Effects of the adenosine A2A receptor antagonist KW6002 on the dopaminergic system, motor performance, and neuroinflammation in a rat model of Parkinson's disease.

Adenosine A2A-receptors (A2AR) and dopamine D2-receptors (D2R) are known to work together in a synergistic manner. Inhibiting A2ARs by genetic or pharmacological means can relief symptoms and have neuroprotective effects in certain conditions. We applied PET imaging to evaluate the impact of the A2AR antagonist KW6002 on D2R availability and neuroinflammation in an animal model of Parkinson's disease. Male Wistar rats with 6-hydroxydopamine-induced damage to the right striatum were given 3 mg/kg of KW6002 daily for 20 days. Motor function was assessed using the rotarod and cylinder tests, and neuroinflammation and dopamine receptor availability were measured using PET scans with the tracers [11C]PBR28 and [11C]raclopride, respectively. On day 7 and 22 following 6-OHDA injection, rats were sacrificed for postmortem analysis. PET scans revealed a peak in neuroinflammation on day 7. Chronic treatment with KW6002 significantly reduced [11C]PBR28 uptake in the ipsilateral striatum [normalized to contralateral striatum] and [11C]raclopride binding in both striata when compared to the vehicle group. These imaging findings were accompanied by an improvement in motor function. Postmortem analysis showed an 84% decrease in the number of Iba-1+ cells in the ipsilateral striatum [normalized to contralateral striatum] of KW6002-treated rats compared to vehicle rats on day 22 (p = 0.007), corroborating the PET findings. Analysis of tyrosine hydroxylase levels showed less dopaminergic neuron loss in the ipsilateral striatum of KW6002-treated rats compared to controls on day 7. These findings suggest that KW6002 reduces inflammation and dopaminergic neuron loss, leading to less motor symptoms in this animal model of Parkinson's disease.

Real-world data of non-invasive stimulation of the human insula-prefrontal cortices using deep TMS to treat anxiety for occupational stress and generalized anxiety disorder.

Activation of the insula is found in all anxiety-related disorders and increased insular-prefrontal cortex (PFC) functional connectivity is associated with reduced anxiety. In this study, the combined stimulation of the insula and PFC using the dTMS H4 (insula+LPFC) and H2 (PFC) coils were used to reduce anxiety in 13 subjects experiencing occupational stress, and 55 participants suffering from generalized anxiety disorder (GAD). The combined HF stimulation of the insula and PFC significantly decreased anxiety scores according to the HARS, CAS, and STAI anxiety scales, leading to a reduction in anxiety according to HARS of 88.7% and 70.7% in participants with occupational stress and the clinical sample of participants diagnosed with GAD, respectively. The findings suggest that the prefrontal-insular axis is critical for the regulation of anxiety and its stimulation can be used for the treatment of anxiety in people suffering from occupational stress and GAD.

Impact of an Adenosine A2A Receptor Agonist and Antagonist on Binding of the Dopamine D2 Receptor Ligand [11C]raclopride in the Rodent Striatum.

Adenosine A2A and dopamine D2 receptors in the basal ganglia form heterotetrameric structures that are involved in the regulation of motor activity and neuropsychiatric functions. The present study examines the A2A receptor-mediated modulation of D2 receptor binding in vivo using positron emission tomography (PET) with the D2 antagonist tracer [11C]raclopride. Healthy male Wistar rats (n = 8) were scanned (60 min dynamic scan) with [11C]raclopride at baseline and 7 days later following an acute administration of the A2A agonist CGS21680 (1 mg/kg), using a MicroPET Focus-220 camera. Nondisplaceable binding potential (BPND) values were calculated using a simplified reference tissue model (SRTM), with cerebellum as the reference tissue. SRTM analysis did not show any significant changes in [11C]raclopride BPND (p = 0.102) in striatum after CGS21680 administration compared to the baseline. As CGS21680 strongly affects hemodynamics, we also used arterial blood sampling and a metabolite-corrected plasma input function for compartment modeling using the reversible two-tissue compartment model (2TCM) to obtain the BPND from the k3/k4 ratio and from the striatum/cerebellum volume of distribution ratio (DVR) in a second group of animals. These rats underwent dynamic [11C]raclopride scans after pretreatment with a vehicle (n = 5), a single dose of CGS21680 (1 mg/kg, n = 5), or a single dose of the A2A antagonist KW6002 (1 mg/kg, n = 5). The parent fraction in plasma was significantly higher in the CGS21680-treated group (p = 0.0001) compared to the vehicle-treated group. GCS21680 administration significantly reduced the striatal k3/k4 ratio (p < 0.01), but k3 and k4 estimates may be less reliable. The BPND (DVR-1) decreased from 1.963 ± 0.27 in the vehicle-treated group to 1.53 ± 0.55 (p = 0.080) or 1.961 ± 0.11 (p = 0.993) after the administration of CGS21680 or KW6002, respectively. Our study suggests that the A2A agonist CGS21680, but not the antagonist KW6002, may reduce the D2 receptor availability in the striatum.

A single dose of ketamine cannot prevent protracted stress-induced anhedonia and neuroinflammation in rats.

Worldwide, millions of people suffer from treatment-resistant depression. Ketamine, a glutamatergic receptor antagonist, can have a rapid antidepressant effect even in treatment-resistant patients. A proposed mechanism for the antidepressant effect of ketamine is the reduction of neuroinflammation. To further explore this hypothesis, we investigated whether a single dose of ketamine can modulate protracted neuroinflammation in a repeated social defeat (RSD) stress rat model, which resembles features of depression. To this end, male animals exposed to RSD were injected with ketamine (20 mg/kg) or vehicle. A combination of behavioral analyses and PET scans of the inflammatory marker TSPO in the brain were performed. Rats submitted to RSD showed anhedonia-like behavior in the sucrose preference test, decreased weight gain, and increased TSPO levels in the insular and entorhinal cortices, as observed by [11C]-PK11195 PET. Whole brain TSPO levels correlated with corticosterone levels in several brain regions of RSD exposed animals, but not in controls. Ketamine injection 1 day after RSD disrupted the correlation between TSPO levels and serum corticosterone levels, but had no effect on depressive-like symptoms, weight gain or the protracted RSD-induced increase in TSPO expression in male rats. These results suggest that ketamine does not exert its effect on the hypothalamic-pituitary-adrenal axis by modulation of neuroinflammation.

Astroglial gliotransmitters released via Cx43 hemichannels regulate NMDAR-dependent transmission and short-term fear memory in the basolateral amygdala.

Astrocytes release gliotransmitters via connexin 43 (Cx43) hemichannels into neighboring synapses, which can modulate synaptic activity and are necessary for fear memory consolidation. However, the gliotransmitters released, and their mechanisms of action remain elusive. Here, we report that fear conditioning training elevated Cx43 hemichannel activity in astrocytes from the basolateral amygdala (BLA). The selective blockade of Cx43 hemichannels by microinfusion of TAT-Cx43L2 peptide into the BLA induced memory deficits 1 and 24 h after training, without affecting learning. The memory impairments were prevented by the co-injection of glutamate and D-serine, but not by the injection of either alone, suggesting a role for NMDA receptors (NMDAR). The incubation with TAT-Cx43L2 decreased NMDAR-mediated currents in BLA slices, effect that was also prevented by the addition of glutamate and D-serine. NMDARs in primary neuronal cultures were unaffected by TAT-Cx43L2, ruling out direct effects of the peptide on NMDARs. Finally, we show that D-serine permeates through purified Cx43 hemichannels reconstituted in liposomes. We propose that the release of glutamate and D-serine from astrocytes through Cx43 hemichannels is necessary for the activation of post-synaptic NMDARs during training, to allow for the formation of short-term and subsequent long-term memory, but not for learning per se.

Prenatal fluoxetine impairs non-hippocampal but not hippocampal memory in adult male rat offspring.

Fluoxetine is often prescribed to treat depression during pregnancy. Rodent studies have shown that fluoxetine exposure during early development can induce persistent changes in the emotional behavior of the offspring. However, the effects of prenatal fluoxetine on memory have not been elucidated. This study evaluates the memory of adult male offspring from rat dams orally administered with a clinically relevant dose of 0.7 mg/kg fluoxetine from 9 weeks before pregnancy to 1 week before delivery. Hippocampal-dependent (Morris Water Maze, MWM) and non-hippocampal-dependent (Novel Object Recognition, NOR) memory paradigms were assessed. Anxiety- and depressive-like symptoms were also evaluated using the Open Field Test, Tail Suspension Test and Sucrose Preference Test. Male rats exposed to fluoxetine during gestation displayed NOR memory impairments during adulthood, as well as increased anxiety- and depressive-like symptoms. In the MWM, the offspring of fluoxetine-treated dams did not show learning deficits. However, a retention impairment was found on remote memory, 15 days after the end of training. Molecular analyses showed increased expression of NMDA subunit NR2B, and a decrease in NR2A-to- NR2B ratio in the temporal cortex, but not in the hippocampus, suggesting changes in NMDA receptor composition. These results suggest that in utero exposure to fluoxetine induces detrimental effects on non-hippocampal memory and in remote retention of hippocampal-dependent memory, which is believed to be stored in the temporal cortex, possibly due to changes in cortical NMDA receptor subunit stoichiometry. The present results warrant the need for studies on potential remote memory deficits in human offspring exposed to fluoxetine in utero.

In Vivo Induction of P-Glycoprotein Function can be Measured with [18F]MC225 and PET.

P-Glycoprotein (P-gp) is an efflux pump located at the blood-brain barrier (BBB) that contributes to the protection of the central nervous system by transporting neurotoxic compounds out of the brain. A decline in P-gp function has been related to the pathogenesis of neurodegenerative diseases. P-gp inducers can increase the P-gp function and are considered as potential candidates for the treatment of such disorders. The P-gp inducer MC111 increased P-gp expression and function in SW480 human colon adenocarcinoma and colo-320 cells, respectively. Our study aims to evaluate the P-gp inducing effect of MC111 in the whole brain in vivo, using the P-gp tracer [18F]MC225 and positron emission tomography (PET). Eighteen Wistar rats were treated with either vehicle solution, 4.5 mg/kg of MC111 (low-dose group), or 6 mg/kg of MC111 (high-dose group). Animals underwent a 60 min dynamic PET scan with arterial-blood sampling, 24 h after treatment with the inducer. Data were analyzed using the 1-tissue-compartment model and metabolite-corrected plasma as the input function. Model parameters such as the influx constant (K1) and volume of distribution (VT) were calculated, which reflect the in vivo P-gp function. P-gp and pregnane xenobiotic receptor (PXR) expression levels of the whole brain were assessed using western blot. The administration of MC111 decreased K1 and VT of [18F]MC225 in the whole brain and all of the selected brain regions. In the high-dose group, whole-brain K1 was decreased by 34% (K1-high-dose = 0.20 ± 0.02 vs K1-control = 0.30 ± 0.02; p < 0.001) and in the low-dose group by 7% (K1-low-dose = 0.28 ± 0.02 vs K1-control = 0.30 ± 0.02; p = 0.42) compared to controls. Whole-brain VT was decreased by 25% in the high-dose group (VT-high-dose = 5.92 ± 0.41 vs VT-control = 7.82 ± 0.38; p < 0.001) and by 6% in the low-dose group (VT-low-dose = 7.35 ± 0.38 vs VT-control = 7.82 ± 0.37; p = 0.38) compared to controls. k2 values did not vary after treatment. The treatment did not affect the metabolism of [18F]MC225. Western blot studies using the whole-brain tissue did not detect changes in the P-gp expression, however, preliminary results using isolated brain capillaries found an increasing trend up to 37% in treated rats. The decrease in K1 and VT values after treatment with the inducer indicates an increase in the P-gp functionality at the BBB of treated rats. Moreover, preliminary results using brain endothelial cells also sustained the increase in the P-gp expression. In conclusion, the results verify that MC111 induces P-gp expression and function at the BBB in rats. An increasing trend regarding the P-gp expression levels is found using western blot and an increased P-gp function is confirmed with [18F]MC225 and PET.

Chronic harmine treatment has a delayed effect on mobility in control and socially defeated rats.

INTRODUCTION: Depression is characterized by behavioral, cognitive and physiological changes, imposing a major burden on the overall wellbeing of the patient. Some evidence indicates that social stress, changes in growth factors (e.g., brain-derived neurotrophic factor (BDNF)), and neuroinflammation are involved in the development and progression of the disease. The monoamine oxidase A inhibitor drug harmine was suggested to have both antidepressant and anti-inflammatory properties and may, therefore, be a potential candidate for treatment of depression. AIM: The goal of this study was to assess the effects of harmine on behavior, brain BDNF levels, and microglia activation in control rats and a rat model of social stress. MATERIAL AND METHODS: Rats were submitted to 5 consecutive days of repeated social defeat (RSD) or control conditions. Animals were treated daily with harmine (15 mg/kg) or vehicle from day 3 until the end of the experiment. To assess the effects of harmine treatment on behavior, the sucrose preference test (SPT) was performed on days 1, 6, and 15, the open field test (OFT) on days 6 and 14, and the novel object recognition test (NOR) on day 16. Brain microgliosis was assessed using [11C]PBR-28 PET on day 17. Animals were terminated on day 17, and BDNF protein concentrations in the hippocampus and frontal cortex were analyzed using ELISA. RESULTS: RSD significantly decreased bodyweight and increased anxiety and anhedonia-related parameters in the OFT and SPT on day 6, but these behavioral effects were not observed anymore on day 14/15. Harmine treatment caused a significant reduction in bodyweight gain in both groups, induced anhedonia in the SPT on day 6, and significantly reduced the mobility and exploratory behavior of the animals in the OFT mainly on day 14. PET imaging and the NOR test did not show any significant effects on microglia activation and memory, respectively. BDNF protein concentrations in the hippocampus and frontal cortex were not significantly affected by either RSD or harmine treatment. DISCUSSION: Harmine was not able to reverse the acute effects of RSD on anxiety and anhedonia and even aggravated the effect of RSD on bodyweight loss. Moreover, harmine treatment caused unexpected side effects on general locomotion, both in RSD and control animals, but did not influence glial activation status and BDNF concentrations in the brain. In this model, RSD-induced stress was not strong enough to induce long-term effects on the behavior, neuroinflammation, or BDNF protein concentration. Thus, the efficacy of harmine treatment on these delayed parameters needs to be further evaluated in more severe models of chronic stress.

Effect of Dopamine D2 Receptor Antagonists on [18F]-FEOBV Binding.

The interaction of dopaminergic and cholinergic neurotransmission in, e.g., Parkinson's disease has been well established. Here, D2 receptor antagonists were used to assess changes in [18F]-FEOBV binding to the vesicular acetylcholine transporter (VAChT) in rodents using positron emission tomography (PET). After pretreatment with either 10 mg/kg haloperidol, 1 mg/kg raclopride, or vehicle, 90 min dynamic PET scans were performed with arterial blood sampling. The net influx rate (Ki) was obtained from Patlak graphical analysis, using a metabolite-corrected plasma input function and dynamic PET data. [18F]-FEOBV concentration in whole-blood or plasma and the metabolite-corrected plasma input function were not significantly changed by the pretreatments (adjusted p > 0.07, Cohen's d 0.28-1.89) while the area-under-the-curve (AUC) of the parent fraction of [18F]-FEOBV was significantly higher after haloperidol treatment (adjusted p = 0.022, Cohen's d = 2.51) than in controls. Compared to controls, the AUC of [18F]-FEOBV, normalized for injected dose and body weight, was nonsignificantly increased in the striatum after haloperidol (adjusted p = 0.4, Cohen's d = 1.77) and raclopride (adjusted p = 0.052, Cohen's d = 1.49) treatment, respectively. No changes in the AUC of [18F]-FEOBV were found in the cerebellum (Cohen's d 0.63-0.74). Raclopride treatment nonsignificantly increased Ki in the striatum 1.3-fold compared to control rats (adjusted p = 0.1, Cohen's d = 1.1) while it reduced Ki in the cerebellum by 28% (adjusted p = 0.0004, Cohen's d = 2.2) compared to control rats. Pretreatment with haloperidol led to a nonsignificant reduction in Ki in the striatum (10%, adjusted p = 1, Cohen's d = 0.44) and a 40-50% lower Ki than controls in all other brain regions (adjusted p < 0.0005, Cohen's d = 3.3-4.7). The changes in Ki induced by the selective D2 receptor antagonist raclopride can in part be quantified using [18F]-FEOBV PET imaging. Haloperidol, a nonselective D2/σ receptor antagonist, either paradoxically decreased cholinergic activity or blocked off-target [18F]-FEOBV binding to σ receptors. Hence, further studies evaluating the binding of [18F]-FEOBV to σ receptors using selective σ receptor ligands are necessary.

Modeling of [18F]FEOBV Pharmacokinetics in Rat Brain.

PURPOSE: [18F]Fluoroethoxybenzovesamicol ([18F]FEOBV) is a radioligand for the vesicular acetylcholine transporter (VAChT), a marker of the cholinergic system. We evaluated the quantification of [18F]FEOBV in rats in control conditions and after partial saturation of VAChT using plasma and reference tissue input models and test-retest reliability. PROCEDURE: Ninety-minute dynamic [18F]FEOBV PET scans with arterial blood sampling were performed in control rats and rats pretreated with 10 µg/kg FEOBV. Kinetic analyses were performed using one- (1TCM) and two-tissue compartmental models (2TCM), Logan and Patlak graphical analyses with metabolite-corrected plasma input, reference tissue Patlak with cerebellum as reference tissue, standard uptake value (SUV) and SUV ratio (SUVR) using 60- or 90-min acquisition. To assess test-retest reliability, two dynamic [18F]FEOBV scans were performed 1 week apart. RESULTS: The 1TCM did not fit the data. Time-activity curves were more reliably estimated by the irreversible than the reversible 2TCM for 60 and 90 min as the influx rate Ki showed a lower coefficient of variation (COV, 14-24 %) than the volume of distribution VT (16-108 %). Patlak graphical analysis showed a good fit to the data for both acquisition times with a COV (12-27 %) comparable to the irreversible 2TCM. For 60 min, Logan analysis performed comparably to both irreversible models (COV 14-32 %) but showed lower sensitivity to VAChT saturation. Partial saturation of VAChT did not affect model selection when using plasma input. However, poor correlations were found between irreversible 2TCM and SUV and SUVR in partially saturated VAChT states. Test-retest reliability and intraclass correlation for SUV were good. CONCLUSION: [18F]FEOBV is best modeled using the irreversible 2TCM or Patlak graphical analysis. SUV should only be used if blood sampling is not possible.

The Role of the Rodent Insula in Anxiety.

The human insula has been consistently reported to be overactivated in all anxiety disorders, activation which has been suggested to be proportional to the level of anxiety and shown to decrease with effective anxiolytic treatment. Nonetheless, studies evaluating the direct role of the insula in anxiety are lacking. Here, we set out to investigate the role of the rodent insula in anxiety by either inactivating different insular regions via microinjections of glutamatergic AMPA receptor antagonist CNQX or activating them by microinjection of GABA receptor antagonist bicuculline in rats, before measuring anxiety-like behavior using the elevated plus maze. Inactivation of caudal and medial insular regions induced anxiogenic effects, while their activation induced anxiolytic effects. In contrast, inactivation of more rostral areas induced anxiolytic effects and their activation, anxiogenic effects. These results suggest that the insula in the rat has a role in the modulation of anxiety-like behavior in rats, showing regional differences; rostral regions have an anxiogenic role, while medial and caudal regions have an anxiolytic role, with a transition area around bregma +0.5. The present study suggests that the insula has a direct role in anxiety.

Cx46 hemichannel modulation by nitric oxide: Role of the fourth transmembrane helix cysteine and its possible involvement in cataract formation.

Under normal conditions, connexin (Cx) hemichannels have a low open probability, which can increase under pathological conditions. Since hemichannels are permeable to relatively large molecules, their exacerbated activity has been linked to cell damage. Cx46 is highly expressed in the lens and its mutations have been associated to cataract formation, but it is unknown whether Cx46 has a role in non-genetic cataract formation (i.e. aging and diabetes). Nitric oxide (NO) is a key element in non-genetic cataract formation and Cx46 hemichannels have been shown to be sensitive to NO. The molecular mechanisms of the effects of NO on Cx46 are unknown, but are likely to result from Cx46 S-nitrosation (also known as S-nitrosylation). In this work, we found that lens opacity was correlated with Cx46 S-nitrosation in an animal model of cataract. Consistent with this result, a NO donor increased Cx46 S-nitrosation and hemichannel opening in HLE-B3 cells (cell line derived from human lens epithelial cells). Mutagenesis studies point to the cysteine located in the fourth transmembrane helix (TM4; human C212, rat C218) as the NO sensor. Electrophysiological studies performed in Xenopus oocytes revealed that rat Cx46 hemichannels are sensitive to different NO donors, and that the presence of C218 is necessary to observe the NO donors' effects. Unexpectedly, gap junctions formed by Cx46 were insensitive to NO or the reducing agent dithiothreitol. We propose that increased hemichannel opening and/or changes in their electrophysiological properties of human Cx46 due to S-nitrosation of the cysteine in TM4 could be an important factor in cataract formation.

Role of Astroglial Hemichannels and Pannexons in Memory and Neurodegenerative Diseases.

Under physiological conditions, astroglial hemichannels and pannexons allow the release of gliotransmitters from astrocytes. These gliotransmitters are critical in modulating synaptic transmission, plasticity and memory. However, recent evidence suggests that under pathological conditions, they may be central in the development of various neurodegenerative diseases. Here we review current literature on the role of astroglial hemichannels and pannexons in memory, stress and the development of neurodegenerative diseases, and propose that they are not only crucial for normal brain function, including memory, but also a potential target for the treatment of neurodegenerative diseases.

Dopamine receptor D5 deficiency results in a selective reduction of hippocampal NMDA receptor subunit NR2B expression and impaired memory.

Pharmacological evidence associates type I dopamine receptors, including subtypes D1 and D5, with learning and memory. Analyses using genetic approaches have determined the relative contribution of dopamine receptor D1 (D1R) in cognitive tasks. However, the lack of drugs that can discriminate between D1R and D5R has made the pharmacological distinction between the two receptors difficult. Here, we aimed to determine the role of D5R in learning and memory. In this study we tested D5R knockout mice and wild-type littermates in a battery of behavioral tests, including memory, attention, locomotion, anxiety and motivational evaluations. Our results show that genetic deficiency of D5R significantly impairs performance in the Morris water maze paradigm, object location and object recognition memory, indicating a relevant role for D5R in spatial memory and recognition memory. Moreover, the lack of D5R resulted in decreased exploration and locomotion. In contrast, D5R deficiency had no impact on working memory, anxiety and depressive-like behavior, measured using the spontaneous alternation, open-field, tail suspension test, and forced swimming test. Electrophysiological analyses performed on hippocampal slices showed impairment in long-term-potentiation in mice lacking D5R. Further analyses at the molecular level showed that genetic deficiency of D5R results in a strong and selective reduction in the expression of the NMDA receptor subunit NR2B in the hippocampus. These findings demonstrate the relevant contribution of D5R in memory and suggest a functional interaction of D5R with hippocampal glutamatergic pathways.

Retrospective Evaluation of Deep Transcranial Magnetic Stimulation as Add-On Treatment for Parkinson's Disease.

OBJECTIVE: To evaluate the safety and assess the different symptom improvements found after a combined low-frequency primary motor cortex and high-frequency prefrontal cortex (PFC) stimulation using the deep TMS (dTMS) H-coil, as an add-on treatment for Parkinson's disease (PD). METHODS: Forty-five PD patients underwent 14 dTMS sessions; each consisting of 1 Hz stimulation of the primary motor cortex for 15 min, followed by 10 Hz stimulation of the PFC for 15 min. Clinical assessments were performed, BEFORE, at the MIDDLE, and END of therapy as well as at FOLLOW-UP after 30 days, using Movement Disorder Society-Unified Parkinson's Disease Rating Scale, TINETTI, UP&GO, SCOPA, HDRS21, Beck Depression Inventory, and self-applied daily motor assessment scales. RESULTS: Treatment was well-tolerated, without serious adverse effects. dTMS-induced significant PD symptom improvements at END and at FOLLOW-UP, in all subscales of the UPDRS, gait speed, depressive symptoms, balance, autonomic symptoms, and a 73% increase in daily ON time. CONCLUSION: In the cohort of PD patients treated, dTMS was well-tolerated with only minor adverse effects. The dTMS-induced significant improvements in motor, postural, and motivational symptoms of PD patients and may potentiate concurrent levodopa treatment. SIGNIFICANCE: The present study demonstrates that dTMS may have a much wider spectrum of beneficial effects than previously reported for TMS, including enhancement of levodopa effects, suggesting that future clinical trials with dTMS should include a broader range of symptom measurements.

The insula modulates arousal-induced reluctance to try novel tastes through adrenergic transmission in the rat.

Reluctance to try novel tastes (neophobia) can be exacerbated in arousing situations, such as when children are under social stress or in rodents, when the new taste is presented in a high arousal context (HA) compared to a low arousal context (LA). The present study aimed at determining whether adrenergic transmission at the Insula regulates the reluctance to try novel tastes induced by arousing contexts. To this end, a combination of systemic and intra-insular manipulations of adrenergic activity was performed before the novel taste (saccharin 0.1%) was presented either in LA or HA contexts in rats. Our results show that systemic adrenergic activity modulates reluctance to try novel tastes. Moreover, intra-insular microinjections of propranolol or norepinephrine (NE) were found to modulate the effects of arousing contexts on reluctance to try novel tastes. Finally, intra-insular propranolol blocked epinephrine-induced increased reluctance, while intra-insular NE blocked oral propranolol-induced decreases in reluctance and increased the reluctance to try novel tastes presented in low arousing contexts. In conclusion, our results suggest that the insula is a critical site for regulating the effects of arousal in the reluctance to try novel tastes via the adrenergic system.

Restraint stress increases hemichannel activity in hippocampal glial cells and neurons.

Stress affects brain areas involved in learning and emotional responses, which may contribute in the development of cognitive deficits associated with major depression. These effects have been linked to glial cell activation, glutamate release and changes in neuronal plasticity and survival including atrophy of hippocampal apical dendrites, loss of synapses and neuronal death. Under neuro-inflammatory conditions, we recently unveiled a sequential activation of glial cells that release ATP and glutamate via hemichannels inducing neuronal death due to activation of neuronal NMDA/P2X7 receptors and pannexin1 hemichannels. In the present work, we studied if stress-induced glia activation is associated to changes in hemichannel activity. To this end, we compared hemichannel activity of brain cells after acute or chronic restraint stress in mice. Dye uptake experiments in hippocampal slices revealed that acute stress induces opening of both Cx43 and Panx1 hemichannels in astrocytes, which were further increased by chronic stress; whereas enhanced Panx1 hemichannel activity was detected in microglia and neurons after acute/chronic and chronic stress, respectively. Moreover, inhibition of NMDA/P2X7 receptors reduced the chronic stress-induced hemichannel opening, whereas blockade of Cx43 and Panx1 hemichannels fully reduced ATP and glutamate release in hippocampal slices from stressed mice. Thus, we propose that gliotransmitter release through hemichannels may participate in the pathogenesis of stress-associated psychiatric disorders and possibly depression.

Dopamine receptor D3 deficiency results in chronic depression and anxiety.

Over the last decade accumulating evidence suggests that brain dopamine (DA) has a role in depression, particularly given the high comorbidity of depression with Parkinson's Disease (PD) and the antidepressant effects of the DA receptor subtype 3 (D3R) agonist pramipexole. The present study assesses the role of D3R in depression. Here we hypothesized that D3R mediates the antidepressant effects of DA. Thus, genetic deficiency of D3R in D3R knockout (D3RKO) mice would yield animals with chronic depressive symptoms. Whereas D3R deficient mice did not show significant alterations in locomotion when tested in the openfield, these animals showed anxiety-like symptoms measured as a significant increase in thigmotaxis at the openfield and a significantly lower time spent in the lit compartment at the light/dark exploration test. D3RKO animals also showed depressive-like symptoms as measured by increased immobility time in the Porsolt forced swim test and the tail suspension test, as well as anhedonia measured in the non-motor dependent sucrose test. In conclusion, D3R deficiency results in anxiety-like and depressive-like symptoms that cannot be attributed to motor dysfunction.

Role of the insular cortex in taste familiarity.

Determining the role of the main gustatory cortical area within the insular cortex (IC), in conditioned taste aversion (CTA) has been elusive due to effective compensatory mechanisms that allow animals to learn in spite of lacking IC. IC lesions performed before CTA training induces mild if any memory impairments, while IC lesions done weeks after CTA produce amnesia. IC lesions before taste presentation have also been shown not to affect taste familiarity learning (attenuation of neophobia). This lack of effect could be either explained by compensation from other brain areas or by a lack of involvement of the IC in taste familiarity. To assess this issue, rats were bilaterally IC lesioned with ibotenic acid (200-300 nl.; 15 mg/ml) one week before or after taste familiarity, using either a preferred (0.1%) or a non-preferred (0.5%) saccharin solution. Rats lesioned before familiarity showed a decrease in neophobia to both solutions but no difference in their familiarity curve or their slope. When animals were familiarized and then IC lesioned, both IC lesioned groups treated the solutions as familiar, showing no differences from sham animals in their retention of familiarity. However, both lesioned groups showed increased latent inhibition (or impaired CTA) when CTA trained after repeated pre-exposures. The role of the IC in familiarity was also assessed using temporary inactivation of the IC, using bilateral micro-infusions of sodium channel blocker bupivacaine before each of 3 saccharin daily presentations. Intra-insular bupivacaine had no effects on familiarity acquisition, but did impair CTA learning in a different group of rats micro-infused before saccharin presentation in a CTA training protocol. Our data indicate that the IC is not essentially involved in acquisition or retention of taste familiarity, suggesting regional dissociation of areas involved in CTA and taste familiarity.

Release of gliotransmitters through astroglial connexin 43 hemichannels is necessary for fear memory consolidation in the basolateral amygdala.

Recent in vitro evidence indicates that astrocytes can modulate synaptic plasticity by releasing neuroactive substances (gliotransmitters). However, whether gliotransmitter release from astrocytes is necessary for higher brain function in vivo, particularly for memory, as well as the contribution of connexin (Cx) hemichannels to gliotransmitter release, remain elusive. Here, we microinfused into the rat basolateral amygdala (BLA) TAT-Cx43L2, a peptide that selectively inhibits Cx43-hemichannel opening while maintaining synaptic transmission or interastrocyte gap junctional communication. In vivo blockade of Cx43 hemichannels during memory consolidation induced amnesia for auditory fear conditioning, as assessed 24 h after training, without affecting short-term memory, locomotion, or shock reactivity. The amnesic effect was transitory, specific for memory consolidation, and was confirmed after microinfusion of Gap27, another Cx43-hemichannel blocker. Learning capacity was recovered after coinfusion of TAT-Cx43L2 and a mixture of putative gliotransmitters (glutamate, glutamine, lactate, d-serine, glycine, and ATP). We propose that gliotransmitter release from astrocytes through Cx43 hemichannels is necessary for fear memory consolidation at the BLA. Thus, the present study is the first to demonstrate a physiological role for astroglial Cx43 hemichannels in brain function, making these channels a novel pharmacological target for the treatment of psychiatric disorders, including post-traumatic stress disorder.