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From trypanosomes to the nervous system, from molecules to behavior: a survey, on the occasion of the 90th anniversary of Castellani's discovery of the parasites in sleeping sickness
No full textThe observation of Trypanosomes in patients affected by sleeping sickness has been reported in 1903 by Aldo Castellani. On the occasion of the 90th anniversary of this discovery, we here present the findings recently obtained in and experimental model of African trypanosomiasis in the rat. The molecular and cellular mechanisms of the interplay between the parasite and the host have been largely clarified: a bidirectional signalling occurs between trypanosomes and CD8+ T cells of the host animal. This new pathogenetic mechanism of infection involves a lymphocyte triggering factor released by the parasite and interferon-gamma. A recently isolated neuronal interferon-gamma could also play a role in the disease. The selective induction of major histocompatibility antigens class I has revealed the involvement of the hypothalamic paraventricular and supraoptic nuclei in trypanosomiasis. Finally, studies based on the expression of the immediate early gene c-fos have pointed out during the infection a selective d ysregulation of the suprachiasmatic nucleus of the hypothalamus, that plays the role of biological clock. The latter finding could account for the disruption of endogenous rhythms in sleeping sickness
Fos-related protein expression in the midline paraventricular nucleus of the rat thalamus: basal oscillation and relationship with limbic efferents
No full textThe expression of Fos-related protein, encoded by the proto-oncogene c-fos, was investigated by means of immunohistochemistry in the paraventricular nucleus of the thalamic midline (PV) during nighttime and daytime in rats entrained to a 12-h light/12-h dark cycle. In the first step of this study the animal's physiological state preceding perfusion was monitored with electro-encephalographic recording. It was thus detected that the PV contained a considerable number of Fos-like-immunostained neurons during the hours of darkness, when the rats had been awake, and that the number of Fos-like-immunoreactive neurons was significantly lower during the hours of light, after a period of sleep. In the second step of this study Fos immunohistochemistry was combined with the retrograde transport of a gold-labeled tracer injected either in the amygdala or in the nucleus accumbens. This strategy enabled us to determine that in the rats perfused during nighttime Fos-related protein was spontaneously induced in PV cell s projecting to these targets, with a significant prevalence of neurons projecting to the amygdala in the anterior portion of the PV and of neurons projecting to the nucleus accumbens in the posterior part of the nucleus. In addition, a significant reduction of Fos-like-immunoreactive cells was detected in the PV ipsilaterally to the injection, indicating that tracer administration and axonal transport may interfere with c-fos expression in neurons. Altogether the present data indicate that Fos-related protein expression undergoes a marked oscillation in the PV during 24 h in basal conditions, and that c-fos is induced in the PV relay neuronal subsets when the animal is awake.(ABSTRACT TRUNCATED AT 250 WORDS
Melatonin and its new agonist S-20098 restore synchronized sleep fragmented by experimental trypanosome infection in the rat
No full textThe experimental infection with the parasite Trypanosoma brucei in the rat provides a unique model of dysfunction of the sleep regulatory mechanisms, because the length of synchronized sleep episodes is selectively and dramatically reduced in the advanced stages of the disease. In the present study, melatonin was acutely administered (3 mg/kg SC) to trypanosome-infected rats, before the sleep onset. This treatment resulted in a significant increase of the length of synchronized sleep episodes in respect to the infected animals and to those that had received only the vehicle. Thus, melatonin restored a normal sleep pattern during the infection. Similar findings were obtained with the new melatonin agonist S-20098. The sleep parameters were not significantly modified by either melatonin or S-20098 acute administration to noninfected animals. These findings indicate that exogenous melatonin and S-20098 exert a selective regulatory action on sleep fragmentation during experimental trypanosomiasis
Circadian rhythms of DNA content in brain and kidney: effects of environmental stimulation
No full textMale adult Wistar rats kept under natural lighting show circadian rhythms of DNA content and DNA synthesis in the cerebral cortex, cerebellum and kidney. In the cerebral cortex the acrophases of the 2 rhythms almost coincide during the dark period. On the other hand, in kidney, the acrophase of DNA synthesis is phase-advanced by about 14 h with respect to the acrophase of DNA content, which occurs in the dark span, as in the cerebral cortex. Comparable results were obtained in 5 week-old rats raised under artificial lighting conditions (LD 7:19) and exposed for a few days to sensory and social stimulation (an enriched sensory environment). At variance with the latter data, the circadian changes of DNA content and synthesis flattened out and were not statistically significant in the cerebral cortex of 5 week-old rats kept for a few days under conditions of sensory and social deprivation (an impoverished sensory environment). A similar effect occurred in kidney with regard to the rhythm of DNA content, while the circadian rhythm of DNA synthesis remained statistically significant but was phase-delayed by about 6h with respect to the corresponding rhythm occurring in the enriched environment. In sensory impoverished rats, MESOR values of kidney wet weight and DNA specific activity were significantly higher than in sensory enriched rats, while MESOR values of DNA content were significantly lower. The data demonstrate the striking dependence of the circadian variations of DNA content and synthesis on the nature of environmental stimulation
Brain-derived neurotrophic factor undergoes physiological changes during a 24 hours cycle in rat brain
No full textIn vivo visualization of dendritic cells in the mouse brain cortex by multiphoton microscopy in normal conditions and parasitic infection
No full textWe 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
Circadian variation in expression of the trkB receptor in adult rat hyppocampus
No full textThe expression of brain-derived neurotrophic factor (BDNF) in the central nervous system (CNS) and the expression of its high-affinity trkB receptor on neuron surfaces are known to depend on neuron activity. The expression of BDNF (mRNA and protein) and trkB mRNA shows circadian oscillations in rat hippocampal homogenates. We investigated circadian variations in trkB expression in specific areas of the adult rat hippocampal formation by immunohistochemistry. In sets of two experiments performed in the spring, 39 2-month-old male Wistar rats were accustomed to a 12-h light-12-h dark cycle for 2 weeks. Three animals were then sacrificed every 4 h. Forty-micrometer- thick coronal sections of hippocampal formation were obtained and processed for trkB immunohistochemistry. Cell staining intensity was assessed by image analysis of different hippocampal areas on five sections per animal. Circadian rhythmicity was evaluated by the cosinor method. Statistically significant circadian variations in trkB expression were found in dentate gyrus, entorhinal cortex, and the CA3 and hilar regions of the hippocampus, with highest expression during the first half of the dark (activity) period. These findings suggest a relationship between trkB expression and the physiological neuronal activation of wakefulness. TrkB receptor expression in the hippocampal regions studied was continuous and changes were gradual over the 24-h cycle, suggesting that more complex regulatory mechanisms also intervened
Trypanosomes cause dysregulation of c-fos expression in the rat suprachiasmatic nucleus
No full textRats infected with the parasite Trypanosoma brucei brucei showed selective changes of c-fos expression in the suprachiasmatic nucleus of the hypothalamus (SCN) during spontaneous sleep (S) and wakefulness (W) under a basal 12 h/12 h light-dark (L-D) cycle. In the vast majority of W (D phase) control animals the SCN was devoid of cells displaying Fos-related immunopositivity, while Fos-like-immunoreactive (ir) neurones were detected in most S (L phase) control rats. In most infected animals, on the other hand, Fos-ir neurones were detected in the SCN during W, but not during the S period, with a significant difference between control and infected S rats. Thus, these data indicate that the basal c-fos expression in the SCN during the L-D and S-W cycles is considerably altered in experimental trypanosomiasis. This is the first observation of a selective change in the SCN in trypanosome-infected rat brains. Since the SCN plays an important role as a pace-maker for biological rhythms, this finding may provide a basis for understanding the pathogenesis behind endogenous rhythm dyregulation and changes in sleeping pattern in human trypanosomiasis (African sleeping sickness)
Migratory behavior of dendritic cells in the mouse brain cortex in normal and pathological conditions by multiphoton microscopy
No full textDendritic cells (DCs) have an immune surveillance role in the central nervous system. It is well
known that in normal conditions they maintain the immuno-tolerance by T-cell suppression and that
their presence is limited to the meninges, choroid plexus and rarely to the brain parenchyma. During
inflammation, DCs infiltrate the brain parenchyma, enhance their antigen processing capacity and
promote the initiation of immune responses by T-cell activation. Information on DCs in vivo in the
CNS is very limited.
We are investigating the dynamic behavior of the DCs in the mouse brain cortex in vivo in normal
conditions and in a model of chronic neuroinflammation represented by infection with the parasite
Trypanosoma brucei (Tb), the causative agent of African trypanosomiasis. This disease evolves in
humans and experimental animals in two stages: an early stage, in which Tb invade peripheral organs
through the hemolymphatic system; and a late meningoencephalitic stage with severe consequences
on nervous system functions.
In order to visualize dynamic cell processes in healthy mice and in mice infected with the non-human
pathogenic subspecies Tb brucei, a little craniotomy was performed leaving the dura mater
unperturbed and the bone flap was replaced by a coverglass. This chronic implantation provides an
excellent optical access for multiphoton acquisition in vivo, allowing high-resolution imaging over
time. Blood vessels were visualized by iv injection of TRITC-conjugated dextran. Four-dimensional
reconstruction (x,y,z,t) of transcranial images were analyzed by Imaris software to track the
migratory route of DCs, their position with respect to the vasculature over time, and their movement
features (crawling, rolling, etc.).
The initial results of this study show that in the healthy brain DCs are mainly localized in the
meninges, they are motionless and exhibit a round shape. In addition, with the progression of Tb
brucei infection DCs have been observed to penetrate the brain parenchyma from the meninges, their
number increases and their motility is enhanced. At an advanced phase of the infection, phagosomelike
structures are also observed in the brain parenchyma.
The
data suggest an involvement of DCs in pathogenetic mechanisms and
severity of Tb
brain
infection.
In
vivo
experiments with fluorescent Tb
brucei
are in progress to visualize in the brain
potential
interactions between the parasite and host immune cells represented by DCs
Sleep and timekeeping changes, and dysregulation of the biological clock in experimental trypanosomiasis
No full textThe rest-activity and body temperature 24 hours cycles, as well as the pattern of spontaneous sleep, were investigated in rats after infection with Trypanosoma brucei brucei. In the infected rats, which were entrained to a 12 hours/12 hours photoperiod, a considerable hypokinesia was detected during the hours of darkness. In most of the infected animals, the body temperature cycle displayed a lower amplitude and an advance of about 3 hours in respect to control rats; in addition, the body temperature rhythm was not significant in some infected rats. The relative proportion of slow wave synchronized sleep, as well as the rapid-eye movement (REM) latency, were significantly reduced in the infected animals, in which sleep was considerably fragmented. The induction of Fos (the protein encoded by the immediate early gene c-fos), in response to light stimulation during the early subjective night, was severely impaired in the hypothalamic suprachiasmatic nuclei in trypanosome-infected rats. Altogether these data point out a disruption of locomotor activity and body temperature 24 hours cycle and a major disorganization of sleep during experimental trypanosomiasis. In addition, our findings indicate that the molecular and functional correlates of the synchronizing action of the suprachiasmatic nuclei, which play a major role of biological clock of endogenous biological rhythms, could be altered during trypanosome infection
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