BACKGROUND:Many neuropsychiatric disorders involve dysregulation of the dopaminergic (DA) input to the forebrain. DA afferents from the midbrain ventral tegmental area (VTA) are particularly relevant. A key neuromodulatory influence on DAVTA neurons arises from lateral hypothalamic area hypocretin/orexin (OX) neurons. Despite being a major input, the differential actions of OX peptides A and B on their receptors (OX1R and OX2R) in DA neurons are poorly understood. METHODS:Using genetically engineered mice whose DA cells selectively lack OX input via Hcrtr1 (DAOx1R-KO) or Hcrtr2 (DAOx2R-KO), we assessed DAVTA neuron intrinsic excitability ex vivo and evaluated behavioral phenotypes across socioemotional and cognitive domains. RESULTS:We discovered previously unrecognized effects of OX peptides on DAVTA cell response. While OXA enhanced DAVTA neuron firing via OX1Rs, OXB diminished firing via OX2Rs. Behaviorally, DA OX1R loss generated anxiety-like responding and context-dependent hyperactivity, while DA OX2R loss decreased sociability and compromised aversion-driven learning. Loss of either OX1Rs or OX2Rs in DA cells elicited impulsivity and compulsivity-like behavioral patterns. CONCLUSIONS:We evidenced distinct functions of OX1R versus OX2R signaling in modulating the intrinsic excitability of DAVTA neurons and influencing DA-related behaviors. Our data implicate OX→DA signaling pathways in neuropsychiatric endophenotypes relevant to obsessive-compulsive, attention-deficit/hyperactivity, and autism spectrum disorders and inform therapeutic strategies targeting OX receptors.

Postsynaptic density (PSD) is a tightly interconnected protein network ensuring synaptic function through the interaction of neurotransmitter receptors, structural adaptor proteins, and signaling molecules. Disruption of PSD may cause neurological diseases, including autism spectrum disorders and cognitive impairment. We demonstrate that the SKT adaptor distinctly localizes within dendritic spines as an integral component of the synaptic network, binding PSD-95 and SHANK3. SKT-knockout (KO) mice show significant abnormalities in dendritic spine density and morphology, consistent with RhoA and Rac1 GTPase dysregulated activity. KO-derived neuronal cultures display delayed neuronal synchronization and maturation associated with glutamatergic pre- and postsynaptic impairment. Behavioral tests on KO mice reveal increased self-grooming activity and impaired motor coordination, with altered cognitive and executive functions compared to wild-type mice. Overall, SKT emerges as a key contributor to the structural and functional PSD organization, regulating synaptic function through its interactions with PSD components.

Sleep stages resembling the REM and non-REM (NREM) phases observed in mammals and birds were recently found in other vertebrates, cephalopods, and euarthropods. REM sleep-like stages could mark a leap in cognitive evolution, by regulating the visuomotor elaboration that Coombs and Trestman deem essential for the cognitively complex lineages. The more widespread NREM sleep-like stages likely modulate metabolism and homeostasis of synaptic circuits.

Plasma membrane macromolecular complexes function as signaling hubs that regulate cell behavior, which is particularly relevant in cancer. Our study provides evidence that the complex formed by the hERG1 potassium channel and the β1 subunit of integrin receptors preferentially localizes in Lipid Rafts (LRs) in Pancreatic Ductal Adenocarcinoma (PDAC) cell lines and primary samples. The complex recruits the p85 subunit of phosphatidyl-inositol-3-kinase (PI3K), activating phosphoinositide metabolism and triggering an intracellular signaling pathway centered on Akt. This pathway ultimately affects cancer cell proliferation through cyclins and p21, and cell migration through the small GTPase Rac-1 and f-actin organization. The hERG1/β1 integrin complex in LRs can be dissociated and the downstream signaling pathway can be inhibited by either disrupting LRs through methyl-beta-cyclodextrin (MβCD) or inhibiting cholesterol synthesis by statins. Treatment with a single chain bispecific antibody—scDb-hERG1-β1—specifically targeting the complex significantly potentiates the effects of both MβCD and statins on intracellular signaling. Consequently, these treatments decrease PDAC cell proliferation and motility in vitro. From a pharmacological perspective, different statins produce anti-neoplastic effects in synergy with scDb-hERG1-β1. Such combination also enhances tumor sensitivity to chemotherapeutic drugs, such as gemcitabine and oxaliplatin. The efficacy of these combination treatments depends on the amount of the hERG1/β1 integrin complex present on the plasma membrane of cancer cells. Finally, the combined treatment with statins and scDb-hERG1-β1 significantly reduces tumor growth and improves survival in vivo, in a preclinical mouse model. These results suggest that the combination of scDb-hERG1-β1 and statins represent a potential novel strategy for treating PDAC patients.

K+ channel modulation is essential to regulate the Ca2+ signals that trigger T and B lymphocyte activation. The K+ channel complement is however deeply altered in cancer cells, as it is implicated in the neoplastic progression. We investigated the functional expression of K+ channels in different diffuse large B cell lymphoma (DLBCL) cell lines (SU-DHL-4, WSU-DLCL2, U2932) and EBV-infected lymphocytes, as controls of normally proliferating lymphocytes. We studied the K+ channels best characterized in lymphocytes (the voltage-gated KV1.3 and the Ca2+-activated KCa3.1) and two members of the ether-à-go-go (EAG) family (KV11.1, or hERG1; KV12.2, or hELK2) that are often overexpressed in cancer cells. All the above channel types were found in lymphoma cell lines. However, compared to normal lymphocytes, KV1.3 and KCa3.1 tended to be substituted by EAG channels, and especially by hERG1. In all DLBCL models, cell adhesion to fibronectin stimulated the formation of the cancer-specific macromolecular complex between hERG1 and the β1 subunit of integrin receptors. No such complex was formed by hELK2. A strong reduction of lymphoma proliferation was produced when the hERG1-β1 integrin complex was disrupted by a bispecific antibody (scDb-hERG1-β1), or by the hERG1-binding macrolide antibiotic clarithromycin. Finally, in a cohort of mucosa-associated lymphoid tissue (MALT) lymphoma patients treated with clarithromycin, a trend for an improved overall survival probability was observed in patients expressing the hERG1-β1 integrin complex. Our results suggest that the hERG1-β1 integrin complex promotes lymphoma cells' proliferation. Disrupting the complex by specific targeting agents represents a new therapeutic approach to be explored.

Mounting evidence underline the relevance of macromolecular complexes in cancer. Integrins frequently recruit ion channels and transporters within complexes which behave as signaling hubs. A complex composed by β1 integrin, hERG1 K+ channel, the neonatal form of the Na+ channel NaV 1.5 (nNaV1.5) and the Na+/H+ antiporter NHE1 (NHE1/hERG1/β1/nNaV1.5 complex) has been recently described to be expressed and regulate relevant cancer related behaviors in Breast Cancer (BCa) cells. We analyzed the expression and impact on outcome of the genes encoding the four proteins forming the NHE1/hERG1/β1/nNaV1.5 complex (SLC9A1, KCNH2, ITGB1 and SCN5A) in public datasets. The corresponding proteins were also evaluated by immunohistochemistry and their expression was correlated with clinic-pathological and molecular characteristics and patients’ survival. The expression of KCNH2 and SCN5A was significantly correlated in primary BCa as occurs in the heart, although with a broader distribution, forming a functional network which also included ITGB1 and SLC9A1. The co-expression proteins emerged from the immunohistochemistry analysis. Interestingly, hERG1, nNav1.5 and the hERG1/β1 integrin complex associated with several clinical features, including molecular subtype and hormone receptor status. Moreover, hERG1 and the combination of hERG1 and nNav1.5 had impact on prognosis, contributing to identifying a group of patients with worse prognosis. hERG1 and nNav1.5 channels along with β1 integrins and the NHE1 antiporter are co-expressed in BCa both at gene and protein levels, assembling into a macromolecular complex. The NHE1/hERG1/β1/nNaV1.5 complex can be considered a novel biomarker and potential target for therapy for BCa patients.

The cross talk between the Microenvironment (TME) and cancer cells controls proliferation, migration and pro-metastatic cancer behavior, often through integrin-mediated cell adhesion to the Extracellular Matrix (ECM). Integrin receptors coordinate signaling hubs constituted by multiprotein plasma membrane complexes often comprising ion channels and transporters. We here describe a novel signaling pathway triggered by (1 integrin activation by the ECM protein fibronectin (FN). This pathway involves a (1 integrin-centered plasma membrane complex formed by different transport proteins: the hERG1 K+ channel, the neonatal form of the Na+ channel NaV 1.5 (nNaV1.5) and the Na+/H+ antiporter NHE1. The NHE1/hERG1/(1/nNaV1.5 complex is present on the plasma membrane of Breast Cancer (BCa) cells, in particular Triple Negative Breast Cancer (TNBCa). The expression of the two ion channels present in the complex is mutually regulated. The complex engagement by cell adhesion to FN stimulates a NHE1-mediated cytoplasmic alkalinization, which modulates f-actin organization. This in turn controls TNBCa migration and invasiveness. All this pathway is impaired by blocking either hERG1 or nNaV1.5 which also cause complex disassembly. The same result is obtained by harnessing the hERG1/(1 integrin complex through a single chain bispecific antibody (scDb-hERG1-(1). In conclusion, we uncovered a plasma membrane complex that recruits different ion transport proteins that regulate cellular K+, Na+ and H+ fluxes and cooperate in modulating downstream signals and thus malignant behavior. This complex could be targeted to develop novel therapeutic strategies for one of the most difficult-to-treat cancers, i.e. TNBCa. ### Competing Interest Statement The authors have declared no competing interest.

We combine experimental data and mathematical modeling to offer a coherent explanation of the dynamics of hERG1 interaction with integrins, which governs f-actin organization and migration in cancer cells. The hERG1 potassium channel is aberrantly over expressed in tumors and regulates the cancer cell response to integrin-dependent adhesion. We unravel a novel signaling pathway by which integrin engagement by the ECM protein fibronectin promotes hERG1 translocation to the plasma membrane and its association with β1 integrins, by activating girdin-dependent Gαi3 proteins and protein kinase B (Akt). By sequestering hERG1, β1 integrins make it avoid Rab5-mediated endocytosis, where unbound channels are degraded. The cycle of hERG1 expression determines the resting potential (V rest ) oscillations and drives the cortical f-actin dynamics and thus cell motility. To interpret the slow biphasic kinetics of hERG1/β1 integrin interplay, we developed a mathematical model based on a generic balanced inactivation–like module. Integrin-mediated cell adhesion triggers two contrary responses: a rapid stimulation of hERG1/β1 complex formation, followed by a slow inhibition which restores the initial condition. The protracted hERG1/β1 integrin cycle determines the slow time course and cyclic behavior of cell migration in cancer cells.

Pharmacological studies aimed at the development of newly synthesized drugs directed against ion channels (as well as genetic studies of ion channel mutations) involve the development and use of transfected cells. However, the identification of the best clone, in terms of transfection efficiency, is often a time consuming procedure when performed through traditional methods such as manual patch-clamp. On the other hand, the use of other faster techniques, such as for example the IF, are not informative on the effective biological functionality of the transfected ion channel(s). In the present work, we used the high throughput automated ion channel reader (ICR) technology (ICR8000 Aurora Biomed Inc.) that combine atomic absorption spectroscopy with a patented microsampling process to accurately measure ion flux in cell-based screening assays. This technology indeed helped us to evaluate the transfection efficiency of hERG1 and hKv1.3 channels respectively on the HEK-293 and CHO cellular models. Moreover, as proof of the validity of this innovative method, we have corroborated these data with the functional characterization of the potassium currents carried out by the same clones through patch-clamp recordings. The results obtained in our study are promising and represent a valid methodological strategy to screen a large number of clones simultaneously and to pharmacologically evaluate their functionality within an extremely faster timeframe.

Despite recent advances in understanding the causes of epilepsy, especially the genetic, comprehending the biological mechanisms that lead to the epileptic phenotype remains difficult. A paradigmatic case is constituted by the epilepsies caused by altered neuronal nicotinic acetylcholine receptors (nAChRs), which exert complex physiological functions in mature as well as developing brain. The ascending cholinergic projections exert potent control of forebrain excitability, and wide evidence implicates nAChR dysregulation as both cause and effect of epileptiform activity. First, tonic-clonic seizures are triggered by administration of high doses of nicotinic agonists, whereas non-convulsive doses have kindling effects. Second, sleep-related epilepsy can be caused by mutations on genes encoding nAChR subunits widely expressed in the forebrain (CHRNA4, CHRNB2, CHRNA2). Third, in animal models of acquired epilepsy, complex time-dependent alterations in cholinergic innervation are observed following repeated seizures. Heteromeric nAChRs are central players in epileptogenesis. Evidence is wide for autosomal dominant sleep-related hypermotor epilepsy (ADSHE). Studies of ADSHE-linked nAChR subunits in expression systems suggest that the epileptogenic process is promoted by overactive receptors. Investigation in animal models of ADSHE indicates that expression of mutant nAChRs can lead to lifelong hyperexcitability by altering i) the function of GABAergic populations in the mature neocortex and thalamus, ii) synaptic architecture during synaptogenesis. Understanding the balance of the epileptogenic effects in adult and developing networks is essential to plan rational therapy at different ages. Combining this knowledge with a deeper understanding of the functional and pharmacological properties of individual mutations will advance precision and personalized medicine in nAChR-dependent epilepsy.

The cytoplasmic Ca2+ concentration and the activity of K+ channels on the plasma membrane regulate cellular processes ranging from mitosis to oriented migration. The interplay between Ca2+ and K+ signals is intricate, and different cell types rely on peculiar cellular mechanisms. Derangement of these mechanisms accompanies the neoplastic progression. The calcium signals modulated by voltage-gated (KV) and calcium-dependent (KCa) K+ channel activity regulate progression of the cell division cycle, the release of growth factors, apoptosis, cell motility and migration. Moreover, KV channels regulate the cell response to the local microenvironment by assembling with cell adhesion and growth factor receptors. This chapter summarizes the pathophysiological roles of Ca2+ and K+ fluxes in normal and cancer cells, by concentrating on several biological systems in which these functions have been studied in depth, such as early embryos, mammalian cell lines, T lymphocytes, gliomas and colorectal cancer cells. A full understanding of the underlying mechanisms will offer a comprehensive view of the ion channel implication in cancer biology and suggest potential pharmacological targets for novel therapeutic approaches in oncology.

Hypocretin/Orexin (HCRT/OX) and dopamine (DA) are two key effectors of salience processing, reward and stress-associated behavior and motivational states, yet their respective roles and interactions are poorly delineated. We inactivated HCRT-to-DA connectivity by genetic disruption of Hypocretin receptor type-1 (Hcrtr1), Hypocretin receptor type-2 (Hcrtr2), or both receptors (Hcrtr1&2) in dopamine neurons and analyzed the consequences on vigilance states, brain oscillations, and cognitive performance in freely behaving mice. Unexpectedly, loss of Hcrtr2, but not Hcrtr1 or Hcrtr1&2, led to dramatic increases in theta (7-11 Hz) electroencephalographic (EEG) activity during both wakefulness and rapid-eye-movement (REM) sleep. Compared to controls, DAHcrtr2-deficient mice spent more time in an active (or theta activity-enriched) substate of wakefulness, as well as exhibited prolonged REM sleep. Additionally, both wake and REM sleep displayed enhanced theta-gamma phase-amplitude coupling. The baseline waking EEG of DAHcrtr2-deficient mice exhibited diminished infra-theta, but increased theta power, two hallmarks of EEG hyperarousal, which however were found to be uncoupled from the mice’ locomotor activity. Upon exposure to novel, either rewarding or stress-inducing environments, DAHcrtr2-deficient mice’ waking state featured more pronounced surges in theta and fast-gamma (52-80 Hz) EEG activities compared to their littermate controls, further suggesting increased alertness. Cognition was next evaluated using an operant conditioning paradigm, demonstrating that DAHcrtr2-ablated mice exhibit faster learning, and once performance was stable and attentional demands were increased, they manifested higher attentional capabilities. Concomitantly, the mice however displayed maladaptive patterns of reward-seeking, with behavioral indices of increased impulsivity as well as compulsivity. None of the EEG changes observed in DAHcrtr2-deficient mice were seen in dopaminergic Hcrtr1-ablated mice, which tended to show opposite EEG phenotypes. Our findings establish a clear, genetically-defined link between monosynaptic HCRT-to-dopaminergic neurotransmission and theta oscillations, with a differential and novel role of HCRTR2 in cross-frequency coupling, attentional processes, and executive functions, relevant to disorders including narcolepsy, attention-deficit/hyperactivity disorder, and Parkinson’s disease.

Voltage-gated potassium channel KV1.3 inhibitors have been shown to be effective in preventing T-cell proliferation and activation by affecting intracellular Ca2+ homeostasis. Here, we present the structure-activity relationship, KV1.3 inhibition, and immunosuppressive effects of new thiophene-based KV1.3 inhibitors with nanomolar potency on K+ current in T-lymphocytes and KV1.3 inhibition on Ltk- cells. The new KV1.3 inhibitor trans-18 inhibited KV1.3 -mediated current in phytohemagglutinin (PHA)-activated T-lymphocytes with an IC50 value of 26.1 nM and in mammalian Ltk- cells with an IC50 value of 230 nM. The KV1.3 inhibitor trans-18 also had nanomolar potency against KV1.3 in Xenopus laevis oocytes (IC50 = 136 nM). The novel thiophene-based KV1.3 inhibitors impaired intracellular Ca2+ signaling as well as T-cell activation, proliferation, and colony formation.

Abstract Background A considerable proportion of patients do not fully recover from COVID-19 infection and report symptoms that persist beyond the initial phase of infection: this condition is defined long-COVID-19 syndrome (LCS). LCS can involve lungs as well as several extrapulmonary organs, including the cardiovascular system. The risk and 1-year burden of cardiovascular diseases (CVD) is increased in COVID-19 survivors, even in subjects at low risk of CVD. Recently, we documented that acute COVID-19 infection induces altered platelet activation state characterized by a prothrombotic phenotype and by the formation of platelet-leukocyte aggregates (PLA), that may be involved in the pulmonary microthrombi found in autoptic specimens. No data are yet available on the contribution of platelet activation to residual pulmonary impairment and procoagulant potential in LCS patients. Purpose To study platelet activation status, microvesicle (MV) profile, platelet thrombin generation capacity (pTGC) in LCS patients enrolled at 6 months after resolution of the acute phase (6mo-FU), compared to acute COVID-19 infection patients. Methods 6mo-FU COVID-19 patients (n=24) with established LCS were enrolled at Centro Cardiologico Monzino. Residual pulmonary impairment was assessed by Cardiopulmonary Exercise Testing (CPET) and 64-rows-CT scan evaluation. Platelet activation (P-selectin, Tissue Factor [TF] and PLA) and MV profile were assessed by flow cytometry; pTGC by calibrated automated thrombogram. 46 patients enrolled during acute COVID-19 infection and 46 healthy subjects (HS) were used for comparison. Results Dispnea in LCS patients was confirmed by CPET showing compromised alveolus-capillary membrane diffusion and residual pulmonary impairment. TF+-platelet and -MV levels were 3-fold (1.5% [1.2–2.9] vs 2.4% [1.6–5.7]) and 2-fold (217/μl [137–275] vs 435/μl [275–633]) lower at 6mo-FU compared to acute phase, being comparable to HS. pTGC behaved similarly. At 6mo-FU, the MV profile, in terms of total number and cell origin, returned to physiological levels. Conversely, although lower than that measured in acute phase, a 2.5-fold higher platelet P-selectin expression (6.9% [3–13.5] vs 11.7% [5.2–18.9]) and PLA formation (35.5% [27.4–46.8] vs 67.7% [45.7–85.3]) was observed at 6mo-FU compared to HS. Interestingly, a significant correlation between PLA formation and residual pulmonary impairment was observed (r=−0.423; p=0.02). Conclusion These data strengthen the hypothesis that the presence of PLA in the bloodstream, and thus also in the pulmonary microcirculation, may contribute to support pulmonary dysfunction still observed in LCS patients. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Health (Ricerca Corrente 2020 MPP COVID4)

Ion channels are implicated in various diseases, including cancer, in which they modulate different aspects of cancer progression. In particular, potassium channels are often aberrantly expressed in cancers, a major example being provided by hERG1. The latter is generally complexed with β1 integrin in tumour cells, and such a molecular complex represents a new druggable hub. The present study focuses on the characterization of the functional consequences of the interaction between hERG1 and β1 integrins on different substrates over time. To this purpose, we studied the interplay alteration on the plasma membrane through patch clamp techniques in a cellular model consisting of human embryonic kidney (HEK) cells stably transfected with hERG1 and in a cancer cell model consisting of SH-SY5Y neuroblastoma cells, endogenously expressing the channel. Cells were seeded on different substrates known to stimulate β1 integrins, such as fibronectin (FN) for HEK-hERG1 and laminin (LMN) for SH-SY5Y. In HEK cells stably overexpressing hERG1, we observed a hERG1 current density increase accompanied by Vrest hyperpolarization after cell seeding onto FN. Notably, a similar behaviour was shown by SH-SY5Y neuroblastoma cells plated onto LMN. Interestingly, we did not observe this phenomenon when plating the cells on substrates such as Bovine Serum Albumin (BSA) or Polylysine (PL), thus suggesting a crucial involvement of ECM proteins as well as of β1 integrin activation.

Mutant subunits of the neuronal nicotinic ACh receptor (nAChR) can cause Autosomal Dominant Sleep-related Hypermotor Epilepsy (ADSHE), characterized by frontal seizures during non-rapid eye movement (NREM) sleep. We studied the cellular bases of the pathogenesis in brain slices from mice conditionally expressing the ADSHE-linked β2V287L nAChR subunit. β2V287L mice displayed minor structural alterations, except for a ~10% decrease of prefrontal cortex thickness. However, they showed a substantial decrease of the excitatory input to layer V fast-spiking (FS) interneurons, despite a concomitant increase in the number of glutamatergic terminals around the cell soma. Hence, prefrontal hyperexcitability may depend on a permanent impairment of surround inhibition. The effect disappeared when β2V287L was silenced until postnatal day 15th, suggesting that the transgene selectively affects the maturation of glutamatergic synapses on FS neurons. The other main population of interneurons in layer V was constituted by somatostatin-expressing regular spiking cells. When tested with 10 µM nicotine, these displayed larger somatic nicotinic currents in transgenic mice. Thus, during wakefulness, activation of β2V287L-containing nAChRs by the high cholinergic tone may counteract hyperexcitability by promoting local inhibition by somatostatin-expressing cells and decreasing the effect of glutamatergic deficit in FS neurons. This interpretation was tested in networks disinhibited by 2 μM bicuculline. Slices expressing β2V287L were more susceptible to develop synchronized activity in the absence of nicotine. Addition of the drug boosted excitability in the controls, but had little effect in β2V287L. Our findings suggest why NREM sleep favors ADSHE seizures and nicotine can be palliative in patients.

The cellular functions are regulated by a complex interplay of diffuse and local signals. Studying the latter is challenging, but experimental work in cell physiology has led to recognize that understanding a cell's dynamics requires a deep comprehension of local fluctuations of cytosolic regulators. Macromolecular complexes are major determinants of local signaling. Multienzyme assemblies limit the diffusion restriction to reaction kinetics by direct exchange of metabolites. Likewise, close coupling of ion channels and transporters modulates the ion concentration around a channel mouth or transporter binding site. Extreme signal locality is brought about by conformational coupling between membrane proteins, as is typical of mechanotransduction. A paradigmatic case is integrin-mediated cell adhesion. Sensing the extracellular microenvironment and providing an appropriate response are essential in growth and development and have innumerable pathological implications. The process involves bidirectional signal transduction by complex supramolecular structures that link integrin receptors to ion channels and transporters, growth factor receptors, cytoskeletal elements, and other regulatory elements. The dynamics of such complexes are only beginning to be understood. A thoroughly studied example is the association between integrin receptors and the voltage-gated K+ channels Kv11.1. These channels are widely expressed in early embryos, where their physiological roles are poorly understood and apparently different from the shaping of action potential firing in the adult. Hints about these roles come from studies in cancer cells, where Kv11.1 is often overexpressed and appears to reassume functions it presumably exerts during embryogenesis, such as controlling cell proliferation/differentiation, apoptosis, and migration. Kv11.1 is implicated in these processes through its linking to integrin subunits, which in turn regulates channel expression. Specific cellular functions, such as proliferation and migration, appear to be modulated by distinct conformational states of the channel (e.g., open and closed), whose balance is affected by the link with integrin subunits.

Both dopaminergic (DA) and orexinergic (OX) systems establish brain-wide neuromodulatory circuits that profoundly influence brain states and behavioral outputs. To unravel their interactions, we inactivated OX-to-DA neurotransmission by selective disruption of HcrtR1/OxR1 , or HcrtR2/OxR2 , or both receptors, in DA neurons. Chronic loss of OXR2 in DA neurons ( OxR2Dat-CKO mice) dramatically increased electrocorticographic (EcoG) theta rhythms in wakefulness and REM sleep. Episode duration and total times spent in ‘active’ wakefulness and REMS were prolonged, and theta/fast-gamma wave coupling was enhanced in both states. Increased theta in OxR2DatCKO mice baseline wake was accompanied by diminished infra-theta and increased fast-gamma activities, i.e. the mice exhibited signs of constitutive electrocortical hyperarousal, albeit uncoupled with locomotor activity. These effects were not seen in OxR1 -ablated dopaminergic mutants, which tended to show opposite phenotypes, resembling those caused by the loss of both receptors. Our data establish a clear, genetically-defined link between monosynaptic orexin-to-dopaminergic connectivity and the power of theta oscillations, with a differential role of OXR2 in cross-frequency wave coupling and attentional processes.### Competing Interest StatementThe authors have declared no competing interest.