Diana Millán-Aldaco

Neurogenesis in the subventricular zone following transcranial magnetic field stimulation and nigrostriatal lesions

Neurogenesis continues at least in two regions of the mammalian adult brain, the subventricular zone (SVZ) and the subgranular zone in hippocampal dentate gyrus. Neurogenesis in these regions is subjected to physiological regulation and can be modified by pharmacological and pathological events. Here we report the induction of neurogenesis in the SVZ and the differentiation after nigrostriatal pathway lesion along with transcranial magnetic field stimulation (TMFS) in adult rats. Significant numbers of proliferating cells demonstrated by bromodeoxyuridine‐positive reaction colocalized with the neuronal marker NeuN were detected bilaterally in the SVZ, and several of these cells also expressed tyrosine hydroxylase. Transplanted chromaffin cells into lesioned animals also induced bilateral appearance of subependymal cells. These results show for the first time that unilateral lesion, transplant, and/or TMFS induce neurogenesis in the SVZ of rats and also that TMFS prevents the motor alterations induced by the lesion.

Modafinil enhances extracellular levels of dopamine in the nucleus accumbens and increases wakefulness in rats

Modafinil (MOD) is a wakefulness-promoting drug that improves the alertness levels in narcolepsy; however, the molecular mechanism of action remains to be elucidated. We found that after a single icv injection of MOD (10 microg/5 microl) the extracellular levels of dopamine (DA) and l-DOPA collected from the nucleus accumbens were increased and decreased, respectively. Separately, the icv administration of MOD (10 microg/5 microl) to rats enhanced wakefulness (W) whereas diminished sleep during 4h. Lastly, the alertness induced by MOD was partially antagonized by the sleep-inducing endocannabinoid anandamide (ANA). We conclude that MOD enhances the extracellular levels of DA, promotes W and its effects on sleep are partially blocked by ANA.

Cannabidiol, a constituent of Cannabis sativa, modulates sleep in rats

Δ9‐tetrahydrocannabinol (Δ9‐THC) and cannabidiol (CBD) are two major constituents of Cannabis sativa. Δ9‐THC modulates sleep, but no clear evidence on the role of CBD is available. In order to determine the effects of CBD on sleep, it was administered intracerebroventricular (icv) in a dose of 10 μg/5 μl at the beginning of either the lights‐on or the lights‐off period. We found that CBD administered during the lights‐on period increased wakefulness (W) and decreased rapid eye movement sleep (REMS). No changes on sleep were observed during the dark phase. Icv injections of CBD (10 μg/5 μl) induced an enhancement of c‐Fos expression in waking‐related brain areas such as hypothalamus and dorsal raphe nucleus (DRD). Microdialysis in unanesthetized rats was carried out to characterize the effects of icv administration of CBD (10 μg/5 μl) on extracellular levels of dopamine (DA) within the nucleus accumbens. CBD induced an increase in DA release. Finally, in order to test if the waking properties of CBD could be blocked by the sleep‐inducing endocannabinoid anandamide (ANA), animals received ANA (10 μg/2.5 μl, icv) followed 15 min later by CBD (10 μg/2.5 μl). Results showed that the waking properties of CBD were not blocked by ANA. In conclusion, we found that CBD modulates waking via activation of neurons in the hypothalamus and DRD. Both regions are apparently involved in the generation of alertness. Also, CBD increases DA levels as measured by microdialysis and HPLC procedures. Since CBD induces alertness, it might be of therapeutic value in sleep disorders such as excessive somnolence.

The anandamide membrane transporter inhibitor, VDM-11, modulates sleep and c-Fos expression in the rat brain

Endogenous cannabinoids or endocannabinoids are lipid molecules that have a variety of biological actions, most notably via activation of the cannabinoid receptors. The family of endocannabinoids includes arachidonoylethanolamide (ANA) which modulates different behaviors, such as sleep. However, it is unknown whether pharmacological elevation of ANA endogenous levels might induce sleep. VDM 11 [(5 Z,8 Z,11 Z,14 Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide] is commonly used as an inhibitor of ANA cellular uptake, and thereby to potentiate its actions. In this study we have examined whether VDM-11 exerts any effect on the sleep-wake cycle and c-Fos expression in brain areas. When assayed alone in rats, VDM-11 (10 or 20 microg/5 microL, i.c.v.) at the beginning of the lights-off period, reduced wakefulness and increased sleep. The CB(1) cannabinoid receptor antagonist, SR141716A, partially reversed the effects of VDM-11 on sleep. Additionally, VDM-11 enhanced c-Fos expression in sleep-related brain areas such as the anterior hypothalamic area, paraventricular thalamic nucleus, and pedunculopontine tegmental nucleus. It is concluded that VDM-11 displays sleep-inducing properties and these effects slightly, albeit significantly, are reversed using SR141716A. Furthermore, c-Fos data suggest a possible underlying neuroanatomical substrate of the sleep-inducing properties of VDM-11. We report evidence suggesting that VDM-11 might be considered for the development of new pharmacological and pharmaceutical approaches to treat sleep disorders such as insomnia.

Growth hormone improves hippocampal adult cell survival and counteracts the inhibitory effect of prolonged sleep deprivation on cell proliferation

Sleep deprivation (SD) produces numerous deleterious changes in brain cells, including apoptosis. It has been demonstrated that growth hormone (GH) stimulates cell growth and counteracts apoptosis, although this anti-apoptotic effect has not been tested against SD. To determine the protective effect of GH administration on cell proliferation and survival in the dentate gyrus (DG) of the hippocampus after sleep deprivation; we injected Wistar adult rats with a low dose of recombinant human GH (rhGH 5 ng/kg) per seven days and then we gently sleep deprived the animals for 48 consecutive hours. 5-Bromodeoxiuridine (BrdU) was administered to assess cell proliferation after the GH treatment and NeuN was used as marker of cell fate. Our results indicate that GH produced a three fold increase in the number of BrdU positive cells within the DG [Control = 1044 ± 106.38 cells, rhGH = 2952 ± 99.84 cells, P<0.01]. In contrast, 48 h of SD significantly reduced cell proliferation but this effect was antagonized by the GH administration [SD = 540 ± 18.3 cells, rhGH + SD = 1116 ± 84.48 cells, P<0.004]. Paradoxically, SD and GH administration increased cell survival separately but no significantly compared with control animals. However, cell survival was increased in animals treated with rhGH+SD compared to rats injected with saline solution [P<0.04]. Within the survival cells, the percentage of neurons was higher in SD animals [95%] compared with saline group, while this percentage (NeuN positive cells) was increased in animals treated with rhGH+SD [120%] compared with rhGH [25%] alone. Our findings indicate that GH strongly promotes cell proliferation in the adult brain and also protects the hippocampal neuronal precursors against the deleterious effect of prolonged sleep loss.

Administration of URB597, Oleoylethanolamide or Palmitoylethanolamide Increases Waking and Dopamine in Rats

Background Oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) are amides of fatty acids and ethanolamine named N-acylethanolamines or acylethanolamides. The hydrolysis of OEA and PEA is catalyzed by the fatty acid amide hydrolase (FAAH). A number of FAAH inhibitors that increase the levels of OEA and PEA in the brain have been developed, including URB597. In the present report, we examined whether URB597, OEA or PEA injected into wake-related brain areas, such as lateral hypothalamus (LH) or dorsal raphe nuclei (DRN) would promote wakefulness (W) in rats. Methodology and Principal Findings Male Wistar rats (250–300 g) were implanted for sleep studies with electrodes to record the electroencephalogram and electromyogram as well as a cannulae aimed either into LH or into DRN. Sleep stages were scored to determine W, slow wave sleep (SWS) and rapid eye movement sleep (REMS). Power spectra bands underly neurophysiological mechanisms of the sleep-wake cycle and provide information about quality rather than quantity of sleep, thus fast Fourier transformation analysis was collected after the pharmacological trials for alpha (for W; α = 8–12 Hz), delta (for SWS; δ = 0.5–4.0 Hz) and theta (for REMS; θ = 6.0–12.0 Hz). Finally, microdialysis samples were collected from a cannula placed into the nucleus accumbens (AcbC) and the levels of dopamine (DA) were determined by HPLC means after the injection of URB597, OEA or PEA. We found that microinjection of compounds (10, 20, 30 µg/1 µL; each) into LH or DRN during the lights-on period increased W and decreased SWS as well as REMS and enhanced DA extracellular levels. Conclusions URB597, OEA or PEA promoted waking and enhanced DA if injected into LH or DRN. The wake-promoting effects of these compounds could be linked with the enhancement in levels of DA and indirectly mediated by anandamide.

Effects on sleep and dopamine levels of microdialysis perfusion of cannabidiol into the lateral hypothalamus of rats

AIMS The major non-psychoactive component of Cannabis sativa, cannabidiol (CBD), displays a plethora of actions including wakefulness. In the present study, we addressed whether perfusing CBD via microdialysis into lateral hypothalamus (LH) during the lights-on period would modify the sleep-wake cycle of rats as well as the contents of dopamine (DA) collected from nucleus accumbens (AcbC). Additionally, we tested whether perfusion of CBD into LH would block the sleep rebound after a sleep deprivation period. MAIN METHODS Electroencephalogram and electromyogram electrodes were implanted in rats as well as a guide-cannula aimed to LH or AcbC. CBD perfusion was carried out via cannulae placed into LH whereas contents of DA were collected from AcbC and analyzed using HPLC means. KEY FINDINGS We found that microdialysis perfusion of CBD (30, 60, or 90 nM) into LH of rat enhances alertness and suppresses sleep. This effect was accompanied with an increase in DA extracellular levels collected from the AcbC. Furthermore, perfusion of CBD into LH after total sleep deprivation prevented the sleep rebound. SIGNIFICANCE These findings enhance the investigation about the neurobiological properties of CBD on sleep modulation.

Epidermal langerhans cells in the terrestrial turtle, Kinosternum integrum

In mammalian epidermis, Langerhans cells (LC) are the only antigen-presenting dendritic cells that possess the ectoenzyme adenosine triphosphase (ATPase) and constitutively express class II molecules encoded by the major histocompatibility complex. Recently, we demonstrated the presence of LC in chicken epidermis. The aim of the present study is to demonstrate the presence of LC-like cells in turtle Kinosternum integrum, epidermis by light and ultrastructural ATPase histochemistry. ATPase-positive dendritic cells were observed in epidermal sheets whose maximum mean number was 192 cells/mm2. Electron microscopy for ATPase stained sections showed an electrondense precipitate in the plasma membrane of dendritic clear cells located among basal and suprabasal keratinocytes, ultrastructurally similar to LC. In serial sections, some dendritic cells showed LC (Birbeck) granules. The present study demonstrates for the first time ATPase-positive dendritic cells, morphologically similar to LC, in reptilian epidermis.

Aspects of the narcolepsy-cataplexy syndrome in O/E3-null mutant mice.

Orexins (hypocretins) are peptide neurotransmitters produced by a small group of neurons located exclusively in the lateral hypothalamus (LH). Orexins modulate arousal, and as a result, have profound effects on feeding behavior and the sleep-wake cycle. Loss of orexin producing neurons leads to a narcoleptic phenotype, characterized by sudden transitions from vigilance to rapid eye movement (REM) sleep (direct transition to REM, DREM) observed in electroencephalogram (EEG) and electromyogram (EMG) recordings. In this study, we demonstrate that mice lacking the basic helix-loop-helix transcription factor O/E3 (also known as ebf2) have a decrease in orexin-producing cells in the LH, in addition to a severely impaired orexinergic innervation of the pons. These changes in the orexinergic circuit of O/E3-null animals induce a narcoleptic phenotype, similar to the one arising in orexin-deficient and orexin-ataxin-3 mice. Taken together, our results suggest that O/E3 plays a central role during the establishment of a functional orexinergic circuit by controlling the expression of essential hypothalamic neurotransmitter and the correct development of the nerve fibers arising from the hypothalamus. This is the first report regarding the narcolepsy-cataplexy syndrome in O/E3-null mice, which adds the importance of transcription factors in the regulation of neural subpopulations that control the sleep-wake cycle.

Langerhans Cell-Like Dendritic Cells in the Cornea, Tongue and Oesophagus of the Chicken (Gallus gallus)

Langerhans cells are dendritic leucocytes which reside mainly within stratified squamous epithelia of skin and mucosa. Their visualization requires the use of ATPase histochemistry, electron microscopy for identifying the unique trilaminar cytoplasmic organelles (the Langerhans cell granules or Birbeck granules), and the expression of major histocompatibility complex class II molecules. Following uptake of antigen, Langerhans cells migrate via the afferent lymphatics to the lymph nodes and undergo differentiation from an antigen-processing cell to an antigen-presenting cell. Using the same approach as that employed in previous studies for the identification of chicken epidermal Langerhans cells, we show here the presence of ATPase-positive and major histocompatibility complex class II-positive Langerhans cell-like dendritic cells at the mucosal surface of the eye, tongue and oesophagus of the chicken. Ultrastructurally, these cells qualified as Langerhans cells except that they lack Langerhans cell granules. Thus, as in mammalian skin and mucosa, chicken mucosa contains mucosal dendritic cells with morphological and phenotypical features for the engagement of incoming antigens within epithelium and lamina propria.