are currently reported. In the context of an issue dedicated to Camillo Golgi, historical notes on seminal discoveries on these diseases are also presented. Neuroinflammation is currently closely associated with pathogenetic mechanisms of chronic neurodegenerative diseases. Neuroinflammatory signaling in brain infections is instead relatively neglected in the neuroscience community, despite the fact that the above infections provide paradigmatic examples of alterations of the intercellular crosstalk between neurons and non-neuronal cells. In rabies, strategies of immune evasion of the host lead to silencing neuroinflammatory signaling. In the intravascular pathology which characterizes cerebral malaria, leukocytes and Plasmodium do not enter the brain parenchyma. In sleeping sickness, leukocytes and African trypanosomes invade the brain parenchyma at an advanced stage of infection. Both the latter pathologies leave open many questions on the targeting of neuronal functions and on the pathogenetic role of non-neuronal cells, and in particular astrocytes and microglia, in these diseases. All three infections are hallmarked by very severe clinical pictures and relative sparing of neuronal structure. Multidisciplinary approaches and a concerted action of the neuroscience community are needed to shed light on intercellular crosstalk in these dreadful brain diseases. Such effort could also lead to new knowledge on non-neuronal mechanisms which determine neuronal death or survival.

The chemical heterogeneity of cortical interneurons: nitric oxide synthase vs. calbindin and parvalbumin immunoreactivity in the rat.

Neurons that contain nitric oxide synthase (NOS) type I and the calcium binding proteins calbindin D28k or parvalbumin were simultaneously visualized by means of double immunohistofluorescence in the cerebral cortex of Wistar and Sprague-Dawley rats. All the three immunoreactive cell populations were primarily represented by nonpyramidal neurons. NOS-immunoreactive cells were less numerous than the calbindin- or parvalbumin-immunoreactive ones, and were intermingled with the neurons containing these calcium binding proteins. NOS-immunoreactive cells were separate from the parvalbumin-immunoreactive ones, whereas a minor proportion of them was found to be colocalized with calbindin. The cortical neurons in which NOS and calbindin coexisted were more numerous in the Sprague-Dawley than in the Wistar rats, and displayed an anteroposterior gradient of density, with the highest concentration in the medial prefrontal, frontal, and cingulate cortices. Double NOS-calbindin-immunoreactive neurons prevailed in the deep cortical layers and they were relatively numerous in the cingulate cortex. The present data indicate a selectivity in the expression of NOS vs. calbindin and parvalbumin in cortical cells, and further support the chemical heterogeneity of GABAergic interneurons in the cerebral cortex.

H1N1 influenza virus induces narcolepsy-like sleep disruption and targets sleep–wake regulatory neurons in mice

Significance Influenza A virus infections are risk factors for narcolepsy, a disease in which autoimmunity has been implicated. We tested experimentally whether influenza virus infections could be causally related to narcolepsy. We found that mice infected with a H1N1 influenza A virus strain developed over time sleep–wake changes described in murine models of narcolepsy and narcolepsy patients. In the brain, the virus infected orexin/hypocretin-producing neurons, which are destroyed in human narcolepsy, and other cells in the distributed sleep–wake-regulating neuronal network. The findings, obtained in mice lacking an adaptive autoimmune response, thus provide new avenues for research on infection-related mechanisms in narcolepsy. An increased incidence in the sleep-disorder narcolepsy has been associated with the 2009–2010 pandemic of H1N1 influenza virus in China and with mass vaccination campaigns against influenza during the pandemic in Finland and Sweden. Pathogenetic mechanisms of narcolepsy have so far mainly focused on autoimmunity. We here tested an alternative working hypothesis involving a direct role of influenza virus infection in the pathogenesis of narcolepsy in susceptible subjects. We show that infection with H1N1 influenza virus in mice that lack B and T cells (Recombinant activating gene 1-deficient mice) can lead to narcoleptic-like sleep–wake fragmentation and sleep structure alterations. Interestingly, the infection targeted brainstem and hypothalamic neurons, including orexin/hypocretin-producing neurons that regulate sleep–wake stability and are affected in narcolepsy. Because changes occurred in the absence of adaptive autoimmune responses, the findings show that brain infections with H1N1 virus have the potential to cause per se narcoleptic-like sleep disruption.