Oral Fentanyl Consumption Alters Sleep Rhythms, Promotes Avoidance Behaviors, Impairs Fear Extinction Learning, and Alters Basolateral Amygdala Physiology in Male and Female Mice.

The number of opioid overdose deaths has increased over the past several years, mainly driven by an increase in the availability of highly potent synthetic opioids, like fentanyl, in the illicit drug supply. While many previous studies on fentanyl and other opioids have focused on intravenous administration, other routes of administration remain relatively understudied. Here, we used a drinking in the dark (DiD) paradigm to model oral fentanyl self-administration using increasing fentanyl concentrations in male and female mice over 5 weeks. Fentanyl consumption peaked in both female and male mice at the 30 µg/mL dose, with female mice consuming significantly more fentanyl than male mice. Mice consumed sufficient fentanyl such that withdrawal was precipitated with naloxone, with males having more severe withdrawal symptoms, despite lower pharmacological exposure. Fentanyl consumption disrupted normal sleep rhythms in both male and female mice. We also performed behavioral assays to measure avoidance behavior and reward-seeking during fentanyl abstinence. Female mice displayed more avoidance behaviors in the open field assay, whereas male mice showed evidence of these behaviors in the light/dark box assay. Female mice also exhibited increased reward-seeking in the sucrose preference test. Fentanyl-consuming mice of both sexes showed impaired cued fear extinction learning following fear conditioning and increased excitatory synaptic drive and increased excitability of BLA principal neurons. Our experiments demonstrate that long-term oral fentanyl consumption results in wide-ranging physiological and behavioral disruptions. This model could be useful to further study fentanyl withdrawal syndrome, fentanyl seeking, and behaviors associated with protracted fentanyl withdrawal.

Striatal Subregion-selective Dysregulated Dopamine Receptor-mediated Intracellular Signaling in a Model of DOPA-responsive Dystonia.

Although the mechanisms underlying dystonia are largely unknown, dystonia is often associated with abnormal dopamine neurotransmission. DOPA-responsive dystonia (DRD) is a prototype disorder for understanding dopamine dysfunction in dystonia because it is caused by mutations in genes necessary for the synthesis of dopamine and alleviated by the indirect-acting dopamine agonist l-DOPA. Although adaptations in striatal dopamine receptor-mediated intracellular signaling have been studied extensively in models of Parkinson's disease, another movement disorders associated with dopamine deficiency, little is known about dopaminergic adaptations in dystonia. To identify the dopamine receptor-mediated intracellular signaling associated with dystonia, we used immunohistochemistry to quantify striatal protein kinase A activity and extracellular signal-related kinase (ERK) phosphorylation after dopaminergic challenges in a knockin mouse model of DRD. l-DOPA treatment induced the phosphorylation of both protein kinase A substrates and ERK largely in D1 dopamine receptor-expressing striatal neurons. As expected, this response was blocked by pretreatment with the D1 dopamine receptor antagonist SCH23390. The D2 dopamine receptor antagonist raclopride also significantly reduced the phosphorylation of ERK; this contrasts with models of parkinsonism in which l-DOPA-induced ERK phosphorylation is not mediated by D2 dopamine receptors. Further, the dysregulated signaling was dependent on striatal subdomains whereby ERK phosphorylation was largely confined to dorsomedial (associative) striatum while the dorsolateral (sensorimotor) striatum was unresponsive. This complex interaction between striatal functional domains and dysregulated dopamine-receptor mediated responses has not been observed in other models of dopamine deficiency, such as parkinsonism, suggesting that regional variation in dopamine-mediated neurotransmission may be a hallmark of dystonia.

Chronic Ethanol Exposure Modulates Periaqueductal Gray to Extended Amygdala Dopamine Circuit.

The bed nucleus of the stria terminalis (BNST) is a component of the extended amygdala that regulates motivated behavior and affective states and plays an integral role in the development of alcohol-use disorder (AUD). The dorsal subdivision of the BNST (dBNST) receives dense dopaminergic input from the ventrolateral periaqueductal gray (vlPAG)/dorsal raphe (DR). To date, no studies have examined the effects of chronic alcohol on this circuit. Here, we used chronic intermittent ethanol exposure (CIE), a well-established rodent model of AUD, to functionally interrogate the vlPAG/DR-BNST dopamine (DA) circuit during acute withdrawal. We selectively targeted vlPAG/DRDA neurons in tyrosine hydroxylase-expressing transgenic adult male mice. Using ex vivo electrophysiology, we found hyperexcitability of vlPAG/DRDA neurons in CIE-treated mice. Further, using optogenetic approaches to target vlPAG/DRDA terminals in the dBNST, we revealed a CIE-mediated shift in the vlPAG/DR-driven excitatory-inhibitory (E/I) ratio to a hyperexcitable state in dBNST. Additionally, to quantify the effect of CIE on endogenous DA signaling, we coupled optogenetics with fast-scan cyclic voltammetry to measure pathway-specific DA release in dBNST. CIE-treated mice had significantly reduced signal half-life, suggestive of faster clearance of DA signaling. CIE treatment also altered the ratio of vlPAG/DRDA-driven cellular inhibition and excitation of a subset of dBNST neurons. Overall, our findings suggest a dysregulation of vlPAG/DR to BNST dopamine circuit, which may contribute to pathophysiological phenotypes associated with AUD.SIGNIFICANCE STATEMENT The dorsal bed nucleus of the stria terminalis (dBNST) is highly implicated in the pathophysiology of alcohol-use disorder and receives dopaminergic inputs from ventrolateral periaqueductal gray/dorsal raphe regions (vlPAG/DR). The present study highlights the plasticity within the vlPAG/DR to dBNST dopamine (DA) circuit during acute withdrawal from chronic ethanol exposure. More specifically, our data reveal that chronic ethanol strengthens vlPAG/DR-dBNST glutamatergic transmission while altering both DA transmission and dopamine-mediated cellular inhibition of dBNST neurons. The net result is a shift toward a hyperexcitable state in dBNST activity. Together, our findings suggest chronic ethanol may promote withdrawal-related plasticity by dysregulating the vlPAG/DR-dBNST DA circuit.

Tauopathy and alcohol consumption interact to alter locus coeruleus excitatory transmission and excitability in male and female mice.

Alcohol use disorder is a major public health concern in the United States. Recent work has suggested a link between chronic alcohol consumption and the development of tauopathy disorders, such as Alzheimer's disease and frontotemporal dementia. However, relatively little work has investigated changes in neural circuitry involved in both tauopathy disorders and alcohol use disorder. The locus coeruleus (LC) is the major noradrenergic nucleus in the brain and is one of the earliest sites to be affected by tau lesions. The LC is also implicated in the rewarding effects of ethanol and alcohol withdrawal. In this study we assessed effects of long-term ethanol consumption and tauopathy on the physiology of LC neurons. Male and female P301S mice, a humanized transgenic mouse model of tauopathy, underwent 16 weeks of intermittent access to 20% ethanol from 3 to 7 months of age. We observed higher total alcohol consumption in female mice regardless of genotype. Male P301S mice consumed more ethanol and had a greater preference for ethanol than wild-type (WT) males. At the end of the drinking study, LC function was assessed using ex vivo whole cell electrophysiology. We found significant changes in excitatory inputs to the LC due to both ethanol and genotype. We found significantly increased excitability of the LC due to ethanol with greater effects in female P301S mice than in female WT mice. Our study identifies significant changes in the LC due to interactions between tauopathy and long-term ethanol use. These findings could have important implications regarding LC activity and changes in behavior due to both ethanol- and tauopathy-related dementia.

Blockade of M4 muscarinic receptors on striatal cholinergic interneurons normalizes striatal dopamine release in a mouse model of TOR1A dystonia.

Trihexyphenidyl (THP), a non-selective muscarinic receptor (mAChR) antagonist, is commonly used for the treatment of dystonia associated with TOR1A, otherwise known as DYT1 dystonia. A better understanding of the mechanism of action of THP is a critical step in the development of better therapeutics with fewer side effects. We previously found that THP normalizes the deficit in striatal dopamine (DA) release in a mouse model of TOR1A dystonia (Tor1a+/ΔE knockin (KI) mice), revealing a plausible mechanism of action for this compound, considering that abnormal DA neurotransmission is consistently associated with many forms of dystonia. However, the mAChR subtype(s) that mediate the rescue of striatal dopamine release remain unclear. In this study we used a combination of pharmacological challenges and cell-type specific mAChR conditional knockout mice of either sex to determine which mAChR subtypes mediate the DA release-enhancing effects of THP. We determined that THP acts in part at M4 mAChR on striatal cholinergic interneurons to enhance DA release in both Tor1a+/+ and Tor1a+/ΔE KI mice. Further, we found that the subtype selective M4 antagonist VU6021625 recapitulates the effects of THP. These data implicate a principal role for M4 mAChR located on striatal cholinergic interneurons in the mechanism of action of THP and suggest that subtype selective M4 mAChR antagonists may be effective therapeutics with fewer side effects than THP for the treatment of TOR1A dystonia.

Noradrenergic circuits and signaling in substance use disorders.

The central noradrenergic system innervates almost all regions of the brain and, as such, is well positioned to modulate many neural circuits implicated in behaviors and physiology underlying substance use disorders. Ample pharmacological evidence demonstrates that α1, α2, and ß adrenergic receptors may serve as therapeutic targets to reduce drug -seeking behavior and drug withdrawal symptoms. Further, norepinephrine is a key modulator of the stress response, and stress has been heavily implicated in reinstatement of drug taking. In this review, we discuss recent advances in our understanding of noradrenergic circuitry and noradrenergic receptor signaling in the context of opioid, alcohol, and psychostimulant use disorders.

Differential expression of striatal proteins in a mouse model of DOPA-responsive dystonia reveals shared mechanisms among dystonic disorders.

Dystonia is characterized by involuntary muscle contractions that cause debilitating twisting movements and postures. Although dysfunction of the basal ganglia, a brain region that mediates movement, is implicated in many forms of dystonia, the underlying mechanisms are unclear. The inherited metabolic disorder DOPA-responsive dystonia is considered a prototype for understanding basal ganglia dysfunction in dystonia because it is caused by mutations in genes necessary for the synthesis of the neurotransmitter dopamine, which mediates the activity of the basal ganglia. Therefore, to reveal abnormal striatal cellular processes and pathways implicated in dystonia, we used an unbiased proteomic approach in a knockin mouse model of DOPA-responsive dystonia, a model in which the striatum is known to play a central role in the expression of dystonia. Fifty-seven of the 1805 proteins identified were differentially regulated in DOPA-responsive dystonia mice compared to control mice. Most differentially regulated proteins were associated with gene ontology terms that implicated either mitochondrial or synaptic dysfunction whereby proteins associated with mitochondrial function were generally over-represented and proteins associated with synaptic function were largely under-represented. Remarkably, nearly 20% of the differentially regulated striatal proteins identified in our screen are associated with pathogenic variants that cause inherited disorders with dystonia as a sign in humans suggesting shared mechanisms across many different forms of dystonia.

Cell-intrinsic effects of TorsinA(ΔE) disrupt dopamine release in a mouse model of TOR1A dystonia.

TOR1A-associated dystonia, otherwise known as DYT1 dystonia, is an inherited dystonia caused by a three base-pair deletion in the TOR1A gene (TOR1AΔE). Although the mechanisms underlying the dystonic movements are largely unknown, abnormalities in striatal dopamine and acetylcholine neurotransmission are consistently implicated whereby dopamine release is reduced while cholinergic tone is increased. Because striatal cholinergic neurotransmission mediates dopamine release, it is not known if the dopamine release deficit is mediated indirectly by abnormal acetylcholine neurotransmission or if Tor1a(ΔE) acts directly within dopaminergic neurons to attenuate release. To dissect the microcircuit that governs the deficit in dopamine release, we conditionally expressed Tor1a(ΔE) in either dopamine neurons or cholinergic interneurons in mice and assessed striatal dopamine release using ex vivo fast scan cyclic voltammetry or dopamine efflux using in vivo microdialysis. Conditional expression of Tor1a(ΔE) in cholinergic neurons did not affect striatal dopamine release. In contrast, conditional expression of Tor1a(ΔE) in dopamine neurons reduced dopamine release to 50% of normal, which is comparable to the deficit in Tor1a+/ΔE knockin mice that express the mutation ubiquitously. Despite the deficit in dopamine release, we found that the Tor1a(ΔE) mutation does not cause obvious nerve terminal dysfunction as other presynaptic mechanisms, including electrical excitability, vesicle recycling/refilling, Ca2+ signaling, D2 dopamine autoreceptor function and GABAB receptor function, are intact. Although the mechanistic link between Tor1a(ΔE) and dopamine release is unclear, these results clearly demonstrate that the defect in dopamine release is caused by the action of the Tor1a(ΔE) mutation within dopamine neurons.

Cholinergic leukocytes in sepsis and at the neuroimmune junction in the spleen.

The spleen is a key participant in the pathophysiology of sepsis and inflammatory disease. Many splenocytes exhibit a cholinergic phenotype, but our knowledge regarding their cholinergic biology and how they are affected by sepsis is incomplete. We evaluated effects of acute sepsis on the spleen using the cecal ligation and puncture (CLP) model in C57BL/6 and ChATBAC-eGFP mice. Quantification of cholinergic gene expression showed that choline acetyltransferase and vesicular acetylcholine transporter (VAChT) are present and that VAChT is upregulated in sepsis, suggesting increased capacity for release of acetylcholine (ACh). High affinity choline transporter is not expressed but organic acid transporters are, providing additional mechanisms for release. Flow cytometry studies identified subpopulations of cholinergic T and B cells as well as monocytes/macrophages. Neither abundance nor GFP intensity of cholinergic T cells changed in sepsis, suggesting that ACh synthetic capacity was not altered. Spleens have low acetylcholinesterase activity, and the enzyme is localized primarily in red pulp, characteristics expected to favor cholinergic signaling. For cellular studies, ACh was quantified by mass spectroscopy using d4-ACh internal standard. Isolated splenocytes from male mice contain more ACh than females, suggesting the potential for gender-dependent differences in cholinergic immune function. Isolated splenocytes exhibit basal ACh release, which can be increased by isoproterenol (4 and 24 h) or by T cell activation with antibodies to CD3 and CD28 (24 h). Collectively, these data support the concept that sepsis enhances cholinergic function in the spleen and that release of ACh can be triggered by stimuli via different mechanisms.

The neurobiological basis for novel experimental therapeutics in dystonia.

Dystonia is a movement disorder characterized by involuntary muscle contractions, twisting movements, and abnormal postures that may affect one or multiple body regions. Dystonia is the third most common movement disorder after Parkinson's disease and essential tremor. Despite its relative frequency, small molecule therapeutics for dystonia are limited. Development of new therapeutics is further hampered by the heterogeneity of both clinical symptoms and etiologies in dystonia. Recent advances in both animal and cell-based models have helped clarify divergent etiologies in dystonia and have facilitated the identification of new therapeutic targets. Advances in medicinal chemistry have also made available novel compounds for testing in biochemical, physiological, and behavioral models of dystonia. Here, we briefly review motor circuit anatomy and the anatomical and functional abnormalities in dystonia. We then discuss recently identified therapeutic targets in dystonia based on recent preclinical animal studies and clinical trials investigating novel therapeutics.

Trihexyphenidyl rescues the deficit in dopamine neurotransmission in a mouse model of DYT1 dystonia.

Trihexyphenidyl, a nonselective muscarinic receptor antagonist, is the small molecule drug of choice for the treatment of DYT1 dystonia, but it is poorly tolerated due to significant side effects. A better understanding of the mechanism of action of trihexyphenidyl is needed for the development of improved treatments. Because DTY1 dystonia is associated with both abnormal cholinergic neurotransmission and abnormal dopamine regulation, we tested the hypothesis that trihexyphenidyl normalizes striatal dopamine release in a mouse model of DYT1 dystonia using ex vivo fast scan cyclic voltammetry and in vivo microdialysis. Trihexyphenidyl increased striatal dopamine release and efflux as assessed by ex vivo voltammetry and in vivo microdialysis respectively. In contrast, ʟ-DOPA, which is not usually effective for the treatment of DYT1 dystonia, did not increase dopamine release in either Dyt1 or control mice. Trihexyphenidyl was less effective at enhancing dopamine release in Dyt1 mice relative to controls ex vivo (mean increase WT: 65% vs Dyt1: 35%). Trihexyphenidyl required nicotinic receptors but not glutamate receptors to increase dopamine release. Dyt1 mice were more sensitive to the dopamine release decreasing effects of nicotinic acetylcholine receptor antagonism (IC50: WT = 29.46 nM, Dyt1 = 12.26 nM) and less sensitive to acetylcholinesterase inhibitors suggesting that nicotinic acetylcholine receptor neurotransmission is altered in Dyt1 mice, that nicotinic receptors indirectly mediate the differential effects of trihexyphenidyl in Dyt1 mice, and that nicotinic receptors may be suitable therapeutic targets for DYT1 dystonia.

Deletion of neurturin impairs development of cholinergic nerves and heart rate control in postnatal mouse hearts.

The neurotrophic factor neurturin is required for normal cholinergic innervation of adult mouse heart and bradycardic responses to vagal stimulation. Our goals were to determine effects of neurturin deletion on development of cardiac chronotropic and dromotropic functions, vagal baroreflex response, and cholinergic nerve density in nodal regions of postnatal mice. Experiments were performed on postnatal C57BL/6 wild-type (WT) and neurturin knockout (KO) mice. Serial electrocardiograms were recorded noninvasively from conscious pups using an ECGenie apparatus. Mice were treated with atenolol to evaluate and block sympathetic effects on heart rate (HR) and phenylephrine (PE) to stimulate the baroreflex. Immunohistochemistry was used to label cholinergic nerves in paraffin sections. WT and KO mice showed similar age-dependent increases in HR and decreases in PR interval between postnatal days (P) 2.5 and 21. Treatment with atenolol reduced HR significantly in WT and KO pups at P7.5. PE caused a reflex bradycardia that was significantly smaller in KO pups. Cholinergic nerve density was significantly less in nodal regions of P7.5 KO mice. We conclude that cholinergic nerves have minimal influence on developmental changes in HR and PR, QRS, and QTc intervals in mouse pups. However, cholinergic nerves mediate reflex bradycardia by 1 week postnatally. Deletion of neurturin impairs cholinergic innervation of the heart and the vagal efferent component of the baroreflex early during postnatal development.

Remodeling of stellate ganglion neurons after spatially targeted myocardial infarction: Neuropeptide and morphologic changes.

BACKGROUND: Myocardial infarction (MI) induces remodeling in stellate ganglion neurons (SGNs). OBJECTIVE: We investigated whether infarct site has any impact on the laterality of morphologic changes or neuropeptide expression in stellate ganglia. METHODS: Yorkshire pigs underwent left circumflex coronary artery (LCX; n = 6) or right coronary artery (RCA; n = 6) occlusion to create left- and right-sided MI, respectively (control: n = 10). At 5 ± 1 weeks after MI, left and right stellate ganglia (LSG and RSG, respectively) were collected to determine neuronal size, as well as tyrosine hydroxylase (TH) and neuropeptide Y immunoreactivity. RESULTS: Compared with control, LCX and RCA MIs increased mean neuronal size in the LSG (451 ± 25 vs 650 ± 34 vs 577 ± 55 µm(2), respectively; P = .0012) and RSG (433 ± 22 vs 646 ± 42 vs 530 ± 41 µm(2), respectively; P = .002). TH immunoreactivity was present in the majority of SGNs. Both LCX and RCA MIs were associated with significant decreases in the percentage of TH-negative SGNs, from 2.58% ± 0.2% in controls to 1.26% ± 0.3% and 0.7% ± 0.3% in animals with LCX and RCA MI, respectively, for LSG (P = .001) and from 3.02% ± 0.4% in controls to 1.36% ± 0.3% and 0.68% ± 0.2% in LCX and RCA MI, respectively, for RSG (P = .002). Both TH-negative and TH-positive neurons increased in size after LCX and RCA MI. Neuropeptide Y immunoreactivity was also increased significantly by LCX and RCA MI in both ganglia. CONCLUSION: Left- and right-sided MIs equally induced morphologic and neurochemical changes in LSG and RSG neurons, independent of infarct site. These data indicate that afferent signals transduced after MI result in bilateral changes and provide a rationale for bilateral interventions targeting the sympathetic chain for arrhythmia modulation.

Base pair stability of 8-chloro- and 8-iodo-2'-deoxyguanosine opposite 2'-deoxycytidine: implications regarding the bioactivity of 8-oxo-2'-deoxyguanosine.

8-Oxo-2'-deoxyguanosine (OdG) is an abundant and promutagenic damaged nucleotide that has been linked to aging and disease. To gain insight into the alternate base pairings of OdG, 8-chloro- and 8-iodo-2'-deoxyguanosine were incorporated into oligonucleotides and, along with 2'-deoxyguanosine and 8-bromo-2'-deoxyguanosine, were tested for their stability in base pairs opposite dC. We found a strong correlation between increased atomic radius and bond length at C8 and decreased base pair stability. These findings along with NMR studies on the base conformation of the corresponding nucleosides support the theory that the steric bulk of the 8-oxygen plays a role in OdG mutation and disease.

Managing head lice in an era of increasing resistance to insecticides.

Head lice are present in all age groups, however, the peak age for infestation is 7-8 years and the incidence varies throughout the year with higher incidence during the winter. Different insecticides have been used over the past 60 years to manage this condition. There is now strong evidence of insecticide resistance established in many countries to such an extent that some of these chemicals have become obsolete. Resistance to some pediculicides can vary from country to country and region to region within a country. The lack of a local monitoring system of resistance patterns means that parents and pupils are hampered in making an informed decision regarding how to treat head lice. One should no longer assume that treatment failure is due to poor treatment compliance or re-infestation. Clear treatment guidelines drawn up by healthcare professionals with an interest in head lice and taking into account regional/national resistance patterns should be implemented. These guidelines should combine chemical and non-chemical approaches to treatment and be coordinated and regularly reviewed by local public health departments. Drug companies should be made to provide up-to-date efficacy of their products.