Interaction Between Ca<Subscript>v</Subscript>2.1α<Subscript>1</Subscript> and CaMKII in Ca<Subscript>v</Subscript>2.1α<Subscript>1</Subscript> Mutant Mice, <Emphasis Type="Italic">Rolling Nagoya</Emphasis>

It has been reported earlier that interactions between Ca_v2.1α₁ and calcium/calmodulin-dependent protein kinase II (CaMKII) in the presynaptic fraction and between the NMDA receptor subunit NR2B and CaMKII in the postsynaptic density (PSD) fraction are important for neuronal function. Ca_v2.1α₁, CaMKII, and NR2B are predominantly expressed in the hippocampus. To examine the above interactions and CaMKII activity in the hippocampal presynapse and PSD of Rolling Nagoya mice carrying a mutation in Ca_v2.1α₁ subunit, we performed immunoprecipitation and Western blot analyses. In the presynapse, the interaction between Ca_v2.1α₁ and CaMKII and the phosphorylation of CaMKII (at Thr286) and its substrate Synapsin I (at Ser603) were decreased in mutant mice compared to wild-type mice. In the PSD, a similar pattern was observed for the interaction between NR2B and CaMKII and the phosphorylation of CaMKII (at Thr286) and its substrate AMPA receptor subunit glutamate receptor 1 (at Ser831) between mutant and wild-type mice. Our data indicate that disruption of the interaction between Ca_v2.1α₁ and CaMKII may down-regulate presynaptic CaMKII activity and that Rolling Nagoya mice would be a useful model for examining presynaptic function.     

Receptor–receptor interactions involving adenosine A<Subscript>1</Subscript> or dopamine D<Subscript>1</Subscript> receptors and accessory proteins

Summary. The molecular basis for the known intramembrane receptor–receptor interactions among heptahelical receptors (G protein coupled receptors, GPCR) was postulated to be heteromerization based on receptor subtype specific interactions between different types of homomers of GPCR. Adenosine and dopamine receptors in the basal ganglia have been fundamental to demonstrate the existence of receptor heteromers and the functional consequences of such molecular interactions. The heterodimer is only one type of heteromeric complex and the evidence is equally compatible with the existence of higher order heteromeric complexes, where also adapter proteins such as homer proteins and scaffolding proteins can exist, assisting in the process of linking the GPCR and ion channel receptors together in a receptor mosaic that may have special integrative value and may constitute the molecular basis for learning and memory. Heteromerization of D₂ dopamine and A_2A adenosine receptors is reviewed by Fuxe in another article in this special issue. Here, heteromerization between D₁ dopamine and A₁ adenosine receptors is reviewed. Heteromers formed by dopamine D₁ and D₂ receptors and by adenosine A₁ and A_2A receptors also occur in striatal cells and open new perspectives to understand why two receptors with apparently opposite effects are expressed in the same neuron and in the nerve terminals. The role of accessory proteins also capable of interacting with receptor–receptor heteromers in regulating the traffic and the molecular physiology of these receptors is also discussed. Overall, the knowledge of the reason why such complex networks of receptor–receptor and receptor–protein interactions occur in striatal cells is crucial to develop new strategies to combat neurological and neuropsychiatric diseases.     

CSF Aβ<Subscript>1–42</Subscript>, but not p-Tau<Subscript>181</Subscript>, Predicted Progression from Amnestic MCI to Alzheimer’s Disease Dementia

The purpose of the study was to determine whether Aβ_1–42 and p-Tau₁₈₁ cerebral spinal fluid (CSF) levels can predict progression from amnestic mild cognitive impairment (aMCI) to Alzheimer’s disease dementia (ADD) in a 3-year follow-up study. All participants were evaluated blindly by a behavioral neurologist and a neuropsychologist, and classified according to the Petersen criteria for aMCI and according to the Clinical Dementia Rating (CDR) scale. Individuals were also submitted to lumbar puncture at baseline. Levels of Aβ_1–42 and p-Tau₁₈₁ were measured by immunoenzymatic assay. Values were adjusted for age and sex. Thirty-one of 33 (93.9%) participants completed follow-up. Approximately 39% of aMCI individuals progressed to ADD. The relative risk of developing ADD in those with Aβ_1–42 CSF levels lower than 618.5 pg/mL was 17.4 times higher than in those whose levels were higher than 618.5 pg/mL (P?=?0.003). p-Tau₁₈₁ alone did not predict progression to ADD (P?=?0.101). The relative risk in those with a p-Tau₁₈₁/Aβ_1–42 ratio higher than 0.135 was 5.7 times greater (P?<?0.001). Aβ_1–42 and p-Tau₁₈₁ explained 40.1% of the verbal memory test subscore of the Consortium to Establish a Registry for Alzheimer’s Disease (ΔCERADs) variance (P?=?0.008). Aβ_1–42 strongly predicted progression from aMCI to ADD. p-Tau₁₈₁ alone, or its relation to Aβ_1–42, was inferior than Aβ_1–42 alone as a predictor of progression to ADD.     

An antihypokinesic action of <Emphasis Type="Bold">α</Emphasis><Subscript>2</Subscript>-adrenoceptors upon MPTP-induced behaviour deficits in mice

The effects of co-administration of either the dopamine precursor, L-Dopa, or the directly-acting, mixed dopamine (DA) agonist, apomorphine, with the alpha-adrenoceptor agonists, clonidine and guanfacine, upon the motor activity of hypoactive L-Dopa-tolerant MPTP-treated C57 BL/6 mice were measured in four experiments. In each case, MPTP (2 x 40 mg/kg, s.c., separated by a 24-hr interval) was administered eight-to-ten weeks before behavioural testing. It was found that clonidine co-administered with L-Dopa (20 mg/kg) restored motor activity in a dose- and parameter-related manner: locomotion and total activity were restored by the 1 mg/kg dose, rearing behaviour by the 0.3 and 1 mg/kg doses. The restorative effects of clonidine (1 mg/kg), co-administered with L-Dopa, were antagonised completely by pretreatment with yohimbine (1 mg/kg), but not by prazosin (1 mg/kg). Guanfacine (1 mg/kg) co-administered with L-Dopa (20 mg/kg) restored locomotor, but not rearing, behaviour in L-Dopa-tolerant MPTP-treated mice. The antikinesic action of guanfacine was antagonised completely by yohimbine (1 mg/kg), but not prazosin (1 mg/kg). Clonidine (1 or 3 mg/kg) co-administered with apomorphine (0.1, 0.3, 1.0 or 3.0 mg/kg), directly-acting DA agonist, did not restore motor behaviour in the hypokinesic L-Dopa-tolerant MPTP-treated mice. Nor did apomorphine, by itself, affect the motor activity of these animals. Neurochemical analysis indicated marked DA, DOPAC and HVA depletions in the striatum, and to a much lesser extent in the frontal cortex, of MPTP-treated mice. The synergistic antiparkinsonian action of clonidine with L-Dopa, but not apomorphine, in hypokinetic MPTP mice for the restoration of responding to a suprathreshold dose of L-Dopa, to which "wearing-off" had been induced previously, is discussed.     

Increased infections with β-blocker use in ischemic stroke,a β<Subscript>2</Subscript>-receptor mediated process?

Strokes promote immunosuppression, partially from increased sympathetic activity. Altering sympathetic drive with β-blockers has variably been shown to improve stroke outcomes. This study adds to this literature using propensity score matching to limit confounding and by examining the effects of selective and non-selective β-blockers. Prospective data from acute ischemic stroke admissions at a single center from July 2010–June 2015 were analyzed. Outcomes included infection (urinary tract infection [UTI], pneumonia, or bacteremia), discharge modified Rankin Score (mRS), and in-hospital death. Any selective and non-selective β-blocker use during the first 3 days of admission were investigated with propensity score matching. A sensitivity analysis was also performed. This study included 1431 admissions. Any β-blocker use was associated with increased infections (16.4 vs. 10.7%, p = 0.030). Non-selective β-blocker use was associated with increased infections (18.9 vs. 9.7%, p = 0.005) and UTIs (13.0 vs. 5.5%, p = 0.009). Selective β-blocker use was not associated with infection. There were no associations between β-blocker use and in-hospital death or discharge mRS. In the sensitivity analysis, the association between non-selective β-blocker use and urinary tract infections persisted (12.5 vs. 4.2%, p = 0.044). No associations with death or mRS were found. Early β-blocker use after ischemic stroke may increase the risk of infection but did not change disability or mortality risk. The mechanism may be mediated by β₂-adrenergic receptor antagonism given the different effects seen with selective versus non-selective β-blocker use.     

Changes in the mRNA Levels of α<Subscript>2A</Subscript> and α<Subscript>2C</Subscript> Adrenergic Receptors in Rat Models of Parkinson’s Disease and <Emphasis Type="SmallCaps">l</Emphasis>-DOPA-Induced Dyskinesia

The changes in the mRNA levels of α_2A and α_2C adrenoceptors were investigated in unilateral 6-OHDA-lesioned rat model of Parkinson’s disease and l-DOPA-induced dyskinesia using in situ hybridization. In the untreated 6-OHDA-lesioned rats, α_2A expression was elevated in the locus coeruleus (160 ± 8% and 142 ± 8% in lesioned and unlesioned sides compared to the comparable side in sham-operated rats). Following long-term (21 days, twice daily) treatment with l-DOPA (25 mg/kg l-DOPA methyl ester plus benserazide 6.25 mg/kg) in 6-OHDA-lesioned rats, levels of α_2A adrenoceptor mRNA in the locus coeruleus were decreased, compared to the 6-OHDA-lesioned rats, returning to the levels of α_2A mRNA in the sham-operated rats. α_2A adrenoceptor expression was not changed in other brain regions in any treatment group. There was no change in α_2C expression in the rostral or caudal striatum in which the highest density of α_2C mRNA is present. In conclusion, the data presented in this study demonstrate an increase in α_2A adrenoceptor mRNA in the locus coeruleus in the 6-OHDA-lesioned rat model of Parkinson’s disease. In addition, the data show that repeated treatment with l-DOPA in 6-OHDA-lesioned rats, which induces dyskinesia, restores α_2A mRNA levels. These changes of α_2A mRNA expression, observed in the locus coeruleus, might be of importance to basal ganglia transmission and motor function.     

Fast Inactivation of Ca<Subscript>V</Subscript>2.2 Channels Is Prevented by the Gβ<Subscript>1</Subscript> Subunit in Rat Sympathetic Neurons

Voltage-dependent regulation of Ca_V2.2 channels by G-proteins is performed by the β (Gβ) subunit. Most studies of regulation by G-proteins have focused on channel activation; however, little is known regarding channel inactivation. This study investigated inactivation of Ca_V2.2 channels in superior cervical ganglion neurons that overexpressed Gβ subunits. Ca_V2.2 currents were recorded by whole-cell patch clamping configuration. We found that the Gβ₁ subunit reduced inactivation, while Gβ₅ subunit did not alter at all inactivation kinetics compared to control recordings. Ca_V2.2 current decay in control neurons consisted of both fast and slow inactivation; however, Gβ₁-overexpressing neurons displayed only the slow inactivation. Fast inactivation was restored by a strong depolarization of Gβ₁-overexpressing neurons, therefore, through a voltage-dependent mechanism. The Gβ₁ subunit shifted the voltage dependence of inactivation to more positive voltages and reduced the fraction of Ca_V2.2 channels resting in the inactivated state. These results support that the Gβ₁ subunit inhibits the fast inactivation of Ca_V2.2 channels in SCG neurons. They explain the long-observed sustained Ca²⁺ current under G-protein modulation.     

Serum α<Subscript>1</Subscript>-antitrypsin level and phenotype associated with familial moyamoya disease

Background Obstructive vascular lesions at the terminal portion of the internal carotid arteries are thought to be the primary and essential lesions in moyamoya disease. The etiology remains unknown. To detect possible mediators of the thickened intima of moyamoya disease, we measured serum alpha-1-antitrypsin (1-AT) levels and characterized the phenotype of patients with familial moyamoya disease.Patients and methods Fifty-six individuals were examined, including 29 patients with moyamoya disease from 14 families. Serum 1-AT levels were analyzed by electroimmunoassay and genomic phenotype by isoelectric focusing.Results All individuals had a normal 1-AT phenotype. The average serum 1-AT level in moyamoya disease patients was significantly higher than that of normal individuals, although both were within the normal range.Conclusions These findings suggest that serum 1-AT level may be a marker, rather than an etiologic factor, indicating the progression of moyamoya disease.     

IRF-8 is Involved in Amyloid-β<Subscript>1–40</Subscript> (Aβ<Subscript>1–40</Subscript>)-induced Microglial Activation: a New Implication in Alzheimer’s Disease

Using microarray analysis, we detected microRNA-124 (miR-124) to be abundantly expressed in the olfactory bulb (OB). miR-124 regulates adult neurogenesis in the subventricular zone (SVZ). However, much less is known about its role in newborn OB neurons. Here, using both gain-of-function and loss-of-function approaches, we demonstrate that brain-specific miR-124 affects dendritic morphogenesis and spine density in newborn OB neurons. Functional Annotation Clustering of miR-124 targets was enriched in “cell morphogenesis involved in neuron differentiation.”     

GluR2/3, NMDAε1 and GABA<Subscript>A</Subscript> receptors in Creutzfeldt-Jakob disease

The excitatory ionotropic glutamate receptors N-methyl-d-aspartate (NMDA) and -amino-3-hydro-5methyl-4-isoxazole propionic acid (AMPA) receptors, and the inhibitory -aminobutyric acid (GABA) receptors are major regulators of synaptic transmission in the central nervous system. Glutamate receptors AMPA GluR2/3 and NMDA R2A: NR2A (NMDA1), and GABA_A (GABA_A R1) receptors were examined by immunohistochemistry in the cerebral cortex (frontal cortex) entorhinal cortex, hippocampus and cerebellar cortex in nine patients with sporadic Creutzfeldt-Jakob disease (CJD) and eight age-matched controls obtained 3–8 h after death. All patients with CJD showed methionine/methionine in codon 129 of the prion protein gene. Decreased GluR2/3 immunoreactivity was found in the frontal cortex, entorhinal cortex and Purkinje cells; reduced NMDA1 immunoreactivity was found in the frontal cortex, entorhinal cortex, and molecular and granular cell layers of the cerebellum. Decreased GluR2/3 and NMDA1 immunoreactivity was also observed in the molecular layer of the dentate gyrus, but not in the hippocampus proper in cases with hippocampal involvement. GABA_A R1 expression was markedly decreased in the granular cell layer of the cerebellum in CJD. Decreased GluR2/3 and NMDA1 expression correlated with prion protein deposition, neuron loss and spongiform degeneration in the cerebral cortex in every case. However, reduced GluR2/3 immunoreactivity in Purkinje cells was apparently independent of these parameters. In contrast to ionotropic glutamate receptors, GABA_A R1 immunoreactivity was moderately increased in the frontal cortex, entorhinal cortex and molecular layer of the cerebellum in CJD. The present results show marked and selective abnormalities in the expression of crucial neurotransmitter receptors in CJD, ionotropic glutamate receptors being more severely affected than ionotropic GABA receptors. These findings stress selective vulnerability of glutamate receptors versus GABA receptors in CJD.     

Platelet phospholipase A<Subscript>2</Subscript> activity in Alzheimer’s disease and mild cognitive impairment

Summary. Phospholipase A₂ (PLA₂) controls the metabolism of phospholipids in cell membranes. In the brain, PLA₂ influences the processing of the amyloid precursor protein (APP) and thus the production of the amyloid-beta peptides (A), which are the major components of the senile plaques in Alzheimers disease (AD). Reduced PLA₂ activity has been reported in brain and in platelets of AD patients. In the present study we investigated PLA₂ activity in platelets from 21 AD patients as compared to 17 healthy elderly controls and 11 individuals with mild cognitive impairment (MCI). Subjects were cognitively assessed by the Mini-Mental State Examination (MMSE) and the CAMDEX schedule. Platelet PLA₂ activity was determined by radio-enzymatic assay, which mainly detected a calcium-independent form of the enzyme present also in the brain (iPLA₂). PLA₂ activity was significantly lower in AD than in controls (p<0.001). Mean PLA₂ activity in MCI individuals was between the values of AD patients and controls, with a subgroup showing PLA as low as the lowest AD patients, but the differences from MCI were not significant from AD and control groups. Lower PLA₂ activity was significantly correlated with a worse cognitive performance both at the MMSE (p=0.001) and the cognitive sub-scale of the CAMDEX inventory (p=0.002). Our data replicate previous findings of reduced platelet PLA₂ activity in AD. Both reduced PLA₂ activity and the correlation with impaired cognition were also reported in brain tissue of AD patients, suggesting thus that the present determinations in platelets may be related to a reduction in the brain. In the brain the inhibition of PLA₂ inhibits the physiological secretion of the APP, a mechanism that increases A formation. Further longitudinal studies should investigate whether those MCI individuals with the lowest PLA₂ values in platelets would be at a higher risk to develop AD during a longitudinal follow up.     

Platelet phospholipase A<Subscript>2</Subscript> activity in patients with Alzheimer’s disease,vascular dementia and ischemic stroke

Summary Phospholipase A₂ (E.C. 3.1.1.4, PLA₂) plays an essential role in metabolism of membrane phospholipids, it is related to inflammatory reactions, secretion of amyloid precursor protein and activation of NMDA receptor after ischemia. In the present study we investigated PLA₂ activity in platelets from 37 Alzheimer’s disease (AD) patients, 32 vascular dementia (VaD) patients and 32 individuals with ischemic stroke as compared to 27 healthy elderly controls. PLA₂ activity was determined using radiometric assay. Mean platelet PLA₂ activity was increased in individuals with Alzheimer’s disease (p < 0.001). In VaD group the enzyme activity was between the values in AD and controls, these differences being significant from both groups. In the group of patients with ischemic stroke mean PLA₂ activity was higher either 48 h after the stroke or 7 days later (in both cases p < 0.001). The results may be particularly interesting in light of the fact, that inhibitors of PLA₂ activity are known.     

Receptor–receptor interactions in central cardiovascular regulation. Focus on neuropeptide/α<Subscript>2</Subscript>-adrenoreceptor interactions in the nucleus tractus solitarius

Summary. The nucleus tractus solitarii (NTS) is a key nucleus in central cardiovascular control. In this mechanism it is well known the role of the α₂-adrenoreceptors for the modulation of the autonomic pathways. Moreover a number of neuropeptides described in the NTS, including Neuropeptide Y (NPY), Galanin (GAL) and Angiotensin II (Ang II), have different roles in regulating the cardiovascular function within this nucleus. We show in this review several data which help to understand how these neuropeptides (NPY, GAL and Ang II) could modulate the cardiovascular responses mediated through α₂-adrenoreceptors in the NTS. Also we show for the first time the interactions between neuropeptides in the brain, specifically the interactions between NPY, GAL, and Ang II, and its functional relevance for central cardiovascular regulation. These data strength the role of neuropeptides on central autonomic control and provide some evidences to understand the neurochemical mechanisms involved in the cardiovascular responses from the NTS.     

Characterization of brain tumours with spin–spin relaxation: pilot case study reveals unique <Emphasis Type="Italic">T</Emphasis><Subscript>2</Subscript> distribution profiles of glioblastoma,oligodendroglioma and meningioma

Prolonged spin–spin relaxation times in tumour tissue have been observed since some of the earliest nuclear magnetic resonance investigations of the brain. Over the last three decades, numerous studies have sought to characterize tumour morphology and malignancy using quantitative assessment of T ₂ relaxation times, although attempts to categorize and differentiate tumours have had limited success. However, previous work must be interpreted with caution as relaxation data were typically acquired using a variety of multiple echo sequences with a range of echoes and T ₂ decay curves and were frequently fit with monoexponential analysis. We defined the distribution of T ₂ components in three different human brain tumours (glioblastoma, oligodendroglioma, meningioma) using a multi-echo sequence with a greater number of echoes and a longer acquisition window than previously used (48 echoes, data collection out to 1120 ms) with no a priori assumptions about the number of exponential components contributing to the T ₂ decay. T ₂ relaxation times were increased in tumour tissue and each tumour showed a distinct T ₂ distribution profile. Tumours have complex and unique compartmentalization characteristics. Quantitative assessment of T ₂ relaxation in brain cancer may be useful in evaluating different grades of brain tumours on the basis of their T ₂ distribution profile, and has the potential to be a non-invasive diagnostic tool which may also be useful in monitoring therapy. Further study with a larger sample size and varying grades of tumours is warranted.     

Selective loss of P2Y<Subscript>2</Subscript> nucleotide receptor immunoreactivity is associated with Alzheimer’s disease neuropathology

The uridine nucleotide-activated P2Y(2), P2Y(4) and P2Y(6) receptors are widely expressed in the brain and are involved in many CNS processes, including those which malfunction in Alzheimer's disease (AD). However, the status of these receptors in the AD neocortex, as well as their putative roles in the pathogenesis of neuritic plaques and neurofibrillary tangles, remain unclear. In this study, we used immunoblotting to measure P2Y(2), P2Y(4) and P2Y(6) receptors in two regions of the postmortem neocortex of neuropathologically assessed AD patients and aged controls. P2Y(2) immunoreactivity was found to be selectively reduced in the AD parietal cortex, while P2Y(4) and P2Y(6) levels were unchanged. In contrast, all three receptors were preserved in the occipital cortex, which is known to be minimally affected by AD neuropathology. Furthermore, reductions in parietal P2Y(2) immunoreactivity correlated both with neuropathologic scores and markers of synapse loss. These results provide a basis for considering P2Y(2) receptor changes as a neurochemical substrate of AD, and point towards uridine nucleotide-activated P2Y receptors as novel targets for disease-modifying AD pharmacotherapeutic strategies.     

Preprothyrotropin-releasing hormone<Subscript>178–199</Subscript> affects tyrosine hydroxylase biosynthesis in hypothalamic neurons

ProThyrotropin-releasing hormone (proTRH) is a prohormone widely distributed in many areas of the brain. After biosynthesis, proTRH is subjected to post-translational processing to generate TRH and seven non-TRH peptides. Among these non-TRH sequences, we found previously that preproTRH_178–199 could regulate the secretion of prolactin in suckled rats by their pups. Dopamine (DA), the main regulator of prolactin secretion, is produced in dopaminergic tyrosine hydroxylase (TH)-positive neurons in the hypothalamic arcuate nucleus (ARC). In this study we investigated whether prolactin release during the estrous sexual cycle is regulated by prepro TRH_178–199 through its effecton DA neurons of the ARC. We observed that biotinylated prepro TRH_178–199 bound to neurons in the ARC; this was higher during proestrus than during diestrus. Binding of preproTRH_178–199 to DA neurons was seen only during proestrus in the ARC. Using primary neuronal hypothalamic cultures we found that preproTRH_178–199peptide decreased TH levels in a dose-responsive manner, whereas intra-ARC administration of preproTRH_178–199 induced a 20-fold increase in plasma prolactin levels. Together, these results suggest a potential role for preproTRH_178–199 in regulating dopaminergic neurons involved in the inhibition of pituitary prolactin release.     

Expression of α<Subscript>2</Subscript>-macroglobulin,neutrophil elastase,and interleukin-1α differs in early-stage and late-stage atherosclerotic lesions in the arteries of the circle of Willis

Different types of atherosclerotic (AS) lesions can be distinguished histologically and represent different stages of AS plaque development. Late-stage lesions more frequently develop complications such as plaque rupture and thrombosis with vessel occlusion than early AS lesions. To clarify whether protective, destructive, and inflammatory proteins are differentially expressed in early-stage and late-stage AS plaques we examined the proteinase inhibitor α₂-macroglobulin (A2M), the neutrophil elastase (NE)—an enzyme degrading elastin and collagen fibers—and the proinflammatory protein interleukin-1α (IL-1α) in all types of AS plaques in the arteries of the circle of Willis from 78 human autopsy cases of both genders (61–91 years of age). Paraffin sections of AS plaques were immunostained with antibodies directed against A2M, NE and IL-1α. In initial AS lesions A2M was found, whereas NE and IL-1α were absent. NE and IL-1α became detectable as soon as a significant number of macrophages occurred within AS lesions. With increasing histopathological type of AS lesions, a marked increase of the area of the plaque exhibiting NE and IL-1α was observed. The area which exhibits A2M in AS plaques, on the other hand, did not vary significantly between the different stages. Thus, our results indicate a disproportionately high increase of the destructive enzyme NE and the proinflammatory protein IL-1α in relation to A2M with the progression of the grade of AS lesions pointing to the transgression of the protective capacity of A2M by NE and IL-1α in late-stage plaques. Therefore, our findings support the hypothesis that NE-induced tissue damage in late-stage AS plaques contributes to the development of plaque rupture and subsequent thrombosis.     

P2X<Subscript>7</Subscript> Receptor–Pannexin 1 Hemichannel Association: Effect of Extracellular Calcium on Membrane Permeabilization

Activation of P2X₇ receptor (P2X₇R) and pannexin have been implicated in membrane permeabilization associated with ischemic cell death and many other inflammatory processes. P2X₇R has a unique property of forming large pore upon repeated or prolonged application of agonist like ATP or 2′, 3′-(4-benzoyl) benzoyl ATP. It has been proposed that pannexin 1 (panx1) hemichannel associates with P2X₇R to form large pore, though the actual mechanism is not yet understood. Calcium concentration in extracellular milieu drops in many patho-physiological conditions, e.g. ischemia, when P2X₇R/pannexin is also known to be activated. Therefore, we hypothesize that extracellular calcium ([Ca²⁺]_o) plays an important role in the coupling of P2X₇R–panx1 and subsequent membrane permeabilization. In this study we show that membrane permeability of the P2X₇R and panx1 expressing N2A cell increases in ([Ca²⁺]_o)-free solution. In [Ca²⁺]_o-free solution, fluorescent dye calcein trapped cells exhibited time-dependent dye leakage resulting in about 50% decrease of fluorescence intensity in 30 min. Control cells in 2 mM [Ca²⁺]_o did not show such leakage. Like N2A cells, mixed culture of neuron and glia, derived from hippocampal progenitor cells showed similar dye leakage. Dye leakage was blocked either by pannexin-specific blocker, carbenoxolone or P2X₇R antagonists, Brilliant Blue G, and oxidized ATP. Furthermore P2X₇R and panx1 were co-immunoprecipitated. The amount of P2X₇R protein pulled-down with panx1, increased by twofold when cells were incubated 30 min in [Ca²⁺]_o-free buffer. Taken together, the results of this study demonstrate the activation and association of P2X₇R–panx1, triggered by the removal of [Ca²⁺]_o.