Changes In Receptor Density Research Articles

Abstract 1650: Non-invasive imaging tool to predict BRCA2 silencing in the context of prostate cancer

Abstract Background: We recently demonstrated that biallelic as well as monoallelic loss of the DNA damage response (DDR) gene BRCA2 in prostate cancer (PC) cell lines and mCRPC organoids leads to an aggressive form of PC and early androgen deprivation therapy (ADT) resistance (Chakraborty et al.). Clinical studies have established that DDR deficiency is generally associated with a poor prognosis. Very recently, immunohistochemical analysis of mCRPC patient samples by Paschalis et al. revealed that defects in DDR genes (in particular BRCA2 and ATM) are associated with increased prostate-specific membrane antigen (PSMA; folate hydrolase, FOLH1) expression on the cell membrane. PSMA expression can be measured non-invasively in pre-clinical models and human subjects using one of the several positron emission tomography (PET) imaging agents such as [68Ga]-PSMA11 or [124I]-MSK-PSMA11 that are being evaluated in pre-clinical and/or clinical setting. Therefore, we hypothesized that upregulation of PSMA expression can be a marker for BRCA2 loss and this increased expression can be quantified using PET agents both in vitro and in vivo. Experimental design: We investigated the effect of BRCA2 deletion on PSMA expression in the castration sensitive human PC cell line LNCaP at the transcriptional and translational level and quantified the changes using saturation binding assays with [124I]-MSK-PSMA11. Using CRISPR-Cas9 and RNAi-based methods, we silenced BRCA2 in the castration sensitive cell line LNCaP and evaluated its effect on PSMA at the transcriptional and translational level. We carried out saturation binding assay using [124I]-MSK-PSMA11 to measure changes in cell surface PSMA receptor density. Results: BRCA2 knockout was achieved successfully using CRISPR-Cas9 based methods. Immunoblotting analysis revealed that BRCA2 loss resulted in a significant increase in PSMA levels when compared to control LNCaP cell line. Immunohistochemical analysis confirmed this obervation. Cell binding assays demonstrated that BRCA2 null LNCaP cell lines have about 5-6 fold higher uptake of the PET tracer [124I]-MSK-PSMA11. We will be conducting in vivo studies to demonstrate that BRCA2 deletion leads to a significant increase in PSMA signal in mice xenograft models. Conclusions: Our results indicate that BRCA2 silencing leads to significant upregulation of PSMA expression in PC cell lines, which can be imaged using a PSMA targeted PET tracer. These studies were partly supported by DOD-PCRP-Grant # W81XWH-19-1-0536 and PCF Young Investigator Award to Goutam Chakraborty.

Inhibition of Colony-Stimulating Factor 1 Receptor by PLX3397 Prevents Amyloid Beta Pathology and Rescues Dopaminergic Signaling in Aging 5xFAD Mice.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease. In this study, to investigate the effect of microglial elimination on AD progression, we administered PLX3397, a selective colony-stimulating factor 1 receptor inhibitor, to the mouse model of AD (5xFAD mice). Amyloid-beta (Aβ) deposition and amyloid precursor protein (APP), carboxyl-terminal fragment β, ionized calcium-binding adaptor molecule 1, synaptophysin, and postsynaptic density (PSD)-95 levels were evaluated in the cortex and hippocampus. In addition, the receptor density changes in dopamine D2 receptor (D2R) and metabotropic glutamate receptor 5 were evaluated using positron emission tomography (PET). D2R, tyrosine hydroxylase (TH), and dopamine transporter (DAT) levels were analyzed in the brains of Tg (5xFAD) mice using immunohistochemistry. PLX3397 administration significantly decreased Aβ deposition following microglial depletion in the cortex and hippocampus of Tg mice. In the neuro-PET studies, the binding values for D2R in the Tg mice were lower than those in the wild type mice; however, after PLX3397 treatment, the binding dramatically increased. PLX3397 administration also reversed the changes in synaptophysin and PSD-95 expression in the brain. Furthermore, the D2R and TH expression in the brains of Tg mice was significantly lower than that in the wild type; however, after PLX3397 administration, the D2R and TH levels were significantly higher than those in untreated Tg mice. Thus, our findings show that administering PLX3397 to aged 5xFAD mice could prevent amyloid pathology, concomitant with the rescue of dopaminergic signaling, suggesting that targeting microglia may serve as a useful therapeutic option for neurodegenerative diseases, including AD.

Identifying Musculoskeletal Pain Generators Using Clinical PET.

Identifying the source of a person's pain is a significant clinical challenge because the physical sensation of pain is believed to be subjective and difficult to quantify. The experience of pain is not only modulated by the individual's threshold to painful stimuli but also a product of the person's affective contributions, such as fear, anxiety, and previous experiences. Perhaps then to quantify pain is to examine the degree of nociception and pro-nociceptive inflammation, that is, the extent of cellular, chemical, and molecular changes that occur in pain-generating processes. Measuring changes in the local density of receptors, ion channels, mediators, and inflammatory/immune cells that are involved in the painful phenotype using targeted, highly sensitive, and specific positron emission tomography (PET) radiotracers is therefore a promising approach toward objectively identifying peripheral pain generators. Although several preclinical radiotracer candidates are being developed, a growing number of ongoing clinical PET imaging approaches can measure the degree of target concentration and thus serve as a readout for sites of pain generation. Further, when PET is combined with the spatial and contrast resolution afforded by magnetic resonance imaging, nuclear medicine physicians and radiologists can potentially identify pain drivers with greater accuracy and confidence. Clinical PET imaging approaches with fluorine-18 fluorodeoxyglucose, fluorine-18 sodium fluoride, and sigma-1 receptor PET radioligand and translocator protein radioligands to isolate the source of pain are described here.

Accounting for red blood cell accessibility reveals distinct invasion strategies in Plasmodium falciparum strains.

The growth of the malaria parasite Plasmodium falciparum in human blood causes all the symptoms of malaria. To proliferate, non-motile parasites must have access to susceptible red blood cells, which they invade using pairs of parasite ligands and host receptors that define invasion pathways. Parasites can switch invasion pathways, and while this flexibility is thought to facilitate immune evasion, it may also reflect the heterogeneity of red blood cell surfaces within and between hosts. Host genetic background affects red blood cell structure, for example, and red blood cells also undergo dramatic changes in morphology and receptor density as they age. The in vivo consequences of both the accessibility of susceptible cells, and their heterogeneous susceptibility, remain unclear. Here, we measured invasion of laboratory strains of P. falciparum relying on distinct invasion pathways into red blood cells of different ages. We estimated invasion efficiency while accounting for red blood cell accessibility to parasites. This approach revealed different tradeoffs made by parasite strains between the fraction of cells they can invade and their invasion rate into them, and we distinguish “specialist” strains from “generalist” strains in this context. We developed a mathematical model to show that generalist strains would lead to higher peak parasitemias in vivo compared to specialist strains with similar overall proliferation rates. Thus, the ecology of red blood cells may play a key role in determining the rate of P. falciparum parasite proliferation and malaria virulence.

Supplemental taurine during adolescence and early adulthood has sex-specific effects on cognition, behavior and neurotransmitter levels in C57BL/6J mice dependent on exposure window

The mammalian brain goes through final maturation during late adolescence and early adulthood with sex differences in timing. The key cellular processes, including changes in neurotransmitter receptor density and synaptic pruning, make this age uniquely vulnerable to neurotoxic insults. Teenagers and young adults are the major consumers of energy drinks, which contain high levels of taurine and caffeine. Taurine is one of the most abundant amino acids in the central nervous system, but the effects of supplemental taurine consumption during adolescence has not been well studied. We conducted an initial short-term exposure study with 0.12% taurine in drinking water and a long-term exposure dose-response study using 0.06 and 0.12% taurine in male and female C57BL/6J mice. We examined a broad range of cognitive functions and behaviors and measured neurotransmitter levels. We found no significant differences in anxiety, open field locomotor activity, or sensorimotor gating. However, we found impairments in novel object recognition and sex differences in Morris water maze. When taurine treatment stopped before behavioral experiments began, male mice had significant impairments in spatial learning and memory. In the dose-response study when taurine treatment continued throughout behavioral experiments, females had significant impairments. We also found sex differences in neurotransmitter levels with females having higher levels of glutamate, DOPAC and 5-HIAA. We conclude that both females and males are at risk from excess taurine consumption during final brain maturation.

Adenosine kinase and adenosine receptors A1R and A2AR in temporal lobe epilepsy and hippocampal sclerosis and association with risk factors for SUDEP

The "adenosine hypothesis of SUDEP" (sudden unexpected death in epilepsy) predicts that a seizure-induced adenosine surge combined with impaired metabolic clearance can foster lethal apnea or cardiac arrest. Changes in adenosine receptor density and adenosine kinase (ADK) occur in surgical epilepsy patients. Our aim was to correlate the distribution of ADK and adenosine A2A and A1 receptors (A2A R and A1 R) in surgical tissue from patients with temporal lobe epilepsy and hippocampal sclerosis (TLE/HS) with SUDEP risk factors. In 75 cases, patients were stratified into high-risk (n=16), medium-risk (n=11) and low-risk (n=48) categories according to the frequency of generalized seizures before surgery. Using whole-slide scanning Definiens image analysis we quantified the labeling index (LI) for ADK, A2A R, and A1 R in seven regions of interest: temporal cortex, temporal lobe white matter, CA1, CA4, dentate gyrus, subiculum, and amygdala and relative to glial and neuronal densities with glial fibrillary acidic protein (GFAP) and neuronal nuclear antigen (NeuN). A1 R showed predominant neuronal, A2A R astroglial, and ADK nuclear labeling in all regions but with significant variation. Compared with the low-risk group, the high-risk group had significantly lower A2A R LI in the temporal cortex. In HS cases with severe neuronal cell loss and gliosis predominantly in the CA1 and CA4 regions, significantly higher A1 R was present in the amygdala in high-risk than in low-risk cases. There was no significant difference in neuronal loss or gliosis between the risk groups or differences for ADK labeling. Reduced cortical A2A R suggests glial dysfunction and impaired adenosine modulation in response to seizures in patients at higher risk for SUDEP. Increased neuronal A1 R in the high-risk group could contribute to periictal amygdala dysfunction in SUDEP.

The luminescent HiBiT peptide enables selective quantitation of G protein–coupled receptor ligand engagement and internalization in living cells

G protein-coupled receptors (GPCRs) are prominent targets to new therapeutics for a range of diseases. Comprehensive assessments of their cellular interactions with bioactive compounds, particularly in a kinetic format, are imperative to the development of drugs with improved efficacy. Hence, we developed complementary cellular assays that enable equilibrium and real-time analyses of GPCR ligand engagement and consequent activation, measured as receptor internalization. These assays utilize GPCRs genetically fused to an N-terminal HiBiT peptide (1.3 kDa), which produces bright luminescence upon high-affinity complementation with LgBiT, an 18-kDa subunit derived from NanoLuc. The cell impermeability of LgBiT limits signal detection to the cell surface and enables measurements of ligand-induced internalization through changes in cell-surface receptor density. In addition, bioluminescent resonance energy transfer is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent tracers. The sensitivity and dynamic range of these assays benefit from the specificity of bioluminescent resonance energy transfer and the high signal intensity of HiBiT/LgBiT without background luminescence from receptors present in intracellular compartments. These features allow analyses of challenging interactions having low selectivity or affinity and enable studies using endogenously tagged receptors. Using the β-adrenergic receptor family as a model, we demonstrate the versatility of these assays by utilizing the same HiBiT construct in analyses of multiple aspects of GPCR pharmacology. We anticipate that this combination of target engagement and proximal functional readout will prove useful to the study of other GPCR families and the development of new therapeutics.

Developmental Changes of Glutamate and GABA Receptor Densities in Wistar Rats.

Neurotransmitters and their receptors are key molecules of signal transduction and subject to various changes during pre- and postnatal development. Previous studies addressed ontogeny at the level of neurotransmitters and expression of neurotransmitter receptor subunits. However, developmental changes in receptor densities to this day are not well understood. Here, we analyzed developmental changes in excitatory glutamate and inhibitory γ-aminobutyric acid (GABA) receptors in adjacent sections of the rat brain by means of quantitative in vitro receptor autoradiography. Receptor densities of the ionotropic glutamatergic receptors α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA) as well as of the ionotropic GABAA and metabotropic GABAB receptors were investigated using specific high-affinity ligands. For each receptor binding site, significant density differences were demonstrated in the investigated regions of interest [olfactory bulb, striatum, hippocampus, and cerebellum] and developmental stages [postnatal day (P) 0, 10, 20, 30 and 90]. In particular, we showed that the glutamatergic and GABAergic receptor densities were already present between P0 and P10 in all regions of interest, which may indicate the early relevance of these receptors for brain development. A transient increase of glutamatergic receptor densities in the hippocampus was found, indicating their possible involvement in synaptic plasticity. We demonstrated a decline of NMDA receptor densities in the striatum and hippocampus from P30 to P90, which could be due to synapse elimination, a process that redefines neuronal networks in postnatal brains. Furthermore, the highest increase in GABAA receptor densities from P10 to P20 coincides with the developmental shift from excitatory to inhibitory GABA transmission. Moreover, the increase from P10 to P20 in GABAA receptor densities in the cerebellum corresponds to a point in time when functional GABAergic synapses are formed. Taken together, the present data reveal differential changes in glutamate and GABA receptor densities during postnatal rat brain development, which may contribute to their specific functions during ontogenesis, thus providing a deeper understanding of brain ontogenesis and receptor function.

Особенности свободнорадикального окисления и динамики содержания кортикостероидных рецепторов в легких у животных с разной чувствительностью к гипоксии в постреанимационном периоде

Цель исследования - изучение особенностей динамики показателей свободнорадикального окисления и уровня кортикостероидных рецепторов в легких у животных с разной чувствительностью к гипоксии в постреанимационном периоде. Методика. Эксперимент выполнен на самцах беспородных белых крыс, разделенных по устойчивости к гипоксии на 2 группы: неустойчивые и высокоустойчивые. Остановку системного кровообращения продолжительностью 5 мин моделировали под общим эфирным наркозом (через 1 нед после тестирования на устойчивость к гипоксии) интраторакальным пережатием сосудистого пучка сердца с последующей реанимацией. Через 1, 3, 5, 7, 14, 21 и 35 сут животных под эфирным наркозом декапитировали. В плазме крови определяли содержание кортикостерона и альдостерона, в гомогенатах легких - концентрацию глюкокортикоидных и минералокортикоидных рецепторов, уровни продуктов, реагирующих с тиобарбитуровой кислотой, карбонилированных белков, железозависимое образование битирозина, активность супероксиддисмутазы, каталазы, содержание восстановленного глутатиона. Результаты. Установлено, что для неустойчивых к гипоксии животных характерна высокая интенсивность свободнорадикального окисления, проявляющаяся развитием карбонильного стресса на фоне снижения активности ключевых антиоксидантов и сопряженная с выраженными изменениями динамики кортикостероидных рецепторов: в первые 3 сут постреанимационного периода наблюдалось снижение уровня кортикостероидных рецепторов, а к концу 35-суточного мониторинга значительное нарастание концентрации минералокортикоидных рецепторов. Высокую степень устойчивости к гипоксии отличает относительно низкая интенсивность свободнорадикального окисления, характеризующаяся только усилением липопероксидации за счет адекватной емкости и сохранности водорастворимых антиоксидантных систем, экранирующих белки. В раннем восстановительном периоде это сопровождается сохранением уровня кортикостероидных рецепторов, в позднем - существенным ростом концентрации глюкокортикоидных рецепторов. Заключение. Увеличение содержания глюкокортикоидных рецепторов у высокоустойчивых к гипоксии животных в позднем восстановительном периоде (14-е - 35-е сут) играет важную роль, предположительно путем модулирования течения воспалительного процесса, ограничения неконтролируемых иммунных реакции и повреждение тканей. Следствием значительного нарастания концентрации минералокортикоидных рецепторов у животных с низкой устойчивостью к гипоксии может быть усиление сосудистого ремоделирования и развитие фиброза в легких. Aim. The aim of the study was to characterize changes in free radical oxidation and corticosteroid receptor density in the lungs of animals with different sensitivity to hypoxia in the postresuscitation period. Methods. Experiments were performed on mongrel male white rats divided into two groups based on their hypoxia resistance, non-resistant and highly resistant. One week after testing the rats for resistance to hypoxia, a 5-min arrest of systemic circulation was modeled under ether anesthesia by intrathoracic compression of the vascular bundle of the heart with subsequent resuscitation. The follow-up period was 35 days. At 1, 3, 5, 7, 14, 21, and 35 days, the animals were sacrificed by decapitation under ether anesthesia, and blood and tissue samples were collected. Concentrations of corticosterone and aldosterone were measured in plasma; concentrations of glucocorticoid and mineralocorticoid receptors, thiobarbituric acid reactive substances, and carbonylated proteins, iron-dependent formation of dityrosine, activities of superoxide dismutase and catalase, and concentration of reduced glutathione were measured in lung homogenates. Results. Hypoxia non-resistant rats had a high intensity of free radical oxidation as evident from the development of carbonyl stress associated with decreased activities of key antioxidants and pronounced changes in the dynamics of corticosteroid receptors. A reduced level of corticosteroid receptors was observed in the first three days of resuscitation period and followed by a significant increase in mineralocorticoid receptors at the end of 35-day monitoring. High resistance to hypoxia was characterized by a relatively low intensity of free radical oxidation, evident only from increased lipid peroxidation, due to an adequate capacity and preservation of water-soluble antioxidants. During the early recovery period, high resistance to hypoxia was associated with preserved level of corticosteroid receptors whereas during late recovery, the high hypoxia resistance was associated with a significant increase in the concentration of glucocorticoid receptors. Conclusion. The increased density of glucocorticoid receptors in hypoxia high-resistant animals during the late recovery period (days 14-35) plays an important role, presumably by modulating the inflammatory process and restricting uncontrolled immune responses and tissue damage. The significant increase in mineralocorticoid receptors in hypoxia low-resistance animals may result in stimulation of vascular remodeling and development of pulmonary fibrosis.

Activity-Based Training Reverses Spinal Cord Injury-Induced Changes in Kidney Receptor Densities and Membrane Proteins.

Complications in upper and lower urinary function arise after spinal cord injury (SCI), which creates a significant impact on quality of life for those affected. One upper urinary complication is SCI-induced polyuria, or the overproduction of urine, of which the underlying mechanisms have yet to be elucidated. Activity-based training (ABT) has been utilized in both animal and clinical settings as a rehabilitative therapy to improve many issues that arise after SCI, including more recently urogenital function. The goal of the current study was to identify potential mechanisms contributing to previously identified improvements in polyuria with ABT, using a male rat moderate-severe spinal contusion model. Although ABT had no significant effect on reversing injury-induced alterations of serum arginine vasopressin and urinary atrial natriuretic peptide levels, there was a dramatic effect upon the receptors of these fluid balance hormones (vasopressin receptor 2 and natriuretic peptide A receptor), as well as kidney aquaporin 2 and sodium channels. ABT changes in densities of key receptors and kidney membrane proteins involved in fluid balance after chronic SCI support the likelihood of multiple mechanisms through which exercise can positively influence urinary tract function after SCI. By understanding the mechanisms, amount, and timing regarding how ABT improves different aspects of urinary function, more targeted training strategies can be developed to optimize the functional gains within the SCI population.

Extinction training following cocaine or MDMA self-administration produces discrete changes in D2-like and mGlu5 receptor density in the rat brain.

Several studies strongly support the role of the dopamine D2-like and glutamate mGlu5 receptors in psychostimulant reward and relapse. The present study employed cocaine or MDMA self-administration with yoked-triad procedure in rats to explore whether extinction training affects the drug-seeking behavior and the D2-like and mGlu5 receptor Bmax and Kd values in several regions of the animal brain. Both cocaine and MDMA rats developed maintenance of self-administration, but MDMA evoked lower response rates and speed of self-administration acquisition. During reinstatement tests, cocaine or MDMA seeking behavior was produced by either exposure to the drug-associated cues or drug-priming injections. The extinction training after cocaine self-administration did not alter significantly D2-like receptor expression the in the limbic and subcortical brain areas, while MDMA yoked rats showed a decrease of the D2-like binding density in the nucleus accumbens and increase in the hippocampus and a rise of affinity in the striatum and hippocampus. Interestingly, in the prefrontal cortex a reduction in the mGlu5 receptor density in cocaine- or MDMA-abstinent rats was demonstrated, with significant effects being observed after previous MDMA exposure. Moreover, rats self-administered cocaine showed a rise in the density of mGlu5 receptor for the nucleus accumbens. This study first time shows that abstinence followed extinction training after cocaine or MDMA self- or passive-injections changes the D2-like and mGlu5 density and affinity. The observed changes in the expression of both receptors are brain-region specific and related to either pharmacological and/or motivational features of cocaine or MDMA.

Astrocytic modulation of glutamatergic synaptic transmission is reduced in NTS of rats submitted to short-term sustained hypoxia.

Sustained hypoxia (SH) activates chemoreceptors to produce cardiovascular and respiratory responses to bring the arterial partial pressure of O2 back to the physiological range. We evaluated the effect of SH (fraction of inspired O2 = 0.10, 24 h) on glutamatergic synaptic transmission and the interaction neuron-astrocyte in neurons of the nucleus tractus solitarii (NTS). Tractus solitarius (TS) fiber stimulation induced glutamatergic currents in neurons and astrocytes. SH increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/kainate) [-183 ± 122 pA (n = 10) vs. -353 ± 101 pA (n = 10)] and N-methyl-d-aspartate (NMDA) current amplitude [61 ± 10 pA (n = 7) vs. 102 ± 37 pA (n = 10)]. To investigate the effects of SH, we used fluoroacetate (FAC), an astrocytic inhibitor, which revealed an excitatory modulation on AMPA/kainate current and an inhibitory modulation of NMDA current in control rats. SH blunted the astrocytic modulation of AMPA [artificial cerebrospinal fluid (aCSF): -353 ± 101 pA vs. aCSF + FAC: -369 ± 76 pA (n = 10)] and NMDA currents [aCSF: 102 ± 37 pA vs. aCSF + FAC: 108 ± 32 pA (n = 10)]. SH increased AMPA current density [control: -6 ± 3.5 pA/pF (n = 6) vs. SH: -20 ± 12 pA/pF (n = 7)], suggesting changes in density, conductance, or affinity of AMPA receptors. SH produced no effect on astrocytic resting membrane potential, input resistance, and AMPA/kainate current. We conclude that SH decreased the neuron-astrocyte interaction at the NTS level, facilitating the glutamatergic transmission, which may contribute to the enhancement of cardiovascular and respiratory responses to baro- and chemoreflexes activation in SH rats. NEW & NOTEWORTHY Using an electrophysiological approach, we have shown that in nucleus tractus solitarii (NTS) from control rats, astrocytes modulate the AMPA and NMDA currents in NTS neurons, changing their excitability. Sustained hypoxia (SH) increased both glutamatergic currents in NTS neurons due to 1) a reduction in the astrocytic modulation and 2) an increase in the density of AMPA receptors. These new findings show the importance of neuron-astrocyte modulation in the excitatory synaptic transmission in NTS of control and SH rats.

Allosteric modulation of sigma receptors by BH3 mimetics ABT-737, ABT-263 (Navitoclax) and ABT-199 (Venetoclax)

ABT-737, ABT-263 (Navitoclax) and ABT-199 (Venetoclax) are under intensive preclinical and clinical investigation as treatments for hematologic and other malignancies. These small molecules mimic pro-death B-cell lymphoma-2 (Bcl-2) Homology 3 (BH3) domain-only proteins. They also bear a structural resemblance to certain sigma (σ) receptor ligands. Moreover, the Bcl-2 and σ receptor protein families are both located primarily at the endoplasmic reticulum, mediate cell death and survival through protein–protein interactions, and physically associate. Accordingly, we examined the ability of the ABT series of BH3 mimetics to interact with σ receptors using radioligand-binding techniques. Negative allosteric modulation of [3H](+)-pentazocine, an agonist, binding to σ1 receptors in guinea pig brain membranes was observed for ABT-737, ABT-263 and ABT-199. Findings included reduction of specific binding to distinct plateaus in concentration-dependent fashion, significant slowing of radioligand dissociation kinetics, and decreases in radioligand affinity with no or modest changes in maximal receptor densities. Using a ternary complex model, dissociation constants (KX) for modulator binding to the σ1 receptor ranged from 1 to 2.5 μM, while negative cooperativity factors (α), representing the changes in affinity of ligand and modulator when bound as a ternary complex with the receptor, ranged from 0.15 to 0.42. These observations were extended and reinforced by studies using intact small cell (NCI-H69) and non-small cell (NCI-H23) lung cancer cells, and by using an antagonist σ1 receptor radioligand, E-N-1-(3′-[125I]iodoallyl)-N′-4-(3″,4″-dimethoxyphenethyl)piperazine, in mouse brain membranes. By contrast, exploratory studies indicate marked enhancement of the σ2 receptor binding of [3H]1,3-di-(o-tolyl)guanidine/(+)-pentazocine in NCI-H23 cells and guinea pig brain membranes. These findings raise intriguing questions regarding mechanism and potential functional outcomes.

Intranasal oxytocin administration promotes emotional contagion and reduces aggression in a mouse model of callousness

Deficits in empathy, the ability to share an emotion of another individual, constitute a hallmark of several psychopathological conditions, including conduct disorder. The co-occurrence of excess rates of aggression, general violation of societal norms and callous-unemotional traits confers specific risk for adult psychopathy. In the present study, we relied on a recently devised experimental model of conduct disorder in mice to test the potential efficacy of intranasal oxytocin administration. Two subgroups of BALB/cJ male mice exhibiting opposite profiles in emotional contagion (i.e. socially transmitted adoption of another's emotional states) underwent a series of tests mapping onto reactive aggression, information processing, perseverative behaviour, punishment-related emotional memory, physiological arousal and hormonal stress reactivity, with or without intranasal oxytocin administration (5.0 or 20.0 μg/kg). Collectively, our data indicate that a trait of markedly reduced emotional contagion is associated with a behavioural syndrome of sensorimotor gating deficits, impaired emotional memory, increased aggression and stereotyped behaviours, dysregulations in the circadian rhythms of activity and body temperature and dampened physiological reactivity to external stressors. Moreover, in the absence of changes in oxytocin receptor density in the neural network involved in empathy-like behaviour, we showed that oxytocin administration normalised emotional contagion, aggression and behavioural stereotypies, thereby ameliorating the phenotype of mice characterised by deficient empathy-like behaviour. Besides, oxytocin led to a lower, more prolonged neuroendocrine response of the HPA-axis to stress in all mice. Ultimately, current data support the notion that oxytocin may constitute a valid therapeutic approach in disturbances characterised by abnormal aggression and excess callousness.

Endocannabinoid and Muscarinic Signaling Crosstalk in the 3xTg-AD Mouse Model of Alzheimer's Disease.

The endocannabinoid system, which modulates emotional learning and memory through CB1 receptors, has been found to be deregulated in Alzheimer's disease (AD). AD is characterized by a progressive decline in memory associated with selective impairment of cholinergic neurotransmission. The functional interplay of endocannabinoid and muscarinic signaling was analyzed in seven-month-old 3xTg-AD mice following the evaluation of learning and memory of an aversive stimulus. Neurochemical correlates were simultaneously studied with both receptor and functional autoradiography for CB1 and muscarinic receptors, and regulations at the cellular level were depicted by immunofluorescence. 3xTg-AD mice exhibited increased acquisition latencies and impaired memory retention compared to age-matched non-transgenic mice. Neurochemical analyses showed changes in CB1 receptor density and functional coupling of CB1 and muscarinic receptors to Gi/o proteins in several brain areas, highlighting that observed in the basolateral amygdala. The subchronic (seven days) stimulation of the endocannabinoid system following repeated WIN55,212-2 (1 mg/kg) or JZL184 (8 mg/kg) administration induced a CB1 receptor downregulation and CB1-mediated signaling desensitization, normalizing acquisition latencies to control levels. However, the observed modulation of cholinergic neurotransmission in limbic areas did not modify learning and memory outcomes. A CB1 receptor-mediated decrease of GABAergic tone in the basolateral amygdala may be controlling the limbic component of learning and memory in 3xTg-AD mice. CB1 receptor desensitization may be a plausible strategy to improve behavior alterations associated with genetic risk factors for developing AD.

GABAA Receptor Density Is Not Altered by a Novel Herbal Anxiolytic Treatment.

Anxiety disorders are highly prevalent and considered a major public health concern worldwide. Current anxiolytics are of limited efficacy and associated with various side effects. Our novel herbal treatment (NHT), composed of four constituents, was shown to reduce anxiety-like behavior while precluding a common side effect caused by current anxiolytics, i.e., sexual dysfunction. Nevertheless, NHT's mechanism of action is yet to be determined. There is evidence that some medicinal herbs interact with the GABAergic system. Therefore, we aimed to examine whether NHT's anxiolytic-like effect is exerted by alterations in GABAA receptor density in the hippocampus, prefrontal cortex, and hypothalamus. The effects of 3-weeks treatment with NHT on anxiety-like behavior and locomotion were assessed using the elevated plus maze (EPM) and the open field test (OFT), respectively. Regional GABAA receptor levels were analyzed using [3H] RO15-1788 high-affinity binding assays. In stressed mice, NHT reduced anxiety-like behavior similarly to the benzodiazepine, clonazepam, while locomotion remained intact. Lack of changes or minor changes in regional GABAA receptor density in the brain were induced by NHT or clonazepam. In naive mice, performance in the EPM, locomotion and GABAA receptor densities were not altered by treatment with NHT or clonazepam. These findings support NHT as an efficacious and safe anxiolytic, although the GABAergic involvement remains to be further elucidated.

GABAA Receptor Availability Changes Underlie Symptoms in Isolated Cervical Dystonia.

GABAA receptor availability changes within sensorimotor regions have been reported in some isolated forms of dystonia. Whether similar abnormalities underlie symptoms in cervical dystonia is not known. In the present study, a total of 15 cervical dystonia patients and 15 age- and sex-matched controls underwent 11C-flumazenil PET/CT scanning. The density of available GABAA receptors was estimated using a Simplified Reference Tissue Model 2. Group differences were evaluated using a two-sample T-test, and correlations with dystonia severity, as measured by the Toronto Western Spasmodic Torticollis Rating Scale, and disease duration were evaluated using a regression analysis. Voxel-based analyses revealed increased GABAA availability within the right precentral gyrus in brain motor regions previously associated with head turning and the left parahippocampal gyrus. GABAA availability within the bilateral cerebellum was negatively correlated with dystonia severity, and GABAA availability within the right thalamus and a variety of cerebellar and cortical regions were negatively correlated with disease duration. While GABAA availability changes within primary motor areas could represent a partial compensatory response to loss of inhibition within sensorimotor network, GABAergic signaling impairment within the cerebellum may be a key contributor to dystonia severity.

Low empathy-like behaviour in male mice associates with impaired sociability, emotional memory, physiological stress reactivity and variations in neurobiological regulations.

Deficits in empathy have been proposed to constitute a hallmark of several psychiatric disturbances like conduct disorder, antisocial and narcissistic personality disorders. Limited sensitivity to punishment, shallow or deficient affect and reduced physiological reactivity to environmental stressors have been often reported to co-occur with limited empathy and contribute to the onset of antisocial phenotypes. Empathy in its simplest form (i.e. emotional contagion) is addressed in preclinical models through the evaluation of the social transmission of emotional states: mice exposed to a painful stimulus display a higher response if in the presence of a familiar individual experiencing a higher degree of discomfort, than in isolation. In the present study, we investigated whether a reduction of emotional contagion can be considered a predictor of reduced sociality, sensitivity to punishment and physiological stress reactivity. To this aim, we first evaluated emotional contagion in a group of Balb/cJ mice and then discretised their values in four quartiles. The upper (i.e. Emotional Contagion Prone, ECP) and the lower (i.e. Emotional Contagion Resistant, ECR) quartiles constituted the experimental groups. Our results indicate that mice in the lower quartile are characterized by reduced sociability, impaired memory of negative events and dampened hypothalamic-pituitary-adrenocortical reactivity to external stressors. Furthermore, in the absence of changes in oxytocin receptor density, we show that these mice exhibit elevated concentrations of oxytocin and vasopressin and reduced density of BDNF receptors in behaviourally-relevant brain areas. Thus, not only do present results translate to the preclinical investigation of psychiatric disturbances, but also they can contribute to the study of emotional contagion in terms of its adaptive significance.

Dopamine and μ-opioid receptor dysregulation in the brains of binge-eating female rats - possible relevance in the psychopathology and treatment of binge-eating disorder.

Adult, female rats given irregular, limited access to chocolate develop binge-eating behaviour with normal bodyweight and compulsive/perseverative and impulsive behaviours similar to those in binge-eating disorder. We investigated whether (a) dysregulated central nervous system dopaminergic and opioidergic systems are part of the psychopathology of binge-eating and (b) these neurotransmitter systems may mediate the actions of drugs ameliorating binge-eating disorder psychopathology. Binge-eating produced a 39% reduction of striatal D1 receptors with 22% and 23% reductions in medial and lateral caudate putamen and a 22% increase of striatal μ-opioid receptors. There was no change in D1 receptor density in nucleus accumbens, medial prefrontal cortex or dorsolateral frontal cortex, striatal D2 receptors and dopamine reuptake transporter sites, or μ-opioid receptors in frontal cortex. There were no changes in ligand affinities. The concentrations of monoamines, metabolites and estimates of dopamine (dopamine/dihydroxyphenylacetic acid ratio) and serotonin/5-hydroxyindolacetic acid ratio turnover rates were unchanged in striatum and frontal cortex. However, turnover of dopamine and serotonin in the hypothalamus was increased ~20% and ~15%, respectively. Striatal transmission via D1 receptors is decreased in binge-eating rats while μ-opioid receptor signalling may be increased. These changes are consistent with the attenuation of binge-eating by lisdexamfetamine, which increases catecholaminergic neurotransmission, and nalmefene, a μ-opioid antagonist.

Cocaine self-administration, extinction training and drug-induced relapse change metabotropic glutamate mGlu5 receptors expression: Evidence from radioligand binding and immunohistochemistry assays

Several behavioral findings highlight the importance of glutamatergic transmission and its metabotropic receptor type 5 (mGlu5) in the controlling of cocaine reward and seeking behaviors. The molecular or neurochemical nature of such interactions is not well recognized, so in the present paper we determine if cocaine self-administration and extinction/reinstatement models with the yoked triad control procedure alter mGlu5 receptor density in rats. [³H]MPEP was used to evaluate mGlu5 receptors density and affinity in selected brain structures, while immunofluorescence analysis was used to detect changes in mGlu5 receptors’ brain location.Cocaine self-administration and yoked cocaine delivery evoked a significant elevation in mGlu5 receptors’ density in the dorsal striatum, while receptor protein expression was importantly elevated in the substantia nigra and reduced in the nucleus accumbens shell. Cocaine administration followed by 10 extinction training sessions resulted in biphasic mGlu5 receptor density changes in the prefrontal cortex-nucleus accumbens pathway. mGlu5 receptors’ up-regulation was noted for cocaine self-administration and extinction training in the hippocampus and in yoked cocaine controls following drug abstinence in the dorsal striatum. A cocaine priming dose (but not a saline priming) resulted in a significant decrease of mGlu5 receptors’ density in the nucleus accumbens of rats previously treated with the drug and in the hippocampus of rats previously self-administered cocaine. The latter decrease in mGlu5 receptors’ density and protein expression in the hippocampus was parallel to an increase in [³H]MPEP affinity and opposite to a rise observed after single cocaine administration (ip) to drug-naïve yoked saline controls. Additionally, we also observed a significant elevation in the protein expression of the tested receptors in the limbic cortex in both cocaine groups. The present results shown modality dependent and brain-region specific changes in mGlu5 receptors’ localization and membrane specific binding.