Prefrontal cortical distribution of muscarinic M2 and cannabinoid-1 (CB1) receptors in adult male mice with or without chronic adolescent exposure to Δ9-tetrahydrocannabinol.

Chronic adolescent administration of marijuana's major psychoactive compound, ∆9-tetrahydrocannabinol (Δ9-THC), produces adaptive changes in adult social and cognitive functions sustained by prelimbic prefrontal cortex (PL-PFC). Memory and learning processes in PL-PFC neurons can be regulated through cholinergic muscarinic-2 receptors (M2R) and modulated by activation of cannabinoid-1 receptors (CB1Rs) targeted by Δ9-THC. Thus, chronic exposure to Δ9-THC during adolescence may alter the expression and/or distribution of M2Rs in PL-PFC neurons receiving CB1R terminals. We tested this hypothesis by using electron microscopic dual CB1R and M2R immunolabeling in adult C57BL/6 J male mice that had received vehicle or escalating dose of Δ9-THC through adolescence. In vehicle controls, CB1R immunolabeling was mainly localized to axonal profiles virtually devoid of M2R but often apposing M2R-immunoreactive dendrites and dendritic spines. The dendrites received inputs from CB1R-labeled or unlabeled terminals, whereas spines received asymmetric synapses exclusively from axon terminals lacking CB1Rs. Adolescent Δ9-THC significantly increased plasmalemmal M2R-immunogold density exclusively in large dendrites receiving input from CB1R-labeled terminals. In contrast, cytoplasmic M2R-immunogold density decreased in small spines of the Δ9-THC-treated adult mice. We conclude that Δ9-THC engagement of CB1Rs during adolescence increases M2R plasmalemmal accumulation in large proximal dendrites and decreases M2R cytoplasmic expression in small spines of PL-PFC.

Hypocretin1/orexinA-immunoreactive axons form few synaptic contacts on rat ventral tegmental area neurons that project to the medial prefrontal cortex.

BACKGROUND: Hypocretins/orexins (Hcrt/Ox) are hypothalamic neuropeptides involved in sleep-wakefulness regulation. Deficiency in Hcrt/Ox neurotransmission results in the sleep disorder narcolepsy, which is characterized by an inability to maintain wakefulness. The Hcrt/Ox neurons are maximally active during wakefulness and project widely to the ventral tegmental area (VTA). A dopamine-containing nucleus projecting extensively to the cerebral cortex, the VTA enhances wakefulness. In the present study, we used retrograde tracing from the medial prefrontal cortex (mPFC) to examine whether Hcrt1/OxA neurons target VTA neurons that could sustain behavioral wakefulness through their projections to mPFC. RESULTS: The retrograde tracer Fluorogold (FG) was injected into mPFC and, after an optimal survival period, sections through the VTA were processed for dual immunolabeling of anti-FG and either anti-Hcrt1/OxA or anti-TH antisera. Most VTA neurons projecting to the mPFC were located in the parabrachial nucleus of the ipsilateral VTA and were non-dopaminergic. Only axonal profiles showed Hcrt1/OxA-immunoreactivity in VTA. Hcrt1/OxA reactivity was observed in axonal boutons and many unmyelinated axons. The Hcrt1/OxA immunoreactivity was found filling axons but it was also observed in parts of the cytoplasm and dense-core vesicles. Hcrt1/OxA-labeled boutons frequently apposed FG-immunolabeled dendrites. However, Hcrt1/OxA-labeled boutons rarely established synapses, which, when they were established, were mainly asymmetric (excitatory-type), with either FG-labeled or unlabeled dendrites. CONCLUSIONS: Our results provide ultrastructural evidence that Hcrt1/OxA neurons may exert a direct synaptic influence on mesocortical neurons that would facilitate arousal and wakefulness. The paucity of synapses, however, suggest that the activity of VTA neurons with cortical projections might also be modulated by Hcrt1/OxA non-synaptic actions. In addition, Hcrt1/OxA could modulate the postsynaptic excitatory responses of VTA neurons with cortical projections to a co-released excitatory transmitter from Hcrt1/OxA axons. Our observation of Hcrt1/OxA targeting of mesocortical neurons supports Hcrt1/OxA wakefulness enhancement in the VTA and could help explain the characteristic hypersomnia present in narcoleptic patients.

Hypocretin1/OrexinA axon targeting of laterodorsal tegmental nucleus neurons projecting to the rat medial prefrontal cortex.

Cortical activation and goal-directed behaviors characterize wakefulness. One cortical region especially involved in these phenomena is the medial prefrontal cortex (mPFC), which receives many inputs from cholinergic-containing neurons in brain stem structures implicated in arousal and wakefulness, such as the laterodorsal tegmental nucleus (LDT). Hypocretins/orexins (Hcrt/Ox), whose dysfunction is linked to narcolepsy, maintains arousal and stabilizes sleep-wakefulness states. We aim to determine if Hcrt1/OxA axons (1) innervate LDT neurons projecting to the mPFC, a target that would allow them to sustain arousal and wakefulness, and (2) target preferentially cholinergic versus noncholinergic LDT neurons. The retrograde tracer Fluorogold (FG) was injected in the rat mPFC, and dual immunolabeling of anti-FG and either anti-choline acetyltransferase (ChAT) or anti-Hcrt1/OxA antisera was determined in LDT. Also, actual Hcrt1/OxA targeting of cholinergic LDT neurons was ascertained by dual anti-Hcrt1/OxA and anti-ChAT detection in additional noninjected animals. Many LDT FG-labeled neurons were cholinergic (52.05 ± 3.72%). Hcrt1/OxA immunoprecipitate was observed in cytoplasm and granular vesicles within axons. Some Hcrt1/OxA-containing axons established asymmetric excitatory-type synapses with either unlabeled (46/438) or FG-labeled (7/438) dendrites. One-third of the target neurons were ChAT labeled. Hcrt1/OxA excitatory input to LDT neurons projecting to mPFC probably contributes to the wakefulness-enhancing actions of Hcrt/Ox impaired in narcoleptics.

Plasticity of the hypocretinergic/orexinergic system after a chronic treatment with suvorexant in rats. Role of the hypocretinergic/orexinergic receptor 1 as an autoreceptor.

The hypothalamic hypocretinergic/orexinergic (Hcrt/Ox) system is involved in many physiological and pathophysiological processes. Malfunction of Hcrt/Ox transmission results in narcolepsy, a sleep disease caused in humans by progressive neurodegeneration of hypothalamic neurons containing Hcrt/Ox. To explore the Hcrt/Ox system plasticity we systemically administered suvorexant (a dual Hcrt/Ox receptor antagonist) in rats to chronically block Hcrt/Ox transmission without damaging Hcrt/Ox cells. Three groups of eight rats (four males and four females) received daily i.p. injections of suvorexant (10 or 30 mg/kg) or vehicle (DMSO) over a period of 7 days in which the body weight was monitored. After the treatments cerebrospinal fluid (CSF) Hcrt1/OxA concentration was measured by ELISA, and hypothalamic Hcrt/OxR1 and Hcrt/OxR2 levels by western blot. The systemic blockade of the Hcrt/Ox transmission with the suvorexant high dose produced a significant increase in body weight at the end of the treatment, and a significant decrease in CSF Hcrt1/OxA levels, both features typical in human narcolepsy type 1. Besides, a significant overexpression of hypothalamic Hcrt/OxR1 occurred. For the Hcrt/OxR2 two very close bands were detected, but they did not show significant changes with the treatment. Thus, the plastic changes observed in the Hcrt/Ox system after the chronic blockade of its transmission were a decrease in CSF Hcrt1/OXA levels and an overexpression of hypothalamic Hcrt/OxR1. These findings support an autoregulatory role of Hcrt/OxR1 within the hypothalamus, which would induce the synthesis/release of Hcrt/Ox, but also decrease its own availability at the plasma membrane after binding Hcrt1/OxA to preserve Hcrt/Ox system homeostasis.

Functional anatomy of the sleep-wakefulness cycle: wakefulness.

Sleep is a necessary, diverse, periodic, and an active condition circadian and homeostatically regulated and precisely meshed with waking time into the sleep-wakefulness cycle (SWC). Photic retinal stimulation modulates the suprachiasmatic nucleus, which acts as the pacemaker for SWC rhythmicity. Both the light period and social cues adjust the internal clock, making the SWC a circadian, 24-h period in the adult human. Bioelectrical and behavioral parameters characterize the different phases of the SWC. For a long time, lesions and electrical stimulation of brain structures, as well as connection studies, were the main methods used to decipher the foundations of the functional anatomy of the SWC. That is why the first section of this review presents these early historical studies to then discuss the current state of our knowledge based on our understanding of the functional anatomy of the structures underlying the SWC. Supported by this description, we then present a detailed review and update of the structures involved in the phase of wakefulness (W), including their morphological, functional, and chemical characteristics, as well as their anatomical connections. The structures for W generation are known as the "ascending reticular activating system", and they keep and maintain the "thalamo-cerebral cortex unit" awake. This system originates from the neuronal groups located within the brainstem, hypothalamus, and basal forebrain, which use known neurotransmitters and whose neurons are more active during W than during the other SWC states. Thus, synergies among several of these neurotransmitters are necessary to generate the cortical and thalamic activation that is characteristic of the W state, with all the plastic qualities and nuances present in its different behavioral circumstances. Each one of the neurotransmitters exerts powerful influences on the information and cognitive processes as well as attentional, emotional, motivational, behavioral, and arousal states. The awake "thalamo-cerebral cortex unit" controls and adjusts the activation pattern through a top-down action on the subcortical cellular groups that are the origin of the "ascending reticular activating system".

Hypocretin1/OrexinA-containing axons innervate locus coeruleus neurons that project to the Rat medial prefrontal cortex. Implication in the sleep-wakefulness cycle and cortical activation.

The Hypocretin1/OrexinA (Hcrt1/OxA) neuropeptides are found in a group of posterolateral hypothalamus neurons and are involved in sleep-wakefulness cycle regulation. Hcrt1/OxA neurons project widely to brainstem aminergic structures, such as the locus coeruleus (LC), which are involved in maintenance of wakefulness and EEG activation through intense projections to the medial prefrontal cortex (mPFC). Moreover, defects of the Hcrt1/OxA system are linked to narcolepsy, a disorder characterized by excessive diurnal hypersomnia and REM state disturbance. We aimed to determine whether Hcrt1/OxA neurons innervate LC neurons (noradrenergic and nonnoradrenergic) that project to the mPFC, thereby sustaining behavioral wakefulness. To assess this, we used retrograde tracing from mPFC injections and either Hcrt1/OxA or tyrosine hydroxylase (TH) immunohistochemical labeling in single sections of rat LC. The retrograde tracer Fluorogold (FG) was microinjected into mPFC and, at optimal survival periods, sections through the LC were processed for dual immunolabeling of anti-FG and either anti-Hcrt1/OxA or anti-TH antisera. Many LC neurons projecting to mPFC were nonnoradrenergic. Electron microscopy revealed a prominent localization of Hcrt1/OxA in unmyelinated axons and axon boutons (varicosities and axon terminals) within the LC. Hcrt1/OxA-immunoreactive axon boutons frequently apposed (104/1907) or made asymmetric excitatory-type synapses (60/1907) with FG-immunolabeled dendrites, indicating that Hcrt1/OxA can modulate the activity of LC neurons with cortical projections. Our results show that Hcrt1/OxA hypothalamic neurons likely excite LC neurons that project to the mPFC, and thus activate EEG and facilitate wakefulness. In narcoleptics, who are deficient in Hcrt1/OxA, impairment of this Hcrt1/OxA hypothalamic input to LC might contribute to the appearance of excessive daytime sleepiness.

Megalin interacts with APP and the intracellular adapter protein FE65 in neurons.

Increasing evidence has implicated megalin, a low-density lipoprotein receptor-related protein, in the pathogenesis of Alzheimer's disease (AD). In the brain, megalin is expressed in brain capillaries, ependymal cells and choroid plexus, where it participates in the clearance of brain amyloid ß-peptide (Aß) complex. Recently, megalin has also been detected in oligodendrocytes and astrocytes. In this study we demonstrate that megalin is widely distributed in neurons throughout the brain. Additionally, given that FE65 mediates the interaction between the low density lipoprotein receptor-related protein-1 and the amyloid precursor protein (APP) to modulate the rate of APP internalization from the cell surface, we hypothesize that megalin could also interact with APP in neurons. Our results confirm that megalin interacts with APP and FE65, suggesting that these three proteins form a tripartite complex. Moreover, our findings imply that megalin may participate in neurite branching. Taken together, these results indicate that megalin has an important role in Aß-mediated neurotoxicity, and therefore may be involved in the neurodegenerative processes that occur in AD.

Ultrastructural evidence for mu and delta opioid receptors at noradrenergic dendrites and glial profiles in the cat locus coeruleus.

The Locus Coeruleus (LC) is a pontine nucleus involved in many physiological processes, including the control of the sleep/wake cycle (SWC). At cellular level, the LC displays a high density of opioid receptors whose activation decreases the activity of LC noradrenergic neurons. Also, microinjections of morphine administered locally in the LC of the cat produce sleep associated with synchronized brain activity in the electroencephalogram (EEG). Even though much of the research on sleep has been done in the cat, the subcellular location of opioid receptors in the LC and their relationship with LC noradrenergic neurons is not known yet in this species. Therefore, we conducted a study to describe the ultrastructural localization of mu-opioid receptors (MOR), delta-opioid receptors (DOR) and tyrosine hydroxylase (TH) in the cat LC using high resolution electron microscopy double-immunocytochemical detection. MOR and DOR were localized mainly in dendrites (45% and 46% of the total number of profiles respectively), many of which were noradrenergic (35% and 53% for MOR and DOR, respectively). TH immunoreactivity was more frequent in dendrites (65% of the total number of profiles), which mostly also expressed opioid receptors (58% and 73% for MOR and DOR, respectively). Because the distribution of MORs and DORs are similar, it is possible that a substantial sub-population of neurons co-express both receptors, which may facilitate the formation of MOR-DOR heterodimers. Moreover, we found differences in the cat subcellular DOR distribution compared with the rat. This opens the possibility to the existence of diverse mechanisms for opioid modulation of LC activity.

Adolescent administration of Δ9-THC decreases the expression and function of muscarinic-1 receptors in prelimbic prefrontal cortical neurons of adult male mice.

Long-term cannabis use during adolescence has deleterious effects in brain that are largely ascribed to the activation of cannabinoid-1 receptors (CB1Rs) by delta-9-tetrahydrocannabinol (∆9-THC), the primary psychoactive compound in marijuana. Systemic administration of ∆9-THC inhibits acetylcholine release in the prelimbic-prefrontal cortex (PL-PFC). In turn, PL-PFC acetylcholine plays a role in executive activities regulated by CB1R-targeting endocannabinoids, which are generated by cholinergic stimulation of muscarinic-1 receptors (M1Rs). However, the long-term effects of chronic administration of increasing doses of ∆9-THC in adolescent males on the distribution and function of M1 and/or CB1 receptors in the PL-PFC remains unresolved. We used C57BL\6J male mice pre-treated with vehicle or escalating daily doses of ∆9-THC to begin filling this gap. Electron microscopic immunolabeling showed M1R-immunogold particles on plasma membranes and in association with cytoplasmic membranes in varying sized dendrites and dendritic spines. These dendritic profiles received synaptic inputs from unlabeled, CB1R- and/or M1R-labeled axon terminals in the PL-PFC of both treatment groups. However, there was a size-dependent decrease in total (plasmalemmal and cytoplasmic) M1R gold particles in small dendrites within the PL-PFC of mice receiving ∆9-THC. Whole cell current-clamp recording in PL-PFC slice preparations further revealed that adolescent pretreatment with ∆9-THC attenuates the hyperpolarization and increases the firing rate produced by local muscarinic stimulation. Repeated administration of ∆9-THC during adolescence also reduced spontaneous alternations in a Y-maze paradigm designed for measures of PFC-dependent memory function in adult mice. Our results provide new information implicating M1Rs in cortical dysfunctions resulting from adolescent abuse of marijuana.

Circadian control of hepatitis B virus replication.

Chronic hepatitis B virus (HBV) infection is a major cause of liver disease and cancer worldwide for which there are no curative therapies. The major challenge in curing infection is eradicating or silencing the covalent closed circular DNA (cccDNA) form of the viral genome. The circadian factors BMAL1/CLOCK and REV-ERB are master regulators of the liver transcriptome and yet their role in HBV replication is unknown. We establish a circadian cycling liver cell-model and demonstrate that REV-ERB directly regulates NTCP-dependent hepatitis B and delta virus particle entry. Importantly, we show that pharmacological activation of REV-ERB inhibits HBV infection in vitro and in human liver chimeric mice. We uncover a role for BMAL1 to bind HBV genomes and increase viral promoter activity. Pharmacological inhibition of BMAL1 through REV-ERB ligands reduces pre-genomic RNA and de novo particle secretion. The presence of conserved E-box motifs among members of the Hepadnaviridae family highlight an evolutionarily conserved role for BMAL1 in regulating this family of small DNA viruses.

Dopamine innervation in the thalamus: monkey versus rat.

We recently identified the thalamic dopaminergic system in the human and macaque monkey brains, and, based on earlier reports on the paucity of dopamine in the rat thalamus, hypothesized that this dopaminergic system was particularly developed in primates. Here we test this hypothesis using immunohistochemistry against the dopamine transporter (DAT) in adult macaque and rat brains. The extent and density of DAT-immunoreactive (-ir) axons were remarkably greater in the macaque dorsal thalamus, where the mediodorsal association nucleus and the ventral motor nuclei held the densest immunolabeling. In contrast, sparse DAT immunolabeling was present in the rat dorsal thalamus; it was mainly located in the mediodorsal, paraventricular, ventral medial, and ventral lateral nuclei. The reticular nucleus, zona incerta, and lateral habenular nucleus held numerous DAT-ir axons in both species. Ultrastructural analysis in the macaque mediodorsal nucleus revealed that thalamic interneurons are a main postsynaptic target of DAT-ir axons; this suggests that the marked expansion of the dopamine innervation in the primate in comparison to the rodent thalamus may be related to the presence of a sizable interneuron population in primates. We remark that it is important to be aware of brain species differences when using animal models of human brain disease.

Discovery of Highly Selective Inhibitors of Calmodulin-Dependent Kinases That Restore Insulin Sensitivity in the Diet-Induced Obesity in Vivo Mouse Model.

Polymorphisms in the region of the calmodulin-dependent kinase isoform D (CaMK1D) gene are associated with increased incidence of diabetes, with the most common polymorphism resulting in increased recognition by transcription factors and increased protein expression. While reducing CaMK1D expression has a potentially beneficial effect on glucose processing in human hepatocytes, there are no known selective inhibitors of CaMK1 kinases that can be used to validate or translate these findings. Here we describe the development of a series of potent, selective, and drug-like CaMK1 inhibitors that are able to provide significant free target cover in mouse models and are therefore useful as in vivo tool compounds. Our results show that a lead compound from this series improves insulin sensitivity and glucose control in the diet-induced obesity mouse model after both acute and chronic administration, providing the first in vivo validation of CaMK1D as a target for diabetes therapeutics.

The Transition Between Slow-Wave Sleep and REM Sleep Constitutes an Independent Sleep Stage Organized by Cholinergic Mechanisms in the Rostrodorsal Pontine Tegmentum.

There is little information on either the transition state occurring between slow-wave sleep (SWS) and rapid eye movement (REM) sleep, as well as about its neurobiological bases. This transition state, which is known as the intermediate state (IS), is well-defined in rats but poorly characterized in cats. Previous studies in our laboratory demonstrated that cholinergic stimulation of the perilocus coeruleus α nucleus (PLCα) in the pontine tegmentum of cats induced two states: wakefulness with muscle atonia and a state of dissociated sleep we have called the SPGO state. The SPGO state has characteristics in common with the IS, such including the presence of ponto-geniculo-occipital waves (PGO) and EEG synchronization with δ wave reduction. Therefore, the aims of the present study were (1) to characterize the IS in the cat and, (2), to study the analogy between the SPGO and the different sleep stages showing PGO activity, including the IS. Polygraphic recordings of 10 cats were used. In seven cats carbachol microinjections (20-30 nL, 0.01-0.1 M) were delivered in the PLCα. In the different states, PGO waves were analyzed and power spectra obtained for the δ, θ, α, and ß bands of the EEG from the frontal and occipital cortices, and for the θ hippocampal band. Statistical comparisons were made between the values obtained from the different states. The results indicate that the IS constitutes a state with characteristics that are distinct from both the preceding SWS and the following REM sleep, and that SPGO presents a high analogy with the IS. Therefore, the SPGO state induced by administering carbachol in the PLCα nucleus seems to be an expression of the physiological IS of the cat. Consequently, we propose that the PLCα region, besides being involved in the mechanisms of muscle atonia, may also be responsible for organizing the transition from SWS to REM sleep.

Sleep-wakefulness effects after microinjections of hypocretin 1 (orexin A) in cholinoceptive areas of the cat oral pontine tegmentum.

Hypocretinergic/orexinergic neurons, which are known to be implicated in narcolepsy, project to the pontine tegmentum areas involved in the control of rapid eye movement (REM) sleep. Here, we report the effects on sleep-wakefulness produced by low-volume microinjections of hypocretin (Hcrt)1 (20-30 nL, 100, 500 and 1000 microm) and carbachol (20-30 nL, 0.1 m) delivered in two areas of the oral pontine tegmentum of free-moving cats with electrodes for chronic sleep recordings: in the dorsal oral pontine tegmentum (DOPT) and in the ventral part of the oral pontine reticular nucleus (vRPO). Carbachol in the DOPT produced dissociate polygraphic states, with some but not all REM sleep signs. In contrast, carbachol in the vRPO produced a shift with short latency from wakefulness (W) to REM sleep with all of its polygraphic and behavioral signs. Hcrt-1 in the DOPT increased W and decreased both slow-wave sleep (SWS) and REM sleep during the first 3 h post-drug. The same doses of Hcr-1 in the vRPO produced a significant suppression of REM sleep without a definitive trend for changes in the other states. Both groups showed significant decreases in the number of transitions from SWS to REM sleep. Thus, Hcrt-1 produced distinct effects in cholinoceptive areas of the oral pontine tegmentum; in the DOPT it promoted W, suppressed SWS and probably defacilitated REM sleep, and in the vRPO it directly inhibited REM sleep. Hypocretinergic/orexinergic signaling is lost in narcoleptics and this absence would mean that pontine defacilitation/inhibition of REM sleep would also be absent, explaining why these patients can fall directly into REM sleep from W.

Numerical-Experimental Study of the Consolidation Phenomenon in the Selective Laser Melting Process with a Thermo-Fluidic Coupled Model.

One of the main limiting factors for a widespread industrial use of the Selective Laser Melting Process it its lack of productivity, which restricts the use of this technology just for high added-value components. Typically, the thickness of the metallic powder that is used lies on the scale of micrometers. The use of a layer up to one millimeter would be necessarily associated to a dramatic increase of productivity. Nevertheless, when the layer thickness increases, the complexity of consolidation phenomena makes the process difficult to be governed. The present work proposes a 3D finite element thermo-coupled model to study the evolution from the metallic powder to the final consolidated material, analyzing specifically the movements and loads of the melt pool, and defining the behavior of some critical thermophysical properties as a function of temperature and the phase of the material. This model uses advanced numerical tools such as the Arbitrary Lagrangean⁻Eulerian formulation and the Automatic Remeshing technique. A series of experiments have been carried out, using a high thickness powder layer, allowing for a deeper understanding of the consolidation phenomena and providing a reference to compare the results of the numerical calculations.

Subcellular distribution of M2 muscarinic receptors in relation to dopaminergic neurons of the rat ventral tegmental area.

Acetylcholine can affect cognitive functions and reward, in part, through activation of muscarinic receptors in the ventral tegmental area (VTA) to evoke changes in mesocorticolimbic dopaminergic transmission. Among the known muscarinic receptor subtypes present in the VTA, the M2 receptor (M2R) is most implicated in autoregulation and also may play a heteroreceptor role in regulation of the output of the dopaminergic neurons. We sought to determine the functionally relevant sites for M2R activation in relation to VTA dopaminergic neurons by examining the electron microscopic immunolabeling of M2R and the dopamine transporter (DAT) in the VTA of rat brain. The M2R was localized to endomembranes in DAT-containing somatodendritic profiles but showed a more prominent, size-dependent plasmalemmal location in nondopaminergic dendrites. M2R also was located on the plasma membrane of morphologically heterogenous axon terminals contacting unlabeled as well as M2R- or DAT-labeled dendrites. Some of these terminals formed asymmetric synapses resembling those of cholinergic terminals in the VTA. The majority, however, formed symmetric, inhibitory-type synapses or were apposed without recognized junctions. Our results provide the first ultrastructural evidence that the M2R is expressed, but largely not available for local activation, on the plasma membrane of VTA dopaminergic neurons. Instead, the M2R in this region has a distribution suggesting more indirect regulation of mesocorticolimbic transmission through autoregulation of acetylcholine release and changes in the physiological activity or release of other, largely inhibitory transmitters. These findings could have implications for understanding the muscarinic control of cognitive and goal-directed behaviors within the VTA.

Cellular and subcellular distributions of delta opioid receptor activation sites in the ventral oral pontine tegmentum of the cat.

The ventral division of the reticular oral pontine nucleus (vRPO) is a pontine tegmentum region critically involved in REM sleep generation. Previous reports of morphine microinjections in the cat pontine tegmentum have shown that opioid receptor activation in this region modulates REM sleep. Even though opiate administration has marked effects on sleep-wake cycle architecture, the distribution of opioid receptors in vRPO has only been partially described. Using an antiserum directed against delta opioid receptor (DOR), to which morphine binds, in the present study, we use (1) light microscopy to determine DOR cellular distribution in the rostral pontine tegmentum and (2) electron microscopy to determine DOR subcellular distribution in the cat vRPO. In the dorsal pons, DOR immunoreactivity was evenly distributed throughout the neuropil of the reticular formation and was particularly intense in the parabrachial nuclei and locus coeruleus; the ventral and central areas of the RPO and locus coeruleus complex were especially rich in DOR-labeled somata. Within the vRPO, DOR was localized mainly in the cytoplasm and on plasma membranes of medium to large dendrites (47.8% of DOR-labeled profiles), which received both symmetric and asymmetric synaptic contacts mainly from non-labeled (82% of total inputs) axon terminals. Less frequently, DOR was distributed presynaptically in axon terminals (19% of DOR-labeled profiles). Our results suggest that DOR activation in vRPO regulates REM sleep occurrence by modulating postsynaptic responses to both excitatory and inhibitory afferents. DOR activation in vRPO could have, however, an additional role in direct modulation of neurotransmitter release from axon terminals.

Actinobacillus actinomycetemcomitans lipopolysaccharide stimulates the phosphorylation of p44 and p42 MAP kinases through CD14 and TLR-4 receptor activation in human gingival fibroblasts.

Tyrosine phosphorylation is an early step in lipopolysaccharide (LPS) stimulated monocytes and macrophages that appears to play a key role in signal transduction. We have demonstrated that LPS purified from Actinobacillus actinomycetemcomitans also increases protein tyrosine phosphorylation in human gingival fibroblasts (HGF). This effect was elicited rapidly after LPS stimulation at concentrations that stimulate anti-bacterial responses in human gingival fibroblasts. Two main proteins, with an apparent molecular weight of 44 and 42 kDa, were phosphorylated after LPS stimulation of the human gingival fibroblasts. The phosphorylation was detected after 5 to 15 min and reached the maximum at 30 min of treatment. The increase in tyrosine phosphorylation was apparent following stimulation with LPS at 10 ng/ml and the response was dose dependent up to 10 microg/ml. Pretreatment with the tyrosine kinase inhibitors, herbimycin A and genistein inhibited the LPS-stimulated phosphorylation of p44 and p42 MAP kinases in a dose dependent manner. Pretreatment of human gingival fibroblasts with antibodies anti-CD14 or anti-TLR-4 but not anti-TLR-2 inhibited the LPS-induced tyrosine phosphorylation of p44 and p42. Additionally, LPS-induced p44 and p42 phosphorylation was inhibited by polymyxin treatment. These findings demonstrate that LPS from A. actinomycetemcomintans increases rapidly p44 and p42 phosphorylation (ERK 1 and ERK 2, respectively) in human gingival fibroblasts. Our data also suggest that CD14 and TLR-4 receptors are involved in the LPS effects in human gingival fibroblasts.

Electron microscopic localization of M2-muscarinic receptors in cholinergic and noncholinergic neurons of the laterodorsal tegmental and pedunculopontine nuclei of the rat mesopontine tegmentum.

Muscarinic m2 receptors (M2Rs) are implicated in autoregulatory control of cholinergic output neurons located within the pedunculopontine (PPT) and laterodorsal tegmental (LTD) nuclei of the mesopontine tegmentum (MPT). However, these nuclei contain many noncholinergic neurons in which activation of M2R heteroceptors may contribute significantly to the decisive role of the LTD and PPT in sleep-wakefulness. We examined the electron microscopic dual immunolabeling of M2Rs and the vesicular acetylcholine transporter (VAchT) in the MPT of rat brain to identify the potential sites for M2R activation. M2R immunogold labeling was predominately seen in somatodendritic profiles throughout the PPT/LTD complex. In somata, M2R immunogold particles were often associated with Golgi lamellae and cytoplasmic endomembrannes, but were rarely in contact with the plasma membrane, as was commonly seen in dendrites. Approximately 36% of the M2R-labeled somata and 16% of the more numerous M2R-labeled dendrites coexpressed VAchT. M2R and M2R/VAchT-labeled dendritic profiles received synapses from inhibitory- and excitatory-type axon terminals, over 88% of which were unlabeled and others contained exclusively M2R or VAchT immunoreactivity. In axonal profiles M2R immunogold was localized to plasmalemmal and cytoplasmic regions and showed a similar distribution in many VAchT-negative glial profiles. These results provide ultrastructural evidence suggestive of somatic endomembrane trafficking of M2Rs, whose activation serves to regulate the postsynaptic excitatory and inhibitory responses in dendrites of cholinergic and noncholinergic neurons in the MPT. They also suggest the possibility that M2Rs in this brain region mediate the effects of acetylcholine on the release of other neurotransmitters and on glial signaling. J. Comp. Neurol. 524:3084-3103, 2016. © 2016 Wiley Periodicals, Inc.

The disconnected brain stem does not support rapid eye movement sleep rebound following selective deprivation.

STUDY OBJECTIVES: To determine whether the brain stem can independently support the processes of rapid eye movement sleep rebound and pressure that follow deprivation. DESIGN: Cats with a brain-stem separation from the forebrain were compared to intact subjects on their response to rapid eye movement sleep deprivation. PARTICIPANTS: Eight adult mongrel cats of both sexes. INTERVENTIONS: All cats had electrodes implanted for polygraphic recordings, and 4 subjects sustained a mesencephalic transection. Weeks later, a 24-hour undisturbed sleep-wakefulness recording session was performed, and the next day, a similar session started with a 6-hour deprivation period, which was followed by 18 hours of undisturbed sleep. MEASUREMENTS AND RESULTS: Deprivation produced 90.1% and 87.8 % losses of rapid eye movement sleep time in intact and decerebrate cats, respectively. However, no significant changes in non-rapid eye movement sleep, drowsiness, or waking time percentages were seen in either group of animals when comparing the 6-hour time blocks of the deprivation and baseline sessions, indicating selective rapid eye movement sleep deprivation. During the 6-hour block following deprivation, rapid eye movement sleep time increased a significant 34.6% in intact cats while, in contrast, there was no rapid eye movement sleep rebound in decerebrate animals. The number of aborted episodes of rapid eye movement sleep during deprivation exceeded the number of episodes during the same period of the baseline day by 3 and 5 folds in intact and decerebrate cats, respectively, indicating an increase in rapid eye movement sleep pressure. CONCLUSIONS: Rebound in rapid eye movement sleep after deprivation cannot be sustained by the brain stem alone; in contrast, rapid eye movement sleep pressure persisted in the decerebrate cat, demonstrating that this process does not depend on descending forebrain influences. This indicates that rebound and pressure are 2 different components of the recovery process after rapid eye movement sleep deprivation and that, as such, are likely controlled by different mechanisms.