Cell-specific expression of plasma membrane calcium ATPase isoforms in retinal neurons

Ca(2+) extrusion by high-affinity plasma membrane calcium ATPases (PMCAs) is a principal mechanism for the clearance of Ca(2+) from the cytosol. The PMCA family consists of four isoforms (PMCA1-4). Little is known about the selective expression of these isoforms in brain tissues or about the physiological function conferred upon neurons by any given isoform. We investigated the cellular and subcellular distribution of PMCA isoforms in a mammalian retina. Mouse photoreceptors, cone bipolar cells and horizontal cells, which respond to light with a graded polarization, express isoform 1 (PMCA1) of the PMCA family. PMCA2 is localized to rod bipolar cells, horizontal cells, amacrine cells, and ganglion cells, and PMCA3 is predominantly expressed in spiking neurons, including both amacrine and ganglion cells but is also found in horizontal cells. PMCA4 was found to be selectively expressed in both synaptic layers. Optical measurements of Ca(2+) clearance showed that PMCAs mediate Ca(2+) extrusion in both rod and cone bipolar cells. In addition, we found that rod bipolar cells, but not cone bipolar cells possess a prominent Na(+)/Ca(2+) exchange mechanism. We conclude that PMCA isoforms are selectively expressed in retinal neurons and that processes of Ca(2+) clearance are different in rod and cone bipolar cells.     

Perisynaptic organization of plasma membrane calcium pumps in cerebellar cortex

Calcium, a ubiquitous intracellular messenger, regulates numerous intracellular signaling pathways. To permit specificity of signal transduction and prevent unwanted cross-talk between pathways, sites of calcium entry in neurons are localized to specific membrane domains. To test whether Ca(2+) extrusion pumps might exhibit analogous compartmentalization, we used immunohistochemistry to determine the subcellular localization of the two main plasma membrane Ca(2+)-ATPase (PMCA) isoforms in the cortex of the rat cerebellum. We find that both PMCA2 and PMCA3 are targeted to distinct compartments within the plasma membrane. In the molecular layer, both isoforms were at highest levels within synaptic profiles, but PMCA2 was postsynaptic and PMCA3 was presynaptic. Moreover, inside these compartments, both pumps exhibited nonuniform distributions. These data imply that cerebellar neurons possess remarkably effective mechanisms to target and restrict PMCA2 and -3 to specific membrane domains, raising the possibility that calcium pumps contribute to local Ca(2+) signaling.     

Isoform-specific distribution of the plasma membrane Ca2+ ATPase in the rat brain

Regulation of cytoplasmic calcium is crucial both for proper neuronal function and cell survival. The concentration of Ca2+ in cytoplasm of a neuron at rest is 10,000 times lower than in the extracellular space, pointing to the importance of the transporters that extrude intracellular Ca2+. The family of plasma membrane calcium-dependent ATPases (PMCAs) represent a major component of the Ca2+ regulatory system. However, little information is available on the regional and cellular distribution of these calcium pumps. We used immunohistochemistry to investigate the distribution of each of the four PMCA isoforms (PMCA1-4) in the rat brain. Each isoform exhibited a remarkably precise and distinct pattern of distribution. In many cases, PMCA isoforms in a single brain structure were differentially expressed within different classes of neurons, and within different subcellular compartments. These data show that each isoform is independently organized and suggest that PMCAs may play a more complex role in calcium homeostasis than generally recognized.     

Astrocytes selectively enhance N-type calcium current in hippocampal neurons

Astrocytes influence neuronal development, synapse formation, and synaptic transmission, partly through affecting neuronal calcium signals. In order to elucidate the extent to which astrocytes modulate neuronal voltage-gated calcium currents, we performed a whole-cell patch clamp analysis of neurons in astrocyte-deplete and astrocyte-enriched conditions. We demonstrate that hippocampal neurons in an astrocyte-enriched environment show augmentation of voltage-gated calcium current at 1-3 days in vitro. Further study in pairs of adjacent neurons showed that the augmentation in calcium current was dependent on direct contact with the astrocyte. Pharmacological analysis demonstrated the augmentation is selective for the N-type calcium current, although immunochemical labeling of the alpha1(B) subunit of the N-type calcium channel was unchanged. These findings show that astrocytes regulate neuronal voltage-gated calcium currents in a contact-dependent manner. The specificity of the effect for the N-type calcium current at early days in culture has special significance regarding the role of astrocytes in hippocampal synaptogenesis.     

Subcellular calcium dynamics during juvenile development in mouse hippocampal astrocytes

Astrocytes generate calcium signals throughout their fine processes, which are assumed to locally regulate neighbouring neurotransmission and blood flow. The intercellular morphological relationships mature during juvenile periods when astrocytes elongate highly ramified processes. In this study, we examined developmental changes in calcium activity patterns of single hippocampal astrocytes using a transgenic mouse line in which astrocytes selectively express a genetically encoded calcium indicator, Yellow Cameleon‐Nano50. Compared with postnatal day 7, astrocytes at postnatal day 30 showed larger subcellular calcium events and a greater proportion of somatic events. At both ages, the calcium activity was abolished by removal of extracellular calcium ions. Calcium events in late juvenile astrocytes were not affected by spontaneously occurring sharp waves that trigger synchronized neuronal spikes, implying the independence of astrocyte calcium signals from neuronal synchronization. These results demonstrate that astrocytes undergo dynamic changes in their activity patterns during juvenile development.     

Formaldehyde increases intracellular calcium concentration in primary cultured hippocampal neurons partly through NMDA receptors and T-type calcium channels

Objective Formaldehyde at high concentrations is a contributor to air pollution.It is also an endogenous metabolic product in cells,and when beyond physiological concentrations,has pathological effects on neurons.Formaldehyde induces mis-folding and aggregation of neuronal tau protein,hippocampal neuronal apoptosis,cognitive impairment and loss of memory functions,as well as excitation of peripheral nociceptive neurons in cancer pain models.Intracellular calcium([Ca²⁺]_i) is an important intracellular messenger,and plays a key role in many pathological processes.The present study aimed to investigate the effect of formaldehyde on[Ca²⁺]_i and the possible involvement of N-methyl-Z)-aspartate receptors （NMDARs） and T-type Ca²⁺ channels on the cell membrane.Methods Using primary cultured hippocampal neurons as a model,changes of[Ca²⁺]_i in the presence of formaldehyde at a low concentration were detected by confocal laser scanning microscopy.Results Formaldehyde at 1 mmol/L approximately doubled[Ca²⁺]_i.（2R）-amino-5-phosphonopentanoate （AP5,25μmol/L,an NMDAR antagonist） and mibefradil(MIB,1 umol/L,a T-type Ca²⁺channel blocker),given 5 min after formaldehyde perfusion,each partly inhibited the formaldehyde-induced increase of[Ca²⁺]_i,and this inhibitory effect was reinforced by combined application of AP5 and MIB.When applied 3 min before formaldehyde perfusion,AP5 （even at 50μmol/L） did not inhibit the formaldehyde-induced increase of[Ca²⁺]_i but MIB（1 umol/L） significantly inhibited this increase by 70%.Conclusion These results suggest that formaldehyde at a low concentration increases[Ca²⁺]_i in cultured hippocampal neurons;NMDARs and T-type Ca²⁺ channels may be involved in this process.     

Nimodipine decreases calcium action potentials in rabbit hippocampal CA1 neurons in an age-dependent and concentration-dependent manner

Intracellular recordings were made from rabbit hippocampal CA1 neurons in vitro using slices from aging and young adult rabbits. Calcium action potentials were studied in the presence of 4 μm tetrodotoxin using electrodes filled with 2M CsCl. Increasing concentrations of the dihydropyridine L-type calcium channel antagonist nimodipine were tested on the amplitude and time course of calcium action potentials. The calcium action potential (AP) consisted of two components: an initial fast phase followed by a slower plateau phase. No difference in the peak amplitude of the initial fast phase was observed between age groups. The amplitude and duration of the slower plateau phase of the calcium AP was significantly larger in aging neurons. Switching to a zero Ca²⁺ medium in the presence of 200 μm CdCl₂ completely blocked the calcium AP. Nimodipine decreased the plateau phase of the calcium AP at concentrations as low as 100 nm in aging neurons and 10 μm in young neurons. Switching to higher concentrations of nimodipine did not reveal any substantially increased block of the calcium AP plateau phase. These data suggest that enhanced calcium influx through L-type calcium channels is largely responsible for the enhanced calcium action potentials observed in aging CA1 neurons. The action of nimodipine in reducing the plateau phase of the calcium action potential may underlie the drug's notable ability to improve learning in hippocampally dependent tasks in aging animals.     

Increased expression of Nogo-A in hippocampal neurons of patients with temporal lobe epilepsy

Mesial temporal lobe epilepsy (TLE) is associated with pronounced anatomical and biochemical changes in the hippocampal formation including extensive neurodegeneration, reorganization of mossy fibres and sprouting of interneurons. Although the anatomical features and some of the physiological consequences of hippocampal remodeling have been well documented, the molecular mechanisms underlying the profound and orientated outgrowth of hippocampal neurons in TLE are not yet understood. The reticulon protein Nogo-A has been associated with an inhibitory action on axon growth and plasticity. Using immunohistochemistry and in situ hybridization, we investigated the expression of Nogo-A in specimens obtained at surgery from patients with TLE compared with those obtained from autopsy controls. In control specimens, Nogo-A immunoreactivity and mRNA were mainly confined to oligodendrocytes. Only approximately 40% of the specimens revealed low expression of Nogo-A mRNA in neurons. In contrast, in TLE patients with and without Ammon's horn sclerosis, Nogo-A mRNA and immunoreactivity were markedly up-regulated in most neurons (3.6- and 4.4-fold increases in Nogo-A mRNA in granule cells of sclerotic and nonsclerotic specimens) and their processes throughout the hippocampal formation. Similar elevations in Nogo-A mRNA and protein levels were determined by quantitative RT-PCR and Western blotting. Since Nogo-A expression was also up-regulated in specimens without hippocampal sclerosis, it may be induced by seizures prior to progressing neurodegeneration.     

Axonal remodeling during postnatal maturation of CA3 hippocampal pyramidal neurons

Anatomical substrates were investigated for local circuit hyperexcitability that occurs in the CA3 subfield of the rat hippocampus during postnatal week 2. A transient excess of excitatory local circuit connectivity was hypothesized to underlie this hyperexcitability. To test this hypothesis, recurrent excitatory axon arbors from single biocytin-filled CA3 pyramidal cells were reconstructed. Arbors were analyzed in segments of area CA3 comparable in size to in vitro minislice preparations, which were shown to reproduce the developmental hyperexcitability seen in intact slices during postnatal week 2. Segments were then adjusted for hippocampal growth, based on age-dependent changes in neuron density in stratum pyramidale. Axon arbors were found to be short and possessed very few branches during the first postnatal week. By the second postnatal week, arbors had undergone dramatic growth and were much longer and more complex in their branching patterns. By adulthood, a significant decrease in all measures of arbor length and complexity was observed. Following growth adjustment, measures of axon length and varicosity number during week 2 were not significantly different from that of adulthood. However, the number of axon branches decreased by 50%. These results suggest that, during early postnatal life, there is exuberant outgrowth of local CA3 recurrent axons, and with maturation these recurrent collaterals are remodeled. Short-ranging, profusely branched axons appear to be replaced by longer-ranging arbors that possess fewer branches. Maturational changes in the dendritic location rather than the number of early-formed recurrent excitatory synapses may explain developmental hyperexcitability of the hippocampal CA3 subfield. J. Comp. Neurol. 384:165-180, 1997. © 1997 Wiley-Liss, Inc.     

Effects of opioids on glutamate induced calcium intracellular increase in individually perfused rat hippocampal neurons in vitro

1. 1. Superfusion of cultured hippocampal cells with glutamate (Glu) 0.5 mM for 5 min induces an increase of [Ca²⁺_i] that is quickly followed by a recovery to control level. Addition of dynorphin or D-pen²-D-pen⁵-enkephalin (DPDPE) induces a persistence of the elevated [Ca²⁺_i], while [D-Ala²,N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO) does not influence it.

2. 2. Superfusion with Glu for 10 min induces a persistent increase of [Ca²⁺_i], that is partially reverted by DAMGO, but not affected by dynorphin or DPDPE.

3. 3. The author suggests a differential influence of selective opioids on the Glu-induced [Ca²⁺_i] increase.

     

Changes in protein kinase C isozymes in the rat hippocampal formation following hippocampal lesion

The cellular and regional distribution of the four protein kinase C (PKC) isoforms in the rat hippocampal formation and the response of PKC to lesions were determined by employing immunohistochemical and immunochemical techniques with antibodies specific to PKC (α), -(β I), -(β II), and -(γ). PKC (α) intensely stained the periphery of the pyramidal cell in the stratum pyramidale. The granule cells, glial cells, and mossy fibers were anti-PKC (α) negative. The cytoplasm, axons, and dendrites of basket cells and interneurons in the hilus were labeled with anti-PKC (α). Anti-PKC (β I) immunoreactivity was localized on the periphery of pyramidal cells and interneurons of the hilus, as well as the oriens, radiatum, and molecular layers of the CA regions. Anti-PKC (β II) immunoreactivity was mainly cytoplasmic, extending into the dendrites in the hippocampal pyramidal cells and the dentate granule cells, and also in some glial cells. In the stratum radiatum of the CA1, anti-PKC (γ) immunoreactivity localized to the pyramidal cell cytoplasm, extending into the dendrites. Following fimbria-fornix (FF) lesions, the anti-PKC (α) and -(β I) staining of the pyramidal cell periphery was markedly reduced. The anti-PKC (γ) staining of the pyramidal and granular cells of the dentate gyrus was reduced whereas the interneuron staining in the hilus was increased. In the FF-lesioned hippocampus, anti-PKC (α) and antiαPKC (β II) labeled reactive glial cells, whereas anti-PKC (β I) and -(γ) did not. Quantitative Western blot analysis revealed a dramatic increase in the particulate/total PKC for all isozyme forms, although the total levels of PKC, except PKC (γ), did not change following FF lesions. The PKC (γ) concentration doubled after FF lesions. Perforant path lesions resulted in a marked alteration in the neuronal staining in dentate gyrus with anti-PKC (α) and -(β I) and in increased numbers of anti-PKC (α)- and anti-PKC (β II)-positive glial cells. Anti-PKC (γ) staining did not change noticeably. The total PKC concentration did not change for isozymes α, β I, and γ, but PKC (βII) concentration increased by 48% following perforant path lesions as detected by Western blot analysis. The particulate/total PKC decreased for all four isozymes although the reduction in PKC (β I) concentration was not statistically significant. This change in PKC compartmentalization is in marked contrast to an increased level of particulate PKC following FF lesions. Thus, the effects of deefferentation and deafferentation for each PKC isoform were different.     

Calcium extrusion protein expression in the hippocampal formation of chronic epileptic rats after kainate-induced status epilepticus

PURPOSE: The plasma membrane Ca2+ -adenosine triphosphatase (ATPase) (PMCA) and (potassium-dependent) sodium-calcium exchange [NC(K)X] represent two main calcium-extrusion mechanisms that are important for the restoration of [Ca2+]i levels after electrical activity. We investigated whether the expression of these calcium-extrusion proteins is altered in the course of epileptogenesis. METHODS: Hippocampal-parahippocampal protein expression of NCX1, 2, and 3, PMCA1-4, and NCKX2 at an early and late stage after kainate-induced status epilepticus (SE) was compared with that in control rats by using immunocytochemistry. RESULTS: Several alterations were found in chronic epileptic rats: (a) NCX1 expression was permanently decreased in the inner molecular layer (IML) of the dentate gyrus (DG) and entorhinal cortex layer III (ECIII), related to neuronal loss in hilus and ECIII, respectively; (b) PMCA and NCKX2 expression was transiently upregulated in the IML, and decreased in several areas where cell loss had occurred, (c) NCX3 expression, which in control rats is abundant in presynaptic terminals of mossy fibers (MF), was extensively and permanently decreased in stratum lucidum and hilar region. In addition, newly formed MF sprouts that project to the DG iml did not noticeably express NCX3; (d) NCX2 and NCKX2 were (transiently) upregulated in astrocytes of epileptic rats throughout the hippocampal formation, including ECIII. CONCLUSIONS: These region-specific changes in calcium-extrusion proteins reflect a change in calcium regulation. Whether these regional-specific changes of calcium-extrusion proteins are associated with an abnormal calcium homeostasis must be determined. Because some alterations of calcium-extrusion protein expression are already present at an early stage of epileptogenesis, they could be involved in this process.     

Dynamic regulation of spine-dendrite coupling in cultured hippocampal neurons

We investigated the role of dendritic spine morphology in spine-dendrite calcium communication using novel experimental and theoretical approaches. A transient rise in [Ca2+]i was produced in individual spine heads of Fluo-4-loaded cultured hippocampal neurons by flash photolysis of caged calcium. Following flash photolysis in the spine head, a delayed [Ca2+]i transient was detected in the parent dendrites of only short, but not long, spines. Delayed elevated fluorescence in the dendrite of the short spines was also seen with a membrane-bound fluorophore and fluorescence recovery from bleaching of a calcium-bound fluorophore had a much slower kinetics, indicating that the dendritic fluorescence change reflects a genuine diffusion of free [Ca2+]i from the spine head to the parent dendrite. Calcium diffusion between spine head and the parent dendrite was regulated by calcium stores as well as by a Na-Ca exchanger. Spine length varied with the recent history of the [Ca2+]i variations in the spine, such that small numbers of calcium transients resulted in elongation of spines whereas large numbers of calcium transients caused shrinkage of the spines. Consequently, spine elongation resulted in a complete isolation of the spine from the dendrite, while shrinkage caused an enhanced coupling with the parent dendrite. These studies highlight a dynamically regulated coupling between a dendritic spine head and its parent dendrite.     

Hypoxic preconditioning failure in aging hippocampal neurons: impaired gene expression and rescue with intracellular calcium chelation

Hypoxic preconditioning in the brain (HPC), a phenomenon whereby noninjurious hypoxia induces resistance to cell death following ischemia, requires the expression of specific genes. Declines in signal transduction pathway activity with aging may decrease the genomic response to HPC and limit its neuroprotective efficacy. To test this, we determined how signal transduction gene expression, intracellular Ca(2+) levels, and phosphorylation of the survival-associated kinase Akt differ in hippocampal slice cultures (HSCs) made from postnatal day 7-10 (P7-10) and 2-year-old rats following HPC. HPC neuroprotection decreased with increasing source animal age, and HPC could not be demonstrated in HCSs made from animals >6 months of age, despite adjusting the duration of hypoxic exposure. Preconditioning protection required the survival kinase Akt in P10 hippocampal slices cultures. In P9 cultures, HPC increased Akt phosphorylation and the expression of prosurvival genes, including Bcl-2, heat shock proteins, protein kinases, c-jun, and NfκB. Lack of increased Akt phosphorylation and a greatly diminished signaling pathway gene response were found in HSCs from aging animals. Moderate and transient increases in [Ca(2+) ](i) during HPC occurred in P7-10 HSCs, but [Ca(2+) ](i) was persistently increased at 1 and 24 hr after preconditioning in HSCs from 2-year-old rats. The intracellular Ca(2+) chelator BAPTA-AM facilitated HPC neuroprotection in 2-year-old HSCs and restored the pattern of post-HPC gene expression seen in immature animals. We conclude that age-related loss of preconditioning may be due to altered intracellular Ca(2+) homeostasis (excess and sustained increase in [Ca(2+) ](i) ) and is a lesion that prevents critical elements of neuroprotective signal transduction.     

Cellular and subcellular localization of the neuron‐specific plasma membrane calcium ATPase PMCA1a in the rat brain

Regulation of intracellular calcium is crucial both for proper neuronal function and survival. By coupling ATP hydrolysis with Ca²⁺ extrusion from the cell, the plasma membrane calcium‐dependent ATPases (PMCAs) play an essential role in controlling intracellular calcium levels in neurons. In contrast to PMCA2 and PMCA3, which are expressed in significant levels only in the brain and a few other tissues, PMCA1 is ubiquitously distributed, and is thus widely believed to play a “housekeeping” function in mammalian cells. Whereas the PMCA1b splice variant is predominant in most tissues, an alternative variant, PMCA1a, is the major form of PMCA1 in the adult brain. Here, we use immunohistochemistry to analyze the cellular and subcellular distribution of PMCA1a in the brain. We show that PMCA1a is not ubiquitously expressed, but rather is confined to neurons, where it concentrates in the plasma membrane of somata, dendrites, and spines. Thus, rather than serving a general housekeeping function, our data suggest that PMCA1a is a calcium pump specialized for neurons, where it may contribute to the modulation of somatic and dendritic Ca²⁺ transients. J. Comp. Neurol. 518:3169–3183, 2010. © 2010 Wiley‐Liss, Inc.     

Modification of the responses of hippocampal neurons in the monkey during the learning of a conditional spatial response task

In order to analyze the function of the hippocampus in learning, the activity of single neurons was recorded while monkeys learned a task of the type known to be impaired by damage to the hippocampus. In the conditional response task, the monkey had to learn to make one response when one stimulus was shown, and a different response when a different stimulus was shown. It had previously been shown that there are neurons in the hippocampal formation that respond in this task, to, for example, a combination of a particular visual stimulus that had been associated in previous learning with a particular behavioral response. In the present study, it was found that during such conditional response learning, the activity of 22% of the neurons in the hippocampus and parahippocampal gyrus with activity specifically related to the task altered their responses so that their activity, which was initially equal to the two new stimuli, became progressively differential to the two stimuli when the monkey learned to make different responses to the two stimuli. These changes occurred for different neurons just before, at, or just after the time when the monkey learned the correct response to make to the stimuli. In addition to these neurons, which had differential responses that were sustained for as long as the recordings continued, another population of neurons (45% of those with activity specifically related to the task) developed differential activity to the two new stimuli, yet showed such differential responses transiently for only a small number of trials at about the time when the monkey learned. These findings are consistent with the hypothesis that some synapses on hippocampal neurons modify during this type of learning so that some neurons come to respond to particular stimulus-response associations that are being learned. Further, the finding that many hippocampal neurons started to reflect the new learning, but then stopped responding differentially (the transient neurons), is consistent with the hypothesis that the hippocampal neurons with large sustained changes in their activity inhibited the transient neurons, which then underwent reverse learning, thus providing a competitive mechanism by which not all neurons are allocated to any one learned association or event.     

Expression of embryonic tau protein isoforms persist during adult neurogenesis in the hippocampus

Tau is a microtubule-associated protein with a developmentally regulated expression of multiple isoforms. The neonatal isoform is devoid of two amino terminal inserts and contains only three instead of four microtubule-binding repeats (0N/3R-tau). We investigated the temporal expression pattern of 0N-tau and 3R-tau in the rat hippocampus. After the decline of 0N- and 3R-tau immunoreactivity during the postnatal development both isoforms remain highly expressed in a few cells residing beneath the granule cell layer. Coexpression of the polysialylated neuronal cell adhesion molecule, doublecortin, and incorporated bromodeoxyuridine showed that these cells are proliferating progenitor cells. In contrast mature granule cells express the adult tau protein isoform containing one aminoterminal insert domain (1N-tau). Therefore a shift in tau isoform expression takes place during adult neurogenesis, which might be related to migration, differentiation, and integration in the granule cell layer. A model for studying shifts in tau isoform expression in a defined subset of neurons might help to understand the etiology of tauopathies, when isoform composition is crucial for neurodegeneration, as in Pick's disease or FTDP-17.     

Calcium uptake and retention during long-term potentiation of neuronal activity in the rat hippocampal slice preparation

Alterations in⁴⁵Ca uptake and retention as well as total intracellular calcium content were measured in hippocampal ‘in vitro’ slice preparations following the induction of long-term potentiation (LTP) in CA1 pyramidal neurones. The results indicate that tetanic stimulation of the Schaffer-commissural input produces a significant increase in the uptake and retention of calcium which parallels LTP of the CA1 population spike response.