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 Alzheimer's disease in particular. Issues in the interpretation of such preclinical data and their predictive value for clinical translation are also discussed

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

The expression of immediate early genes in the brain during sleep and wake

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The pioneering experimental studies on sleep deprivation

The experimental studies on sleep deprivation were initiated by the Russian physician and scientist, Mafie de Manaceine, who studied sleep-deprived puppies kept in constant activity. She reported in 1894 that the complete absence of sleep was fatal in a few days, pointing out that the most severe lesions occurred in the brain. In 1898, the Italian physiologists Lamberto Daddi and Giulio Tarozzi also kept dogs awake by walking them; the animals died after 9-17 days, and their survival was unrelated to food consumption. In the histological study performed by Daddi, degenerative alterations, mainly represented by chromatolytic changes, were observed in neurons of the spinal ganglia, Purkinje cells of the cerebellum, and neurons of the frontal cortex. Daddi ascribed these changes to a state of autointoxication of the brain during insomnia. In 1898, the psychiatrist Cesare Agostini, interested in the psychic phenomena caused by prolonged insomnia in humans, sleep deprived dogs by keeping them in a metallic cag e in order to avoid fatigue. The dogs survived about 2 weeks, and degenerative changes were observed in their brains. In these experimental paradigms, the effect of sleep loss was confounded by motor exhaustion and/or intense sensory stimulation. In spite of the absence of adequate controls, the pioneering studies performed at the end of the 19th century represented the first experimental attempts to relate sleep with neural centers and suggested that sleep is a vital function and that the brain may be affected by insomnia

Chapter 15: Immediate early gene expression in sleep and wakefulness

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Sleeping with the clock: pacemaker neurons enter the scene

Theoretical contributions provided by Giuseppe Moruzzi in 1972 and Giovanni Berlucchi in 1970 are here revisited, highlighting an itinerary of knowledge on the relationships between sleep-wake alternation and biological clocks, and on the role of pacemaker neurons. With a modern insight, Moruzzi dealt with the role of homeostatic mechanisms and of "timing devices" in sleep and wake, and he referred to a theory formulated by Berlucchi. This theory, which has remained hidden in a book chapter, stemmed from a careful critical evaluation of previous experimental approaches and theories. With a remarkable intuition, Berlucchi proposed that the sleep-wake cycle is an endogenous biological rhythm, as other body rhythmic functions with which it interacts, and that this cycle is generated, as other rhythms, by a functional group of pacemaker neurons, endowed with endogenous rhythmic properties. Berlucchi viewed pacemaker neurons as hierarchically organized cells, entrained by the environment, controlled by intercellular, synaptic and nonsynaptic communication. All these hypotheses have been subsequently confirmed by discoveries that are here summarized. These issues are still at the forefront of research; many questions, however, are still open

Brain metabolic DNA in memory processing and genome turnover

Sophisticated methods are currently used to investigate the properties of brain DNA and clarify its role under physiological conditions and in neurological and psychiatric disorders. Attention is now called on a DNA fraction present in the adult rat brain that is characterized by an elevated turnover and is not involved in cell division or DNA repair. The fraction, known as brain metabolic DNA (BMD), is modulated by strain, stress, circadian oscillations, exposure to enriched or impoverished environment, and notably by several training protocols and post-trial sleep. BMD is frequently localized in glial cells but is also present in neurons, often in the perinucleolar region. Its distribution in repetitive and non-repetitive DNA fractions shows that BMD differs from native DNA and that in learning rats its profile differs from that of control rats. More detailed knowledge of the molecular, cellular, and time-dependent BMD features will be necessary to define its role in memory acquisition and processing and in the pathogenesis of neurologic disorders

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

The 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

Trypanosoma brucei and the nervous system

African sleeping sickness, characterized by a peculiar pain syndrome and prominent neuropsychiatric symptoms, is caused by the parasite Trypanosoma brucei (T.b.). In experimental T.b. infections, a molecule released from the trypanosomes has been isolated that binds to the CD8 molecule of T cells, whereby T cells are activated to secrete interferon gamma. This cytokine binds to the parasites and triggers them to proliferate, establishing a peculiar bidirectional activating signal system. The hypothesis is presented that the molecules involved in these bidirectional signals might also interact with neurons, thus causing brain dysfunctions. Studies on the molecular interactions between parasites and the nervous system in sleeping sickness might reveal basic mechanisms underlying other neuropsychiatric diseases

Fos-related protein expression in the midline paraventricular nucleus of the rat thalamus: basal oscillation and relationship with limbic efferents

The 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