Subthreshold Dose Research Articles

Meningeal KATP channels contribute to behavioral responses in preclinical migraine models

Abstract Human experimental studies have shown that levcromakalim, an ATP-sensitive potassium (KATP) channel opener, induces migraine attacks in people with migraine but not in healthy volunteers. However, the exact site of action for KATP channels in migraine pathophysiology remains unclear. This study investigates the role of these channels in the meninges in eliciting behavioral hypersensitivity responses in mice. The effects of KATP channel signaling were assessed using preclinical migraine models induced by repetitive stress or dural stimulation. Prolactin, CGRP, sodium nitroprusside (SNP), and KATP channel openers or blockers were administered systemically or onto the dura of mice followed by behavioral testing using periorbital von Frey or facial grimace measurements. Repetitive stress sensitized mice to a normally subthreshold systemic dose of levcromakalim. The KATP blocker glibenclamide significantly reduced responses to systemic SNP following repetitive stress. In naive mice, direct dural application of levcromakalim or SNP elicited periorbital hypersensitivity. Responses to dural levcromakalim were inhibited by coinjection with glibenclamide or sumatriptan. By contrast, injection of levcromakalim in the periorbital skin did not induce hypersensitivity. Moreover, repetitive stress sensitized mice to dural injection of normally subthreshold doses of levcromakalim or SNP. Finally, dural coinjection of glibenclamide inhibited periorbital hypersensitivity induced by CGRP or prolactin in female mice. These studies demonstrate that the meninges can be one site of action for the migraine-triggering effects of KATP channel openers. They also show that NO donors, CGRP, and prolactin can produce behavioral hypersensitivity through opening of KATP channels in the meninges.

Joint administration of sub-threshold doses of the acetylcholinesterase inhibitor donepezil with those of the NMDA receptor antagonist ketamine improved rats’ recognition memory abilities

Alzheimer’s Disease (AD) is a serious progressive neurodegenerative illness conducting to the decay of cognitive functions. A few drugs have been approved for the therapy of AD, including the acetylcholinesterase inhibitors (AChEIs) like donepezil. Their efficiency, however, is modest and their application is associated with toxicity. Recently, the N-methyl-D-aspartate (NMDA) receptor antagonist ketamine, a rapidly acting antidepressant, has been proposed as a potential agent for the treatment of AD. The present study was designed to investigate the effects exerted by the combination of sub-threshold doses of donepezil with those of ketamine on rats’ recognition memory abilities. For these experiments, the object recognition task (ORT) and the object location task (OLT), two procedures assessing non-spatial and spatial recognition memory respectively in rodents were used. Post-training acute administration of inactive doses of donepezil (0.3 mg/kg) and ketamine (1 mg/kg) counteracted non-spatial and spatial recognition memory impairments. The present findings, although preliminary, propose that the combined administration of ketamine and donepezil could represent a new strategy for the therapy of memory disorders, a common feature of AD patients.

Anxiolytic- and antidepressive-like effects of harmaline in mice are mediated via histamine H3 receptor blockade

Many neuropsychiatric disorders can be caused by neurotransmitter dysfunction. Experimental studies have demonstrated that histamine and the harmaline affect physiological processes through interaction with other neurotransmitter systems. The objective of these experiments was to investigate the involvement of the histaminergic system in the effects of harmaline on anxiety- and depressive-related effects in male NMRI mice. Behavioral tests were employed to evaluate anxiety-related symptoms (elevated plus maze; EPM), depressive-like symptoms (forced swim test; FST), and cognitive decline (step-down test). The histamine H3 receptor (H3R) agonist α-methylhistamine dihydrobromide (α-MH; 5 mg/kg, i.p.) had anxiolytic- and depressive-like effects, while the H3R antagonist thioperamide (10 mg/kg, i.p.) showed an antidepressive-like property. The subthreshold dose of α-MH resulted in an increase in the tendency of mice treated with the harmaline (2.5 mg/kg) to remain in the EPM open-arms. A subthreshold dose of thioperamide (5 mg/kg) increased the time spent in the open-arms in mice treated with harmaline (2.5 and 5 mg/kg) while a high dose of harmaline decreased the immobility time. Furthermore, two higher doses of harmaline resulted in a reduction in the number of open-arm entries. Similarly, mice administered with thioperamide and a low dose of harmaline decreased locomotor activity in the EPM. Ultimately, the combined thioperamide and harmaline did not impair memory retrieval of mice. These experiments demonstrate that the histaminergic system is implicated in the anxiety- and depressive-related effects of harmaline. The combination of thioperamide and harmaline is effective in treating anxiety and depression without having an adverse effect on memory formation.

Subthreshold Cannabidiol Potentiates Levetiracetam in the Kainic Acid Model of Temporal Lobe Epilepsy: A Pilot Study.

Refractoriness to antiseizure medications is still a major concern in the pharmacotherapy of epilepsy. For this reason, we decided to evaluate the combination of levetiracetam and cannabidiol, administered at a subthreshold dose, to limit the possible adverse effects of this phytocannabinoid. We administered levetiracetam (300 mg/kg/day, via osmotic minipumps), cannabidiol (120 mg/kg/day, injected once a day subcutaneously), or their combination for one week in epileptic rats. Saline-treated epileptic rats were the control group. Animals were monitored with video electroencephalography the week before and after the treatment. No changes were found in the controls. Levetiracetam did not significantly reduce the total seizure number or the overall seizure duration. Still, the overall number of seizures (p < 0.001, Duncan's new multiple range test) and their total duration (p < 0.01) increased in the week following treatment withdrawal. Cannabidiol did not change seizures when administered as a single drug. Instead, levetiracetam combined with cannabidiol resulted in a significant reduction in the overall number and duration of seizures (p < 0.05), when comparing values measured during treatment with both pre- and post-treatment values. These findings depended on changes in convulsive seizures, while non-convulsive seizures were stable. These results suggest that cannabidiol determined a remarkable potentiation of levetiracetam antiseizure effects at a subthreshold dose.

Coronaridine congeners induce anticonvulsant activity in rodents by hippocampal mechanisms involving mainly potentiation of GABAA receptors

The coronaridine congeners catharanthine and 18-methoxycoronaridine (18-MC) display sedative, anxiolytic, and antidepressant properties by acting on mechanisms involving GABAergic and/or monoaminergic transmissions. Here, we expanded their pharmacological properties by studying their anticonvulsant activity in male and female mice using the pentylenetetrazole (PTZ)-induced seizure test. To determine potential neurochemical mechanisms, the effect of congeners on monoamine content and kainic acid (KA)-induced epileptiform discharge was studied in the hippocampus. The behavioral results showed that coronaridine congeners induce acute anticonvulsant activity in a dose-dependent but sex-independent manner. Repeated treatment with a subthreshold dose (20 mg/kg) of each congener produced anticonvulsant activity in a sex-independent manner, but was significantly higher in male mice when compared to its acute effect. Using a behaviourally relevant regimen, we found that PTZ increased dopamine metabolites and serotonin tissue content. Coronaridine congeners, which induced distinct effects on monoamines, blunted the effect of PTZ instead of potentiating it, suggesting the existence of another mechanism in their anticonvulsant activity. The electrophysiological results indicated that both congeners inhibit KA-induced epileptiform discharges in hippocampal slices. A key aspect of this study is that the activity of both congeners was observed only in the presence of GABA, supporting the notion that hippocampal GABAAR potentiation plays an important role. Our study showed that coronaridine congeners induce acute anticonvulsant activity in a sex-independent manner. However, a comparatively higher susceptibility was observed in male mice after repeated treatment. The underlying hippocampal mechanisms mainly involve GABAAR potentiation, whereas monoamines play a minor role in the anticonvulsive action.

Spinal cord microglia drive sex differences in ethanol-mediated PGE2-induced allodynia

The mechanisms of how long-term alcohol use can lead to persistent pain pathology are unclear. Understanding how earlier events of short-term alcohol use can lower the threshold of non-painful stimuli, described as allodynia could prove prudent to understand important initiating mechanisms. Previously, we observed that short-term low-dose alcohol intake induced female-specific allodynia and increased microglial activation in the spinal cord dorsal horn. Other literature describes how chronic ethanol exposure activates Toll-like receptor 4 (TLR4) to initiate inflammatory responses. TLR4 is expressed on many cell types, and we aimed to investigate whether TLR4 on microglia is sufficient to potentiate allodynia during a short-term/low-dose alcohol paradigm. Our study used a novel genetic model where TLR4 expression is removed from the entire body by introducing a floxed transcriptional blocker (TLR4-null background (TLR4LoxTB)), then restricted to microglia by breeding TLR4LoxTB animals with Cx3CR1:CreERT2 animals. As previously reported, after 14 days of ethanol administration alone, we observed no increased pain behavior. However, we observed significant priming effects 3 hrs post intraplantar injection of a subthreshold dose of prostaglandin E2 (PGE2) in wild-type and microglia-TLR4 restricted female mice. We also observed a significant female-specific shift to pro-inflammatory phenotype and morphological changes in microglia of the lumbar dorsal horn. Investigations in pain priming-associated neuronal subtypes showed an increase of c-Fos and FosB activity in PKCγ interneurons in the dorsal horn of female mice directly corresponding to increased microglial activity. This study uncovers cell- and female-specific roles of TLR4 in sexual dimorphisms in pain induction among non-pathological drinkers.

Hyperglycemia sensitizes female mice to stress-induced depressive-like behavior in an inflammation-independent manner

BackgroundDepression is a multifaceted disorder that represents one of the most common causes of disability. The risk for developing depression is increased in women and among individuals with chronic diseases. For example, individuals in the United States with diabetes mellitus (DM) are at a twofold increased risk of developing depression compared to the general population and approximately one-quarter of women with diabetes have comorbid depression. The neurobiological mechanisms underlying this association between diabetes and depression is not fully understood and is particularly under-investigated in female models. We sought to explore the role of neuroinflammation in diabetes-induced depression in a female mouse model of hyperglycemia. MethodsTo this end, we utilized female C57BL/6 J mice to (1) characterize the depressive-like symptoms in response to 75 mg/kg/day dose of streptozotocin (STZ) over 5 days, a dose reported to induce hyperglycemia in female mice (n=20), (2) determine if female hyperglycemic mice are sensitized to unpredictable chronic mild stress (UCMS)-induced depressive-like behavior and neuroinflammation (n=28), and (3) investigate if female hyperglycemic mice are primed to respond to a subthreshold dose of lipopolysaccharide (LPS), an acute inflammatory challenge (n=21). ResultsOur results demonstrate that female mice exhibit robust hyperglycemia but limited evidence of depressive-like behavior in response to 75 mg/kg STZ. Additionally, we observe that healthy female mice have limited response to our stress protocol; however, hyperglycemic mice display increased stress-sensitivity as indicated by increased immobility in the forced swim test. While STZ mice show evidence of mild neuroinflammation, this effect was blunted by stress. Further, STZ mice failed to display a sensitization to inflammation-induced depressive-like behavior. ConclusionWe interpret this data to indicate that while STZ-induced hyperglycemia does increase vulnerability to stress-induced depressive-like behavior, this effect is not a consequence of neuroinflammatory priming. Future studies will seek to better understand the mechanisms underlying this sensitization.

Repetitive Administration of Low-Dose Lipopolysaccharide Improves Repeated Social Defeat Stress-Induced Behavioral Abnormalities and Aberrant Immune Response.

Repetitive exposure of innate immune cells to a subthreshold dosage of endotoxin components may modulate inflammatory responses. However, the regulatory mechanisms in the interactions between the central nervous system (CNS) and the immune system remain unclear. This study aimed to investigate the effects of lipopolysaccharide (LPS) preconditioning in repeated social defeat stress (RSDS)-induced abnormal immune responses and behavioral impairments. This study aimed to elucidate the mechanisms that underlie the protective effects of repeated administration of a subthreshold dose LPS on behavioral impairments using the RSDS paradigm. LPS preconditioning improved abnormal behaviors in RSDS-defeated mice, accompanied by decreased monoamine oxidases and increased glucocorticoid receptor expression in the hippocampus. In addition, pre-treated with LPS significantly decreased the recruited peripheral myeloid cells (CD11b+CD45hi), mainly circulating inflammatory monocytes (CD11b+CD45hiLy6ChiCCR2+) into the brain in response to RSDS challenge. Importantly, we found that LPS preconditioning exerts its protective properties by regulating lipocalin-2 (LCN2) expression in microglia, which subsequently induces expressions of chemokine CCL2 and pro-inflammatory cytokine. Subsequently, LPS-preconditioning lessened the resident microglia population (CD11b+CD45intCCL2+) in the brains of the RSDS-defeated mice. Moreover, RSDS-associated expressions of leukocytes (CD11b+CD45+CCR2+) and neutrophils (CD11b+CD45+Ly6G+) in the bone marrow, spleen, and blood were also attenuated by LPS-preconditioning. In particular, LPS preconditioning also promoted the expression of endogenous antioxidants and anti-inflammatory proteins in the hippocampus. Our results demonstrate that LPS preconditioning ameliorates lipocalin 2-associated microglial activation and aberrant immune response and promotes the expression of endogenous antioxidants and anti-inflammatory protein, thereby maintaining the homeostasis of pro-inflammation/anti-inflammation in both the brain and immune system, ultimately protecting the mice from RSDS-induced aberrant immune response and behavioral changes.

Subthreshold activation of the melanocortin system causes generalized sensitization to anorectic agents in mice.

The melanocortin-3 receptor (MC3R) regulates GABA release from agouti-related protein (AgRP) nerve terminals and thus tonically suppresses multiple circuits involved in feeding behavior and energy homeostasis. Here, we examined the role of the MC3R and the melanocortin system in regulating the response to various anorexigenic agents. The genetic deletion or pharmacological inhibition of the MC3R, or subthreshold doses of an MC4R agonist, improved the dose responsiveness to glucagon-like peptide 1 (GLP1) agonists, as assayed by inhibition of food intake and weight loss. An enhanced anorectic response to the acute satiety factors peptide YY (PYY3-36) and cholecystokinin (CCK) and the long-term adipostatic factor leptin demonstrated that increased sensitivity to anorectic agents was a generalized result of MC3R antagonism. We observed enhanced neuronal activation in multiple hypothalamic nuclei using Fos IHC following low-dose liraglutide in MC3R-KO mice (Mc3r-/-), supporting the hypothesis that the MC3R is a negative regulator of circuits that control multiple aspects of feeding behavior. The enhanced anorectic response in Mc3r-/- mice after administration of GLP1 analogs was also independent of the incretin effects and malaise induced by GLP1 receptor (GLP1R) analogs, suggesting that MC3R antagonists or MC4R agonists may have value in enhancing the dose-response range of obesity therapeutics.

Hypersensitivity of the nicotinic acetylcholine receptor subunit (CHRNA2L9′S/L9′S) in female adolescent mice produces deficits in nicotine-induced facilitation of hippocampal-dependent learning and memory

Adolescence is characterized by a critical period of maturation and growth, during which regions of the brain are vulnerable to long-lasting cognitive disturbances. Adolescent exposure to nicotine can lead to deleterious neurological and psychological outcomes. Moreover, the nicotinic acetylcholine receptor (nAChR) has been shown to play a functionally distinct role in the development of the adolescent brain. CHRNA2 encodes for the α2 subunit of nicotinic acetylcholine receptors associated with CA1 oriens lacunosum moleculare GABAergic interneurons and is associated with learning and memory. Previously, we found that adolescent male hypersensitive CHRNA2L9′S/L9′ mice had impairments in learning and memory during a pre-exposure-dependent contextual fear conditioning task that could be rescued by low-dose nicotine exposure. In this study, we assessed learning and memory in female adolescent hypersensitive CHRNA2L9′S/L9′ mice exposed to saline or a subthreshold dose of nicotine using a hippocampus-dependent task of pre-exposure-dependent contextual fear conditioning. We found that nicotine-treated wild-type female mice had significantly greater improvements in learning and memory than both saline-treated wild-type mice and nicotine-treated CHRNA2L9′S/L9′ female mice. Thus, hyperexcitability of CHRNA2 in female adolescent mice ablated the nicotine-mediated potentiation of learning and memory seen in wild-types. Our results indicate that nicotine exposure during adolescence mediates sexually dimorphic patterns of learning and memory, with wild-type female adolescents being more susceptible to the effects of sub-threshold nicotine exposure. To understand the mechanism underlying sexually dimorphic behavior between hyperexcitable CHRNA2 mice, it is critical that further research be conducted.

MODL-12. REPURPOSING SSRI/SNRI TARGETING NEUROTRANSMITTER SIGNALING TO SENSITIZE DMG TO CDK9 INHIBITORS

Abstract BACKGROUND Diffuse Midline Glioma (DMG) comprise a subset of aggressive pediatric brain tumors with few effective therapies. Cyclin-dependent kinase 9 inhibitors (CDK9i) like zotiraciclib (ZTR) are in clinical trials for adult and pediatric glioma, but resistance frequently develops. In this study, we find that co-treatment with CDK9i and brain penetrant, FDA-approved drugs targeting neurotransmitter signaling alters DMG transcription and reduces DMG growth and tumorigenesis. METHODS We conducted drug screens to assess DMG response to a sub-threshold dose of ZTR combined with a library of ~220 bioactive and FDA-approved compounds. Combinatorial effects of CDK9i+SSRI/SNRI treatment were confirmed through in vitro dose curve studies and in orthotopic DMG xenograft models. We identified downstream targets of CDK9i+SSRI/SNRI treatment through phospho-proteomic profiling, RNAseq analysis, immunofluorescence staining, and Western blotting. RESULTS Our targeted drug screens identified SSRIs (selective serotonin reuptake inhibitors) and SNRIs (serotonin and norepinephrine reuptake inhibitors) that increase DMG sensitivity to a low dose of zotiraciclib (ZTR). Follow-up studies confirm that combinations of structurally distinct CDK9i (ZTR, AZD4573) and SSRI/SNRI (sertraline, duloxetine) synergistically reduce DMG growth in neurospheres, induce the apoptotic marker cleaved caspase 3, and reduce the expression of proliferation markers. Additional proteogenomic analyses define downstream targets of CDK9i+SSRI co-treatment, including differential phosphorylation of chromatin regulators and RNA Polymerase 2 (Pol2)-associated elongation factors and splicing machinery. Further RNAseq studies reveal robust transcriptional changes induced by CDK9i+SSRI treatment characterized by altered expression of developmental and synaptic signaling genes. Finally, pilot pre-clinical studies find that CDK9i+SSRI treatment reduces tumor growth and increases progression free survival in a DMG xenograft model. CONCLUSIONS We have identified synergistic CDK9i+SSRI drug combinations that can be applied to alter malignant gene regulation and reduce DMG growth in vitro and in xenografts. These studies provide a basis for a drug-repurposing strategy to combine SSRI/SNRI and CDK9 inhibitors for DMG treatment.

Altered Hippocampal Activation in Seizure-Prone CACNA2D2 Knock-out Mice.

The voltage-gated calcium channel subunit α2δ-2 controls calcium-dependent signaling in neurons, and loss of this subunit causes epilepsy in both mice and humans. To determine whether mice without α2δ-2 demonstrate hippocampal activation or histopathological changes associated with seizure activity, we measured expression of the activity-dependent gene c-fos and various histopathological correlates of temporal lobe epilepsy (TLE) in hippocampal tissue from wild-type (WT) and α2δ-2 knock-out (CACNA2D2 KO) mice using immunohistochemical staining and confocal microscopy. Both genotypes demonstrated similarly sparse c-fos and ΔFosB expressions within the hippocampal dentate granule cell layer (GCL) at baseline, consistent with no difference in basal activity of granule cells between genotypes. Surprisingly, when mice were assayed 1 h after handling-associated convulsions, KO mice had fewer c-fos-positive cells but dramatically increased ΔFosB expression in the dentate gyrus compared with WT mice. After administration of a subthreshold pentylenetetrazol dose, however, KO mice dentate had significantly more c-fos expression compared with WT mice. Other histopathological markers of TLE in these mice, including markers of neurogenesis, glial activation, and mossy fiber sprouting, were similar between WT and KO mice, apart from a small but statistically significant increase in hilar mossy cell density, opposite to what is typically found in mice with TLE. This suggests that the differences in seizure-associated dentate gyrus function in the absence of α2δ-2 protein are likely due to altered functional properties of the network without associated structural changes in the hippocampus at the typical age of seizure onset.

S-3,4-DCPG, a potent orthosteric agonist for the mGlu8 receptor, facilitates extinction and inhibits the reinstatement of morphine-induced conditioned place preference in male rats

The limbic system, particularly the NAc, shows a high concentration of metabotropic glutamate receptors (mGluRs). Recent evidence suggests the significant involvement of mGluRs in mental disorders, including substance abuse and addiction. The objective of this study was to examine the involvement of mGlu8 receptors in the NAc in the mechanisms underlying the extinction and reinstatement of conditioned place preference (CPP) induced by morphine. Male Wistar rats underwent surgical implantation of bilateral cannulas in the NAc and were assessed in a CPP protocol. In study 1 at the same time as the extinction phase, the rats were given varying doses of S-3,4-DCPG (0.03, 0.3, and 3 μg/0.5 μl). In study 2, rats that had undergone CPP extinction were given S-3,4-DCPG (0.03, 0.3, and 3 μg/0.5 μl) five minutes prior to receiving a subthreshold dose of morphine (1 mg/kg) in order to reactivate the previously extinguished morphine response. The findings demonstrated that administering S-3,4-DCPG directly into the accumbens nucleus resulted in a decrease in the duration of the CPP extinction phase. Moreover, dose-dependent administration of S-3,4-DCPG into the NAc inhibited CPP reinstatement. The observations imply that microinjection of S-3,4-DCPG as a potent orthosteric agonist with high selectivity for the mGlu8 receptor into the NAc promotes the process of extinction while concurrently exerting inhibitory effects on the reinstatement of morphine-induced CPP. This effect may be associated with the modulation of glutamate engagement within the NAc and the plasticity of reward pathways at the synaptic level.

Дозовая зависимость смертности от болезней системы кровообращения у работников ядерной индустрии (систематический обзор и pooled-анализ): отсутствие эффекта малых доз и подтверждение порога, установленного НКДАР и МКРЗ при 0,5 Гр

Based on the maintained database (source database) on effects in nuclear workers (NW), a selection of major studies of the relationship between mortality from diseases of the circulatory system (CVD; codes 390–459 according to ICD-9 and I00–I99 according to ICD-10) and external radiation dose. The sample included 30 papers and covered cohorts from 6 countries plus an NW cohort from 15 countries. For the sample, in most cases based on published standardized mortality rates (SMR), the relative risks (RR) of mortality from CVD were calculated for the selected dose groups with subsequent processing of the material for outliers. Initial: n = 207; final sample: n = 199; covers very low (0–10 mSv; 15.8 % of the sample), low (&gt;10–100 mSv; 45.8 %) and moderate (&gt;100–1000 mSv; 36.4 %) doses; data for high doses (&gt;1000 mSv; n = 4; 2 % of the sample), due to dubiousness, were excluded. A systematic review and pooled analysis of the RR for mortality from CVD depending on the dose on an ordinal scale was performed on the final sample. For the entire dose range (0–1000 mSv) and for moderate doses, statistically significant trends in increasing RR were found when expressed in five types of regressions (except for the logarithmic one for the entire range). Although the r values were small (0.230–0.293), the effect was clear. The ERR per 1 Gy (Sv) calculated for moderate doses using linear regression was 0.54. This value is higher than those obtained previously in meta-analyses, but should be considered as the most adequate. No dose relationship was found for the very low + low dose range (0–100 mGy); the r coefficients for the regressions were either negligible or negative at statistical insignificance. For the subthreshold dose range for CVD mortality after exposure (according to UNSCEAR and ICRP: 500 mSv), only a weak trend towards an increase in RR was found, statistically insignificant, despite the large sample size (n = 191), while for the dose range 500–1000 mSv, the highest tendency among the pooled analyzes was revealed to increase the risk depending on the level of exposure (r = 0.297–0.423; statistically insignificant due to the small sample size: n = 8). It is concluded that for mortality from CVD after irradiation, the threshold value of 0.5 Gy established by UNSCEAR and ICRP and confirmed in the present pooled analysis should be strictly adhered to. Due to the lack of effects of low doses, it is inappropriate to raise the issue of low dose effects in the context of these pathologies.

Subthreshold Doses of Inflammatory Mediators potentiate One Another to Elicit Reflex Cardiorespiratory Responses in Anesthetized Rats.

Reflex cardio-vascular and respiratory (CVR) alterations evoked by intraarterial instillation of nociceptive agents are termed vasosensory reflexes. Such responses elicited by optimal doses of inflammatory mediators have been described in our earlier work. The present study was designed to evaluate the interactions between subthreshold doses of inflammatory mediators on perivascular nociceptive afferents in urethane anesthetized rats. Healthy male adult rats (Charles-Foster strain) were anesthetized with an intraperitoneal injection of urethane. After anesthesia, the right femoral artery was cannulated. Respiratory movements, blood pressure, and electrocardiogram were recorded. The interactions between subthreshold doses of algogens in the elicitation of vasosensory reflex responses were studied by instillation of bradykinin (1 nM) and histamine (100 μM) into the femoral artery one after the other, in either temporal combination in separate groups of rats. The CVR responses obtained in these groups were then compared with the responses produced by 100 μM histamine and 1 nM bradykinin in saline-pretreated groups, which served as control. Subthreshold doses of histamine elicited transient tachypnoeic, hyperventilatory, hypotensive, and bradycardiac responses, in rats pretreated with subthreshold doses of bradykinin [p < 0.01, two-sided Dunnett's test] but not in saline pretreated groups [p > 0.05, two-sided Dunnett's test]. Similar responses were elicited by bradykinin after histamine pretreatment compared to the saline-pretreated group. Furthermore, CVR responses produced by histamine in the bradykininpretreated group were greater in magnitude as compared to bradykinin-induced responses in the histamine-pretreated group [p < 0.05, two-sided Dunnett's test]. The present study demonstrates that both bradykinin and histamine potentiate one another in the elicitation of vasosensory reflex responses, and bradykinin is a better potentiator than histamine at the level of perivascular nociceptive afferents in producing reflex CVR changes.

Unraveling the directional relationship of sleep and migraine-like pain.

Migraine and sleep disorders are common co-morbidities. Patients frequently link their sleep to migraine attacks suggesting a potential causal relationship between these conditions. However, whether migraine pain promotes or disrupts sleep or whether sleep disruption can increase the risk of migraine remains unknown. We assessed the potential impact of periorbital allodynia, a measure consistent with migraine-like pain, from multiple preclinical models on sleep quantity and quality. Additionally, we evaluated the possible consequences of sleep deprivation in promoting susceptibility to migraine-like pain. Following the implantation of electroencephalogram/electromyography electrodes to record sleep, mice were treated with either single or repeated systemic injections of nitroglycerin at the onset of their active phase (i.e. nocturnal awake period). Neither single nor repeated nitroglycerin affected the total sleep time, non-rapid eye movement sleep, rapid eye movement sleep, sleep depth or other measures of sleep architecture. To account for the possible disruptive effects of the surgical implantation of electroencephalogram/electromyography electrodes, we used immobility recordings as a non-invasive method for assessing sleep-wake behaviour. Neither single nor repeated nitroglycerin administration during either the mouse sleep (i.e. daylight) or active (i.e. night) periods influenced immobility-defined sleep time. Administration of an inflammatory mediator mixture onto the dura mater at either sleep or active phases also did not affect immobility-defined sleep time. Additionally, inhalational umbellulone-induced migraine-like pain in restraint-stressed primed mice did not alter immobility-defined sleep time. The possible influence of sleep disruption on susceptibility to migraine-like pain was evaluated by depriving female mice of sleep over 6 h with novel objects, a method that does not increase circulating stress hormones. Migraine-like pain was not observed following acute sleep deprivation. However, in sleep-deprived mice, subthreshold doses of systemic nitroglycerin or dural calcitonin gene-related peptide induced periorbital cutaneous allodynia consistent with migraine-like pain. Our data reveal that while migraine-like pain does not significantly disrupt sleep, sleep disruption increases vulnerability to migraine-like pain suggesting that a therapeutic strategy focused on improving sleep may diminish migraine attacks.

Expression of active caspase 3 in the rat lens after in vivo exposure to subthreshold dose of UVR-B

PurposesThe aim of this study is to investigate the time evolution of active caspase 3 within first 120 h in the rat lens after in vivo exposure to subthreshold dose of UVR-B.MethodsTwenty three six-week-old female albino Sprague-Dawley rats were exposed to subthreshold dose (1 kJ/m2) of UVR-B unilaterally and sacrificed at 24, 41, 70 and 120 h after exposure. Lenses were enucleated and active caspase 3 was detected by Western Blot. The time evolution of active caspase 3 was then plotted as a function of relative mean difference in active caspase 3 between exposed and nonexposed lenses.ResultsThere is expression of active caspase 3 in both exposed and nonexposed lenses but there is no difference in relative mean difference in active caspase 3 between exposed and nonexposed lenses in all four postexposure groups.ConclusionsExposure to subthreshold dose of UVR-B does not induce apoptosis in the rat lens in vivo within first 120 h though there is a non-significant increase of active caspase 3 at 120 h. Increase in sample size might reduce the variation level in expression of active caspase 3 in the rat lenses.

Microdosing psychedelics and the risk of cardiac fibrosis and valvulopathy: Comparison to known cardiotoxins.

Though microdosing psychedelics has become increasingly popular, its long-term effects on cardiac health remain unknown. Microdosing most commonly involves ingesting sub-threshold doses of lysergic acid diethylamide (LSD), psilocybin, or other psychedelic drugs 2-4 times a week for at least several weeks, but potentially months or years. Concerningly, both LSD and psilocybin share structural similarities with medications which raise the risk of cardiac fibrosis and valvulopathy when taken regularly, including methysergide, pergolide, and fenfluramine. 3,4-Methylenedioxymethamphetamine, which is also reportedly used for microdosing, is likewise associated with heart valve damage when taken chronically. In this review, we evaluate the evidence that microdosing LSD, psilocybin, and other psychedelics for several months or more could raise the risk of cardiac fibrosis. We discuss the relationship between drug-induced cardiac fibrosis and the 5-HT2B receptor, and we make recommendations for evaluating the safety of microdosing psychedelics in future studies.

Exposure to subthreshold dose of UVR‐B does not induce increase in caspase 3 mRNA expression in the rat lens in vivo within first 24 hours

Aims/Purpose: The aim of this study is to investigate the time evolution of caspase 3 mRNA expression within first 24 h in the rat lens after in vivo exposure to subthreshold dose of UVR‐B.Methods: Twelve six‐week‐old female albino Sprague–Dawley rats were exposed to subthreshold dose (1 kJ/m2) of UVR‐B unilaterally and sacrificed at 1, 8, 16 and 24 h after exposure. Lenses were enucleated and caspase 3 mRNA expression was detected by qRT‐PCR. The time evolution of caspase 3 mRNA expression was then plotted as a function of mean ratios in caspase 3 mRNA expression between exposed and contralateral nonexposed lenses.Results: There is no difference in mean ratios in caspase 3 mRNA expression between exposed and nonexposed lenses in all four postexposure groups.Conclusions: Exposure to subthreshold dose of UVR‐B does not induce increase in caspase 3 mRNA expression in the rat lens in vivo within first 24 h.

Fine-Regional Role of the Claustrum in Anxiety and Higher Sensitivity to Cocaine in Adolescent Cocaine-Exposed Male Mice during Adulthood.

Adolescent cocaine exposure (ACE) induces anxiety and higher sensitivity to substances abuse during adulthood. Here, we show that the claustrum is crucial for controlling these psychiatric problems in male mice. In anxiety-like behavioral tests, the CaMKII-positive neurons in the median portion of the claustrum (MClaustrum) were triggered, and local suppression of these neurons reduced the anxiety-like behavior in ACE mice during adulthood. In contrast, the CaMKII-positive neurons in the anterior portion of the claustrum (AClaustrum) were more activated in response to subthreshold dose of cocaine induced conditioned place preference (CPP), and local suppression of these neurons blocked the acquisition of cocaine CPP in ACE mice during adulthood. Our findings for the first time identified the fine-regional role of the claustrum in regulating the anxiety and susceptibility to cocaine in ACE mice during adulthood, extending our understanding of the claustrum in substance use disorder.