Circadian Kinetics of Cell Cycle Progression in Adult Neurogenic Niches of a Diurnal Vertebrate

The circadian system may regulate adult neurogenesis via intracellular molecular clock mechanisms or by modifying the environment of neurogenic niches, with daily variation in growth factors or nutrients depending on the animal's diurnal or nocturnal lifestyle. In a diurnal vertebrate, zebrafish, we studied circadian distribution of immunohistochemical markers of the cell division cycle (CDC) in 5 of the 16 neurogenic niches of adult brain, the dorsal telencephalon, habenula, preoptic area, hypothalamus, and cerebellum. We find that common to all niches is the morning initiation of G1/S transition and daytime S-phase progression, overnight increase in G2/M, and cycle completion by late night. This is supported by the timing of gene expression for critical cell cycle regulators cyclins D, A2, and B2 and cyclin-dependent kinase inhibitor p20 in brain tissue. The early-night peak in p20, limiting G1/S transition, and its phase angle with the expression of core clock genes, Clock1 and Per1, are preserved in constant darkness, suggesting intrinsic circadian patterns of cell cycle progression. The statistical modeling of CDC kinetics reveals the significant circadian variation in cell proliferation rates across all of the examined niches, but interniche differences in the magnitude of circadian variation in CDC, S-phase length, phase angle of entrainment to light or clock, and its dispersion. We conclude that, in neurogenic niches of an adult diurnal vertebrate, the circadian modulation of cell cycle progression involves both systemic and niche-specific factors. SIGNIFICANCE STATEMENT This study establishes that in neurogenic niches of an adult diurnal vertebrate, the cell cycle progression displays a robust circadian pattern. Common to neurogenic niches located in diverse brain regions is daytime progression of DNA replication and nighttime mitosis, suggesting systemic regulation. Differences between neurogenic niches in the phase and degree of S-phase entrainment to the clock suggest additional roles for niche-specific regulatory mechanisms. Understanding the circadian regulation of adult neurogenesis can help optimize the timing of therapeutic approaches in patients with brain traumas or neurodegenerative disorders and preserve neural stem cells during cytostatic cancer therapies

Genetic structure and drug resistance of <i>Mycobacterium tuberculosis</i> strains in the Kemerovo Region — Kuzbass

Background. Kemerovo Region has a high burden of tuberculosis (TB) with incidence rates twice the national average. The circulating variants of Mycobacterium tuberculosis significantly influence the TB epidemic process. Screening of epidemically significant variants of the pathogen in areas with a high burden of TB underlies epidemiological diagnosis and is necessary for the development of effective prevention measures. However, the population structure of M. tuberculosis in the Kemerovo Region — Kuzbass is poorly understood. Aims: to study genetic heterogeneity and phenotypic resistance to anti-tuberculosis drugs of M. tuberculosis strains in the Kemerovo Region. Materials and methods. The MIRU-VNTR genotyping of 163 M. tuberculosis strains isolated from TB patients in the Kemerovo Region in March–October 2022 was carried out. Cultivation of M. tuberculosis, drug susceptibility testing, and isolation of genomic DNA were carried out by standard methods. Genotypic identification was performed using MIRU-VNTR (24 loci) typing. In parallel, express genotyping was carried out: identification of isolates of the Beijing genotype (by RD105/207) and non-Beijing; subtyping Beijing using real-time PCR tests for detection of Central Asian Russian and B0/W148; identification of the non-Beijing group by real-tine PCR RT tests for LAM, S, Ural. Results. The isolates of the Beijing genotype (67.5%) were found to dominate both among newly diagnosed (64.4%) and previously treated patients (88.5%). MIRU-VNTR typing revealed 75 profiles, of which 94-32 (35.3%) and 100-32 (15.7%) were the most abundant and belonged to the Beijing genotype. Overall, 39.9% and 20.9% of isolates, respectively, were assigned to the Beijing Central Asian Russian and B0/W148 epidemic clusters, which differed significantly in MDR levels (50.8% and 85.3%, respectively; p = 0.005). The second most common were strains of the genetic family of the Euro-American lineage (L4) (31.9%): LAM (6.7%) Ural (7.4%) Haarlem (4.9%) and L4-unclassified (12.9%), MDR among of these minor genotypes was significantly lower than among Beijing genotype strains, and amounted to 11.5% (p 0.001). Strains from HIV-TB patients (56.4% of the total sample) carried an MDR profile more often (54.8%) compared to TB cases without HIV infection (35.2%) (p = 0.005), which may be due to higher proportion of Beijing genotype strains in the HIV-TB group (75.0% vs. 57.7%; p = 0.026). Complete comparability of the SNP analysis (in-house tests) to identify the main genotypes and epidemically significant Beijing subtypes was shown, which made it possible to characterize 75.5% of the sample by the express method. Conclusions. The molecular genetic screening carried out in the Kemerovo Region revealed the heterogeneity of the M. tuberculosis population, which was dominated by strains of the Beijing genotype, with the frequency of subtypes comparable with other territories of the Siberian Federal District

Cognitive Aging in Zebrafish

BACKGROUND: Age-related impairments in cognitive functions represent a growing clinical and social issue. Genetic and behavioral characterization of animal models can provide critical information on the intrinsic and environmental factors that determine the deterioration or preservation of cognitive abilities throughout life. METHODOLOGY/PRINCIPAL FINDINGS: Behavior of wild-type, mutant and gamma-irradiated zebrafish (Danio rerio) was documented using image-analysis technique. Conditioned responses to spatial, visual and temporal cues were investigated in young, middle-aged and old animals. The results demonstrate that zebrafish aging is associated with changes in cognitive responses to emotionally positive and negative experiences, reduced generalization of adaptive associations, increased stereotypic and reduced exploratory behavior and altered temporal entrainment. Genetic upregulation of cholinergic transmission attenuates cognitive decline in middle-aged achesb55/+ mutants, compared to wild-type siblings. In contrast, the genotoxic stress of gamma-irradiation accelerates the onset of cognitive impairment in young zebrafish. CONCLUSIONS/SIGNIFICANCE: These findings would allow the use of powerful molecular biological resources accumulated in the zebrafish field to address the mechanisms of cognitive senescence, and promote the search for therapeutic strategies which may attenuate age-related cognitive decline

The Circadian System Is a Target and Modulator of Prenatal Cocaine Effects

BACKGROUND. Prenatal exposure to cocaine can be deleterious to embryonic brain development, but the results in humans remain controversial, the mechanisms involved are not well understood and effective therapies are yet to be designed. We hypothesize that some of the prenatal effects of cocaine might be related to dysregulation of physiological rhythms due to alterations in the integrating circadian clock function. METHODOLOGY AND PRINCIPLE FINDINGS. Here we introduce a new high-throughput genetically well-characterized diurnal vertebrate model for studying the mechanisms of prenatal cocaine effects by demonstrating reduced viability and alterations in the pattern of neuronal development following repeated cocaine exposure in zebrafish embryos. This effect is associated with acute cocaine-induced changes in the expression of genes affecting growth (growth hormone, zGH) and neurotransmission (dopamine transporter, zDAT). Analysis of circadian gene expression, using quantitative real-time RT-PCR (QPCR), demonstrates that cocaine acutely and dose-dependently changes the expression of the circadian genes (zPer-3, zBmal-1) and genes encoding melatonin receptors (zMelR) that mediate the circadian message to the entire organism. Moreover, the effects of prenatal cocaine depend on the time of treatment, being more robust during the day, independent of whether the embryos are raised under the light-dark cycle or in constant light. The latter suggests involvement of the inherited circadian factors. The principal circadian hormone, melatonin, counteracts the effects of cocaine on neuronal development and gene expression, acting via specific melatonin receptors. CONCLUSIONS/SIGNIFICANCE. These findings demonstrate that, in a diurnal vertebrate, prenatal cocaine can acutely dysregulate the expression of circadian genes and those affecting melatonin signaling, growth and neurotransmission, while repeated cocaine exposure can alter neuronal development. Daily variation in these effects of cocaine and their attenuation by melatonin suggest a potential prophylactic or therapeutic role for circadian factors in prenatal cocaine exposure.National Institutes of Health (DA1541801, MH 065528); National Institute on Drug Abuse (DA-4-7733

F-Spondin/spon1b Expression Patterns in Developing and Adult Zebrafish

F-spondin, an extracellular matrix protein, is an important player in embryonic morphogenesis and CNS development, but its presence and role later in life remains largely unknown. We generated a transgenic zebrafish in which GFP is expressed under the control of the F-spondin (spon1b) promoter, and used it in combination with complementary techniques to undertake a detailed characterization of the expression patterns of F-spondin in developing and adult brain and periphery. We found that F-spondin is often associated with structures forming long neuronal tracts, including retinal ganglion cells, the olfactory bulb, the habenula, and the nucleus of the medial longitudinal fasciculus (nMLF). F-spondin expression coincides with zones of adult neurogenesis and is abundant in CSF-contacting secretory neurons, especially those in the hypothalamus. Use of this new transgenic model also revealed F-spondin expression patterns in the peripheral CNS, notably in enteric neurons, and in peripheral tissues involved in active patterning or proliferation in adults, including the endoskeleton of zebrafish fins and the continuously regenerating pharyngeal teeth. Moreover, patterning of the regenerating caudal fin following fin amputation in adult zebrafish was associated with F-spondin expression in the blastema, a proliferative region critical for tissue reconstitution. Together, these findings suggest major roles for F-spondin in the CNS and periphery of the developing and adult vertebrate

Prenatal and acute cocaine exposure affects neural responses and habituation to visual stimuli

Psychostimulants have many effects on visual function, from adverse, following acute and prenatal exposure to therapeutic, on attention deficit. To determine the impact of prenatal and acute cocaine exposure on visual processing, we studied neuronal responses to visual stimuli in two brain regions of a transgenic larval zebrafish expressing the calcium indicator GCaMP-HS. We found that both red light (LF) and dark (DF) flashes elicited similar responses in the optic tectum neuropil (TOn), while the dorsal telencephalon (dTe) responded only to LF. Acute cocaine (0.5 μM) reduced neuronal responses to LF in both brain regions but did not affect responses to DF. Repeated stimulus presentation led to habituation of dTe neurons to LF. Acute cocaine prevented habituation. TOn habituated to DF, but not LF, and DF habituation was not modified by cocaine. Remarkably, prenatal cocaine exposure prevented the effects of acute cocaine on LF response amplitude and habituation later in development in both brain regions, but did not affect DF responses. We discovered that, in spite of similar neural responses to LF and DF in the TO (superior colliculus in mammals), responses to LF are more complex, involving dTe (homologous to the cerebral cortex), and are more vulnerable to cocaine. Our results demonstrate that acute cocaine exposure affects visual processing differentially by brain region, and that prenatal cocaine exposure modifies zebrafish visual processing in multiple structures in a stimulus-dependent manner. These findings are in accordance with the major role that the optic tectum and cerebral cortex play in sustaining visual attention, and support the hypothesis that modification of these areas by prenatal cocaine exposure may be responsible for visual deficits noted in humans. This model offers new methodological approaches for studying the adverse and therapeutic effects of psychostimulants on attention, and for the development of new pharmacological interventions

Prenatal daytime cocaine exposure dose-dependently alters expression of genes involved in neurotransmission (A) circadian system (B, C) growth (D) and melatonin signaling (E), and these effects are counteracted by melatonin.

<p>(A–E) Melatonin (100 nM for 45 min), Low (0.3 µM) or High (30 µM) cocaine doses (15 min), with or without melatonin pre-treatment (100 nM, 30 min). (F) Melatonin receptor antagonist Luzindole attenuates the counteracting effects of melatonin on cocaine-induced changes in the expression of all six melatonin receptor genes; only one (<i>zMel1a-1</i>) receptor shown. Treatments: luzindole 50 µM for 20 min prior to melatonin; melatonin 100 nM for 20 min prior to cocaine; cocaine 0.3 µM for 15 min. (G) Dose-dependent effects of cocaine (20 min) on melatonin receptor expression. Y axis: fold change relative to control ( = 1). N = 3–4 samples/treatment group, 25 embryos/sample. Mean±SEM, <i>t</i>-test (vs. control), * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001; One way ANOVA (between treatments), a: <i>p</i><0.05, vs. cocaine-only group. Mlt-melatonin; Coc-cocaine.</p

Prenatal exposure to cocaine alters neuronal development in α1-TGFP transgenic zebrafish embryos.

<p>Control embryos (A: 63 hpf; B and C: 37 hpf) show highly similar patterns of GFP reporter expression. Although major anatomical abnormalities were not found in cocaine-treated embryos, they display variable patterns and typically lower levels of GFP reporter expression (D–G) following repeated cocaine administration (1 µM, 15 min every hour for 5 daytime hours, starting at 22–24 hpf). (C) and (G) correspond to the dotted line areas in (B) and (F), respectively, and show GFP reporter expression in the spinal cord (sc) and enteric neurons (arrows). Note that a double string of enteric neurons, separated by a prospective lumen, is visible in both control embryos (c) but only in one of the cocaine-treated embryos (left in F and G). (D) and (E) depict different GFP expression patterns in the forebrain of four cocaine-treated embryos (37 hpf): lateral (D) and semi-dorsal view (e; arrowheads-eyes). All pictures are original photographs of live, responsive-to-touch embryos of the same clutch, placed next to each other on the cover glass. Black spots in (A) and (D) represent pigment. Out-of-focus areas on the close-up whole-embryo pictures (e.g., B, F) are due to the curved position of the embryo around the spherical yolk sack. The pictures are from one of the 12 clutches studied in LD, and are representative of 3 independent experiments. Scale bars: 250 µm. (H and I) Mean (SEM) percent change in fluorescence intensity in the brain and spinal cord of 37 hpf embryos raised in LD (H) or LL (I) conditions, relative to mean Control group intensity in LD (100%). Control (white bar), repeated cocaine (1 µM) administration alone (black bar) or with 30-min melatonin pre-treatment (diagonal bar) at 22 hpf. p<0.05 * relative to LD Control, or # relative to LL Control (one-way ANOVA), n = 28 per treatment group.</p

Normal diurnal variation in <i>zPer3, zBmal1</i> and melatonin receptors is altered in embryos raised under constant light conditions.

<p>Daytime increase in <i>zPer3</i> expression in LD (A) is partially preserved in LL (D; <i>p</i><0.05). Daily variation in <i>zBmal1</i> and <i>zMel1a-1</i> (and other melatonin receptors) is abolished in LL (B vs. E; C vs. F). Y-axis in all plots: fold change relative to LD-Day level ( = 1). N = 3–4 samples/time point in LD or LL group, 25 embryos/sample. Mean±SEM, <i>t</i>-test, * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 indicates difference between LD and LL, at Day or Night, accordingly.</p