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Olfactory epithelium receptor neurons in the nasal cavity, which are exposed to the external environment, reach the olfactory bulb (OB), representing a direct port of entry to the brain. Through retrograde axonal transport, pathogens, toxins and misfolded proteins can reach brain cell groups which innervate the OB and result in functional alterations. Indeed, influenza virus nasal instillation was found to target brainstem and hypothalamic cell groups and result in narcoleptic-like sleep/wake changes [1]. These cell groups included the wake-promoting orexin (OX)-containing neurons, and the sleep-promoting melanin-concentrating hormone (MCH)-containing neurons [1]. Orexinergic innervation of the OB has been reported, but OX and MCH neurons innervating the OB have never been visualized. OX immunoreactivity in the mouse olfactory receptor neurons has been ascribed to the olfactory mucosa. Sources of input to the OB have been studied [2] before the discovery of OX in 1998. Orexinergic innervation of the prefrontal cortex is instead well established. Aim of this study was to reveal OX- and MCH-containing neurons projecting to the OB. Unilateral injections of the retrograde fluorescent tracer Fluoro-Gold (FG) confined to the OB of adult mice were combined with immunophenotyping and quantitative analysis of retrogradely labeled neurons. The findings were compared with those obtained after FG injections in the prefrontal cortex. Following FG injections in the OB, labeled neurons were found in the ipsilateral lateral hypothalamus, and included intermingled OX-A- or MCH-immunoreactive cells. About 8% of orexinergic neurons were labeled when the tracer was confined to the OB. This proportion increased (13±2.49 %) in cases in which a faint halo of tracer diffusion to the lateral portion of the prefrontal cortex was observed. Preliminary data indicate retrograde labeling from the OB of almost 15% of MCH-containing neurons. The findings demonstrate that OX and MCH neurons reach the OB directly, thus providing to environmental agents a route to sleep/wake-regulatory nodes via the nasal cavity., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

As it is well known, Camillo Golgi (1843-1926) reported in 1873 his discovery of the black reaction (reazione nera), based on nervous tissue hardening in potassium dichromate and impregnation with silver nitrate. This method first revealed neurons, including their processes, in their entirety, thus providing the tool for a breakthrough in the knowledge on the structure of the nervous system. Professor of Histology and of General Pathology, Camillo Golgi worked for decades at the University of Pavia, leading a very active laboratory. Most of the original histological preparations of Golgi’s laboratory have unfortunately been lost. However, some slides are still kept at the Museum of the University of Pavia (“Sistema Museale di Ateneo”) but they have not been examined in detail until now. This presentation will provide an account of Golgi’s original slides available nowadays. Images from these preparations (e.g. from the hippocampus, cerebellum, and spinal cord), mostly based on Golgi impregnation, will be shown and compared with Golgi’s drawings and his descriptions of neuronal wiring. The presentation is thus aimed at showing, for the first time, the images which have led to the pioneering observations made by Camillo Golgi, which have opened the field of neurohistology and neuroanatomy and have contributed to the foundations of modern neuroscience., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

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., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

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., Author summary Human African trypanosomiasis, also known as sleeping sickness, is caused by infection with the parasite Trypanosoma brucei. Although the number of reported cases has declined in recent years, the disease still represents a challenge. During the second, meningoencephalitic, stage of the disease trypanosomes invade the brain. Consequently the infection can only be cured with toxic drugs which enter the brain, and is fatal if left untreated. Identifying the onset of this stage is therefore of crucial importance for therapeutic decisions. Using experimental infection in rats, we investigated the expression of interferon-γ and interferon-dependent chemokine genes involved in the disease progression. In parallel, we determined when the characteristic alterations of the sleep/wake cycle emerge in relation to the onset of the encephalitic stage, which was assessed histologically by the detection of parasites in the brain tissue. The results indicate that detection of perturbation of the sleep/wake cycle, especially the intrusion of sleep episodes into wakefulness, followed by an increase in CXCL10 concentration in the cerebrospinal fluid, could provide combined functional and humoral biomarkers of the early encephalitic stage of African trypanosomiasis.

Background:\ud \ud 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.\ud \ud Methodology:\ud \ud 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.\ud \ud Principal findings:\ud \ud 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.\ud \ud Conclusion:\ud \ud 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.

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., Author Summary Human African trypanosomiasis, or sleeping sickness, caused by Trypanosoma brucei infection, evolves from the first, haemolymphatic stage, to the second, meningoencephalitic stage. Classical knowledge sets the initiation of the second stage at the time of parasite invasion of the brain parenchyma; experimental findings have indicated that lymphocytes pave the way for this event. The disease leads to a complex neuropsychiatric syndrome, with sleep-wake alterations considered characteristic of the second stage. Staging criteria are still controversial and rely on cerebrospinal fluid examination. However, recent studies have questioned the route of parasite entry into the neuropil and even the occurrence of parasite neuroinvasion, as well as the correspondence of neurological signs and symptoms with the second stage of disease. In infected rats, we studied for the first time functional signs (body weight changes and sleep structure alterations) together with the presence of parasites and lymphocytes in the neuropil. The data show that parasites and lymphocytes invade the brain parenchyma over time, parasites cross blood vessels to enter the neuropil, and neurological features can precede these events. These findings question the cerebrospinal fluid criteria currently used for disease staging and focus attention on the importance of objective monitoring of clinical disease severity in sleeping sickness.

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., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Dendritic cells (DCs) are a subset of leukocytes highly specialized in antigen-presentation to T cells, thus promoting the immune response. DCs occur in the meninges and choroid plexus. Brain DCs and brain-derived antigens are drained by cerebrospinal fluid in the afferent lymphatic vessels of cervical lymph nodes (cLNs) for antigen presentation. Information on the role of DCs in intracerebral immune response is still limited. We recently demonstrated (Laperchia et al., 2013) that in thy1GFP-M transgenic mice, engineered for the expression of green fluorescent protein (GFP) in a proportion of neurons, also myeloid DCs are GFP-tagged. Our in vivo analysis by two-photon microscopy on young (3-6 months) thy1GFP-M mice showed DCs floating in the cerebrospinal fluid or static at the pia mater/parenchyma interface. We are using this animal model to study brain DCs trafficking by two-photon microscopy in two different inflammatory conditions. The first concerns chronic encephalitis caused by the extracellular parasite Trypanosoma brucei. During the first, hemolymphatic stage of this infection, direct interactions between DCs and parasites were seen within meningeal and cortical microvessels. In the second stage, determined by parasite neuroinvasion, DCs invaded the brain parenchyma, exhibiting a random motion for target antigen recognition. With disease progression, intraparenchymal brain DCs were instead mainly arranged in static clusters which incorporated parasites for efficient antigen capture, and extravasated cytotoxic CD8+ T cells established contact with parasites. Ex vivo analysis on cLNs shown that the subcapsular zone was invaded by migratory DCs, and both migratory and resident DCs preferentially contacted CD8+ T cells. The second condition concerns normal aging (18-20 month old mice), which is known to be associated with low-grade chronic inflammation level and with functional impairments of the immune system, also known as immunosenescence. In these mice we observed that DCs infiltrate the brain parenchyma by transmigration from blood vessels and exhibit a motile behavior suggesting a scanning motion. Moreover, some DCs showed a progressive reduction of their motility until convergence, in about 30 minutes, into clusters whose functional significant has yet to be elucidated. Taken together, our studies enlightens key events of the intracerebral immune response both in presence of pathogens and in physiological aging., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014