Mouse embryonic stem cell-derived cells reveal niches that support neuronal differentiation in the adult rat brain.

A neurogenic niche can be identified by the proliferation and differentiation of its naturally residing neural stem cells. However, it remains unclear whether "silent" neurogenic niches or regions suitable for neural differentiation, other than the areas of active neurogenesis, exist in the adult brain. Embryoid body (EB) cells derived from embryonic stem cells (ESCs) are endowed with a high potential to respond to specification and neuralization signals of the embryo. Hence, to identify microenvironments in the postnatal and adult rat brain with the capacity to support neuronal differentiation, we transplanted dissociated EB cells to conventional neurogenic and non-neurogenic regions. Our results show a neuronal differentiation pattern of EB cells that was dependent on the host region. Efficient neuronal differentiation of EB cells occurred within an adjacent region to the rostral migratory stream. EB cell differentiation was initially patchy and progressed toward an even distribution along the graft by 15-21 days post-transplantation, giving rise mostly to GABAergic neurons. EB cells in the striatum displayed a lower level of neuronal differentiation and derived into a significant number of astrocytes. Remarkably, when EB cells were transplanted to the striatum of adult rats after a local ischemic stroke, increased number of neuroblasts and neurons were observed. Unexpectedly, we determined that the adult substantia nigra pars compacta, considered a non-neurogenic area, harbors a robust neurogenic environment. Therefore, neurally uncommitted cells derived from ESCs can detect regions that support neuronal differentiation within the adult brain, a fundamental step for the development of stem cell-based replacement therapies.

Alpha-Synuclein Gene Alterations Modulate Tyrosine Hydroxylase in Human iPSC-Derived Neurons in a Parkinson's Disease Animal Model.

Parkinson's disease (PD) caused by SNCA gene triplication (3XSNCA) leads to early onset, rapid progression, and often dementia. Understanding the impact of 3XSNCA and its absence is crucial. This study investigates the differentiation of human induced pluripotent stem cell (hiPSC)-derived floor-plate progenitors into dopaminergic neurons. Three different genotypes were evaluated in this study: patient-derived hiPSCs with 3XSNCA, a gene-edited isogenic line with a frame-shift mutation on all SNCA alleles (SNCA 4KO), and a normal wild-type control. Our aim was to assess how the substantia nigra pars compacta (SNpc) microenvironment, damaged by 6-hydroxydopamine (6-OHDA), influences tyrosine hydroxylase-positive (Th+) neuron differentiation in these genetic variations. This study confirms successful in vitro differentiation into neuronal lineage in all cell lines. However, the SNCA 4KO line showed unusual LIM homeobox transcription factor 1 alpha (Lmx1a) extranuclear distribution. Crucially, both 3XSNCA and SNCA 4KO lines had reduced Th+ neuron expression, despite initial successful neuronal differentiation after two months post-transplantation. This indicates that while the SNpc environment supports early neuronal survival, SNCA gene alterations-either amplification or knock-out-negatively impact Th+ dopaminergic neuron maturation. These findings highlight SNCA's critical role in PD and underscore the value of hiPSC models in studying neurodegenerative diseases.

Role of glutamate receptors in the dorsal reticular nucleus in formalin-induced secondary allodynia.

The role of glutamate receptors present in the medullary dorsal reticular nucleus (DRt) in the formalin test and formalin-induced secondary nociception was studied in rats. Secondary mechanical allodynia was assessed with von Frey filaments applied to the rat's hindpaw, and secondary thermal hyperalgesia was evaluated with the tail-immersion test. The selective glutamate receptor antagonists MK801 (N-methyl-D-aspartate receptor antagonist), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (AMPA/KA receptor antagonist) and A841720 (metabotropic glutamate 1 receptor antagonist) were injected into the DRt before or 6 days after formalin injection in the rat. In the formalin test, the three antagonists significantly reduced the number of flinches in both phases of the test. DRt microinjection of MK801 or A841720, but not of CNQX, reduced both secondary nociceptive behaviors. Moreover, pre-treatment with the three antagonists injected into the DRt prevented the development of secondary mechanical allodynia and secondary thermal hyperalgesia. Similarly, in these rats, the number of c-Fos-like immunoreactive neurons were markedly reduced in both the superficial and deep lamina of the dorsal horn. Our findings support the role of DRt as a pain facilitator in acute and chronic pain states, and suggest a key role of glutamate receptors during the development and maintenance of formalin-induced secondary allodynia.

Adrenal Grafts in the Central Nervous System: Chromaffin and Chromaffin Progenitor Cell Transplantation.

Chromaffin cells are neuroendocrine cells that synthesize and release catecholamines and neuroactive molecules. They have been used experimentally in animal models and preclinical studies as a source for cell replacement therapy in Parkinson's disease. The long-term cell survival of these cells in the nervous system is limited, and the observed motor improvements are highly variable. An alternative source for transplantation is chromaffin progenitor cells. These cells have the capacity of self-renewal and to form spheres under low attachment conditions. They release higher quantities of dopamine than chromaffin cells and can differentiate into dopaminergic-like neurons in vitro. The transplantation of these cells into Parkinson's disease animal models has shown to induce stronger motor improvements and better survival rates than chromaffin cells. However, several aspects of chromaffin progenitor cell transplantation remain to be elucidated. Here, we describe methods to isolate and culture chromaffin and chromaffin progenitor cells from the adult cattle adrenal glands. We also describe the procedure for their transplantation into the nervous system and give recommendations for their histological analysis.

Embryoid Body Cells from Human Embryonic Stem Cells Overexpressing Dopaminergic Transcription Factors Survive and Initiate Neurogenesis via Neural Rosettes in the Substantia Nigra.

Transplantation of immature dopaminergic neurons or neural precursors derived from embryonic stem cells (ESCs) into the substantia nigra pars compacta (SNpc) is a potential therapeutic approach for functional restitution of the nigrostriatal pathway in Parkinson's disease (PD). However, further studies are needed to understand the effects of the local microenvironment on the transplanted cells to improve survival and specific differentiation in situ. We have previously reported that the adult SNpc sustains a neurogenic microenvironment. Non-neuralized embryoid body cells (EBCs) from mouse ESCs (mESCs) overexpressing the dopaminergic transcription factor Lmx1a gave rise to many tyrosine hydroxylase (Th+) cells in the intact and damaged adult SNpc, although only for a short-term period. Here, we extended our study by transplanting EBCs from genetically engineered naive human ESC (hESC), overexpressing the dopaminergic transcription factors LMX1A, FOXA2, and OTX2 (hESC-LFO), in the SNpc. Unexpectedly, no graft survival was observed in wild-type hESC EBCs transplants, whereas hESC-LFO EBCs showed viability in the SNpc. Interestingly, neural rosettes, a developmental hallmark of neuroepithelial tissue, emerged at 7- and 15-days post-transplantation (dpt) from the hESC-LFO EBCs. Neural rosettes expressed specification dopaminergic markers (Lmx1a, Otx2), which gave rise to several Th+ cells at 30 dpt. Our results suggest that the SNpc enables the robust initiation of neural differentiation of transplanted human EBCs prompted to differentiate toward the midbrain dopaminergic phenotype.

Dopamine D2 and Adenosine A2A Receptors Interaction on Ca2+ Current Modulation in a Rodent Model of Parkinsonism.

SUMMARY STATEMENT: A2A receptor required previous D2 receptor activation to modulate Ca2+ currents. Istradefylline decreases pramipexole modulation on Ca2+ currents. Istradefylline reduces A2A + neurons activity in striatial microcircuit, but pramipexole failed to further reduce neuronal activity.

Upregulation of D2-class signaling in dopamine-denervated striatum is in part mediated by D3 receptors acting on Ca V 2.1 channels via PIP2 depletion.

The loss of dopaminergic neurons in the substantia nigra compacta followed by striatal dopamine depletion is a hallmark of Parkinson's disease. After dopamine depletion, dopaminergic D(2) receptor (D(2)R)-class supersensitivity develops in striatal neurons. The supersensitivity results in an enhanced modulation of Ca(2+) currents by D(2)R-class receptors. However, the relative contribution of D(2)R, D(3)R, and D(4)R types to the supersensitivity, as well as the mechanisms involved, have not been elucidated. In this study, whole cell voltage-clamp recordings were performed to study Ca(2+) current modulation in acutely dissociated striatal neurons obtained from rodents with unilateral 6-hydroxydopamine lesions in the substantia nigra compacta. Selective antagonists for D(2)R, D(3)R, and D(4)R types were used to identify whether the modulation by one of these receptors experiences a selective change after dopaminergic denervation. It was found that D(3)R-mediated modulation was particularly enhanced. Increased modulation targeted Ca(V)2.1 (P/Q) Ca(2+) channels via the depletion of phosphatidylinositol 4,5-bisphosphate, an intracellular signaling cascade hard to detect in control neurons and hypothesized as being amplified by dopamine depletion. An imbalance in the striatal expression of D(3)R and its splice variant, D(3)nf, accompanied enhanced D(3)R activity. Because Ca(V)2.1 Ca(2+) channels mediate synaptic GABA release from the terminals of striatal neurons, reinforcement of their inhibition by D(3)R may explain in part the profound decrease in synaptic strength in the connections among striatal projection neurons observed in the dopamine-depleted striatum.

In Vivo Wireless Optogenetic Control of Skilled Motor Behavior.

Fine motor skills are essential in everyday life and can be compromised in several nervous system disorders. The acquisition and performance of these tasks require sensory-motor integration and involve precise control of bilateral brain circuits. Implementing unimanual behavioral paradigms in animal models will improve the understanding of the contribution of brain structures, like the striatum, to complex motor behavior as it allows manipulation and recording of neural activity of specific nuclei in control conditions and disease during the performance of the task. Since its creation, optogenetics has been a dominant tool for interrogating the brain by enabling selective and targeted activation or inhibition of neuronal populations. The combination of optogenetics with behavioral assays sheds light on the underlying mechanisms of specific brain functions. Wireless head-mounted systems with miniaturized light-emitting diodes (LEDs) allow remote optogenetic control in an entirely free-moving animal. This avoids the limitations of a wired system being less restrictive for animals' behavior without compromising light emission efficiency. The current protocol combines a wireless optogenetics approach with high-speed videography in a unimanual dexterity task to dissect the contribution of specific neuronal populations to fine motor behavior.

Cannabidiol Partially Blocks the Excessive Sleepiness in Hypocretindeficient Rats: Preliminary Data.

BACKGROUND: Excessive daytime sleepiness and cataplexy are among the symptoms of narcolepsy, a sleep disorder caused by the loss of hypocretin/orexin (HCRT/OX) neurons placed into the Hypothalamus (LH). Several treatments for managing narcolepsy include diverse drugs to induce alertness, such as antidepressants, amphetamine, or modafinil, etc. Recent evidence has shown that cannabidiol (CBD), a non-psychotropic derived from Cannabis sativa, shows positive therapeutic effects in neurodegenerative disorders, including Parkinson´s disease. Furthermore, CBD provokes alertness and enhances wake-related neurochemicals in laboratory animals. Thus, it is plausible to hypothesize that excessive somnolence observed in narcolepsy might be blocked by CBD. OBJECTIVE: Here, we determined whether the systemic injection of CBD (5mg/kg, i.p.) would block the excessive sleepiness in a narcoleptic model. METHODS: To test this idea, the neurotoxin hypocretin-2-saporin (HCRT2/SAP) was bilaterally injected into the LH of rats to eliminate HCRT leading to the establishment of narcoleptic-like behavior. Since excessive somnolence in HCRT2/SAP lesioned rats has been observed during the lights-off period, CBD was administered at the beginning of the dark phase. RESULTS: Hourly analysis of sleep data showed that CBD blocked the sleepiness during the lights-off period across 7h post-injection in lesioned rats. CONCLUSION: Taking together, these preliminary findings suggest that CBD might prevent sleepiness in narcolepsy.

The nonpsychoactive Cannabis constituent cannabidiol is a wake-inducing agent.

Cannabidiol (CBD) is a constituent of Cannabis sativa that induces nonpsychotropic effects, and some of its biological actions in sleep have been described by the authors' group. Here, the authors report that when administered 10 or 20 microg/1 microl during the lights-on period directly into either lateral hypothalamus (LH) or dorsal raphe nuclei (DRN), which are wake-inducing brain areas, CBD enhanced wakefulness and decreased slow wave sleep and REM sleep. Furthermore, CBD increased alpha and theta power spectra but diminished delta power spectra. Additionally, CBD increased c-Fos expression in LH or DRN. These findings suggest that this cannabinoid is a wake-inducing compound that presumably activates neurons in LH and DRN.

Intrastriatal Chromospheres' Transplant Reduces Nociception in Hemiparkinsonian Rats.

The present study evaluates the possible antinociceptive effect of chromosphere transplants in rats injected with 6-hydroxydopamine (6-OHDA), a model of Parkinson's disease. Male adult Wistar rats received 40µg/0.5µl of 6-OHDA or 0.5µl of vehicle into the left substantia nigra (SNc). Rats were evaluated for mechanical allodynia, cold allodynia, thermal hyperalgesia and formalin. Rats with altered nociceptive threshold were transplanted with chromospheres. After transplant, rats were evaluated every week. Our results confirm that 6-OHDA injection into rat's SNc reduces mechanical, thermal, and chemical thresholds. Interestingly, chromospheres' transplant reverted 6-OHDA-induced allodynia and hyperalgesia. The antinociceptive effect induced by chromospheres was dopamine D2- and opioid-receptor dependent since sulpiride or naltrexone reverted its effect.

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.

Effects of the fatty acid amide hydrolase inhibitor URB597 on the sleep-wake cycle, c-Fos expression and dopamine levels of the rat.

Our group has described previously that the endogenous cannabinoid anandamide induces sleep. The hydrolysis of this lipid involves the activity of the fatty acid amide hydrolase (FAAH), which additionally catalyzes the degradation of the satiety factor oleoylethanolamide and the analgesic-inducing lipid palmitoylethanolamide. It has been demonstrated that the inhibition of the FAAH by URB597 increases levels of anandamide, oleoylethanolamide and palmitoylethanolamide in the brain of rats. In order to determinate the physiological properties of the FAAH inhibition on the sleep modulation, we report the pharmacological effects on the sleep-wake cycle of the rat after i.c.v. administrations of URB597, oleoylethanolamide or palmitoylethanolamide (10, 20 microg/5 microl). Separate unilateral i.c.v. injections of 3 compounds during the lights-on period, increased wakefulness and decreased slow wave (SW) sleep in rats in a dose-dependent fashion. We additionally found out that, compared to controls, c-Fos immunoreactivity in hypothalamus and dorsal raphe nucleus was increased in rats that received URB597, oleoylethanolamide or palmitoylethanolamide (10, 20 microg/5 microl, i.c.v.). Next, we found that after an injection of the compounds, levels of dopamine were increased whereas extracellular levels of levodopa (l-DOPA) were decreased. These findings indicate that that inhibition of the FAAH, via URB597, modulates waking. These effects were mimicked separately by the administration of oleoylethanolamide or palmitoylethanolamide. The alertness induced by the compounds tested here activated wake-promoting brain regions and they also induced the release of dopamine. Our results suggest that FAAH activity as well as two molecules that are catalyzed by this enzyme, oleoylethanolamide and palmitoylethanolamide, participate in the regulation of the waking state. Alternative approaches to treat sleep disorders such as excessive somnolence might consider the use of the URB597, oleoylethanolamide or palmitoylethanolamide since all compounds enhance waking.

An iron-deficient diet during development induces oxidative stress in relation to age and gender in Wistar rats.

Iron is a trace element and a structural part of antioxidant enzymes, and its requirements vary according to age and gender. We hypothesized that iron deficiency (ID) leads to an increase in free radicals which mainly affect the brain, and the severity of damage would therefore be dependent on age and gender. Two groups of Wistar rats were evaluated evolutionarily: 100 rats (50 males; 50 females) with ID diet and 100 rats (50 males; 50 females) with standard diet. Both groups were offspring from mothers who were previously under the same dietary intervention. The ages studied roughly correspond to stages of human development: birth (0 postnatal day "PND" in rats), childhood (21 PND), early adolescence (42 PND), late adolescence (56 PND), and adulthood (70 PND). The following biomarkers in the brain, blood, and liver were analyzed: lipid peroxidation products (LPO), protein carbonyl content and activity of the antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase. It was demonstrated that ID subjects are born with high levels of LPO in the brain and low antioxidant activity, the damage being more severe in males. After birth, antioxidant defense focuses on the central level (brain) in ID females and on the peripheral level (blood and liver) in ID males. In two critical stages of development, birth and late adolescence, antioxidant protection is insufficient to counteract oxidative damage in ID subjects. Moreover, we observed that the variability of results in the literature on oxidative stress and ID comes from gender and age of the subjects under study. With this, we can establish patterns and exact moments to carry out studies or treatments.

Recovery of glomerular morphology in the olfactory bulb of young mice after disruption caused by continuous odorant exposure.

Olfactory glomeruli are the first synaptic site of the olfactory system and are formed by the convergence of axons of the same type of sensory neurons onto the olfactory bulbs of the brain. Although the anatomical organization of glomeruli is conserved across species, their particular role in olfactory processing remains uncertain. We studied the composition and maintenance of glomeruli by means of a genetic model, mI7-IRES-tauGFP knock-in young mice, where the cytoskeleton of sensory neurons expressing the mI7 olfactory receptor is tagged with green fluorescent protein. Animals were continuously exposed to heptaldehyde, a cognate ligand of the mI7 receptor, from postnatal days 5-10. We hypothesized that continuous odorant exposure will induce changes in glomerular morphology, and that this can be recovered if the normal odorant environment is reestablished within the early postnatal period. We assessed changes in the distribution of mI7 axons in glomerular morphology, as well as possible changes in the number of the mI7 olfactory sensory neurons. Following odorant exposure the well-defined convergence of mI7 fibers into a single glomerulus was disrupted, producing numerous neighboring glomeruli partially innervated by mI7 fibers. After the normal odor environment was reestablished the number of glomeruli partially innervated by mI7 fibers decreased significantly. Moreover, we found that multiple supernumerary mI7 glomeruli were formed. Our results confirm the significant role of sensory input in glomerular formation and maintenance. Additionally, we show that the developing olfactory system actively maintains glomerular morphology, suggesting the importance of this for olfactory processing.

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

Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and cannabidiol (CBD) are two major constituents of Cannabis sativa. Delta(9)-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 microg/5 microl 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 microg/5microl) 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 microg/5 microl) 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 microg/2.5 microl, icv) followed 15 min later by CBD (10 microg/2.5 microl). 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.

Intrastriatal Grafting of Chromospheres: Survival and Functional Effects in the 6-OHDA Rat Model of Parkinson's Disease.

Cell replacement therapy in Parkinson's disease (PD) aims at re-establishing dopamine neurotransmission in the striatum by grafting dopamine-releasing cells. Chromaffin cell (CC) grafts produce some transitory improvements of functional motor deficits in PD animal models, and have the advantage of allowing autologous transplantation. However, CC grafts have exhibited low survival, poor functional effects and dopamine release compared to other cell types. Recently, chromaffin progenitor-like cells were isolated from bovine and human adult adrenal medulla. Under low-attachment conditions, these cells aggregate and grow as spheres, named chromospheres. Here, we found that bovine-derived chromosphere-cell cultures exhibit a greater fraction of cells with a dopaminergic phenotype and higher dopamine release than CC. Chromospheres grafted in a rat model of PD survived in 57% of the total grafted animals. Behavioral tests showed that surviving chromosphere cells induce a reduction in motor alterations for at least 3 months after grafting. Finally, we found that compared with CC, chromosphere grafts survive more and produce more robust and consistent motor improvements. However, further experiments would be necessary to determine whether the functional benefits induced by chromosphere grafts can be improved, and also to elucidate the mechanisms underlying the functional effects of the grafts.

The administration of endocannabinoid uptake inhibitors OMDM-2 or VDM-11 promotes sleep and decreases extracellular levels of dopamine in rats.

The family of the endocannabinoid system comprises endogenous lipids (such as anandamide [ANA]), receptors (CB(1)/CB(2) cannabinoid receptors), metabolic enzymes (fatty acid amide hydrolase [FAAH]) and a putative membrane transporter (anandamide membrane transporter [AMT]). Although the role of ANA, FAAH or the CB(1) cannabinoid receptor in sleep modulation has been reported, the effects of the inhibition of AMT on sleep remain unclear. In the present study, we show that microdialysis perfusion in rats of AMT inhibitors, (9Z)-N-[1-((R)-4-hydroxbenzyl)-2-hydroxyethyl]-9-octadecenamide (OMDM-2) or N-(4-hydroxy-2-methylphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (VDM-11; 10, 20 or 30 µM; each compound) delivered into the paraventricular thalamic nucleus (PVA) increased sleep and decreased waking. In addition, the infusion of compounds reduced the extracellular levels of dopamine collected from nucleus accumbens. Taken together, these findings illustrate a critical role of AMT in sleep modulation.

Cellular viability effects of fatty acid amide hydrolase inhibition on cerebellar neurons.

The endocannabinoid anandamide (ANA) participates in the control of cell death inducing the formation of apoptotic bodies and DNA fragmentation. The aim of this study was to evaluate whether the ANA degrading enzyme, the fatty acid amide hydrolase (FAAH), would induce cellular death. Experiments were performed in cerebellar granule neurons cultured with the FAAH inhibitor, URB597 (25, 50 or 100 nM) as well as endogenous lipids such as oleoylethanolamide (OEA) or palmitoylethanolamide (PEA) and cellular viability was determined by MTT test. Neurons cultured with URB597 (25, 50 or 100 nM) displayed a decrease in cellular viability. In addition, if cultured with OEA (25 nM) or PEA (100 nM), cellular death was found. These results further suggest that URB597, OEA or PEA promote cellular death.

Infusion of modafinil into anterior hypothalamus or pedunculopontine tegmental nucleus at different time-points enhances waking and blocks the expression of recovery sleep in rats after sleep deprivation.

Clinical studies have indicated that the primary pharmacological activity of modafinil (MOD) is inducing wakefulness; however, the brain targets that underlie its wake-promoting activity have not been described. In the present study, we show that MOD injected into sleep-wake related brain areas promoted alertness. If administered (10, 20, or 30 µg/1 µL) into either anterior hypothalamus (AH) or pedunculopontine tegmental nucleus (PPTg) at 08:00, 12:00 or 16:00 h, MOD enhanced wakefulness whereas diminished slow wave sleep as well as rapid eye movement sleep. In addition, microinjection of MOD (10, 20, or 30 µg/1 µL) either into AH or PPTg after total sleep deprivation prevented the sleep rebound. Taken together, these observations suggest that AH and PPTg play a key role in the wake-inducing effects of MOD and encourage further experimentation to draw a possible mechanism of action.