Why trypanosomes cause sleep disturbances

African trypanosomiasis or sleeping sickness is a complex disease that involves a constellation of symptoms, but is hallmarked, during disease progression, by the onset of disturbances of sleep/wake alternation during 24 h and by alterations in the organization of sleep. In particular, the patients develop frequent and sudden sleep episodes during the day, and wakefulness episodes during night. A striking feature of the illness is represented SOREM (sleep onset rapid eye movement) episodes, in which the patients go directly from wakefulness to REM sleep without passing through non-REM sleep. Similar alterations have been found in Trypanosoma brucei brucei-infected rats, and we are now documenting a relatively early onset of SOREM episodes during the experimental infection. In trypanosome-infected rats, the number of SOREM episodes increases over time and reaches a peak which is coincident with intrusion of sleep into wakefulness and vice versa. We are currently investigating whether the latter changes can help in disease staging by revealing parasite passage across the blood-brain barrier (BBB) and invasion of the brain parenchyma

Tryps and trips: cell trafficking across the 100-year-old blood–brain barrier

One hundred years ago, Edwin E. Goldmann discovered the blood–brain barrier (BBB) using trypan dyes. These dyes were developed and named by Paul Ehrlich during his search for drugs to kill African trypanosomes (extracellular parasites that cause sleeping sickness) while sparing host cells. For Ehrlich, this was the first strategy based on the ‘chemotherapy’ concept he had introduced. The discovery of the BBB revealed, however, the difficulties in drug delivery to the brain. Mechanisms by which parasites enter, dwell, and exit the brain currently provide novel views on cell trafficking across the BBB. These mechanisms also highlight the role of pericytes and endocytosis regulation in BBB functioning and in disrupted BBB gating, which may be involved in the pathogenesis of neurodegeneration

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

Do diencephalic sleep-wake-regulatory systems meet?

A wealth of classical studies on the thalamus pointed out a key role of the reticular thalamic nucleus (Rt) in sleep regulation. Since the discovery of the orexin (Orx)/ hypocretin peptides in 1998, a key role in wakefulness stability has been ascribed to Orx neurons, which reside in the lateral hypothalamus. Rt neuron efferents, which are inhibitory, are distributed to nuclei of the dorsal thalamus. Orx neuron efferents, which are excitatory, are distributed widely in the neuroaxis but concentrated in the thalamus along the midline. Current views seem to consider these systems as separate networks. We here wondered whether these networks meet instead in the diencephalon, and in particular whether Rt and Orx synaptic endings converge on the same neuronal cell bodies or reach separate neurons of the paraventricular thalamic nucleus (PVT) of the thalamic midline. To answer this question, PVT neurons were here investigated in confocal microscopy by means of multiple immunofluorescent labelling: calretinin labelling of PVT cell bodies; Orx + synaptophysin for the labelling of Orx synaptic endings; parvalbumin + synaptophysin for the labelling of Rt synaptic endings. Striking results on the convergence of the two sets of synapses on the same neurons were obtained, since almost 100% of Orx synaptic boutons were apposed to PVT neurons which also received Rt synaptic boutons. Rt axon terminals were more abundant in PVT than Orx ones, and also reached neurons which did not receive Orx input. The present findings on the synaptic wiring of PVT neurons therefore points to the dorsal thalamic midline as a “meeting point” of sleep-wake-regulatory diencephalic networks. PVT efferents reach the prefrontal cortex, and limbic targets represented by the nucleus accumbens and amygdala. The synaptic convergence here demonstrated could thus place PVT neurons at the core of sleep-wake-related modulation of cognitive functions and emotional, affective behaviour

Trypanosoma brucei brucei invasion and T-cell infiltration of the brain parenchyma in experimental sleeping sickness: timing and correlation with functional changes

Background: The timing of Trypanosoma brucei entry into the brain parenchyma to initiate the second, meningoencephalitic stage of human African trypanosomiasis or sleeping sickness is currently debated and even parasite invasion of the neuropil has been recently questioned. Furthermore, the relationship between neurological features and disease stage are unclear, despite the important diagnostic and therapeutic implications. Methodology: Using a rat model of chronic Trypanosoma brucei brucei infection we determined the timing of parasite and T-cell neuropil infiltration and its correlation with functional changes. Parasite DNA was detected using trypanosome-specific PCR. Body weight and sleep structure alterations represented by sleep-onset rapid eye movement (SOREM) periods, reported in human and experimental African trypanosomiasis, were monitored. The presence of parasites, as well as CD4+ and CD8+ T-cells in the neuropil was assessed over time in the brain of the same animals by immunocytochemistry and quantitative analyses. Principal findings: Trypanosome DNA was present in the brain at day 6 post-infection and increased more than 15-fold by day 21. Parasites and T-cells were observed in the parenchyma from day 9 onwards. Parasites traversing blood vessel walls were observed in the hypothalamus and other brain regions. Body weight gain was reduced from day 7 onwards. SOREM episodes started in most cases early after infection, with an increase in number and duration after parasite neuroinvasion. Conclusion: These findings demonstrate invasion of the neuropil over time, after an initial interval, by parasites and lymphocytes crossing the blood-brain barrier, and show that neurological features can precede this event. The data thus challenge the current clinical and cerebrospinal fluid criteria of disease staging

Circumventricular Organs and Parasite Neurotropism: Neglected Gates to the Brain?

Circumventricular organs (CVOs), neural structures located around the third and fourth ventricles, harbor, similarly to the choroid plexus, vessels devoid of a blood-brain barrier (BBB). This enables them to sense immune-stimulatory molecules in the blood circulation, but may also increase chances of exposure to microbes. In spite of this, attacks to CVOs by microbes are rarely described. It is here highlighted that CVOs and choroid plexus can be infected by pathogens circulating in the bloodstream, providing a route for brain penetration, as shown by infections with the parasites Trypanosoma brucei. Immune responses elicited by pathogens or systemic infections in the choroid plexus and CVOs are briefly outlined. From the choroid plexus trypanosomes can seed into the ventricles and initiate accelerated infiltration of T cells and parasites in periventricular areas. The highly motile trypanosomes may also enter the brain parenchyma from the median eminence, a CVO located at the base of the third ventricle, by crossing the border into the BBB-protected hypothalamic arcuate nuclei. A gate may, thus, be provided for trypanosomes to move into brain areas connected to networks of regulation of circadian rhythms and sleep-wakefulness, to which other CVOs are also connected. Functional imbalances in these networks characterize human African trypanosomiasis, also called sleeping sickness. They are distinct from the sickness response to bacterial infections, but can occur in common neuropsychiatric diseases. Altogether the findings lead to the question: does the neglect in reporting microbe attacks to CVOs reflect lack of awareness in investigations or of gate-opening capability by microbes

Expression of interferon-inducible chemokines and sleep/wake changes during early encephalitis in experimental African trypanosomiasis

Background: Human African trypanosomiasis or sleeping sickness, caused by the parasite Trypanosoma brucei, leads to neuroinflammation and characteristic sleep/wake alterations. The relationship between the onset of these alterations and the development of neuroinflammation is of high translational relevance, but remains unclear. This study investigates the expression of interferon (IFN)-γ and IFN-inducible chemokine genes in the brain, and the levels of CXCL10 in the serum and cerebrospinal fluid prior to and during the encephalitic stage of trypanosome infection, and correlates these with sleep/wake changes in a rat model of the disease. Methodology/Principal findings: The expression of genes encoding IFN-γ, CXCL9, CXCL10, and CXCL11 was assessed in the brain of rats infected with Trypanosoma brucei brucei and matched controls using semi-quantitative end-point RT-PCR. Levels of CXCL10 in the serum and cerebrospinal fluid were determined using ELISA. Sleep/wake states were monitored by telemetric recording. Using immunohistochemistry, parasites were found in the brain parenchyma at 14 days post-infection (dpi), but not at 6 dpi. Ifn-γ, Cxcl9, Cxcl10 and Cxcl11 mRNA levels showed moderate upregulation by 14 dpi followed by further increase between 14 and 21 dpi. CXCL10 concentration in the cerebrospinal fluid increased between 14 and 21 dpi, preceded by a rise in the serum CXCL10 level between 6 and 14 dpi. Sleep/wake pattern fragmentation was evident at 14 dpi, especially in the phase of wake predominance, with intrusion of sleep episodes into wakefulness. Conclusions/Significance: The results show a modest increase in Cxcl9 and Cxcl11 transcripts in the brain and the emergence of sleep/wake cycle fragmentation in the initial encephalitic stage, followed by increases in Ifn-γ and IFN-dependent chemokine transcripts in the brain and of CXCL10 in the cerebrospinal fluid. The latter parameter and sleep/wake alterations could provide combined humoral and functional biomarkers of the early encephalitic stage in African trypanosomiasis

The original histological slides of Camillo Golgi and his discoveries on neuronal structure

The metallic impregnation invented by Camillo Golgi in 1873 has allowed the visualization of individual neurons in their entirety, leading to a breakthrough in the knowledge on the structure of the nervous system. Professor of Histology and of General Pathology, Golgi worked for decades at the University of Pavia, leading a very active laboratory. Unfortunately, most of Golgi's histological preparations are lost. The present contribution provides an account of the original slides on the nervous system from Golgi's laboratory available nowadays at the Golgi Museum and Historical Museum of the University of Pavia. Knowledge on the organization of the nervous tissue at the time of Golgi's observations is recalled. Notes on the equipment of Golgi's laboratory and the methodology Golgi used for his preparations are presented. Images of neurons from his slides (mostly from hippocampus, neocortex and cerebellum) are here shown for the first time together with some of Golgi's drawings. The sections are stained with the Golgi impregnation and Cajal stain. Golgi-impregnated sections are very thick (some more than 150 \u3bcm) and require continuous focusing during the microscopic observation. Heterogeneity of neuronal size and shape, free endings of distal dendritic arborizations, axonal branching stand out at the microscopic observation of Golgi-impregnated sections and in Golgi's drawings, and were novel findings at his time. Golgi also pointed out that the axon only originates from cell bodies, representing a constant and distinctive feature of nerve cells which distinguishes them from glia, and subserving transmission at a distance. Dendritic spines can be seen in some cortical neurons, although Golgi, possibly worried about artifacts, did not identify them. The puzzling intricacy of fully impregnated nervous tissue components offered to the first microscopic observations still elicit nowadays the emotion Golgi must have felt looking at his slides

Converging orexinergic and reticular thalamic inputs on thalamic paraventricular neurons in normal conditions and experimental sleeping sickness

A subset of excitatory neurons in the lateral hypothalamus, known to express the peptide orexin/hypocretin (Ox), play a key role in maintaining wakefulness. Projections from Ox neurons are widely distributed in the neuraxis but terminations are heavily concentrated in the thalamus along the midline, especially the paraventricular thalamic nucleus (PVT). The same areas receive afferents from inhibitory, GABAergic neurons expressing parvalbumin (Pv) in the thalamic reticular nucleus (Rt), which has long been considered essential for sleep regulation. While the two circuitries have been regarded as distinct, we tested the hypothesis that PVT neurons represent a common target for both afferent systems by means of confocal microscopy of multiple immunofluorescence labeling in the mouse brain. Calretinin (Cr) was used as marker of PVT neurons. Almost 90% of PVT perikarya were contacted by Pv+ terminals, confirming the prominent role of Rt in modulating PVT activity. Interestingly, about a third of these neurons were also reached by Ox+ terminals, suggesting a key role of the thalamic midline in integrating information pertaining vigilance state control. PVT cells receiving Ox+ but not Pv+ contacts were observed only rarely. In mice infected with the parasite Trypanosoma brucei brucei, the causal agent of the neuroinflammatory disease “sleeping sickness”, Pv+ afferents into PVT were largely preserved, while orexinergic fibers appeared fragmented and reduced in density. Importantly, the fraction of PVT perikarya receiving both Pv+ and Ox+ terminals was reduced by about 50%. The substantially decreased convergence of the two regulatory systems, in association with infection-induced disrupted sleep and sleep/wake cycles, further supports the hypothesis that PVT contributes to vigilance and arousal in physiological conditions

Orexinergic neuron susceptibility to neuroinflammatory and aging-related neurodegenerative diseases

Orexins (a.k.a. hypocretins) play a crucial role in several physiological functions, including energy balance and the maintenance of wakefulness. Deficient orexin signalling is the hallmark of the sleep disorder narcolepsy. Although immune mechanisms have been hypotesized, the pathogenesis of narcolepsy remains to be clarified. Less attention has been devoted to potential orexinergic system alterations in other conditions, and their potential relationships with inflammatory signalling. Neuroinflammation has raised increasing interest in recent years, not only in relation to typical neuroinflammatory diseases, but also with regard to aging-related neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. A role for neuroinflammatory signalling in normal, “healthy” aging is also currently debated, since several lines of evidence have pointed to aging as a chronic low-grade proinflammatory condition. We have examined neurons in the lateral hypothalamus expressing orexin A in different paradigms: i) normal aging in mice, ii) rodent models of a chronic infectious neuroinflammatory condition represented by a parasitic disease that causes sleep/wake alterations, iii) PDAAP mutant mice, a model of Alzheimer’s disease. In these paradigms, we have identified different degrees of neuronal loss in the orexinergic cell population and/or evidence of functional dysregulation of these neurons, together with glial activation in the lateral hypothalamus and sleep/wake changes. Altogether, the data point to a vulnerability of orexin to inflammatory signalling, and potentially place the neuropeptide at the center of neural-immune interactions, drawing attention on the relationships between neuroinflammation, sleep regulation, and orexin neuron damage