The Role of Glutamatergic and Dopaminergic Neurons in the Periaqueductal Gray/Dorsal Raphe: Separating Analgesia and Anxiety.

The periaqueductal gray (PAG) is a significant modulator of both analgesic and fear behaviors in both humans and rodents, but the underlying circuitry responsible for these two phenotypes is incompletely understood. Importantly, it is not known if there is a way to produce analgesia without anxiety by targeting the PAG, as modulation of glutamate or GABA neurons in this area initiates both antinociceptive and anxiogenic behavior. While dopamine (DA) neurons in the ventrolateral PAG (vlPAG)/dorsal raphe display a supraspinal antinociceptive effect, their influence on anxiety and fear are unknown. Using DAT-cre and Vglut2-cre male mice, we introduced designer receptors exclusively activated by designer drugs (DREADD) to DA and glutamate neurons within the vlPAG using viral-mediated delivery and found that levels of analgesia were significant and quantitatively similar when DA and glutamate neurons were selectively stimulated. Activation of glutamatergic neurons, however, reliably produced higher indices of anxiety, with increased freezing time and more time spent in the safety of a dark enclosure. In contrast, animals in which PAG/dorsal raphe DA neurons were stimulated failed to show fear behaviors. DA-mediated antinociception was inhibitable by haloperidol and was sufficient to prevent persistent inflammatory pain induced by carrageenan. In summary, only activation of DA neurons in the PAG/dorsal raphe produced profound analgesia without signs of anxiety, indicating that PAG/dorsal raphe DA neurons are an important target involved in analgesia that may lead to new treatments for pain.

Improvement in patient-reported sleep in type 2 diabetes and prediabetes participants receiving a continuous care intervention with nutritional ketosis.

OBJECTIVE: Sleep disruption is frequently associated with type 2 diabetes (T2D) and hyperglycemia. We recently reported the effectiveness of a continuous care intervention (CCI) emphasizing nutritional ketosis for improving HbA1c, body weight and cardiovascular risk factors in T2D patients. The present study assessed the effect of this CCI approach on sleep quality using a subjective patient-reported sleep questionnaire. METHODS: A non-randomized, controlled longitudinal study; 262 T2D and 116 prediabetes patients enrolled in the CCI and 87 separately recruited T2D patients continued usual care (UC) treatment. Patients completed the Pittsburgh Sleep Quality Index (PSQI) questionnaire. A PSQI score of >5 (scale 0 to 21) was used to identify poor sleepers. RESULTS: Global sleep quality improved in the CCI T2D (p < 0.001) and prediabetes (p < 0.001) patients after one year of intervention. Subjective sleep quality (component 1), sleep disturbance (component 5) and daytime dysfunction (component 7), also showed improvements in the CCI T2D (p < 0.01 for sleep quality and sleep disturbance; and p < 0.001 for daytime dysfunction) and prediabetes patients (p < 0.001 for all three components); compared to the UC T2D group after one year. The proportion of patients with poor sleep quality was significantly reduced after one year of CCI (T2D; from 68.3% at baseline to 56.5% at one year, p = 0.001 and prediabetes; from 77.9% at baseline to 48.7% at one year, p < 0.001). CONCLUSION: This study demonstrates improved sleep quality as assessed by PSQI in patients with T2D and prediabetes undergoing CCI including nutritional ketosis but not in T2D patients receiving UC. The dietary intervention benefited both sleep quality and the severity of T2D symptoms suggesting that nutritional ketosis improves overall health via multiple mechanisms.

Optogenetics and Chemogenetics.

Optogenetics and chemogenetics provide the ability to modulate neurons in a type- and region-specific manner. These powerful techniques are useful to test hypotheses regarding the neural circuit mechanisms of general anesthetic end points such as hypnosis and analgesia. With both techniques, a genetic strategy is used to target expression of light-sensitive ion channels (opsins) or designer receptors exclusively activated by designer drugs in specific neurons. Optogenetics provides precise temporal control of neuronal firing with light pulses, whereas chemogenetics provides the ability to modulate neuronal firing for several hours with the single administration of a designer drug. This chapter provides an overview of neuronal targeting and experimental strategies and highlights the important advantages and disadvantages of each technique.

Optogenetic activation of 5-HT neurons in the dorsal raphe suppresses seizure-induced respiratory arrest and produces anticonvulsant effect in the DBA/1 mouse SUDEP model.

Sudden unexpected death in epilepsy (SUDEP) is a devastating epilepsy complication. Seizure-induced respiratory arrest (S-IRA) occurs in many witnessed SUDEP patients and animal models as an initiating event leading to death. Thus, understanding the mechanisms underlying S-IRA will advance the development of preventive strategies against SUDEP. Serotonin (5-HT) is an important modulator for many vital functions, including respiration and arousal, and a deficiency of 5-HT signaling is strongly implicated in S-IRA in animal models, including the DBA/1 mouse. However, the brain structures that contribute to S-IRA remain elusive. We hypothesized that the dorsal raphe (DR), which sends 5-HT projections to the forebrain, is implicated in S-IRA. The present study used optogenetics in the DBA/1 mouse model of SUDEP to selectively activate 5-HT neurons in the DR. Photostimulation of DR 5-HT neurons significantly and reversibly reduced the incidence of S-IRA evoked by acoustic stimulation. Activation of 5-HT neurons in the DR suppressed tonic seizures in most DBA/1 mice without altering the seizure latency and duration of wild running and clonic seizures evoked by acoustic stimulation. This suppressant effect of photostimulation on S-IRA is independent of seizure models, as optogenetic stimulation of DR also reduced S-IRA induced by pentylenetetrazole, a proconvulsant widely used to model human generalized seizures. The S-IRA-suppressing effect of photostimulation was increased by 5-hydroxytryptophan, a chemical precursor for 5-HT synthesis, and was reversed by ondansetron, a specific 5-HT3 receptor antagonist, indicating that reduction of S-IRA by photostimulation of the DR is specifically mediated by enhanced 5-HT neurotransmission. Our findings suggest that deficits in 5-HT neurotransmission in the DR are implicated in S-IRA in DBA/1 mice, and that targeted intervention in the DR is potentially useful for prevention of SUDEP.

Optogenetic activation of dopamine neurons in the ventral tegmental area induces reanimation from general anesthesia.

Dopamine (DA) promotes wakefulness, and DA transporter inhibitors such as dextroamphetamine and methylphenidate are effective for increasing arousal and inducing reanimation, or active emergence from general anesthesia. DA neurons in the ventral tegmental area (VTA) are involved in reward processing, motivation, emotion, reinforcement, and cognition, but their role in regulating wakefulness is less clear. The current study was performed to test the hypothesis that selective optogenetic activation of VTA DA neurons is sufficient to induce arousal from an unconscious, anesthetized state. Floxed-inverse (FLEX)-Channelrhodopsin2 (ChR2) expression was targeted to VTA DA neurons in DA transporter (DAT)-cre mice (ChR2+ group; n = 6). Optical VTA stimulation in ChR2+ mice during continuous, steady-state general anesthesia (CSSGA) with isoflurane produced behavioral and EEG evidence of arousal and restored the righting reflex in 6/6 mice. Pretreatment with the D1 receptor antagonist SCH-23390 before optical VTA stimulation inhibited the arousal responses and restoration of righting in 6/6 ChR2+ mice. In control DAT-cre mice, the VTA was targeted with a viral vector lacking the ChR2 gene (ChR2- group; n = 5). VTA optical stimulation in ChR2- mice did not restore righting or produce EEG changes during isoflurane CSSGA in 5/5 mice. These results provide compelling evidence that selective stimulation of VTA DA neurons is sufficient to induce the transition from an anesthetized, unconscious state to an awake state, suggesting critical involvement in behavioral arousal.

Electrical stimulation of the parabrachial nucleus induces reanimation from isoflurane general anesthesia.

Clinically, emergence from general anesthesia is viewed as a passive process where anesthetics are discontinued at the end of surgery and anesthesiologists wait for the drugs to wear off. The mechanisms involved in emergence are not well understood and there are currently no drugs that can actively reverse the state of general anesthesia. An emerging hypothesis states that brain regions that control arousal become active during emergence and are a key part of the return to wakefulness. In this study, we tested the hypothesis that electrical activation of the glutamatergic parabrachial nucleus (PBN) in the brainstem is sufficient to induce reanimation (active emergence) during continuous isoflurane general anesthesia. Using c-Fos immunohistochemistry as a marker of neural activity, we first show a selective increase in active neurons in the PBN during passive emergence from isoflurane anesthesia. We then electrically stimulated the PBN to assess whether it is sufficient to induce reanimation from isoflurane general anesthesia. Stimulation induced behavioral arousal and restoration of the righting reflex during continuous isoflurane general anesthesia. In contrast, stimulation of the nearby central inferior colliculus (CIC) did not restore the righting reflex. Spectral analysis of the electroencephalogram (EEG) revealed that stimulation produced a significant decrease in EEG delta power during PBN stimulation. The results are consistent with the hypothesis that the PBN provides critical arousal input during emergence from isoflurane anesthesia.

Physostigmine and Methylphenidate Induce Distinct Arousal States During Isoflurane General Anesthesia in Rats.

BACKGROUND: Although emergence from general anesthesia is clinically treated as a passive process driven by the pharmacokinetics of drug clearance, agents that hasten recovery from general anesthesia may be useful for treating delayed emergence, emergence delirium, and postoperative cognitive dysfunction. Activation of central monoaminergic neurotransmission with methylphenidate has been shown to induce reanimation (active emergence) from general anesthesia. Cholinergic neurons in the brainstem and basal forebrain are also known to promote arousal. The objective of this study was to test the hypothesis that physostigmine, a centrally acting cholinesterase inhibitor, induces reanimation from isoflurane anesthesia in adult rats. METHODS: The dose-dependent effects of physostigmine on time to emergence from a standardized isoflurane general anesthetic were tested. It was then determined whether physostigmine restores righting during continuous isoflurane anesthesia. In a separate group of rats with implanted extradural electrodes, physostigmine was administered during continuous inhalation of 1.0% isoflurane, and the electroencephalogram changes were recorded. Finally, 2.0% isoflurane was used to induce burst suppression, and the effects of physostigmine and methylphenidate on burst suppression probability (BSP) were tested. RESULTS: Physostigmine delayed time to emergence from isoflurane anesthesia at doses ≥0.2 mg/kg (n = 9). During continuous isoflurane anesthesia (0.9% ± 0.1%), physostigmine did not restore righting (n = 9). Blocking the peripheral side effects of physostigmine with the coadministration of glycopyrrolate (a muscarinic antagonist that does not cross the blood-brain barrier) produced similar results (n = 9 each). However, during inhalation of 1.0% isoflurane, physostigmine shifted peak electroencephalogram power from δ (<4 Hz) to θ (4-8 Hz) in 6 of 6 rats. During continuous 2.0% isoflurane anesthesia, physostigmine induced large, statistically significant decreases in BSP in 6 of 6 rats, whereas methylphenidate did not. CONCLUSIONS: Unlike methylphenidate, physostigmine does not accelerate time to emergence from isoflurane anesthesia and does not restore righting during continuous isoflurane anesthesia. However, physostigmine consistently decreases BSP during deep isoflurane anesthesia, whereas methylphenidate does not. These findings suggest that activation of cholinergic neurotransmission during isoflurane anesthesia produces arousal states that are distinct from those induced by monoaminergic activation.

Optogenetic activation of cholinergic neurons in the PPT or LDT induces REM sleep.

Rapid eye movement (REM) sleep is an important component of the natural sleep/wake cycle, yet the mechanisms that regulate REM sleep remain incompletely understood. Cholinergic neurons in the mesopontine tegmentum have been implicated in REM sleep regulation, but lesions of this area have had varying effects on REM sleep. Therefore, this study aimed to clarify the role of cholinergic neurons in the pedunculopontine tegmentum (PPT) and laterodorsal tegmentum (LDT) in REM sleep generation. Selective optogenetic activation of cholinergic neurons in the PPT or LDT during non-REM (NREM) sleep increased the number of REM sleep episodes and did not change REM sleep episode duration. Activation of cholinergic neurons in the PPT or LDT during NREM sleep was sufficient to induce REM sleep.

Electrical stimulation of the ventral tegmental area induces reanimation from general anesthesia.

BACKGROUND: Methylphenidate or a D1 dopamine receptor agonist induces reanimation (active emergence) from general anesthesia. The authors tested whether electrical stimulation of dopaminergic nuclei also induces reanimation from general anesthesia. METHODS: In adult rats, a bipolar insulated stainless steel electrode was placed in the ventral tegmental area (VTA, n = 5) or substantia nigra (n = 5). After a minimum 7-day recovery period, the isoflurane dose sufficient to maintain loss of righting was established. Electrical stimulation was initiated and increased in intensity every 3 min to a maximum of 120 µA. If stimulation restored the righting reflex, an additional experiment was performed at least 3 days later during continuous propofol anesthesia. Histological analysis was conducted to identify the location of the electrode tip. In separate experiments, stimulation was performed in the prone position during general anesthesia with isoflurane or propofol, and the electroencephalogram was recorded. RESULTS: To maintain loss of righting, the dose of isoflurane was 0.9% ± 0.1 vol%, and the target plasma dose of propofol was 4.4 ± 1.1 µg/ml (mean ± SD). In all rats with VTA electrodes, electrical stimulation induced a graded arousal response including righting that increased with current intensity. VTA stimulation induced a shift in electroencephalogram peak power from δ (<4 Hz) to θ (4-8 Hz). In all rats with substantia nigra electrodes, stimulation did not elicit an arousal response or significant electroencephalogram changes. CONCLUSIONS: Electrical stimulation of the VTA, but not the substantia nigra, induces reanimation during general anesthesia with isoflurane or propofol. These results are consistent with the hypothesis that dopamine release by VTA neurons, but not substantia nigra neurons, induces reanimation from general anesthesia.

Active emergence from propofol general anesthesia is induced by methylphenidate.

BACKGROUND: A recent study showed that methylphenidate induces emergence from isoflurane general anesthesia. Isoflurane and propofol are general anesthetics that may have distinct molecular mechanisms of action. The objective of this study was to test the hypothesis that methylphenidate actively induces emergence from propofol general anesthesia. METHODS: Using adult rats, the effect of methylphenidate on time to emergence after a single bolus of propofol was determined. The ability of methylphenidate to restore righting during a continuous target-controlled infusion (TCI) of propofol was also tested. In a separate group of rats, a TCI of propofol was established and spectral analysis was performed on electroencephalogram recordings taken before and after methylphenidate administration. RESULTS: Methylphenidate decreased median time to emergence after a single dose of propofol from 735 s (95% CI: 598-897 s, n = 6) to 448 s (95% CI: 371-495 s, n = 6). The difference was statistically significant (P = 0.0051). During continuous propofol anesthesia with a median final target plasma concentration of 4.0 µg/ml (95% CI: 3.2-4.6, n = 6), none of the rats exhibited purposeful movements after injection of normal saline. After methylphenidate, however, all six rats promptly exhibited arousal and had restoration of righting with a median time of 82 s (95% CI: 30-166 s). Spectral analysis of electroencephalogram data demonstrated a shift in peak power from δ (less than 4 Hz) to θ (4-8 Hz) and ß (12-30 Hz) after administration of methylphenidate, indicating arousal in 4/4 rats. CONCLUSIONS: Methylphenidate decreases time to emergence after a single dose of propofol, and induces emergence during continuous propofol anesthesia in rats. Further study is warranted to test the hypothesis that methylphenidate induces emergence from propofol general anesthesia in humans.

General anesthesia and altered states of arousal: a systems neuroscience analysis.

Placing a patient in a state of general anesthesia is crucial for safely and humanely performing most surgical and many nonsurgical procedures. How anesthetic drugs create the state of general anesthesia is considered a major mystery of modern medicine. Unconsciousness, induced by altered arousal and/or cognition, is perhaps the most fascinating behavioral state of general anesthesia. We perform a systems neuroscience analysis of the altered arousal states induced by five classes of intravenous anesthetics by relating their behavioral and physiological features to the molecular targets and neural circuits at which these drugs are purported to act. The altered states of arousal are sedation-unconsciousness, sedation-analgesia, dissociative anesthesia, pharmacologic non-REM sleep, and neuroleptic anesthesia. Each altered arousal state results from the anesthetic drugs acting at multiple targets in the central nervous system. Our analysis shows that general anesthesia is less mysterious than currently believed.

Adenosine A(1) and A(2A) receptors in mouse prefrontal cortex modulate acetylcholine release and behavioral arousal.

During prolonged intervals of wakefulness, brain adenosine levels rise within the basal forebrain and cortex. The view that adenosine promotes sleep is supported by the corollary that N-methylated xanthines such as caffeine increase brain and behavioral arousal by blocking adenosine receptors. The four subtypes of adenosine receptors are distributed heterogeneously throughout the brain, yet the neurotransmitter systems and brain regions through which adenosine receptor blockade causes arousal are incompletely understood. This study tested the hypothesis that adenosine A(1) and A(2A) receptors in the prefrontal cortex contribute to the regulation of behavioral and cortical arousal. Dependent measures included acetylcholine (ACh) release in the prefrontal cortex, cortical electroencephalographic (EEG) power, and time to waking after anesthesia. Sleep and wakefulness were also quantified after microinjecting an adenosine A(1) receptor antagonist into the prefrontal cortex. The results showed that adenosine A(1) and A(2A) receptors in the prefrontal cortex modulate cortical ACh release, behavioral arousal, EEG delta power, and sleep. Additional dual microdialysis studies revealed that ACh release in the pontine reticular formation is significantly altered by dialysis delivery of adenosine receptor agonists and antagonists to the prefrontal cortex. These data, and early brain transection studies demonstrating that the forebrain is not needed for sleep cycle generation, suggest that the prefrontal cortex modulates EEG and behavioral arousal via descending input to the pontine brainstem. The results provide novel evidence that adenosine A(1) receptors within the prefrontal cortex comprise part of a descending system that inhibits wakefulness.

T47D breast cancer cell growth is inhibited by expression of VACM-1, a cul-5 gene.

Vasopressin-activated calcium-mobilizing (VACM-1), a cul-5 gene, is localized on chromosome 11q22-23 close to the gene for Ataxia Telangiectasia in a region associated with a loss of heterozygosity in breast cancer tumor samples. To examine the biological role of VACM-1, we studied the effect of VACM-1 expression on cellular growth and gene expression in T47D breast cancer cells. Immunocytochemistry studies demonstrated that VACM-1 was expressed in 0.6-6% of the T47D cells and localized to the nucleus of mitotic cells. Overexpressing VACM-1 significantly attenuated cellular proliferation and MAPK phosphorylation when compared to the control cells. In addition, VACM-1 decreased egr-1 and increased Fas-L mRNA levels. Further, egr-1 protein levels were significantly lower in the nuclear fraction from VACM-1 transfected cells when compared to controls. These data indicate that VACM-1 is involved in the regulation of cellular growth.