Validation of DREADD agonists and administration route in a murine model of sleep enhancement.

BACKGROUND: Chemogenetics is a powerful tool to study the role of specific neuronal populations in physiology and diseases. Of particular interest, in mice, acute and specific activation of parafacial zone (PZ) GABAergic neurons expressing the Designer Receptors Activated by Designer Drugs (DREADD) hM3Dq (PZGABA-hM3Dq) enhances slow-wave-sleep (SWS), and this effect lasts for up to 6 h, allowing prolonged and detailed study of SWS. However, the most widely used DREADDs ligand, clozapine N-oxide (CNO), is metabolized into clozapine which has the potential of inducing non-specific effects. In addition, CNO is usually injected intraperitoneally (IP) in mice, limiting the number and frequency of repeated administration. NEW METHODS: The present study is designed to validate the use of alternative DREADDs ligands-deschloroclozapine (DCZ) and compound 21 (C21)-and a new administration route, the voluntary oral administration. RESULTS: We show that IP injections of DCZ and C21 dose-dependently enhance SWS in PZGABA-hM3Dq mice, similar to CNO. We also show that oral administration of CNO, DCZ and C21 induces the same sleep phenotype as compared with IP injection. COMPARISON WITH EXISTING METHODS AND CONCLUSION: Therefore, DCZ and C21 are powerful alternatives to the use of CNO. Moreover, the voluntary oral administration is suitable for repeated dosing of DREADDs ligands.

Two novel mouse models of slow-wave-sleep enhancement in aging and Alzheimer's disease.

Aging and Alzheimer's disease (AD) are both associated with reduced quantity and quality of the deepest stage of sleep, called slow-wave-sleep (SWS). Slow-wave-sleep deficits have been shown to worsen AD symptoms and prevent healthy aging. However, the mechanism remains poorly understood due to the lack of animal models in which SWS can be specifically manipulated. Notably, a mouse model of SWS enhancement has been recently developed in adult mice. As a prelude to studies assessing the impact of SWS enhancement on aging and neurodegeneration, we first asked whether SWS can be enhanced in animal models of aging and AD. The chemogenetic receptor hM3Dq was conditionally expressed in GABAergic neurons of the parafacial zone of aged mice and AD (APP/PS1) mouse model. Sleep-wake phenotypes were analyzed in baseline condition and following clozapine-N-oxide (CNO) and vehicle injections. Both aged and AD mice display deficits in sleep quality, characterized by decreased slow wave activity. Both aged and AD mice show SWS enhancement following CNO injection, characterized by a shorter SWS latency, increased SWS amount and consolidation, and enhanced slow wave activity, compared with vehicle injection. Importantly, the SWS enhancement phenotypes in aged and APP/PS1 model mice are comparable to those seen in adult and littermate wild-type mice, respectively. These mouse models will allow investigation of the role of SWS in aging and AD, using, for the first time, gain-of SWS experiments.

Paternal exposure to morphine during adolescence potentiates morphine withdrawal in male offspring: Involvement of the lateral paragigantocellularis nucleus.

BACKGROUND: Opiate exposure during adolescence perturbs the brain's maturation process and potentially confers long-term adverse consequences, not only in exposed individuals but also in their posterity. Here, we investigate the outcomes of adolescent paternal morphine exposure on morphine withdrawal profile in male offspring. METHODS: Male Wistar rats were chronically subjected to 10 days of an escalating regimen of morphine during adolescence. After a 20-day washout period, adult males were allowed to copulate with naïve females. The adult male offspring were tested for somatic and affective components of naloxone-precipitated morphine withdrawal using conditioned place aversion. Moreover, electrical activity of the lateral paragigantocellularis (LPGi) nucleus, which is involved in development of opiate dependence, was recorded in response to a challenge dose of morphine via extracellular single-unit recordings. RESULTS: Morphine-sired offspring exhibited augmented expression of naloxone-induced somatic and affective signs of opiate withdrawal compared to the control saline-sired counterparts. In vivo recording revealed that LPGi neurons displayed heterogeneous responses (inhibitory, excitatory, and no change) to acute morphine administration in both morphine- and saline-sired animals. The morphine-induced discharge inhibition was potentiated in morphine-sired offspring. However, the extent of discharge excitation in response to morphine did not reach significance in these subjects. Moreover, the lack of alteration in maternal behavior toward morphine-sired offspring indicates that this is due to germline-dependent transmission of epigenetic traits across generations. CONCLUSIONS: Preconception paternal exposure to morphine during adolescence potentiates opiate withdrawal signs in male offspring which is mediated, at least in part, by epigenetic alteration of LPGi-related brain circuitry.

The neuroanatomy and neurochemistry of sleep-wake control.

Sleep-wake control is dependent upon multiple brain areas widely distributed throughout the neural axis. Historically, the monoaminergic and cholinergic neurons of the ascending arousal system were the first to be discovered, and it was only relatively recently that GABAergic and glutamatergic wake- and sleep-promoting populations have been identified. Contemporary advances in molecular-genetic tools have revealed both the complexity and heterogeneity of GABAergic NREM sleep-promoting neurons as well as REM sleep-regulating populations in the brainstem such as glutamatergic neurons in the sublaterodorsal nucleus. The sleep-wake cycle progresses from periods of wakefulness to non-rapid eye movement (NREM) sleep and subsequently rapid eye movement (REM) sleep. Each vigilance stage is controlled by multiple neuronal populations, via a complex regulation that is still incompletely understood. In recent years the field has seen a proliferation in the identification and characterization of new neuronal populations involved in sleep-wake control thanks to newer, more powerful molecular genetic tools that are able to reveal neurophysiological functions via selective activation, inhibition and lesion of neuroanatomically defined sub-types of neurons that are widespread in the brain, such as GABAergic and glutamatergic neurons.1,2.

Differential Role of Pontomedullary Glutamatergic Neuronal Populations in Sleep-Wake Control.

Parafacial zone (PZ) GABAergic neurons play a major role in slow-wave-sleep (SWS), also called non-rapid eye movement (NREM) sleep. The PZ also contains glutamatergic neurons expressing the vesicular transporter for glutamate, isoform 2 (Vglut2). We hypothesized that PZ Vglut2-expressing (PZVglut2) neurons are also involved in sleep control, playing a synergistic role with PZ GABAergic neurons. To test this hypothesis, we specifically activated PZVglut2 neurons using the excitatory chemogenetic receptor hM3Dq. Anatomical inspection of the injection sites revealed hM3Dq transfection in PZ, parabrachial nucleus (PB), sublaterodorsal nucleus (SLD) or various combinations of these three brain areas. Consistent with the known wake- and REM sleep-promoting role of PB and SLD, respectively, chemogenetic activation of PBVglut2 or SLDVglut2 resulted in wake or REM sleep enhancement. Chemogenetic activation of PZVglut2 neurons did not affect sleep-wake phenotype during the mouse active period but increased wakefulness and REM sleep, similar to PBVglut2 and SLDVglut2 activation, during the rest period. To definitively confirm the role of PZVglut2 neurons, we used a specific marker for PZVglut2 neurons, Phox2B. Chemogenetic activation of PZPhox2B neurons did not affect sleep-wake phenotype, indicating that PZ glutamatergic neurons are not sufficient to affect sleep-wake cycle. These results indicate that PZ glutamatergic neurons are not involved in sleep-wake control.

Role of orexin type-1 receptors in paragiganto-coerulear modulation of opioid withdrawal and tolerance: A site specific focus.

Orexin-A and -B neuropeptides are exclusively synthesized in hypothalamic neurons. These have been implicated to play critical roles in the expression of various behavioral manifestations such as feeding, arousal, wakefulness, drug dependence and tolerance. Orexin ligands activate orexin type-1 and orexin type-2 receptors each displaying a distinct selectivity and distribution profile. Orexinergic neurons innervate various brain structures among which the locus coeruleus (LC) and the lateral paragigantocellularis (LPGi) nuclei are well established as the two key mediators of opiate dependence and tolerance. Both nuclei express OX1Rs and the LC receives excitatory and inhibitory inputs from LPGi. Interestingly, the expression of opiate withdrawal signs is temporally associated with the enhanced activity of LC neurons. Numerous studies support the involvement of the orexin system in mediating opiate effects via affecting OX1Rs within the LC and LPGi. Extensive research has long been focused on the role of the ventral tegmental area (VTA) as a critical center in mediating orexin effects as well as reward processing and addiction. However, a growing amount of evidence supports the involvement of some other brain nuclei (such as LC and LPGi) in these phenomena. The mutual contribution of these structures has not been previously addressed in the literature. The present review aims to discuss and piece together the recent findings on the role of OX1Rs in modulating opiate withdrawal and tolerance with an emphasis on the involvement of the putative paragiganto-coerulear pathway. We conclude with a discussion about possible mechanisms of orexin actions within this pathway and its interaction with other neurotransmitter/modulator systems.

Mexneurin is a novel precursor of peptides in the central nervous system of rodents.

Endomorphins (EMs) have been proposed as the endogenous ligand agonists of the µ-opioid receptor; however, no propeptide precursor protein for EMs has been identified. Here, to identify the presumed precursor of EMs, we designed an immunoscreening assay using specific affinity-purified rabbit antisera raised against synthetic EMs in a whole-mouse brain cDNA library. Following this approach, we identify a DNA sequence encoding a protein precursor, which we name proMexneurin, that contains three different peptide sequences: Mexneurin-1 (an EM-like peptide), Mexneurin-2, and Mexneurin-3, a peptide which appears to be unrelated to EMs. RT-PCR analysis and in situ hybridization reveal a widespread distribution of proMexneurin mRNA throughout the mouse brain. Both Mexneurin-1 and Mexneurin-3 peptides display biological activities in the mouse CNS.

Targeted genetic manipulations of neuronal subtypes using promoter-specific combinatorial AAVs in wild-type animals.

Techniques to genetically manipulate the activity of defined neuronal subpopulations have been useful in elucidating function, however applicability to translational research beyond transgenic mice is limited. Subtype targeted transgene expression can be achieved using specific promoters, but often currently available promoters are either too large to package into many vectors, in particular adeno-associated virus (AAV), or do not drive expression at levels sufficient to alter behavior. To permit neuron subtype specific gene expression in wildtype animals, we developed a combinatorial AAV targeting system that drives, in combination, subtype specific Cre-recombinase expression with a strong but non-specific Cre-conditional transgene. Using this system we demonstrate that the tyrosine hydroxylase promoter (TH-Cre-AAV) restricted expression of channelrhodopsin-2 (EF1α-DIO-ChR2-EYFP-AAV) to the rat ventral tegmental area (VTA), or an activating DREADD (hSyn-DIO-hM3Dq-mCherry-AAV) to  the  rat  locus  coeruleus  (LC). High expression levels were achieved in both regions. Immunohistochemistry (IHC) showed the majority of ChR2+ neurons (>93%) colocalized with TH in the VTA, and optical stimulation evoked striatal dopamine release. Activation of TH neurons in the LC produced sustained EEG and behavioral arousal. TH-specific hM3Dq expression in the LC was further compared with: (1) a Cre construct driven by a strong but non-specific promoter (non-targeting); and (2) a retrogradely-transported WGA-Cre delivery mechanism (targeting a specific projection). IHC revealed that the area of c-fos activation after CNO treatment in the LC and peri-LC neurons appeared proportional to the resulting increase in wakefulness (non-targeted > targeted > ACC to LC projection restricted). Our dual AAV targeting system effectively overcomes the large size and weak activity barrier prevalent with many subtype specific promoters by functionally separating subtype specificity from promoter strength.

Impaired circadian photosensitivity in mice lacking glutamate transmission from retinal melanopsin cells.

Intrinsically photoreceptive retinal ganglion cells (ipRGCs) contain the photopigment melanopsin and convey retinal light inputs to the circadian system via the retinohypothalamic tract (RHT) projection to the suprachiasmatic nucleus (SCN). The principal neurotransmitter of this projection is glutamate, and ipRGCs use the vesicular glutamate transporter 2 (VGLUT2) to package glutamate into synaptic vesicles. However, these neurons contain other potential neurotransmitters, such as pituitary adenylate cyclase activating polypeptide (PACAP). To test the role of glutamate in mediating ipRGC light inputs into the SCN, we crossed mice in which Cre-recombinase expression is driven by the melanopsin promotor (Opn4(Cre/+)) with mice in which the second exon of VGLUT2 is flanked by loxP sites (VGLUT2(fl/fl)), producing ipRGCs that are unable to package glutamate into synaptic vesicles. Such mice had free-running circadian rhythms that did not entrain to a 12:12 light-dark (12:12 LD) cycle, nor did they show a phase delay after a 45-min light pulse administered at circadian time (CT) 14. A small subset of the mice did appear to entrain to the 12:12 LD cycle with a positive phase angle to lights-off; a similar entrainment pattern could be achieved in free-running mice if they were exposed to a 12:12 LD cycle with light of a greater intensity. Glutamate transmission from the ipRGCs is necessary for normal light entrainment of the SCN at moderate (0.35 W/m(2)) light levels, but residual transmission (possibly by PACAP in ipRGCs or by other RGCs) can weakly entrain animals, particularly at very high (6.53 W/m(2)) light levels, although it may be less effective at suppressing locomotor activity (light masking).

Brief light stimulation during the mouse nocturnal activity phase simultaneously induces a decline in core temperature and locomotor activity followed by EEG-determined sleep.

Light exerts a variety of effects on mammals. Unexpectedly, one of these effects is the cessation of nocturnal locomotion and the induction of behavioral sleep (photosomnolence). Here, we extend the initial observations in several ways, including the fundamental demonstration that core body temperature (T(c)) drops substantially (about 1.5°C) in response to the light stimulation at CT15 or CT18 in a manner suggesting that the change is a direct response to light rather than simply a result of the locomotor suppression. The results show that 1) the decline of locomotion and T(c) begin soon after nocturnal light stimulation; 2) the variability in the magnitude and onset of light-induced locomotor suppression is very large, whereas the variability in T(c) is very small; 3) T(c) recovers from the light-induced decline in advance of the recovery of locomotion; 4) under entrained and freerunning conditions, the daily late afternoon T(c) increase occurs in advance of the corresponding increase in wheel running; and 5) toward the end of the subjective night, the nocturnally elevated T(c) persists longer than does locomotor activity. Finally, EEG measurements confirm light-induced sleep and, when T(c) or locomotion was measured, show their temporal association with sleep onset. Both EEG- and immobility-based sleep detection methods confirm rapid induction of light-induced sleep. The similarities between light-induced loss of locomotion and drop in T(c) suggest a common cause for parallel responses. The photosomnolence response may be contingent upon both the absence of locomotion and a simultaneous low T(c).

Sleep disturbance induces neuroinflammation and impairment of learning and memory.

Hospitalized patients can develop cognitive function decline, the mechanisms of which remain largely to be determined. Sleep disturbance often occurs in hospitalized patients, and neuroinflammation can induce learning and memory impairment. We therefore set out to determine whether sleep disturbance can induce neuroinflammation and impairment of learning and memory in rodents. Five to 6-month-old wild-type C57BL/6J male mice were used in the studies. The mice were placed in rocking cages for 24 h, and two rolling balls were present in each cage. The mice were tested for learning and memory function using the Fear Conditioning Test one and 7 days post-sleep disturbance. Neuroinflammation in the mouse brain tissues was also determined. Of the Fear Conditioning studies at one day and 7 days after sleep disturbance, twenty-four hour sleep disturbance decreased freezing time in the context test, which assesses hippocampus-dependent learning and memory; but not the tone test, which assesses hippocampus-independent learning and memory. Sleep disturbance increased pro-inflammatory cytokine IL-6 levels and induced microglia activation in the mouse hippocampus, but not the cortex. These results suggest that sleep disturbance induces neuroinflammation in the mouse hippocampus, and impairs hippocampus-dependent learning and memory in mice. Pending further studies, these findings suggest that sleep disturbance-induced neuroinflammation and impairment of learning and memory may contribute to the development of cognitive function decline in hospitalized patients.

Blockade of astrocytic glutamate uptake in the prefrontal cortex induces anhedonia.

Major depression is associated with both dysregulated glutamatergic neurotransmission and fewer astrocytes in limbic areas including the prefrontal cortex (PFC). These deficits may be functionally related. Notably, astrocytes regulate glutamate levels by removing glutamate from the synapse via the glutamate transporter (GLT-1). Previously, we demonstrated that central blockade of GLT-1 induces anhedonia and c-Fos expression in the PFC. Given the role of the PFC in regulating mood, we hypothesized that GLT-1 blockade in the PFC alone would be sufficient to induce anhedonia in rats. We microinjected the GLT-1 inhibitor, dihydrokainic acid (DHK), into the PFC and examined the effects on mood using intracranial self-stimulation (ICSS). At lower doses, intra-PFC DHK produced modest increases in ICSS thresholds, reflecting a depressive-like effect. At higher doses, intra-PFC DHK resulted in cessation of responding. We conducted further tests to clarify whether this total cessation of responding was related to an anhedonic state (tested by sucrose intake), a nonspecific result of motor impairment (measured by the tape test), or seizure activity (measured with electroencephalogram (EEG)). The highest dose of DHK increased latency to begin drinking without altering total sucrose intake. Furthermore, neither motor impairment nor evidence of seizure activity was observed in the tape test or EEG recordings. A decrease in reward value followed by complete cessation of ICSS responding suggests an anhedonic-like effect of intra-PFC DHK; a conclusion that was substantiated by an increased latency to begin sucrose drinking. Overall, these results suggest that blockade of astrocytic glutamate uptake in the PFC is sufficient to produce anhedonia, a core symptom of depression.

Locus ceruleus and anterior cingulate cortex sustain wakefulness in a novel environment.

Locus ceruleus (LC) neuronal activity is correlated with the waking state, yet LC lesions produce only minor alterations in daily wakefulness. Here, we report that sustained elevations in neurobehavioral and EEG arousal in response to exposure to an environment with novel stimuli, including social interaction, are prevented by selective chemical lesions of the LC in rats. Similar results are seen when the anterior cingulate cortex (ACC), which receives especially dense LC innervation, is selectively denervated of LC input or is ablated by the cell-specific neurotoxin ibotenic acid. Anterograde tracing combined with tyrosine hydroxylase immunohistochemistry demonstrates ACC terminals in apposition with the distal dendrites of LC neurons. Our data implicate the ACC as both a source of input to the LC as well as one of its targets and suggests that the two structures engage in a dialog that may provide a critical neurobiological substrate for sustained attention.

3-Monoiodothyronamine: the rationale for its action as an endogenous adrenergic-blocking neuromodulator.

The investigations reported here were designed to gain insights into the role of 3-monoiodothyronamine (T1AM) in the brain, where the amine was originally identified and characterized. Extensive deiodinase studies indicated that T1AM was derived from the T4 metabolite, reverse triiodothyronine (revT3), while functional studies provided well-confirmed evidence that T1AM has strong adrenergic-blocking effects. Because a state of adrenergic overactivity prevails when triiodothyronine (T3) concentrations become excessive, the possibility that T3's metabolic partner, revT3, might give rise to an antagonist of those T3 actions was thought to be reasonable. All T1AM studies thus far have required use of pharmacological doses. Therefore we considered that choosing a physiological site of action was a priority and focused on the locus coeruleus (LC), the major noradrenergic control center in the brain. Site-directed injections of T1AM into the LC elicited a significant, dose-dependent neuronal firing rate change in a subset of adrenergic neurons with an EC(50)=2.7 microM, a dose well within the physiological range. Further evidence for its physiological actions came from autoradiographic images obtained following intravenous carrier-free (125)I-labeled T1AM injection. These showed that the amine bound with high affinity to the LC and to other selected brain nuclei, each of which is both an LC target and a known T3 binding site. This new evidence points to a physiological role for T1AM as an endogenous adrenergic-blocking neuromodulator in the central noradrenergic system.

Role of orexin input in the diurnal rhythm of locus coeruleus impulse activity.

Activation of noradrenergic locus coeruleus (LC) neurons promotes wakefulness and behavioral arousal. In rats, LC neurons receive circadian inputs via a circuit that originates in the suprachiasmatic nucleus (SCN) and relays through the dorsomedial hypothalamus (DMH) to LC; this circuit input increases LC activity during the active period. DMH neurons expressing the peptide neurotransmitter orexin/hypocretin are ideally situated to act as a relay between SCN and LC due to their synaptic inputs from SCN and innervation of LC. Here, we examined the hypothesis that orexin is involved in transmitting circadian signals to LC using single-unit recordings of LC neurons in anesthetized rats maintained in 12:12 light-dark housing. We replicated earlier findings from this lab that LC neurons fire significantly faster on average during the active compared to rest periods. Local microinjection of an orexin antagonist, SB-334867-A attenuated the impulse activities of the fastest firing population of LC neurons during the active period. We also found that DMH orexin neurons project preferentially to LC and express a diurnal rhythm of activation that correlates with LC neuronal firing frequency. Therefore, we propose that DMH orexin neurons play a role in modulating the day-night differences of LC impulse activity.

Retrograde suppression of GABAergic currents in a subset of SCN neurons.

Many postsynaptic neurons release a retrograde transmitter that modulates presynaptic neurotransmitter release. In the suprachiasmatic nucleus (SCN), retrograde signaling is suggested by the presence of dendritic dense-core vesicles. Whole-cell voltage-clamp recordings were made from rat SCN neurons to determine whether a retrograde messenger could modulate the activity of afferent gamma-aminobutyric acid (GABA)ergic inputs. The frequency and amplitude of spontaneous GABAergic currents was significantly reduced in a subpopulation of SCN neurons (eight out of 13) following a postsynaptic depolarization. Similarly, a postsynaptic depolarization significantly reduced the amplitude of evoked GABAergic currents during both day and night recordings. A postsynaptic depolarizing pulse eliminated paired-pulse inhibition of GABAergic currents consistent with a presynaptic mechanism. Muscimol-activated currents were not altered by postsynaptic depolarization, demonstrating that the activity of GABA(A) receptors was not altered. Depolarization-induced inhibition of the GABAergic currents was not observed when a Ca2+ chelator was included in the microelectrode. Postsynaptic depolarization significantly increased the Ca2+ concentration in both the soma and dendrites. The dendritic Ca2+ levels increased faster, to a higher concentration and decayed faster than in the soma. The depolarization-induced inhibition of the evoked GABAergic current was blocked by the G-protein uncoupling agent N-ethylmaleimide, suggesting that the retrograde messenger acts on a pertussis toxin-sensitive G-protein-coupled receptor. Because the majority of SCN neurons receive GABAergic input from neighboring cells, these results describe a retrograde signaling mechanism by which SCN neurons can modulate GABAergic synaptic input.

GABAergic synapses of the suprachiasmatic nucleus exhibit a diurnal rhythm of short-term synaptic plasticity.

We examined synaptic plasticity at intrasuprachiasmatic nucleus (SCN) gamma-aminobutyric acid (GABA)ergic synapses by measuring the paired-pulse ratio between pairs of evoked inhibitory postsynaptic currents (IPSCs). Interstimulus intervals were chosen to represent the range of spontaneous action potential firing frequencies found in SCN neurons. A majority of synapses studied during the day exhibited paired-pulse depression (PPD), whereas a majority of synapses studied during the night showed no PPD. Two types of PPD were found. Type 1 PPD expresses the greatest inhibition at shorter interstimulus intervals, is predominant in the early morning and is likely to be a result of vesicle depletion. Type 2 showed the greatest inhibition at interstimulus intervals between 175 and 225 ms, is found throughout the day yet rarely at night and is likely to be a result of a Ca(2+)-dependent mechanism that is independent of pertussis toxin-sensitive G-proteins. Thus, multiple mechanisms of synaptic plasticity modulate intra-SCN communication throughout the diurnal cycle.

Circadian dynamics of cytosolic and nuclear Ca2+ in single suprachiasmatic nucleus neurons.

Intracellular free Ca(2+) regulates diverse cellular processes, including membrane potential, neurotransmitter release, and gene expression. To examine the cellular mechanisms underlying the generation of circadian rhythms, nucleus-targeted and untargeted cDNAs encoding a Ca(2+)-sensitive fluorescent protein (cameleon) were transfected into organotypic cultures of mouse suprachiasmatic nucleus (SCN), the primary circadian pacemaker. Circadian rhythms in cytosolic but not nuclear Ca(2+) concentration were observed in SCN neurons. The cytosolic Ca(2+) rhythm period matched the circadian multiple-unit-activity (MUA)-rhythm period monitored using a multiple-electrode array, with a mean advance in phase of 4 hr. Tetrodotoxin blocked MUA, but not Ca(2+) rhythms, while ryanodine damped both Ca(2+) and MUA rhythms. These results demonstrate cytosolic Ca(2+) rhythms regulated by the release of Ca(2+) from ryanodine-sensitive stores in SCN neurons.