Identifying genes that interact with Drosophila presenilin and amyloid precursor protein

The γ‐secretase complex is involved in cleaving transmembrane proteins such as Notch and one of the genes targeted in Alzheimer's disease known as amyloid precursor protein (APP). Presenilins function within the catalytic core of γ‐secretase, and mutated forms of presenilins were identified as causative factors in familial Alzheimer's disease. Recent studies show that in addition to Notch and APP, numerous signal transduction pathways are modulated by presenilins, including intracellular calcium signaling. Thus, presenilins appear to have diverse roles. To further understand presenilin function, we searched for Presenilin‐interacting genes in Drosophila by performing a genetic modifier screen for enhancers and suppressors of Presenilin‐dependent Notch‐related phenotypes. We identified 177 modifiers, including known members of the Notch pathway and genes involved in intracellular calcium homeostasis. We further demonstrate that 53 of these modifiers genetically interacted with APP. Characterization of these genes may provide valuable insights into Presenilin function in development and disease. genesis 47:246–260, 2009. © 2009 Wiley‐Liss, Inc.     

Involvement of presenilin holoprotein upregulation in calcium dyshomeostasis of Alzheimer's disease

Mutations in presenilins (PS1 and PS2) account for the vast majority of early onset familial Alzheimer's disease cases. Beside the well investigated role of presenilins as the catalytic unit in γ‐secretase complex, their involvement in regulation of intracellular calcium homeostasis has recently come into more focus of Alzheimer's disease research. Here we report that the overexpression of PS1 full‐length holoprotein forms, in particular familial Alzheimer's disease‐causing forms of PS1, result in significantly attenuated calcium release from thapsigargin‐ and bradykinin‐sensitive stores. Interestingly, treatment of HEK293 cells with γ‐secretase inhibitors also leads to decreased amount of calcium release from endoplasmic reticulum (ER) accompanying elevated PS1 holoprotein levels. Similarly, the knockdown of PEN‐2 which is associated with deficient PS1 endoproteolysis and accumulation of its holoprotein form also leads to decreased ER calcium release. Notably, we detected enhanced PS1 holoprotein levels also in postmortem brains of patients carrying familial Alzheimer's disease PS1 mutations. Taken together, the conditions in which the amount of full length PS1 holoprotein is increased result in reduction of calcium release from ER. Based on these results, we propose that the disturbed ER calcium homeostasis mediated by the elevation of PS1 holoprotein levels may be a contributing factor to the pathogenesis of Alzheimer's disease.     

Effects of transmitters and amyloid-beta peptide on calcium signals in rat cortical astrocytes: Fura-2AM measurements and stochastic model simulations

Background

To better understand the complex molecular level interactions seen in the pathogenesis of Alzheimer''s disease, the results of the wet-lab and clinical studies can be complemented by mathematical models. Astrocytes are known to become reactive in Alzheimer''s disease and their ionic equilibrium can be disturbed by interaction of the released and accumulated transmitters, such as serotonin, and peptides, including amyloid- peptides (A). We have here studied the effects of small amounts of A25–35 fragments on the transmitter-induced calcium signals in astrocytes by Fura-2AM fluorescence measurements and running simulations of the detected calcium signals.

Methodology/Principal Findings

Intracellular calcium signals were measured in cultured rat cortical astrocytes following additions of serotonin and glutamate, or either of these transmitters together with A25–35. A25–35 increased the number of astrocytes responding to glutamate and exceedingly increased the magnitude of the serotonin-induced calcium signals. In addition to A25–35-induced effects, the contribution of intracellular calcium stores to calcium signaling was tested. When using higher stimulus frequency, the subsequent calcium peaks after the initial peak were of lower amplitude. This may indicate inadequate filling of the intracellular calcium stores between the stimuli. In order to reproduce the experimental findings, a stochastic computational model was introduced. The model takes into account the major mechanisms known to be involved in calcium signaling in astrocytes. Model simulations confirm the principal experimental findings and show the variability typical for experimental measurements.

Conclusions/Significance

Nanomolar A25–35 alone does not cause persistent change in the basal level of calcium in astrocytes. However, even small amounts of A25–35, together with transmitters, can have substantial synergistic effects on intracellular calcium signals. Computational modeling further helps in understanding the mechanisms associated with intracellular calcium oscillations. Modeling the mechanisms is important, as astrocytes have an essential role in regulating the neuronal microenvironment of the central nervous system.     

Amyloid β-peptide directly induces spontaneous calcium transients,delayed intercellular calcium waves and gliosis in rat cortical astrocytes

The contribution of astrocytes to the pathophysiology of AD (Alzheimer''s disease) and the molecular and signalling mechanisms that potentially underlie them are still very poorly understood. However, there is mounting evidence that calcium dysregulation in astrocytes may be playing a key role. Intercellular calcium waves in astrocyte networks in vitro can be mechanically induced after Aβ (amyloid β-peptide) treatment, and spontaneously forming intercellular calcium waves have recently been shown in vivo in an APP (amyloid precursor protein)/PS1 (presenilin 1) Alzheimer''s transgenic mouse model. However, spontaneous intercellular calcium transients and waves have not been observed in vitro in isolated astrocyte cultures in response to direct Aβ stimulation in the absence of potentially confounding signalling from other cell types. Here, we show that Aβ alone at relatively low concentrations is directly able to induce intracellular calcium transients and spontaneous intercellular calcium waves in isolated astrocytes in purified cultures, raising the possibility of a potential direct effect of Aβ exposure on astrocytes in vivo in the Alzheimer''s brain. Waves did not occur immediately after Aβ treatment, but were delayed by many minutes before spontaneously forming, suggesting that intracellular signalling mechanisms required sufficient time to activate before intercellular effects at the network level become evident. Furthermore, the dynamics of intercellular calcium waves were heterogeneous, with distinct radial or longitudinal propagation orientations. Lastly, we also show that changes in the expression levels of the intermediate filament proteins GFAP (glial fibrillary acidic protein) and S100B are affected by Aβ-induced calcium changes differently, with GFAP being more dependent on calcium levels than S100B.     

Lysosomal Dysfunction Promotes Cleavage and Neurotoxicity of Tau In Vivo

Expansion of the lysosomal system, including cathepsin D upregulation, is an early and prominent finding in Alzheimer''s disease brain. Cell culture studies, however, have provided differing perspectives on the lysosomal connection to Alzheimer''s disease, including both protective and detrimental influences. We sought to clarify and molecularly define the connection in vivo in a genetically tractable model organism. Cathepsin D is upregulated with age in a Drosophila model of Alzheimer''s disease and related tauopathies. Genetic analysis reveals that cathepsin D plays a neuroprotective role because genetic ablation of cathepsin D markedly potentiates tau-induced neurotoxicity. Further, generation of a C-terminally truncated form of tau found in Alzheimer''s disease patients is significantly increased in the absence of cathepsin D. We show that truncated tau has markedly increased neurotoxicity, while solubility of truncated tau is decreased. Importantly, the toxicity of truncated tau is not affected by removal of cathepsin D, providing genetic evidence that modulation of neurotoxicity by cathepsin D is mediated through C-terminal cleavage of tau. We demonstrate that removing cathepsin D in adult postmitotic neurons leads to aberrant lysosomal expansion and caspase activation in vivo, suggesting a mechanism for C-terminal truncation of tau. We also demonstrate that both cathepsin D knockout mice and cathepsin D–deficient sheep show abnormal C-terminal truncation of tau and accompanying caspase activation. Thus, caspase cleavage of tau may be a molecular mechanism through which lysosomal dysfunction and neurodegeneration are causally linked in Alzheimer''s disease.     

Familial Alzheimer's disease-linked presenilin-1 mutation M146V affects store-operated calcium entry: Does gain look like loss?

Alzheimer's disease (AD) is a neurodegenerative disorder that leads to neuron death and synapse loss in the hippocampus and cortex, with consequent cognitive disability and dementia. Mutations in the presenilin-1 (PS1) gene lead to familial Alzheimer's disease (FAD). Here, we report that the expression of FAD-linked PS1 M146V mutant affects store-operated calcium channel activity (Isoc) in human neuroblastoma SK–N–SH cells. Electrophysiological measurements and calcium imaging experiments have revealed the emergent role of calcium sensor STIM2 in the inhibition of calcium release-activated calcium channel activity (Icrac) and enhancement of intracellular Ca²⁺ stores content due to PS1 M146V mutant expression. In general, the results of this study suggest that the pathological inhibition of one type of store-operated calcium channels caused by FAD PS1 mutant expression may be accounted for by preceding gain of spontaneous activity of store-operated calcium channels driven by STIM2.     

New therapeutic targets in Alzheimer's disease: brain deregulation of calcium and zinc

The molecular determinants of Alzheimer''s (AD) disease are still not completely known; however, in the past two decades, a large body of evidence has indicated that an important contributing factor for the disease is the development of an unbalanced homeostasis of two signaling cations: calcium (Ca²⁺) and zinc (Zn²⁺). Both ions serve a critical role in the physiological functioning of the central nervous system, but their brain deregulation promotes amyloid-β dysmetabolism as well as tau phosphorylation. AD is also characterized by an altered glutamatergic activation, and glutamate can promote both Ca²⁺ and Zn²⁺ dyshomeostasis. The two cations can operate synergistically to promote the generation of free radicals that further intracellular Ca²⁺ and Zn²⁺ rises and set the stage for a self-perpetuating harmful loop. These phenomena can be the initial steps in the pathogenic cascade leading to AD, therefore, therapeutic interventions aiming at preventing Ca²⁺ and Zn²⁺ dyshomeostasis may offer a great opportunity for disease-modifying strategies.     

The Unfolded Protein Response Protects from Tau Neurotoxicity In Vivo

The unfolded protein response is a critical system by which the cell handles excess misfolded protein in the secretory pathway. The role of the system in modulating the effects of aggregation prone cytosolic proteins has received less attention. We use genetic reporters to demonstrate activation of the unfolded protein response in a transgenic Drosophila model of Alzheimer''s disease and related tauopathies. We then use loss of function genetic reagents to support a role for the unfolded protein response in protecting from tau neurotoxicity. Our findings suggest that the unfolded protein response can ameliorate the toxicity of tau in vivo.     

Drosophila as a Model Organism for Investigating Molecular and Cellular Etiologies Underlying Complex Neurological Disorders in Humans

The fruit fly Drosophila has been utilized as a powerful biological system to address fundamental questions concerning neurological disorders in humans, since the related basic molecular components and signal transduction pathways in humans are mostly conserved in Drosophila. In addition, Drosophila offers great experimental advantages in genetics, behavioral analysis and cell and molecular biology. Pathogenesis and etiologies underlying several monogenic neurological disorders including familial Parkinson disease, Alzheimer's disease, or ataxia have been faithfully replicated in Drosophila system when causative mutations of those disorders were transgenically introduced or loss of function mutations of endogenous homologues were made. However, more than 90% of reported cases of neurological disorders are complex forms whose inheritance patterns do not follow a monogenic inheritance. Nevertheless, complex disorders are more often observed among the families or relatives of affected patients, strongly suggesting that they are most likely to be caused by interaction of multiple genetic mutations or combinations of genetic and environmental risk factors. Complex neurological disorders are include sporadic forms of Parkinson disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), dystonia, epilepsy, and mental retardation, etc. Our understanding of the genetic defects and environmental risk factors involved in the onset and progression of complex neurological disorders are, however, still rudimentary. Thus identifying unknown factors involved in the onset and progression of complex neurological disorders in humans is one of the major challenges in medical sciences.     

The Amyloid Precursor Protein Controls PIKfyve Function

While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer’s disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP''s implication in Alzheimer''s disease. Using our recently developed proteo-liposome assay we established the interactome of APP''s intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer''s disease.     

Alterations in both acetylcholinesterase activity and synaptic scaffolding protein localization in the nervous system of Drosophila presenilin mutants

Presenilins are one of two types of critical genetic factors in familial Alzheimer's disease, and they regulate various cellular functions such as intracellular Ca²⁺ homeostasis, the endoplasmic reticulum (ER) stress response, apoptosis, and synaptic transmission. We utilized Drosophila presenilin (psn) mutants as a model for studying the role of this gene in regulating acetylcholinesterase activity (AChE) and synaptic plasticity. Several lines of biochemical evidence indicated that AChE activity in a functionally null psn mutant (psn^B3) was significantly reduced. In addition, we also found that psn^B3 mutant neuromuscular junctions (NMJs) had smaller synaptic boutons and altered localization of Discs large, a synaptic scaffolding protein at the synaptic terminals compared to wild-type controls. These phenotypic defects were completely rescued in transgenic lines expressing the long form of wild-type Psn under an endogenous psn promoter cassette (PEPC-Psn^WT;psn^B3 lines). Taken together, these results indicate that Psn is important for regulating AChE activity, the size of synaptic boutons, and the localization of DLG at synaptic terminals.     

Porphyrin derivatives as inhibitors for acetylcholinesterase from Drosophila melanogaster

The cure for Alzheimer''s disease (AD) is still unknown. According to Cholinergic hypothesis, Alzheimer''s disease is caused by the reduced synthesis of the neurotransmitter, Acetylcholine. Regional cerebral blood flow can be increased in patients with Alzheimer''s disease by Acetylcholinesterase (AChE) inhibitors. In this regard, Tetraphenylporphinesulfonate (TPPS), 5,10,15,20- Tetrakis (4-sulfonatophenyl) porphyrinato Iron(III) Chloride (FeTPPS) and 5,10,15,20-Tetrakis (4-sulfonatophenyl) porphyrinatoIron(III) nitrosyl Chloride (FeNOTPPS) were investigated as candidate compounds for inhibition of Acteylcholinesterase of Drosophila melanogaster (DmAChE) by use of Molecular Docking. The results show that FeNOTPPS forms the most stable complex with DmAChE.     

The actin-binding protein capulet genetically interacts with the microtubule motor kinesin to maintain neuronal dendrite homeostasis

Background

Neurons require precise cytoskeletal regulation within neurites, containing microtubule tracks for cargo transport in axons and dendrites or within synapses containing organized actin. Due to the unique architecture and specialized function of neurons, neurons are particularly susceptible to perturbation of the cytoskeleton. Numerous actin-binding proteins help maintain proper cytoskeletal regulation.

Methodology/Principal Findings

From a Drosophila forward genetic screen, we identified a mutation in capulet-encoding a conserved actin-binding protein-that causes abnormal aggregates of actin within dendrites. Through interaction studies, we demonstrate that simultaneous genetic inactivation of capulet and kinesin heavy chain, a microtubule motor protein, produces elongate cofilin-actin rods within dendrites but not axons. These rods resemble actin-rich structures induced in both mammalian neurodegenerative and Drosophila Alzheimer''s models, but have not previously been identified by loss of function mutations in vivo. We further demonstrate that mitochondria, which are transported by Kinesin, have impaired distribution along dendrites in a capulet mutant. While Capulet and Cofilin may biochemically cooperate in certain circumstances, in neuronal dendrites they genetically antagonize each other.

Conclusions/Significance

The present study is the first molecularly defined loss of function demonstration of actin-cofilin rods in vivo. This study suggests that simultaneous, seemingly minor perturbations in neuronal dendrites can synergize producing severe abnormalities affecting actin, microtubules and mitochondria/energy availability in dendrites. Additionally, as >90% of Alzheimer''s and Parkinson''s cases are sporadic this study suggests mechanisms by which multiple mutations together may contribute to neurodegeneration instead of reliance on single mutations to produce disease.     

Alzheimer's Therapeutics Targeting Amyloid Beta 1–42 Oligomers II: Sigma-2/PGRMC1 Receptors Mediate Abeta 42 Oligomer Binding and Synaptotoxicity

Amyloid beta (Abeta) 1–42 oligomers accumulate in brains of patients with Mild Cognitive Impairment (MCI) and disrupt synaptic plasticity processes that underlie memory formation. Synaptic binding of Abeta oligomers to several putative receptor proteins is reported to inhibit long-term potentiation, affect membrane trafficking and induce reversible spine loss in neurons, leading to impaired cognitive performance and ultimately to anterograde amnesia in the early stages of Alzheimer''s disease (AD). We have identified a receptor not previously associated with AD that mediates the binding of Abeta oligomers to neurons, and describe novel therapeutic antagonists of this receptor capable of blocking Abeta toxic effects on synapses in vitro and cognitive deficits in vivo. Knockdown of sigma-2/PGRMC1 (progesterone receptor membrane component 1) protein expression in vitro using siRNA results in a highly correlated reduction in binding of exogenous Abeta oligomers to neurons of more than 90%. Expression of sigma-2/PGRMC1 is upregulated in vitro by treatment with Abeta oligomers, and is dysregulated in Alzheimer''s disease patients'' brain compared to age-matched, normal individuals. Specific, high affinity small molecule receptor antagonists and antibodies raised against specific regions on this receptor can displace synthetic Abeta oligomer binding to synaptic puncta in vitro and displace endogenous human AD patient oligomers from brain tissue sections in a dose-dependent manner. These receptor antagonists prevent and reverse the effects of Abeta oligomers on membrane trafficking and synapse loss in vitro and cognitive deficits in AD mouse models. These findings suggest sigma-2/PGRMC1 receptors mediate saturable oligomer binding to synaptic puncta on neurons and that brain penetrant, small molecules can displace endogenous and synthetic oligomers and improve cognitive deficits in AD models. We propose that sigma-2/PGRMC1 is a key mediator of the pathological effects of Abeta oligomers in AD and is a tractable target for small molecule disease-modifying therapeutics.     

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca²⁺) often termed a ''Ca²⁺ wave''. While the speed and number of oscillations of the Ca²⁺ wave varies between species, the change in intracellular Ca²⁺ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca²⁺-dependent events. Here we present a protocol for imaging the Ca²⁺ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca²⁺ wave and the downstream changes associated with it.     

Control of Alzheimer's Amyloid Beta Toxicity by the High Molecular Weight Immunophilin FKBP52 and Copper Homeostasis in Drosophila

FK506 binding proteins (FKBPs), also called immunophilins, are prolyl-isomerases (PPIases) that participate in a wide variety of cellular functions including hormone signaling and protein folding. Recent studies indicate that proteins that contain PPIase activity can also alter the processing of Alzheimer''s Amyloid Precursor Protein (APP). Originally identified in hematopoietic cells, FKBP52 is much more abundantly expressed in neurons, including the hippocampus, frontal cortex, and basal ganglia. Given the fact that the high molecular weight immunophilin FKBP52 is highly expressed in CNS regions susceptible to Alzheimer''s, we investigated its role in Aβ toxicity. Towards this goal, we generated Aβ transgenic Drosophila that harbor gain of function or loss of function mutations of FKBP52. FKBP52 overexpression reduced the toxicity of Aβ and increased lifespan in Aβ flies, whereas loss of function of FKBP52 exacerbated these Aβ phenotypes. Interestingly, the Aβ pathology was enhanced by mutations in the copper transporters Atox1, which interacts with FKBP52, and Ctr1A and was suppressed in FKBP52 mutant flies raised on a copper chelator diet. Using mammalian cultures, we show that FKBP52 (−/−) cells have increased intracellular copper and higher levels of Aβ. This effect is reversed by reconstitution of FKBP52. Finally, we also found that FKBP52 formed stable complexes with APP through its FK506 interacting domain. Taken together, these studies identify a novel role for FKBP52 in modulating toxicity of Aβ peptides.     

High-dose of vitamin C supplementation reduces amyloid plaque burden and ameliorates pathological changes in the brain of 5XFAD mice

Blood–brain barrier (BBB) breakdown and mitochondrial dysfunction have been implicated in the pathogenesis of Alzheimer''s disease (AD), a neurodegenerative disease characterized by cognitive deficits and neuronal loss. Besides vitamin C being as one of the important antioxidants, recently, it has also been reported as a modulator of BBB integrity and mitochondria morphology. Plasma levels of vitamin C are decreased in AD patients, which can affect disease progression. However, investigation using animal models on the role of vitamin C in the AD pathogenesis has been hampered because rodents produce with no dependence on external supply. Therefore, to identify the pathogenic importance of vitamin C in an AD mouse model, we cross-bred 5 familial Alzheimer''s disease mutation (5XFAD) mice (AD mouse model) with ι-gulono-γ-lactone oxidase (Gulo) knockout (KO) mice, which are unable to synthesize their own vitamin C, and produced Gulo KO mice with 5XFAD mice background (KO-Tg). These mice were maintained on either low (0.66 g/l) or high (3.3 g/l) supplementation of vitamin C. We found that the higher supplementation of vitamin C had reduced amyloid plaque burden in the cortex and hippocampus in KO-Tg mice, resulting in amelioration of BBB disruption and mitochondrial alteration. These results suggest that intake of a larger amount of vitamin C could be protective against AD-like pathologies.     

Alzheimer's Therapeutics Targeting Amyloid Beta 1–42 Oligomers I: Abeta 42 Oligomer Binding to Specific Neuronal Receptors Is Displaced by Drug Candidates That Improve Cognitive Deficits

Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1–42 oligomers is proposed to underlie cognitive decline in Alzheimer''s disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC₅₀ that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer''s disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors - i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer''s therapeutics.     

Sensitivity changes of photoreceptor cells of Hirudo medicinalis caused by changes in extracellular calcium concentration

Extracellular recordings from the vacoule of photoreceptor cells of Hirudo medicinalis L. were performed using microelectrodes. The cells were adapted by white light flashes given at constant intervals (20 s). Response height versus relative intensity curves obtained from the same cell in physiological saline (PS) and in bathing solutions of either a) lowered calcium contents (2 ΜM/1 or less) or b) raised calcium contents (15 mM/1) were compared. The cells' adaptation state in PS was operationally defined by the ratio Q=h _A /h _S where h _A is the response height evoked by the adapting flashes, and h _S is the corresponding saturation response height. Sensitivity changes were measured by the half saturation intensity shift. Lowering extracellular calcium resulted in:

1. The response height increased and the shape of the response became more rounded and prolonged.
2. The total resistance between the vacuole and outside decreased from 8.2±1.4 MΩ (n=6) in PS to 4.6±0.4 MΩ (n=5). The resistance was independent of the cells' adaptation state.
3. A change of the cells' sensitivity occured either in direction to light adaptation or in direction to dark adaptation. It depended functionally on the ratio Q:

a) if Q was less or equal to about 0.6 the cells' sensitivity increased. b) if Q was greater than 0.6 the cells' sensitivity diminished. Raising extracellular calcium decreased the sensitivity of all cells tested independent of their adaptation states in PS. The results can be interpreted under the assumptions that 1. the sensitivity of leech photoreceptor cells is inversely proportional to the intracellular free calcium concentration and Z. intracellular calcium can interact with extracellular calcium in relatively dark adapted cells whereas in relatively light adapted cells the raise of intracellular free calcium is mainly effected by a release from intracellular stores. It is assumed that a Q value of about 0.6 separates relatively light adapted cells from relatively dark adapted cells.