Generation of iPSC-derived human forebrain organoids assembling bilateral eye primordia.

Induced pluripotent stem cell-derived brain organoids enable the developmental complexities of the human brain to be deconstructed. During embryogenesis, optic vesicles (OVs), the eye primordium attached to the forebrain, develop from diencephalon. However, most 3D culturing methods generate either brain or retinal organoids individually. Here we describe a protocol to generate organoids with both forebrain entities, which we call OV-containing brain organoids (OVB organoids). In this protocol, we first induce neural differentiation (days 0-5) and collect neurospheres, which we culture in a neurosphere medium to initiate their patterning and further self-assembly (days 5-10). Then, upon transfer to spinner flasks containing OVB medium (days 10-30), neurospheres develop into forebrain organoids with one or two pigmented dots restricted to one pole, displaying forebrain entities of ventral and dorsal cortical progenitors and preoptic areas. Further long-term culture results in photosensitive OVB organoids constituting complementary cell types of OVs, including primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections and electrically active neuronal networks. OVB organoids provide a system to help dissect interorgan interactions between the OVs as sensory organs and the brain as a processing unit, and can help model early eye patterning defects, including congenital retinal dystrophy. To conduct the protocol, experience in sterile cell culture and maintenance of human induced pluripotent stem cells is essential; theoretical knowledge of brain development is advantageous. Furthermore, specialized expertise in 3D organoid culture and imaging for the analysis is needed.

ß2-subunit alternative splicing stabilizes Cav2.3 Ca2+ channel activity during continuous midbrain dopamine neuron-like activity.

In dopaminergic (DA) Substantia nigra (SN) neurons Cav2.3 R-type Ca2+-currents contribute to somatodendritic Ca2+-oscillations. This activity may contribute to the selective degeneration of these neurons in Parkinson's disease (PD) since Cav2.3-knockout is neuroprotective in a PD mouse model. Here, we show that in tsA-201-cells the membrane-anchored ß2-splice variants ß2a and ß2e are required to stabilize Cav2.3 gating properties allowing sustained Cav2.3 availability during simulated pacemaking and enhanced Ca2+-currents during bursts. We confirmed the expression of ß2a- and ß2e-subunit transcripts in the mouse SN and in identified SN DA neurons. Patch-clamp recordings of mouse DA midbrain neurons in culture and SN DA neurons in brain slices revealed SNX-482-sensitive R-type Ca2+-currents with voltage-dependent gating properties that suggest modulation by ß2a- and/or ß2e-subunits. Thus, ß-subunit alternative splicing may prevent a fraction of Cav2.3 channels from inactivation in continuously active, highly vulnerable SN DA neurons, thereby also supporting Ca2+ signals contributing to the (patho)physiological role of Cav2.3 channels in PD.

Microgravity-induced stress mechanisms in human stem cell-derived cardiomyocytes.

Exposure to outer space microgravity poses a risk for the development of various pathologies including cardiovascular disease. To study this, we derived cardiomyocytes (CMs) from human-induced pluripotent stem cells and exposed them to simulated microgravity (SMG). We combined different "omics" and chromosome conformation capture technologies with live-cell imaging of various transgenic lines to discover that SMG impacts on the contractile velocity and function of CMs via the induction of senescence processes. This is linked to SMG-induced changes of reactive oxygen species (ROS) generation and energy metabolism by mitochondria. Taken together, we uncover a microgravity-controlled axis causing contractile dysfunctions to CMs. Our findings can contribute to the design of preventive and therapeutic strategies against senescence-associated disease.

L-cysteine modulates visceral nociception mediated by the CaV2.3 R-type calcium channels.

CaV2.3 channels are subthreshold voltage-gated calcium channels that play crucial roles in neurotransmitter release and regulation of membrane excitability, yet modulation of these channels with endogenous molecules and their role in pain processing is not well studied. Here, we hypothesized that an endogenous amino acid l-cysteine could be a modulator of these channels and may affect pain processing in mice. To test this hypothesis, we employed conventional patch-clamp technique in the whole-cell configuration using recombinant CaV2.3 subunit stably expressed in human embryonic kidney (HEK-293) cells. We found in our in vitro experiments that l-cysteine facilitated gating and increased the amplitudes of recombinant CaV2.3 currents likely by chelating trace metals that tonically inhibit the channel. In addition, we took advantage of mouse genetics in vivo using the acetic acid visceral pain model that was performed on wildtype and homozygous Cacna1e knockout male littermates. In ensuing in vivo experiments, we found that l-cysteine administered both subcutaneously and intraperitoneally evoked more prominent pain responses in the wildtype mice, while the effect was completely abolished in knockout mice. Conversely, intrathecal administration of l-cysteine lowered visceral pain response in the wildtype mice, and again the effect was completely abolished in the knockout mice. Our study strongly suggests that l-cysteine-mediated modulation of CaV2.3 channels plays an important role in visceral pain processing. Furthermore, our data are consistent with the contrasting roles of CaV2.3 channels in mediating visceral nociception in the peripheral and central pain pathways.

Human brain organoids assemble functionally integrated bilateral optic vesicles.

During embryogenesis, optic vesicles develop from the diencephalon via a multistep process of organogenesis. Using induced pluripotent stem cell (iPSC)-derived human brain organoids, we attempted to simplify the complexities and demonstrate formation of forebrain-associated bilateral optic vesicles, cellular diversity, and functionality. Around day 30, brain organoids attempt to assemble optic vesicles, which develop progressively as visible structures within 60 days. These optic vesicle-containing brain organoids (OVB-organoids) constitute a developing optic vesicle's cellular components, including primitive corneal epithelial and lens-like cells, retinal pigment epithelia, retinal progenitor cells, axon-like projections, and electrically active neuronal networks. OVB-organoids also display synapsin-1, CTIP-positive myelinated cortical neurons, and microglia. Interestingly, various light intensities could trigger photosensitive activity of OVB-organoids, and light sensitivities could be reset after transient photobleaching. Thus, brain organoids have the intrinsic ability to self-organize forebrain-associated primitive sensory structures in a topographically restricted manner and can allow interorgan interaction studies within a single organoid.

Retinal Vessel Responses to Flicker Stimulation Are Impaired in Ca v 2.3-Deficient Mice-An in-vivo Evaluation Using Retinal Vessel Analysis (RVA).

Objective: Metabolic demand increases with neuronal activity and adequate energy supply is ensured by neurovascular coupling (NVC). Impairments of NVC have been reported in the context of several diseases and may correlate with disease severity and outcome. Voltage-gated Ca2+-channels (VGCCs) are involved in the regulation of vasomotor tone. In the present study, we compared arterial and venous responses to flicker stimulation in Cav2.3-competent (Cav2.3[+/+]) and -deficient (Cav2.3[-/-]) mice using retinal vessel analysis. Methods: The mice were anesthetized and the pupil of one eye was dilated by application of a mydriaticum. An adapted prototype of retinal vessel analyzer was used to perform dynamic retinal vessel analysis. Arterial and venous responses were quantified in terms of the area under the curve (AUCart/AUCven) during flicker application, mean maximum dilation (mMDart/mMDven) and time to maximum dilation (tMDart/tMDven) during the flicker, dilation at flicker cessation (DFCart/DFCven), mean maximum constriction (mMCart/mMCven), time to maximum constriction (tMCart/tMCven) after the flicker and reactive magnitude (RMart/RMven). Results: A total of 33 retinal scans were conducted in 22 Cav2.3[+/+] and 11 Cav2.3[-/-] mice. Cav2.3[-/-] mice were characterized by attenuated and partially reversed arterial and venous responses, as reflected in significantly lower AUCart (p = 0.031) and AUCven (p = 0.047), a trend toward reduced DFCart (p = 0.100), DFCven (p = 0.100), mMDven (p = 0.075), and RMart (p = 0.090) and a trend toward increased tMDart (p = 0.096). Conclusion: To our knowledge, this is the first study using a novel, non-invasive analysis technique to document impairment of retinal vessel responses in VGCC-deficient mice. We propose that Cav2.3 channels could be involved in NVC and may contribute to the impairment of vasomotor responses under pathophysiological conditions.

Non-Mendelian inheritance during inbreeding of Cav3.2 and Cav2.3 deficient mice.

The mating of 77 heterozygous pairs (Cav3.2[+|-] x Cav3.2[+|-]) revealed a significant deviation of genotype distribution from Mendelian inheritance in weaned pups. The mating of 14 pairs (Cav3.2[-|-] female x Cav3.2[+|-] male) and 8 pairs (Cav3.2[+|-] female x Cav3.2[-|-] male) confirmed the significant reduction of deficient homozygous Cav3.2[-|-] pups, leading to the conclusion that prenatal lethality may occur, when one or both alleles, encoding the Cav3.2T-type Ca2+ channel, are missing. Also, the mating of 63 heterozygous pairs (Cav2.3[+|-] x Cav2.3[+|-]) revealed a significant deviation of genotype distribution from Mendelian inheritance in weaned pups, but only for heterozygous male mice, leading to the conclusion that compensation may only occur for Cav2.3[-|-] male mice lacking both alleles of the R-type Ca2+ channel. During the mating of heterozygous parents, the number of female mice within the weaned population does not deviate from the expected Mendelian inheritance. During prenatal development, both, T- and R-type Ca2+ currents are higher expressed in some tissues than postnatally. It will be discussed that the function of voltage-gated Ca2+ channels during prenatal development must be investigated in more detail, not least to understand devastative diseases like developmental epileptic encephalopathies (DEE).

Cav2.3 channel function and Zn2+-induced modulation: potential mechanisms and (patho)physiological relevance.

Voltage-gated calcium channels (VGCCs) are critical for Ca2+ influx into all types of excitable cells, but their exact function is still poorly understood. Recent reconstruction of homology models for all human VGCCs at atomic resolution provides the opportunity for a structure-based discussion of VGCC function and novel insights into the mechanisms underlying Ca2+ selective flux through these channels. In the present review, we use these data as a basis to examine the structure, function, and Zn2+-induced modulation of Cav2.3 VGCCs, which mediate native R-type currents and belong to the most enigmatic members of the family. Their unique sensitivity to Zn2+ and the existence of multiple mechanisms of Zn2+ action strongly argue for a role of these channels in the modulatory action of endogenous loosely bound Zn2+, pools of which have been detected in a number of neuronal, endocrine, and reproductive tissues. Following a description of the different mechanisms by which Zn2+ has been shown or is thought to alter the function of these channels, we discuss their potential (patho)physiological relevance, taking into account what is known about the magnitude and function of extracellular Zn2+ signals in different tissues. While still far from complete, the picture that emerges is one where Cav2.3 channel expression parallels the occurrence of loosely bound Zn2+ pools in different tissues and where these channels may serve to translate physiological Zn2+ signals into changes of electrical activity and/or intracellular Ca2+ levels.

Synapse and Active Zone Assembly in the Absence of Presynaptic Ca2+ Channels and Ca2+ Entry.

Presynaptic CaV2 channels are essential for Ca2+-triggered exocytosis. In addition, there are two competing models for their roles in synapse structure. First, Ca2+ channels or Ca2+ entry may control synapse assembly. Second, active zone proteins may scaffold CaV2s to presynaptic release sites, and synapse structure is CaV2 independent. Here, we ablated all three CaV2s using conditional knockout in cultured hippocampal neurons or at the calyx of Held, which abolished evoked exocytosis. Compellingly, synapse and active zone structure, vesicle docking, and transsynaptic nano-organization were unimpaired. Similarly, long-term blockade of action potentials and Ca2+ entry did not disrupt active zone assembly. Although CaV2 knockout impaired the localization of ß subunits, α2δ-1 localized normally. Rescue with CaV2 restored exocytosis, and CaV2 active zone targeting depended on the intracellular C-terminus. We conclude that synapse assembly is independent of CaV2s or Ca2+ entry through them. Instead, active zone proteins recruit and anchor CaV2s via CaV2 C-termini.

Zn2+-induced changes in Cav2.3 channel function: An electrophysiological and modeling study.

Loosely bound Zn2+ ions are increasingly recognized as potential modulators of synaptic plasticity and neuronal excitability under normal and pathophysiological conditions. Cav2.3 voltage-gated Ca2+ channels are among the most sensitive targets of Zn2+ and are therefore likely to be involved in the neuromodulatory actions of endogenous Zn2+. Although histidine residues on the external side of domain I have been implicated in the effects on Cav2.3 channel gating, the exact mechanisms involved in channel modulation remain incompletely understood. Here, we use a combination of electrophysiological recordings, modification of histidine residues, and computational modeling to analyze Zn2+-induced changes in Cav2.3 channel function. Our most important findings are that multiple high- and low-affinity mechanisms contribute to the net Zn2+ action, that Zn2+ can either inhibit or stimulate Ca2+ influx through Cav2.3 channels depending on resting membrane potential, and that Zn2+ effects may persist for some time even after cessation of the Zn2+ signal. Computer simulations show that (1) most salient features of Cav2.3 channel gating in the absence of trace metals can be reproduced by an obligatory model in which activation of two voltage sensors is necessary to open the pore; and (2) most, but not all, of the effects of Zn2+ can be accounted for by assuming that Zn2+ binding to a first site is associated with an electrostatic modification and mechanical slowing of one of the voltage sensors, whereas Zn2+ binding to a second, lower-affinity site blocks the channel and modifies the opening and closing transitions. While still far from complete, our model provides a first quantitative framework for understanding Zn2+ effects on Cav2.3 channel function and a step toward the application of computational approaches for predicting the complex actions of Zn2+ on neuronal excitability.

Submicromolar copper (II) ions stimulate transretinal signaling in the isolated retina from wild type but not from Cav2.3-deficient mice.

BACKGROUND: So far, only indirect evidence exists for the pharmacoresistant R-type voltage-gated Ca2+ channel (VGCC) to be involved in transretinal signaling by triggering GABA-release onto ON-bipolar neurons. This release of inhibitory neurotransmitters was deduced from the sensitivity of the b-wave to stimulation by Ni2+, Zn2+ and Cu2+. To further confirm the interpretation of these findings, we compared the effects of Cu2+ application and chelation (using kainic acid, KA) on the neural retina from wildtype and Cav2.3-deficient mice. Furthermore, the immediately effect of KA on the ERG b-wave modulation was assessed. METHODS: Transretinal signaling was recorded as an ERG from the superfused murine retina isolated from wildtype and Cav2.3-deficient mice. RESULTS: In mice, the stimulating effect of 100 nM CuCl2 is absent in the retinae from Cav2.3-deficient mice, but prominent in Cav2.3-competent mice. Application of up to 3 mM tricine does not affect the murine b-wave in both genotypes, most likely because of chelating amino acids present in the murine nutrient solution. Application of 27 µM KA significantly increased the b-wave amplitude in wild type and Cav2.3 (-|-) mice. This effect can most likely be explained by the stimulation of endogenous KA-receptors described in horizontal, OFF-bipolar, amacrine or ganglion cells, which could not be fully blocked in the present study. CONCLUSION: Cu2+-dependent modulation of transretinal signaling only occurs in the murine retina from Cav2.3 competent mice, supporting the ideas derived from previous work in the bovine retina that R-type Ca2+ channels are involved in shaping transretinal responses during light perception.

Consequences of hyperphosphorylated tau on the morphology and excitability of hippocampal neurons in aged tau transgenic mice.

The intracellular accumulation of hyperphosphorylated tau characterizes many neurodegenerative diseases such as Alzheimer's disease and frontotemporal dementia. A critical role for tau is supported by studies in transgenic mouse models expressing the P301L mutation with accumulation of hyperphosphorylated human tau in hippocampal pyramidal neurons of aged mice. Especially, the somatodendritic mislocalization of hyperphosphorylated tau seems to affect the neuronal network of the hippocampus. To show the consequences of aggregation of hyperphosphorylated tau within hippocampal neurons of aged mice, the CA1 pyramidal cells were analyzed morphologically and electrophysiologically. Here we demonstrate in the P301L pR5 mouse model that hyperphosphorylated tau leads to an increase in stubby spines and filopodia, as well as a decrease in total dendritic length of hippocampal pyramidal neurons due to a decrease in apical dendritic length and nodes. This atrophy is in line with the significant reduction in CA1 long-term potentiation. Furthermore, mutant tau induced a depolarized threshold for action potential initiation and an increased current of inward rectifying potassium channels, which should lead, together with the long-term potentiation decrease, to a decreased excitability of CA1 neurons.

Experimentally Induced Convulsive Seizures Are Modulated in Part by Zinc Ions through the Pharmacoresistant Cav2.3 Calcium Channel.

BACKGROUND/AIMS: Still in 1999 the first hints were published for the pharmacoresistant Cav2.3 calcium channel to be involved in the generation of epileptic seizures, as transcripts of alpha1E (Cav2.3) and alpha1G (Cav3.1) are changed in the brain of genetic absence epilepsy rats from Strasbourg (GAERS). Consecutively, the seizure susceptibility of mice lacking Cav2.3 was analyzed in great detail by using 4-aminopyridine, pentylene-tetrazol, N-methyl-D-aspartate and kainic acid to induce experimentally convulsive seizures. Further, γ-hydroxybutyrolactone was used for the induction of non-convulsive absence seizures. For all substances tested, Cav2.3-competent mice differed from their knockout counterparts in the sense that for convulsive seizures the deletion of the pharmacoresistant channel was beneficial for the outcome during experimentally induced seizures [1]. The antiepileptic drug lamotrigine reduces seizure activity in Cav2.3-competent but increases it in Cav2.3-deficient mice. In vivo, Cav2.3 must be under tight control by endogenous trace metal cations (Zn2+ and Cu2+). The dyshomeostasis of either of them, especially of Cu2+, may alter the regulation of Cav2.3 severely and its activity for Ca2+ conductance, and thus may change hippocampal and neocortical signaling to hypo- or hyperexcitation. METHODS: To investigate by telemetric EEG recordings the mechanism of generating hyperexcitation by kainate, mice were tested for their sensitivity of changes in neuronal (intracerebroventricular) concentrations of the trace metal cation Zn2+. As the blood-brain barrier limits the distribution of bioavailable Zn2+ or Cu2+ into the brain, we administered micromolar Zn2+ ions intracerebroventricularly in the presence of 1 mM histidine as carrier and compared the effects on behavior and EEG activity in both genotypes. RESULTS: Kainate seizures are more severe in Cav2.3-competent mice than in KO mice and histidine lessens seizure severity in competent but not in Cav2.3-deficient mice. Surprisingly, Zn2+ plus histidine resembles the kainate only control with more seizure severity in Cav2.3-competent than in deficient mice. CONCLUSION: Cav2.3 represents one important Zn2+-sensitive target, which is useful for modulating convulsive seizures.

NCS-1 Deficiency Affects mRNA Levels of Genes Involved in Regulation of ATP Synthesis and Mitochondrial Stress in Highly Vulnerable Substantia nigra Dopaminergic Neurons.

Neuronal Ca2+ sensor proteins (NCS) transduce changes in Ca2+ homeostasis into altered signaling and neuronal function. NCS-1 activity has emerged as important for neuronal viability and pathophysiology. The progressive degeneration of dopaminergic (DA) neurons, particularly within the Substantia nigra (SN), is the hallmark of Parkinson's disease (PD), causing its motor symptoms. The activity-related Ca2+ homeostasis of SN DA neurons, mitochondrial dysfunction, and metabolic stress promote neurodegeneration and PD. In contrast, NCS-1 in general has neuroprotective effects. The underlying mechanisms are unclear. We analyzed transcriptional changes in SN DA neurons upon NCS-1 loss by combining UV-laser microdissection and RT-qPCR-approaches to compare expression levels of a panel of PD and/or Ca2+-stress related genes from wildtype and NCS-1 KO mice. In NCS-1 KO, we detected significantly lower mRNA levels of mitochondrially coded ND1, a subunit of the respiratory chain, and of the neuron-specific enolase ENO2, a glycolytic enzyme. We also detected lower levels of the mitochondrial uncoupling proteins UCP4 and UCP5, the PARK7 gene product DJ-1, and the voltage-gated Ca2+ channel Cav2.3 in SN DA neurons from NCS-1 KO. Transcripts of other analyzed uncoupling proteins (UCPs), mitochondrial Ca2+ transporters, PARK genes, and ion channels were not altered. As Cav channels are linked to regulation of gene expression, metabolic stress and degeneration of SN DA neurons in PD, we analyzed Cav2.3 KO mice, to address if the transcriptional changes in NCS-1 KO were also present in Cav.2.3 KO, and thus probably correlated with lower Cav2.3 transcripts. However, in SN DA neurons from Cav2.3 KO mice, ND1 mRNA as well as genomic DNA levels were elevated, while ENO2, UCP4, UCP5, and DJ-1 transcript levels were not altered. In conclusion, our data indicate a possible novel function of NCS-1 in regulating gene transcription or stabilization of mRNAs in SN DA neurons. Although we do not provide functional data, our findings at the transcript level could point to impaired ATP production (lower ND1 and ENO2) and elevated metabolic stress (lower UCP4, UCP5, and DJ-1 levels) in SN DA neurons from NCS-1 KO mice. We speculate that NCS-1 is involved in stimulating ATP synthesis, while at the same time controlling mitochondrial metabolic stress, and in this way could protect SN DA neurons from degeneration.

Cav2.3 channels contribute to dopaminergic neuron loss in a model of Parkinson's disease.

Degeneration of dopaminergic neurons in the substantia nigra causes the motor symptoms of Parkinson's disease. The mechanisms underlying this age-dependent and region-selective neurodegeneration remain unclear. Here we identify Cav2.3 channels as regulators of nigral neuronal viability. Cav2.3 transcripts were more abundant than other voltage-gated Ca2+ channels in mouse nigral neurons and upregulated during aging. Plasmalemmal Cav2.3 protein was higher than in dopaminergic neurons of the ventral tegmental area, which do not degenerate in Parkinson's disease. Cav2.3 knockout reduced activity-associated nigral somatic Ca2+ signals and Ca2+-dependent after-hyperpolarizations, and afforded full protection from degeneration in vivo in a neurotoxin Parkinson's mouse model. Cav2.3 deficiency upregulated transcripts for NCS-1, a Ca2+-binding protein implicated in neuroprotection. Conversely, NCS-1 knockout exacerbated nigral neurodegeneration and downregulated Cav2.3. Moreover, NCS-1 levels were reduced in a human iPSC-model of familial Parkinson's. Thus, Cav2.3 and NCS-1 may constitute potential therapeutic targets for combatting Ca2+-dependent neurodegeneration in Parkinson's disease.

Intracerebroventricular administration of histidine reduces kainic acid-induced convulsive seizures in mice.

Kainic acid (KA)-induced seizures and other experimental models of epilepsy have been proven to be instrumental in identifying novel targets that could be responsible for human icto- and epileptogenesis. We have previously shown that the ablation of pharmacoresistant voltage-gated Ca2+ channels with Cav2.3 as central ion-conducting pore (R-type Ca2+ channel) reduces the sensitivity towards KA-induced epilepsy in mice. In vivo, Cav2.3 channels are thought to be under tight allosteric control by endogenous loosely bound trace metal cations (Zn2+ and Cu2+) that suppress channel gating via a high-affinity trace metal-binding site. Metal dyshomeostasis in the brain, which is a common feature of (KA-induced) seizures, could therefore alter the normal function of Cav2.3 channels and may shift hippocampal and neocortical signaling towards hyperexcitation. To investigate the role of loosely bound metal ions for KA-induced hyperexcitation in vivo, we examined the effects of manipulating brain trace metal homeostasis in mice. To this end, we developed a murine system for intracerebroventricular administration of trace metal ions and/or histidine (His), which can bind Zn2+ and Cu2+ and is involved in their transendothelial transport at the blood-brain barrier. Unexpectedly, our preliminary findings indicate that application of His alone but not in the presence of Zn2+ has substantial beneficial effects on the outcome of KA-induced epilepsy in mice. As such, our results emphasize previous findings on the complex, two-sided role of loosely bound metal ions with regard to neuronal excitation and degeneration under pathophysiological conditions.

Modulation of Cav2.3 channels by unconjugated bilirubin (UCB) - Candidate mechanism for UCB-induced neuromodulation and neurotoxicity.

Elevated levels of unbound unconjugated bilirubin (UCB) can lead to bilirubin encephalopathy and kernicterus. In spite of a large number of studies demonstrating UCB-induced changes in central neurotransmission, it is still unclear whether these effects involve alterations in the function of specific ion channels. To assess how different UCB concentrations and UCB:albumin (U/A) molar ratios affect neuronal R-type voltage-gated Ca2+ channels, we evaluated their effects on whole-cell currents through recombinant Cav2.3 + ß3 channel complexes and ex-vivo electroretinograms (ERGs) from wildtype and Cav2.3-deficient mice. Our findings show that modestly elevated levels of unbound UCB (U/A = 0.5) produce subtle but significant changes in the voltage-dependence of activation and prepulse inactivation, resulting in a stimulation of currents activated by weak depolarization and inhibition at potentials on the plateau of the activation curve. Saturation of the albumin binding capacity (U/A = 1) produced additional suppression that became significant when albumin was omitted completely and might involve a complete loss of channel function. Acutely administered UCB (U/A = 0.5) has recently been shown to affect transsynaptic signaling in the isolated vertebrate retina. The present report reveals that sustained exposure of the murine retina to UCB significantly suppresses also late responses of the inner retina (b-wave) from wildtype compared to Cav2.3-deficient mice. In addition, recovery during washout was significantly more complete and faster in retinae lacking Cav2.3 channels. Together, these findings show that UCB affects cloned and native Cav2.3 channels at clinically relevant U/A molar ratios and indicate that supersaturation of albumin is not required for modulation but associated with a loss of channel functional that could contribute to chronic neuronal dysfunction.

Non-invasive evaluation of neurovascular coupling in the murine retina by dynamic retinal vessel analysis.

BACKGROUND: Impairment of neurovascular coupling (NVC) was recently reported in the context of subarachnoid hemorrhage and may correlate with disease severity and outcome. However, previous techniques to evaluate NVC required invasive procedures. Retinal vessels may represent an alternative option for non-invasive assessment of NVC. METHODS: A prototype of an adapted retinal vessel analyzer was used to assess retinal vessel diameter in mice. Dynamic vessel analysis (DVA) included an application of monochromatic flicker light impulses in predefined frequencies for evaluating NVC. All retinae were harvested after DVA and electroretinograms were performed. RESULTS: A total of 104 retinal scans were conducted in 21 male mice (90 scans). Quantitative arterial recordings were feasible only in a minority of animals, showing an emphasized reaction to flicker light impulses (8 mice; 14 scans). A characteristic venous response to flicker light, however, could observed in the majority of animals. Repeated measurements resulted in a significant decrease of baseline venous diameter (7 mice; 7 scans, p < 0.05). Ex-vivo electroretinograms, performed after in-vivo DVA, demonstrated a significant reduction of transretinal signaling in animals with repeated DVA (n = 6, p < 0.001). CONCLUSIONS: To the best of our knowledge, this is the first non-invasive study assessing murine retinal vessel response to flicker light with characteristic changes in NVC. The imaging system can be used for basic research and enables the investigation of retinal vessel dimension and function in control mice and genetically modified animals.

Reciprocal modulation of Cav 2.3 voltage-gated calcium channels by copper(II) ions and kainic acid.

Kainic acid (KA) is a potent agonist at non-N-methyl-D-aspartate (non-NMDA) ionotropic glutamate receptors and commonly used to induce seizures and excitotoxicity in animal models of human temporal lobe epilepsy. Among other factors, Cav 2.3 voltage-gated calcium channels have been implicated in the pathogenesis of KA-induced seizures. At physiologically relevant concentrations, endogenous trace metal ions (Cu2+ , Zn2+ ) occupy an allosteric binding site on the domain I gating module of these channels and interfere with voltage-dependent gating. Using whole-cell patch-clamp recordings in human embryonic kidney (HEK-293) cells stably transfected with human Cav 2.3d and ß3 -subunits, we identified a novel, glutamate receptor-independent mechanism by which KA can potently sensitize these channels. Our findings demonstrate that KA releases these channels from the tonic inhibition exerted by low nanomolar concentrations of Cu2+ and produces a hyperpolarizing shift in channel voltage-dependence by about 10 mV, thereby reconciling the effects of Cu2+ chelation with tricine. When tricine was used as a surrogate to study the receptor-independent action of KA in electroretinographic recordings from the isolated bovine retina, it selectively suppressed a late b-wave component, which we have previously shown to be enhanced by genetic or pharmacological ablation of Cav 2.3 channels. Although the pathophysiological relevance remains to be firmly established, we speculate that reversal of Cu2+ -induced allosteric suppression, presumably via formation of stable kainate-Cu2+ complexes, could contribute to the receptor-mediated excitatory effects of KA. In addition, we discuss experimental implications for the use of KA in vitro, with particular emphasis on the seemingly high incidence of trace metal contamination in common physiological solutions.