Early Detection of Alzheimer's Disease From Cortical and Hippocampal Local Field Potentials Using an Ensembled Machine Learning Model.

Early diagnosis of Alzheimer's disease (AD) is a very challenging problem and has been attempted through data-driven methods in recent years. However, considering the inherent complexity in decoding higher cognitive functions from spontaneous neuronal signals, these data-driven methods benefit from the incorporation of multimodal data. This work proposes an ensembled machine learning model with explainability (EXML) to detect subtle patterns in cortical and hippocampal local field potential signals (LFPs) that can be considered as a potential marker for AD in the early stage of the disease. The LFPs acquired from healthy and two types of AD animal models (n = 10 each) using linear multielectrode probes were endorsed by electrocardiogram and respiration signals for their veracity. Feature sets were generated from LFPs in temporal, spatial and spectral domains and were fed into selected machine-learning models for each domain. Using late fusion, the EXML model achieved an overall accuracy of 99.4%. This provided insights into the amyloid plaque deposition process as early as 3 months of the disease onset by identifying the subtle patterns in the network activities. Lastly, the individual and ensemble models were found to be robust when evaluated by randomly masking channels to mimic the presence of artefacts.

Rescue of astrocyte activity by the calcium sensor STIM1 restores long-term synaptic plasticity in female mice modelling Alzheimer's disease.

Calcium dynamics in astrocytes represent a fundamental signal that through gliotransmitter release regulates synaptic plasticity and behaviour. Here we present a longitudinal study in the PS2APP mouse model of Alzheimer's disease (AD) linking astrocyte Ca2+ hypoactivity to memory loss. At the onset of plaque deposition, somatosensory cortical astrocytes of AD female mice exhibit a drastic reduction of Ca2+ signaling, closely associated with decreased endoplasmic reticulum Ca2+ concentration and reduced expression of the Ca2+ sensor STIM1. In parallel, astrocyte-dependent long-term synaptic plasticity declines in the somatosensory circuitry, anticipating specific tactile memory loss. Notably, we show that both astrocyte Ca2+ signaling and long-term synaptic plasticity are fully recovered by selective STIM1 overexpression in astrocytes. Our data unveil astrocyte Ca2+ hypoactivity in neocortical astrocytes as a functional hallmark of early AD stages and indicate astrocytic STIM1 as a target to rescue memory deficits.

Accelerated Aging Characterizes the Early Stage of Alzheimer's Disease.

For Alzheimer's disease (AD), aging is the main risk factor, but whether cognitive impairments due to aging resemble early AD deficits is not yet defined. When working with mouse models of AD, the situation is just as complicated, because only a few studies track the progression of the disease at different ages, and most ignore how the aging process affects control mice. In this work, we addressed this problem by comparing the aging process of PS2APP (AD) and wild-type (WT) mice at the level of spontaneous brain electrical activity under anesthesia. Using local field potential recordings, obtained with a linear probe that traverses the posterior parietal cortex and the entire hippocampus, we analyzed how multiple electrical parameters are modified by aging in AD and WT mice. With this approach, we highlighted AD specific features that appear in young AD mice prior to plaque deposition or that are delayed at 12 and 16 months of age. Furthermore, we identified aging characteristics present in WT mice but also occurring prematurely in young AD mice. In short, we found that reduction in the relative power of slow oscillations (SO) and Low/High power imbalance are linked to an AD phenotype at its onset. The loss of SO connectivity and cortico-hippocampal coupling between SO and higher frequencies as well as the increase in UP-state and burst durations are found in young AD and old WT mice. We show evidence that the aging process is accelerated by the mutant PS2 itself and discuss such changes in relation to amyloidosis and gliosis.

A New Transgenic Mouse Line for Imaging Mitochondrial Calcium Signals.

Mitochondria play a key role in cellular calcium (Ca2+) homeostasis. Dysfunction in the organelle Ca2+ handling appears to be involved in several pathological conditions, ranging from neurodegenerative diseases, cardiac failure and malignant transformation. In the past years, several targeted green fluorescent protein (GFP)-based genetically encoded Ca2+ indicators (GECIs) have been developed to study Ca2+ dynamics inside mitochondria of living cells. Surprisingly, while there is a number of transgenic mice expressing different types of cytosolic GECIs, few examples are available expressing mitochondria-localized GECIs, and none of them exhibits adequate spatial resolution. Here we report the generation and characterization of a transgenic mouse line (hereafter called mt-Cam) for the controlled expression of a mitochondria-targeted, Förster resonance energy transfer (FRET)-based Cameleon, 4mtD3cpv. To achieve this goal, we engineered the mouse ROSA26 genomic locus by inserting the optimized sequence of 4mtD3cpv, preceded by a loxP-STOP-loxP sequence. The probe can be readily expressed in a tissue-specific manner upon Cre recombinase-mediated excision, obtainable with a single cross. Upon ubiquitous Cre expression, the Cameleon is specifically localized in the mitochondrial matrix of cells in all the organs and tissues analyzed, from embryos to aged animals. Ca2+ imaging experiments performed in vitro and ex vivo in brain slices confirmed the functionality of the probe in isolated cells and live tissues. This new transgenic mouse line allows the study of mitochondrial Ca2+ dynamics in different tissues with no invasive intervention (such as viral infection or electroporation), potentially allowing simple calibration of the fluorescent signals in terms of mitochondrial Ca2+ concentration ([Ca2+]).

Familial Alzheimer's disease presenilin-2 mutants affect Ca2+ homeostasis and brain network excitability.

Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Few cases are familial (FAD), due to autosomal dominant mutations in presenilin-1 (PS1), presenilin-2 (PS2) or amyloid precursor protein (APP). The three proteins are involved in the generation of amyloid-beta (Aß) peptides, providing genetic support to the hypothesis of Aß pathogenicity. However, clinical trials focused on the Aß pathway failed in their attempt to modify disease progression, suggesting the existence of additional pathogenic mechanisms. Ca2+ dysregulation is a feature of cerebral aging, with an increased frequency and anticipated age of onset in several forms of neurodegeneration, including AD. Interestingly, FAD-linked PS1 and PS2 mutants alter multiple key cellular pathways, including Ca2+ signaling. By generating novel tools for measuring Ca2+ in living cells, and combining different approaches, we showed that FAD-linked PS2 mutants significantly alter cell Ca2+ signaling and brain network activity, as summarized below.

Presenilin-2 and Calcium Handling: Molecules, Organelles, Cells and Brain Networks.

Presenilin-2 (PS2) is one of the three proteins that are dominantly mutated in familial Alzheimer's disease (FAD). It forms the catalytic core of the γ-secretase complex-a function shared with its homolog presenilin-1 (PS1)-the enzyme ultimately responsible of amyloid-ß (Aß) formation. Besides its enzymatic activity, PS2 is a multifunctional protein, being specifically involved, independently of γ-secretase activity, in the modulation of several cellular processes, such as Ca2+ signalling, mitochondrial function, inter-organelle communication, and autophagy. As for the former, evidence has accumulated that supports the involvement of PS2 at different levels, ranging from organelle Ca2+ handling to Ca2+ entry through plasma membrane channels. Thus FAD-linked PS2 mutations impact on multiple aspects of cell and tissue physiology, including bioenergetics and brain network excitability. In this contribution, we summarize the main findings on PS2, primarily as a modulator of Ca2+ homeostasis, with particular emphasis on the role of its mutations in the pathogenesis of FAD. Identification of cell pathways and molecules that are specifically targeted by PS2 mutants, as well as of common targets shared with PS1 mutants, will be fundamental to disentangle the complexity of memory loss and brain degeneration that occurs in Alzheimer's disease (AD).

ORAI2 Down-Regulation Potentiates SOCE and Decreases Aß42 Accumulation in Human Neuroglioma Cells.

Senile plaques, the hallmarks of Alzheimer's Disease (AD), are generated by the deposition of amyloid-beta (Aß), the proteolytic product of amyloid precursor protein (APP), by ß and γ-secretase. A large body of evidence points towards a role for Ca2+ imbalances in the pathophysiology of both sporadic and familial forms of AD (FAD). A reduction in store-operated Ca2+ entry (SOCE) is shared by numerous FAD-linked mutations, and SOCE is involved in Aß accumulation in different model cells. In neurons, both the role and components of SOCE remain quite obscure, whereas in astrocytes, SOCE controls their Ca2+-based excitability and communication to neurons. Glial cells are also directly involved in Aß production and clearance. Here, we focus on the role of ORAI2, a key SOCE component, in modulating SOCE in the human neuroglioma cell line H4. We show that ORAI2 overexpression reduces both SOCE level and stores Ca2+ content, while ORAI2 downregulation significantly increases SOCE amplitude without affecting store Ca2+ handling. In Aß-secreting H4-APPswe cells, SOCE inhibition by BTP2 and SOCE augmentation by ORAI2 downregulation respectively increases and decreases Aß42 accumulation. Based on these findings, we suggest ORAI2 downregulation as a potential tool to rescue defective SOCE in AD, while preventing plaque formation.

Dampened Slow Oscillation Connectivity Anticipates Amyloid Deposition in the PS2APP Mouse Model of Alzheimer's Disease.

To fight Alzheimer's disease (AD), we should know when, where, and how brain network dysfunctions initiate. In AD mouse models, relevant information can be derived from brain electrical activity. With a multi-site linear probe, we recorded local field potentials simultaneously at the posterior-parietal cortex and hippocampus of wild-type and double transgenic AD mice, under anesthesia. We focused on PS2APP (B6.152H) mice carrying both presenilin-2 (PS2) and amyloid precursor protein (APP) mutations, at three and six months of age, before and after plaque deposition respectively. To highlight defects linked to either the PS2 or APP mutation, we included in the analysis age-matched PS2.30H and APP-Swedish mice, carrying each of the mutations individually. Our study also included PSEN2-/- mice. At three months, only predeposition B6.152H mice show a reduction in the functional connectivity of slow oscillations (SO) and in the power ratio between SO and delta waves. At six months, plaque-seeding B6.152H mice undergo a worsening of the low/high frequency power imbalance and show a massive loss of cortico-hippocampal phase-amplitude coupling (PAC) between SO and higher frequencies, a feature shared with amyloid-free PS2.30H mice. We conclude that the PS2 mutation is sufficient to impair SO PAC and accelerate network dysfunctions in amyloid-accumulating mice.

Early hippocampal hyperexcitability in PS2APP mice: role of mutant PS2 and APP.

Alterations of brain network activity are observable in Alzheimer's disease (AD) together with the occurrence of mild cognitive impairment, before overt pathology. However, in humans as well in AD mouse models, identification of early biomarkers of network dysfunction is still at its beginning. We performed in vivo recordings of local field potential activity in the dentate gyrus of PS2APP mice expressing the human amyloid precursor protein (APP) Swedish mutation and the presenilin-2 (PS2) N141I. From a frequency-domain analysis, we uncovered network hyper-synchronicity as early as 3 months, when intracellular accumulation of amyloid beta was also observable. In addition, at 6 months of age, we identified network hyperactivity in the beta/gamma frequency bands, along with increased theta-beta and theta-gamma phase-amplitude cross-frequency coupling, in coincidence with the histopathological traits of the disease. Although hyperactivity and hypersynchronicity were respectively detected in mice expressing the PS2-N141I or the APP Swedish mutant alone, the increase in cross-frequency coupling specifically characterized the 6-month-old PS2APP mice, just before the surge of the cognitive decline.

When, where and how? Focus on neuronal calcium dysfunctions in Alzheimer's Disease.

Alzheimer's disease (AD), since its characterization as a precise form of dementia with its own pathological hallmarks, has captured scientists' attention because of its complexity. The last 30 years have been filled with discoveries regarding the elusive aetiology of this disease and, thanks to advances in molecular biology and live imaging techniques, we now know that an important role is played by calcium (Ca2+). Ca2+, as ubiquitous second messenger, regulates a vast variety of cellular processes, from neuronal excitation and communication, to muscle fibre contraction and hormone secretion, with its action spanning a temporal scale that goes from microseconds to hours. It is therefore very challenging to conceive a single hypothesis that can integrate the numerous findings on this issue with those coming from the classical fields of AD research such as amyloid-beta (Aß) and tau pathology. In this contribution, we will focus our attention on the Ca2+ hypothesis of AD, dissecting it, as much as possible, in its subcellular localization, where the Ca2+ signal meets its specificity. We will also follow the temporal evolution of the Ca2+ hypothesis, providing some of the most updated discoveries. Whenever possible, we will link the findings regarding Ca2+ dysfunction to the other players involved in AD pathogenesis, hoping to provide a crossover body of evidence, useful to amplify the knowledge that will lead towards the discovery of an effective therapy.

Aß42 oligomers selectively disrupt neuronal calcium release.

Accumulation of amyloid-ß (Aß) peptides correlates with aging and progression of Alzheimer's disease (AD). Aß peptides, which cause early synaptic dysfunctions, spine loss, and memory deficits, also disturb intracellular Ca(2+) homeostasis. By cytosolic and endoplasmic reticulum Ca(2+) measurements, we here define the short-term effects of synthetic Aß42 on neuronal Ca(2+) dynamics. When applied acutely at submicromolar concentration, as either oligomers or monomers, Aß42 did not cause Ca(2+) release or Ca(2+) influx. Similarly, 1-hour treatment with Aß42 modified neither the resting cytosolic Ca(2+) level nor the long-lasting Ca(2+) influx caused by KCl-induced depolarization. In contrast, Aß42 oligomers, but not monomers, significantly altered Ca(2+) release from stores with opposite effects on inositol 1,4,5-trisphosphate (IP3)- and caffeine-induced Ca(2+) mobilization without alteration of the total store Ca(2+) content. Ca(2+) dysregulation by Aß42 oligomers involves metabotropic glutamate receptor 5 and requires network activity and the intact exo-endocytotic machinery, being prevented by tetrodotoxin and tetanus toxin. These findings support the idea that Ca(2+) store dysfunction is directly involved in Aß42 neurotoxicity and represents a potential therapeutic target in AD-like dementia.

Ca2+ dysregulation in neurons from transgenic mice expressing mutant presenilin 2.

Mutations in amyloid precursor protein (APP), and presenilin-1 and presenilin-2 (PS1 and PS2) have causally been implicated in Familial Alzheimer's Disease (FAD), but the mechanistic link between the mutations and the early onset of neurodegeneration is still debated. Although no consensus has yet been reached, most data suggest that both FAD-linked PS mutants and endogenous PSs are involved in cellular Ca2+ homeostasis. We here investigated subcellular Ca2+ handling in primary neuronal cultures and acute brain slices from wild type and transgenic mice carrying the FAD-linked PS2-N141I mutation, either alone or in the presence of the APP Swedish mutation. Compared with wild type, both types of transgenic neurons show a similar reduction in endoplasmic reticulum (ER) Ca2+ content and decreased response to metabotropic agonists, albeit increased Ca2+ release induced by caffeine. In both transgenic neurons, we also observed a higher ER-mitochondria juxtaposition that favors increased mitochondrial Ca2+ uptake upon ER Ca2+ release. A model is described that integrates into a unifying hypothesis the contradictory effects on Ca2+ homeostasis of different PS mutations and points to the relevance of these findings in neurodegeneration and aging.

Presenilin-2 modulation of ER-mitochondria interactions: FAD mutations, mechanisms and pathological consequences.

Presenilin (PS) mutations are the main cause of Familial Alzheimer's Disease (FAD) and have been demonstrated to cause an imbalance of intracellular Ca(2+) homeostasis. Though PS1 and 2 are generally considered to behave similarly in terms of their effects on Ca(2+) handling, we have recently described a novel function, which is unique to PS2, i.e., the modulation of ER-mitochondria juxtaposition. Accordingly, PS2, but not PS1, affects the Ca(2+) cross-talk between these organelles, a key feature in determining cell fate. In particular, PS2 overexpression, and more drastically that of FAD-linked PS2 mutants, strongly increases the interaction between ER and mitochondria, thus facilitating mitochondrial Ca(2+) uptake. The likely mechanisms behind this phenomenon and its potential effects in cell physiology and pathology are discussed.

Presenilin 2 modulates endoplasmic reticulum (ER)-mitochondria interactions and Ca2+ cross-talk.

Presenilin mutations are the main cause of familial Alzheimer's disease (FAD). Presenilins also play a key role in Ca(2+) homeostasis, and their FAD-linked mutants affect cellular Ca(2+) handling in several ways. We previously have demonstrated that FAD-linked presenilin 2 (PS2) mutants decrease the Ca(2+) content of the endoplasmic reticulum (ER) by inhibiting sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) activity and increasing ER Ca(2+) leak. Here we focus on the effect of presenilins on mitochondrial Ca(2+) dynamics. By using genetically encoded Ca(2+) indicators specifically targeted to mitochondria (aequorin- and GFP-based probes) in SH-SY5Y cells and primary neuronal cultures, we show that overexpression or down-regulation of PS2, but not of presenilin 1 (PS1), modulates the Ca(2+) shuttling between ER and mitochondria, with its FAD mutants strongly favoring Ca(2+) transfer between the two organelles. This effect is not caused by a direct PS2 action on mitochondrial Ca(2+)-uptake machinery but rather by an increased physical interaction between ER and mitochondria that augments the frequency of Ca(2+) hot spots generated at the cytoplasmic surface of the outer mitochondrial membrane upon stimulation. This PS2 function adds further complexity to the multifaceted nature of presenilins and to their physiological role within the cell. We also discuss the importance of this additional effect of FAD-linked PS2 mutants for the understanding of FAD pathogenesis.

Presenilin-2 dampens intracellular Ca2+ stores by increasing Ca2+ leakage and reducing Ca2+ uptake.

We have previously shown that familial Alzheimer's disease mutants of presenilin-2 (PS2) and, to a lesser extent, of presenilin-1 (PS1) lower the Ca(2+) concentration of intracellular stores. We here examined the mechanism by which wild-type and mutant PS2 affect store Ca(2+) handling. By using HeLa, SH-SY5Y and MEFs as model cells, and recombinant aequorins as Ca(2+) probes, we show evidence that transient expression of either wild-type or mutant PS2 increases the passive Ca(2+) leakage: both ryanodine- and IP(3)-receptors contribute to Ca(2+) exit out of the ER, whereas the ribosome translocon complex is not involved. In SH-SY5Y cells and MEFs, wild-type and mutant PS2 potently reduce the uptake of Ca(2+) inside the stores, an effect that can be counteracted by over-expression of SERCA-2B. On this line, in wild-type MEFs, lowering the endogenous level of PS2 by RNA interference, increases the Ca(2+)-loading capability of intracellular stores. Furthermore, we show that in PS double knockout MEFs, reduction of Ca(2+) stores is mimicked by the expression of PS2-D366A, a loss-of-function mutant, uncleaved because also devoid of presenilinase activity but not by co-expression of the two catalytic active fragments of PS2. In summary, both physiological and increased levels of wild-type and mutant PS2 reduce the Ca(2+) uptake by intracellular stores. To exert this newly described function, PS2 needs to be in its full-length form, even if it can subsequently be cleaved.

Presenilin mutations linked to familial Alzheimer's disease reduce endoplasmic reticulum and Golgi apparatus calcium levels.

Presenilin-1 and -2 (PS1 and PS2) mutations, the major cause of familial Alzheimer's disease (FAD), have been causally implicated in the pathogenesis of neuronal cell death through a perturbation of cellular Ca(2+) homeostasis. We have recently shown that, at variance with previous suggestions obtained in cells expressing other FAD-linked PS mutations, PS2-M239I and PS2-T122R cause a reduction and not an increase in cytosolic Ca(2+) rises induced by Ca(2+) release from stores. In this contribution we have used different cell models: human fibroblasts from controls and FAD patients, cell lines (SH-SY5Y, HeLa, HEK293, MEFs) and rat primary neurons expressing a number of PS mutations, e.g. P117L, M146L, L286V, and A246E in PS1 and M239I, T122R, and N141I in PS2. The effects of FAD-linked PS mutations on cytosolic Ca(2+) changes have been monitored either by using fura-2 or recombinant cytosolic aequorin as the probe. Independently of the cell model or the employed probe, the cytosolic Ca(2+) increases, caused by agonist stimulation or full store depletion by drug treatment, were reduced or unchanged in cells expressing the PS mutations. Using aequorins, targeted to the endoplasmic reticulum or the Golgi apparatus, we here show that FAD-linked PS mutants lower the Ca(2+) content of intracellular stores. The phenomenon was most prominent in cells expressing PS2 mutants, and was observed also in cells expressing the non-pathogenic, "loss-of-function" PS2-D366A mutation. Taken as a whole, our findings, while confirming the capability of presenilins to modify Ca(2+) homeostasis, suggest a re-evaluation of the "Ca(2+) overload" hypothesis in AD and a new working hypothesis is presented.

Nimodipine selectively stimulates beta-amyloid 1-42 secretion by a mechanism independent of calcium influx blockage.

Several lines of evidence indicate that perturbed cellular Ca2+ homeostasis may play a prominent role in synaptic dysfunction and neuronal death in Alzheimer's disease (AD), suggesting a potential benefit of drugs capable to stabilize Ca2+ homeostasis. We here investigated the effects of a panel of L-type Ca2+ channel antagonists on the secretion of the amyloid beta-peptide (Abeta), which abnormally accumulates in the senile plaques of the brain of AD patients. We found that, in primary and immortalized neuronal cells in culture, nimodipine robustly stimulated secretion (up to about four-fold at 30 microM) of the highly amyloidogenic 42-residue isoform of Abeta (Abeta42), while leaving largely unaffected total Abeta secretion. An analogous effect was also observed in vivo, as the administration of a single dose of nimodipine (10 mg/kg i.p.) induced a significant rise of Abeta42 levels in plasma of Tg2576 mice. The effect of nimodipine was independent of blockage of L-type Ca2+ channels and capacitative calcium entry. Accordingly, nimodipine effect was largely Ca2+-independent, as neither depletion nor rise of extracellular Ca2+ abolished it. Hence, by showing that the effect of nimodipine on Abeta42 production is distinct from its ability to block Ca2+-influx pathways, we provide evidence for a previously uncharacterized effect of this long known molecule also used in clinical practice.

Reduction of Ca2+ stores and capacitative Ca2+ entry is associated with the familial Alzheimer's disease presenilin-2 T122R mutation and anticipates the onset of dementia.

Mutations in the presenilin genes PS1 and PS2, the major cause of familial Alzheimer's disease (FAD), are associated with alterations in Ca2+ signalling. In contrast to the majority of FAD-linked PS1 mutations, which cause an overload of intracellular Ca2+ pools, the FAD-linked PS2 mutation M239I reduces Ca2+ release from intracellular stores [Zatti, G., Ghidoni, R., Barbiero, L., Binetti, G., Pozzan, T., Fasolato, C., Pizzo, P., 2004. The presenilin 2 M239I mutation associated with Familial Alzheimer's Disease reduces Ca2+ release from intracellular stores. Neurobiol. Dis. 15/2, 269-278]. We here show that in human FAD fibroblasts another PS2 mutation (T122R) reduces both Ca2+ release and capacitative Ca2+ entry. The observation, done in two monozygotic twins, is of note since only one of the subjects showed overt signs of disease at the time of biopsy whereas the other one developed the disease 3 years later. This finding indicates that Ca2+ dysregulation anticipates the onset of dementia. A similar Ca2+ alteration occurred in HeLa and HEK293 cells transiently expressing PS2-T122R. Based on these data, the "Ca2+ overload" hypothesis in AD pathogenesis is here discussed and reformulated.

The presenilin 2 M239I mutation associated with familial Alzheimer's disease reduces Ca2+ release from intracellular stores.

Mutations in presenilin (PS) genes account for the majority of the cases of the familial form of Alzheimer's disease (FAD). PS mutations have been correlated with both over-production of the amyloid-beta-42 (Abeta42) peptide and alterations of cellular Ca(2+) homeostasis. We here show, for the first time, the effect of the recently described PS2 FAD-associated M239I mutation on two major parameters of intracellular Ca(2+) homeostasis: the Ca(2+) storing capacity of the endoplasmic reticulum (ER) and the activation level of capacitative Ca(2+) entry (CCE), the Ca(2+) influx pathway activated by depletion of intracellular stores. Ca(2+) release from intracellular stores was significantly reduced in fibroblasts from FAD patients, compared to that found in cells from healthy individuals or patients affected by sporadic forms of Alzheimer's Disease (AD). No significant difference was however found in CCE between FAD and control fibroblasts. Similar results were obtained in two cell lines (HEK293 and HeLa) stably or transiently expressing the PS2 M239I mutation.

Paradoxical Ca2+ rises induced by low external Ca2+ in rat hippocampal neurones.

Confocal Ca2+ imaging of rat hippocampal slices shows a paradoxical effect of acute reductions of the [Ca2+]o. Upon slice perfusion with low-Ca2+ media, a prompt intracellular Ca2+ rise selectively occurs in neurones. This response is observed only in slices challenged with agonists of group I metabotropic glutamate or M1 muscarinic receptors. In contrast, the intracellular Ca2+ level of non-stimulated neurones is insensitive to reductions of [Ca2+]o. The phenomenon is observed in 20-25 % of cultured cortical neurones. Evidence is provided demonstrating that: (1) this paradoxical response is not due to a non-specific decrease in divalent cation concentration but it is selectively activated by a reduction in [Ca2+]o, being maximal with [Ca2+]o between 0.25 and 0.5 mM; (2) upon maximal stimulation, 70-90 % of CA1-CA3 pyramidal neurones sense a reduction in [Ca2+]o; a weaker response is observed in neurones from the neocortex, whereas neurones from the dentate gyrus and granule cells from the cerebellum fail to respond; (3) conditions that elicit paradoxical Ca2+ responses cause depolarisation and increase the firing rate of hippocampal neurones; (4) paradoxical Ca2+ rises depend, primarily, on Ca2+ influx through L-type voltage-operated Ca2+ channels and to a lesser extent on release from intracellular Ca2+ stores. Inhibition of phospholipase C or protein kinase C failed to suppress the neuronal response, whereas a selective inhibitor of the Src-family of tyrosine kinases abolishes the paradoxical neuronal Ca2+ rise. A model is presented to explain how this response is elicited by contemporaneous reduction of the [Ca2+]o and metabotropic receptor stimulation; implications for the pathophysiology of the CNS are also discussed.