Mikael Nygård

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.

Age-related changes in electrophysiological properties of the mouse suprachiasmatic nucleus in vitro

Endogenous biological rhythms are altered at several functional levels during aging. The major pacemaker driving biological rhythms in mammals is the suprachiasmatic nucleus of the hypothalamus. In the present study we used tissue slices from young and old mice to analyze the electrophysiological properties of the retinorecipient ventrolateral part of the suprachiasmatic nucleus. Loose patch and whole-cell recordings were performed during day and night. Both young and old mice displayed a significant variation between day and night in the mean firing rate of suprachiasmatic nucleus neurons. The proportion of cells not firing spontaneous action potentials showed a clear day/night rhythm in young but not in old animals, that had an elevated number of such silent cells during the day compared to young animals. Analysis of firing patterns revealed a more regular spontaneous firing during the day than during the night in the old mice, while there was no difference between day and night in young animals. The frequency of spontaneous inhibitory postsynaptic currents was reduced in ventrolateral suprachiasmatic nucleus neurons in the old animals. Since the inhibitory input to these neurons is mainly derived from within the suprachiasmatic nucleus, this reduction most likely reflects the greater proportion of silent cells found in old animals. The results show that the suprachiasmatic nucleus of old mice is subject to marked electrophysiological changes, which may contribute to physiological and behavioral changes associated with aging.

Suppressors, receptors and effects of cytokines on the aging mouse biological clock

During aging, levels of inflammatory cytokines increase and circadian rhythms are frequently altered. We here investigated neurobiological correlates of neuroinflammation and its age-related variation in the hypothalamic suprachiasmatic nucleus (SCN), the master circadian pacemaker. Day/night variations of transcripts encoding cytokine receptors and suppressors of cytokine signaling (SOCS) were correlated in groups of mice of different ages with Fos induction elicited by intracerebroventricular injections of tumor necrosis factor-alpha and interferon-gamma. Cytokine-elicited Fos induction was high at early night, when SOCS1 and SOCS3 levels were low. Such Fos induction was significantly reduced in the older SCN at early night, and paralleled by reduced expression of interferon-gamma receptor transcripts as compared to the younger SCN. In addition, Fos induction at early night exhibited marked sub-regional differences in the SCN between the age groups. The study shows that SOCS1 and SOCS3 are expressed in the biological clock with a day/night variation that may regulate SCN responsiveness to cytokine exposure, and indicates that effects of pro-inflammatory cytokines on the SCN are markedly altered during senescence.

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.

The aging suprachiasmatic nucleus and cytokines: functional, molecular, and cellular changes in rodents

The aging process brings about a switch to a low‐grade chronic inflammatory condition in the periphery and brain, a condition which may prime brain cells, including those of the hypothalamic suprachiasmatic nucleus (SCN). Little information is available, however, on the responses of the SCN to neuroinflammation and immune‐related challenges, and such responses have not been hitherto investigated during aging. We here provide an overview of these issues and summarize data we obtained in the study of the SCN of young and aged mice. In particular, we analyzed: i) the electrophysiological properties of the SCN core (the retino‐recipient region) in tissue slices; ii) expression and day/night variation of transcripts encoding the receptors for the cytokines interferon‐γ and tumor necrosis factor‐α, as well as the expression of transcripts encoding the proteins “suppressors of cytokine signaling” SOCS1 and SOCS3, by means of quantitative real‐time polymerase chain reaction; levels of mRNAs were correlated with neuronal activation, revealed by Fos induction, elicited in the SCN by intracerebroventricular injections of a mixture of interferon‐γ and tumor necrosis factor‐α during the daytime and nighttime; and iii) response of astrocytes and microglia in the SCN to the same paradigm of cytokine administration. Marked changes of all the above‐mentioned parameters were found in the aged SCN, indicating that the circadian pacemaker is a target of the aging process. In addition, the findings indicate that neurons and glial cells of the biological clock are sensitive to inflammatory signals, and that the response to such signals is altered during senescence.

Rapid nitric oxide-dependent effects of tumor necrosis factor-α on suprachiasmatic nuclei neuronal activity

The effect of tumor necrosis factor-&agr; (TNF-&agr;) on excitability and synaptic function was analyzed in slice preparations of the suprachiasmatic nuclei (SCN), the major mammalian circadian pacemaker. TNF-&agr; caused a rapid increase in the spontaneous firing rate in most SCN neurons examined that was paralleled by an increase of inhibitory postsynaptic currents. The nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester abolished these effects. No effect of TNF-&agr; was found on miniature synaptic currents. The lack of effect on miniature synaptic currents indicates that TNF-&agr; primarily affects neuronal membrane properties to cause the changes in spontaneous firing. TNF-&agr;, levels of which show circadian variation in the brain and increase during inflammatory conditions and aging, may thus through nitric oxide induction modulate SCN electrical output to affect downstream circadian rhythms.

Clock Gene Expression during Chronic Inflammation Induced by Infection with Trypanosoma brucei brucei in Rats

African sleeping sickness is characterized by alterations in rhythmic functions. It is not known if the disease affects the expression of clock genes, which are the molecular basis for rhythm generation. We used a chronic rat model of experimental sleeping sickness, caused by the extracellular parasite Trypanosoma brucei brucei (Tb brucei), to study the effects on clock gene expression. In tissue explants of pituitary glands from Period1-luciferase (Per1-luc) transgenic rats infected with Tb brucei, the period of Per1-luc expression was significantly shorter. In explants containing the suprachiasmatic nuclei (SCN), the Per1-luc rhythms were flat in 21% of the tissues. We also examined the relative expression of Per1, Clock, and Bmal1 mRNA in the SCN, pineal gland, and spleen from control and infected rats using qPCR. Both Clock and Bmal1 mRNA expression was reduced in the pineal gland and spleen following Tb brucei infection. Infected rats were periodic both in core body temperature and in locomotor activity; however, early after infection, we observed a significant decline in the amplitude of the locomotor activity rhythm. In addition, both activity and body temperature rhythms exhibited decreased regularity and “robustness.” In conclusion, although experimental trypanosome infection has previously been shown to cause functional disturbances in SCN neurons, only 21% of the SCN explants had disturbed Per1-luc rhythms. However, our data show that the infection overall alters molecular clock function in peripheral clocks including the pituitary gland, pineal gland, and spleen.

Glial transcripts and immune-challenged glia in the suprachiasmatic nucleus of young and aged mice.

Biological rhythms are frequently disturbed with advancing age, and aging-related changes of glia in the hypothalamic suprachiasmatic nucleus (SCN), the master circadian pacemaker, require special attention. In particular, astrocytes contribute to SCN function, and aging is associated with increased inflammatory activity in the brain, in which microglia could be especially implicated. On this basis, we investigated in the SCN of young and old mice glial transcripts and cell features, and the glial cell response to a central inflammatory challenge. Quantitative real-time reverse transcriptase–polymerase chain reaction (RT-PCR) was used to analyze the expression of mRNAs encoding the astrocytic glial fibrillary acidic protein and the microglial antigen CD11b. Both these transcripts, here investigated in the SCN for the first time, were significantly increased in the old SCN. Glial cell phenotyping with immunohistochemistry revealed hypertrophic and intensely stained astrocytes and microglia in the aged SCN. In both age groups, microglia were scattered throughout the SCN and astrocytes were prominent in the ventral portion, where retinal fibers are densest; in the aged SCN, astrocytes were also numerous in the dorsal portion. After intracerebroventricular injections of a mixture of interferon-γ and tumor necrosis factor-α, or phosphate-buffered saline as control, immunolabeling was evaluated with stereological cell counts and confocal microscopy. Phenotypic features of astrocyte and microglia activation in response to cytokine injections were markedly enhanced in the aged SCN. Subregional variations in glial cell density were also documented in the aged compared to the young SCN. Altogether, the findings show increases in the expression of glial transcripts and hypertrophy of astrocytes and microglia in the aged SCN, as well as age-dependent variation in the responses of immune-challenged SCN glia. The data thus point out an involvement of glia in aging-related changes of the biological clock. (Author correspondence: marina.bentivoglio@univr.it)

Regulation of cytokine signaling and T-cell recruitment in the aging mouse brain in response to central inflammatory challenge

Aging is often accompanied by increased levels of inflammatory molecules in the organism, but age-related changes in the brain response to inflammatory challenges still require clarification. We here investigated in mice whether cytokine signaling and T-cell neuroinvasion undergo age-related changes. We first analyzed the expression of molecules involved in T-cell infiltration and cytokine signaling regulation in the septum and hippocampus of 2-3 months and 20- to 24-month-old mice at 4h after intracerebroventricular injections of tumor necrosis factor (TNF)-alpha or interferon-gammaversus saline injections. Transcripts of the chemokine CXCL9, intercellular adhesion molecule (ICAM)-1 and suppressor of cytokine signaling molecules (SOCS) 1 and 3 were increased in both age groups after cytokine injection; microglia-derived matrix metalloproteinase (MMP) 12 mRNA was induced in old mice also after control saline injections. Age-related changes in ICAM-1 protein expression and T-cell infiltration were then analyzed in mice of 3-4, 8-9 and 15-16 months at 48h after TNF-alpha injections. ICAM-1 immunoreactivity, and Western blotting in striatum, septum, hippocampus and hypothalamus showed progressive age-related enhancement of TNF-alpha-elicited ICAM-1 upregulation. Double immunofluorescence revealed ICAM-1 expression in microglia and astrocytic processes. CD3(+), CD4(+) and CD8(+) T-cells exhibited progressive age-related increases in brain parenchyma and choroid plexus after cytokine exposure. The findings indicate that the brain responses to inflammatory challenges are not only preserved with advancing age, but also include gradual amplification of ICAM-1 expression and T-cell recruitment. The data highlight molecular and cellular correlates of age-related increase of brain sensitivity to inflammatory stimuli, which could be involved in altered brain vulnerability during aging.

PLASMA LEVELS OF INTERFERON-γ CORRELATE WITH AGE-RELATED DISTURBANCES OF CIRCADIAN RHYTHMS AND SURVIVAL IN A NON-HUMAN PRIMATE

Aging can be associated with changes in circadian rhythms and reduction in adaptive immune responses accompanied by expansion of memory T cells and elevated levels of pro-inflammatory cytokines. Recent findings suggest the cytokine interferon-γ (IFN-γ) can affect the function of the hypothalamic suprachiasmatic nucleus (SCN), the master mammalian circadian pacemaker, both in vitro and in vivo. We studied the correlation of plasma levels of IFN-γ and changes in circadian rhythms in a non-human primate species, the nocturnal mouse lemur (Microcebus murinus). Plasma IFN-γ and dehydroepiandrosterone sulfate (DHEA-S), a known biomarker of aging, were determined in middle- to old-age animals by immunoenzymoassay. Daily rhythms of locomotor activity and body temperature as well as survival time of the lemurs were recorded. With aging, mean levels of DHEA-S decreased whereas IFN-γ increased. Aged animals showed biological rhythm alterations characterized by a high percentage of diurnal activity, anticipation of the activity onset relative to lights-off, short free-running period, and delayed occurrence of minimal body temperature. The magnitude of these disturbances was correlated with the plasma level of IFN-γ but not DHEA-S. Most remarkably, in contrast to DHEA-S, increased levels of IFN-γ correlated with duration of the lifetime of the lemurs. These results show the degree of circadian rhythm alterations in an individual is correlated with plasma IFN-γ level during aging, and that plasma IFN-γ level may predict survival, at least in this non-human primate. (Author correspondence: fabienne.aujard@wanadoo.fr)