Different pathways of molecular pathophysiology underlie cognitive and motor tauopathy phenotypes in transgenic models for Alzheimer's disease and frontotemporal lobar degeneration.

A poorly understood feature of the tauopathies is their very different clinical presentations. The frontotemporal lobar degeneration (FTLD) spectrum is dominated by motor and emotional/psychiatric abnormalities, whereas cognitive and memory deficits are prominent in the early stages of Alzheimer's disease (AD). We report two novel mouse models overexpressing different human tau protein constructs. One is a full-length tau carrying a double mutation [P301S/G335D; line 66 (L66)] and the second is a truncated 3-repeat tau fragment which constitutes the bulk of the PHF core in AD corresponding to residues 296-390 fused with a signal sequence targeting it to the endoplasmic reticulum membrane (line 1; L1). L66 has abundant tau pathology widely distributed throughout the brain, with particularly high counts of affected neurons in hippocampus and entorhinal cortex. The pathology is neuroanatomically static and declines with age. Behaviourally, the model is devoid of a higher cognitive phenotype but presents with sensorimotor impairments and motor learning phenotypes. L1 displays a much weaker histopathological phenotype, but shows evidence of neuroanatomical spread and amplification with age that resembles the Braak staging of AD. Behaviourally, the model has minimal motor deficits but shows severe cognitive impairments affecting particularly the rodent equivalent of episodic memory which progresses with advancing age. In both models, tau aggregation can be dissociated from abnormal phosphorylation. The two models make possible the demonstration of two distinct but nevertheless convergent pathways of tau molecular pathogenesis. L1 appears to be useful for modelling the cognitive impairment of AD, whereas L66 appears to be more useful for modelling the motor features of the FTLD spectrum. Differences in clinical presentation of AD-like and FTLD syndromes are therefore likely to be inherent to the respective underlying tauopathy, and are not dependent on presence or absence of concomitant APP pathology.

Synaptic localisation of agmatinase in rat cerebral cortex revealed by virtual pre-embedding.

Light microscopic evidence suggested a synaptic role for agmatinase, an enzyme capable of inactivating the putative neurotransmitter and endogenous anti-depressant agmatine. Using electron microscopy and an alternative pre-embedding approach referred to as virtual pre-embedding, agmatinase was localised pre- and postsynaptically, to dendritic spines, spine and non-spine terminals, and dendritic profiles. In dendritic spines, labelling displayed a tendency towards the postsynaptic density. These results further strengthen a synaptic role for agmatine and strongly suggest a regulatory role for synaptically expressed agmatinase.

Lateral habenular neurons projecting to reward-processing monoaminergic nuclei express hyperpolarization-activated cyclic nucleotid-gated cation channels.

The lateral habenular complex (LHb) is a key signal integrator between limbic forebrain regions and monoaminergic hindbrain nuclei. Major projections of LHb neurons target the dopaminergic ventral tegmental area (VTA) and the serotonergic dorsal (DR) and median raphe nuclei (MnR). Both monoaminergic neurotransmitter systems play a central role in reward processing and reward-related decision-making. Glutamatergic LHb efferents terminate on GABAergic neurons in the VTA, the rostromedial tegmental nucleus (RMTg), and the raphe nuclei, thereby suppressing monoamine release when required by the present behavioral context. Recent studies suggest that the LHb exerts a strong tonic inhibition on monoamine release when no reward is to be obtained. It is yet unknown whether this inhibition is the result of a continuous external activation by other brain areas, or if it is intrinsically generated by LHb projection neurons. To analyze whether the tonic inhibition may be the result of a hyperpolarization-activated cyclic nucleotid-gated cation channel (HCN)-mediated pacemaker activity of LHb projection neurons, we combined retrograde tracing in rats with in situ hybridization of HCN1 to HCN4 mRNAs. In fact, close to all LHb neurons targeting VTA or raphe nuclei are equipped with HCN subunit mRNAs. While HCN1 mRNA is scarce, most neurons display strong expression of HCN2 to HCN4 mRNAs, in line with the potential formation of heteromeric channels. These results are supported by quantitative PCR and immunocytochemical analyses. Thus, our data suggest that the tonic inhibition of monoamine release is intrinsically generated in LHb projection neurons and that their activity may only be modulated by synaptic inputs to the LHb.

Potassium channel expression in adult murine neural progenitor cells.

Neural progenitor cells (NPCs) are a source of new neurons and glia in the adult brain. Most NPCs reside in the forebrain subventricular zone (SVZ) and in the subgranular zone of the dentate gyrus, where they contribute to plasticity in the adult brain. To use their potential for repair, it is essential to identify the molecules that regulate their growth, migration and differentiation. Potassium (K+) channels are promising molecule candidates for NPC regulation as they are important components of signal transduction and their diversity is ideal to cover the complex functions required for cell proliferation and differentiation. There is increasing evidence that K+ channels influence cell growth and neurogenesis, however, very little is known regarding K+ channel distribution in NPCs. We therefore explored the expression of a variety of voltage-gated (Kv), inwardly rectifying (Kir) and two-pore (K2P) K+ channels in the SVZ of adult mice and in neurosphere cultures of NPCs during growth and differentiation. Immunocytochemical analysis revealed a differential expression pattern of K+ channels in nestin+ SVZ precursor cells, early SVZ doublecortin+ neurons and (sub)ependymal cells. These findings were confirmed in neurosphere cultures at the protein and mRNA levels. The expression of some K+ channel proteins, such as Kir4.1, Kir6.1, TREK1 or TASK1, suggests a role of K+ channels in the complex regulation of NPC proliferation, maturation and differentiation.

Morphological and electrophysiological characteristics of neurons within identified subnuclei of the lateral habenula in rat brain slices.

Based on the specificity of its inputs and targets, the lateral habenular complex (LHb) constitutes a pivotal motor-limbic interface implicated in various cerebral functions particularly in regulating monoamine transmission. Despite its functional significance, cellular characteristics underlying LHb functionality have not been examined systematically. The present study aimed to correlate morphological and electrophysiological properties of neurons within the different subnuclei of the LHb using whole-cell recording and neurobiotin labeling in rat slice preparations. Morphological analysis revealed a heterogeneous population of projection neurons randomly distributed throughout the LHb. According to somatodendritic characteristics four main categories were classified including spherical, fusiform, polymorphic and vertical cells. Electrophysiological characterization of neurons within the different categories demonstrated homologous profiles and no significant differences between groups. Typically, LHb neurons possessed high input resistances and long membrane time constants. They also displayed time-dependent inward rectification and distinct afterhyperpolarization. A salient electrophysiological feature of LHb neurons was their ability to generate rebound bursts of action potentials in response to membrane hyperpolarization. Based on the pattern of spontaneous activity, neurons were classified as silent, tonic or bursting. The occurrence of distinctive firing modes was not related to topographic allocation. The patterns of spontaneous firing and evoked discharge were highly sensitive to alterations in membrane potential and merged upon de- and hyperpolarizing current injection and synaptic stimulation. Besides projection neurons, recordings revealed the existence of a subpopulation of cells possessing morphological and physiological properties of neocortical neurogliaform cells. They were considered to be interneurons. Our data suggest that neurons within the different LHb subnuclei behave electrophysiologically more similar than expected, considering their morphological heterogeneity. We conclude that the formation of functional neuronal entities within the LHb may be achieved through defined synaptic inputs to particular neurons, rather than by individual neuronal morphologies and intrinsic membrane properties.

The agmatine-degrading enzyme agmatinase: a key to agmatine signaling in rat and human brain?

Agmatinase, an ureohydrolase belonging to the arginase family, is widely expressed in mammalian tissues including the brain. Here, it may serve two different functions, the inactivation of the arginine derivative agmatine, a putative neurotransmitter, and the formation of the diamine putrescine. In order to identify the cellular sources of agmatinase expression in the brain, we generated a polyclonal monospecific antibody against recombinant rat agmatinase. With immunocytochemistry, selected areas of rat and human brain were screened. Clearly, in both species agmatinase-like immunoreactivity was predominantly detected in distinct populations of neurons, especially cortical interneurons. Also, principal neurons in limbic regions like the habenula and in the cerebellum robustly expressed agmatinase protein. When comparing the overall agmatinase expression with immunocytochemical data available for agmatine and polyamine biosynthetic enzymes, the observed pattern may argue in favor of an agmatine inactivating function rather than fueling the alternative pathway of polyamine synthesis. The putative neurotransmitter agmatine is seemingly involved with mental disorders. Therefore, agmatinase may be similarly important for pathogenesis. The normal expression profile of the protein as described here may therefore be altered under pathological conditions.

Glutamatergic axons from the lateral habenula mainly terminate on GABAergic neurons of the ventral midbrain.

The concept of cortical-subcortical loops emphasizes the importance of the basal ganglia for motor, psychomotor, and emotional cortical functions. These loops are bidirectionally controlled by the midbrain dopaminergic system, predominantly but not exclusively at the level of the striatum including the accumbens nucleus. Successful behaviors increase the activities of the mesostriatal (arising in the complex part of the substantia nigra) and mesolimbic (arising in the ventral tegmental area, VTA) neurons, thereby reinforcing the corresponding actions. In contrast, unsuccessful behaviors result in an increased activation of the lateral habenular complex (LHb), thereby decreasing the activities of mesolimbic neurons. Correspondingly, electrical stimulation of the LHb effectively blocks neuronal activity in the VTA. Whether this block is due to an inhibitory projection from the LHb to the VTA, or whether axons from excitatory LHb neurons target inhibitory neurons within the VTA, is presently not known. Here we show, using in situ hybridization and immunocytochemical double labeling at the light and electron microscopic level, that GABAergic neurons are scarce in the LHb and that glutamatergic axons from the LHb mostly target GABAergic neurons in the VTA and the mesopontine rostromedial tegmental nucleus (RMTg), also known as tail of the VTA (tVTA). These data explain the inhibitory effect of LHb activation on the VTA. In addition, however, a small number of LHb terminals in the VTA actually contacts dopaminergic neurons. The biological importance of these terminals requires further investigation.

Dopaminergic activation excites rat lateral habenular neurons in vivo.

The lateral habenular complex (LHb) of the epithalamus is part of a dorsal diencephalic conduction system connecting basal forebrain with regulatory midbrain nuclei. The LHb has been implicated in the regulation of ascending monoaminergic transmission, particularly midbrain dopaminergic neuronal activity. Here, we have investigated whether the LHb in turn is subject to dopaminergic modulation. Alterations in spontaneous neuronal activity within the LHb following systemic application of dopaminergic drugs have been examined in anesthetized rats using extracellular single unit recordings. The administration of apomorphine (2 mg/kg) resulted in an excitation of individual LHb neurons. On average, the spontaneous action potential firing of the LHb neurons was increased by 39%. However, the apomorphine effect showed marked topographic differences within the LHb. Particularly, a small subset of neurons in the lateral division of the LHb, which was localized within the oval subnucleus, showed an apomorphine-mediated increase in discharge frequency by 96%. In contrast, spontaneous discharge of neurons within other areas of the lateral division was not modified. Likewise, within the medial division of the LHb, a region that preferentially receives projections from dopaminergic midbrain nuclei, the majority of neurons failed to show apomorphine-mediated alterations in action potential firing. However, within the superior subnucleus of this division, an area with yet unclear afferent supply, spontaneous neuronal firing was enhanced by 56%. The apomorphine-mediated excitation of LHb neurons was antagonized by coapplication of haloperidol (2 mg/kg), which alone did not alter spontaneous action potential firing of individual LHb neurons. The present study demonstrates that spontaneous activity of distinct subsets of neurons within the LHb is strongly enhanced by systemic activation of dopaminergic receptors. Despite the small sample size, the data suggest that this dopaminergic modulation shows a topographic specificity. Therefore, the results support the hypothesis of a functional subnuclear organization of the rat habenular complex.

IL-10 overexpression differentially affects cartilage matrix gene expression in response to TNF-alpha in human articular chondrocytes in vitro.

Cartilage-specific extracellular matrix synthesis is the prerequisite for chondrocyte survival and cartilage function, but is affected by the pro-inflammatory cytokine TNF-alpha in arthritis. The aim of the present study was to characterize whether the immunoregulatory cytokine IL-10 might modulate cartilage matrix and cytokine expression in response to TNF-alpha. Primary human articular chondrocytes were treated with either recombinant IL-10, TNF-alpha or a combination of both (at 10ng/mL each) or transduced with an adenoviral vector overexpressing human IL-10 and subsequently stimulated with 10ng/ml TNF-alpha for 6 or 24h. The effects of IL-10 on the cartilage-specific matrix proteins collagen type II, aggrecan, matrix-metalloproteinases (MMP)-3, -13 and pro-inflammatory cytokines were evaluated by real-time RT-PCR and immunohistochemistry. Transduced chondrocytes overexpressed high levels of IL-10 which significantly up-regulated collagen type II expression. TNF-alpha suppressed collagen type II and aggrecan, but increased MMP and cytokine expression in chondrocytes compared to the non-stimulated controls. The TNF-alpha mediated down-regulation of aggrecan expression was significantly antagonized by IL-10 overexpression, whereas the suppression of collagen type II was barely affected. The MMP-13 and IL-1beta expression by TNF-alpha was slightly reduced by IL-10. These results suggest that IL-10 overexpression modulates some catabolic features of TNF-alpha in chondrocytes.

Cellular and subcellular rat brain spermidine synthase expression patterns suggest region-specific roles for polyamines, including cerebellar pre-synaptic function.

In the brain, the polyamines spermidine (Spd) and spermine (Spm) serve highly specific functions by interacting with various ion channel receptors intimately involved with synaptic signaling. Both, glial cells and neurons contain Spd/Spm, but release and uptake mechanisms could re-distribute polyamines between cell types. The cellular and subcellular localization of polyamine biosynthetic enzymes may therefore offer a more appropriate tool to identify local sources of enhanced Spd/Spm synthesis, which may be related with specific roles in neuronal circuits and synaptic function. A recently characterized antibody against Spd synthase was therefore used to screen the rat brain for compartment-specific peaks in enzyme expression. The resulting labeling pattern indicated a clearly heterogeneous expression predominantly localized to neurons and neuropil. The highest levels of Spd synthase expression were detected in the accumbens nucleus, taenia tecta, cerebellar cortex, cerebral cortical layer I, hippocampus, hypothalamus, mesencephalic raphe nuclei, central and lateral amygdala, and the circumventricular organs. Besides a diffuse labeling of the neuropil in several brain areas, the distinct labeling of mossy fiber terminals in the cerebellar cortex directly indicated a synaptic role for Spd synthesis. Electron microscopy revealed a preferential distribution of the immunosignal in synaptic vesicle containing areas. A pre-synaptic localization was also observed in parallel and climbing fiber terminals. Electrophysiological recordings in acute cerebellar slices revealed a Spd-induced block of evoked extracellular field potentials resulting from mossy fiber stimulation in a dose-dependent manner.

Differential distribution of voltage-gated potassium channels Kv 1.1-Kv1.6 in the rat retina during development.

The discharge behavior of neurons depends on a variable expression and sorting pattern of voltage-dependent potassium (Kv) channels that changes during development. The rodent retina represents a neuronal network whose main functions develop after birth. To obtain information about neuronal maturation we analyzed the expression of subunits of the Kv1 subfamily in the rat retina during postnatal development using immunocytochemistry and immunoelectron microscopy. At postnatal day 5 (P5) all the alpha-subunits of Kv1.1-Kv1.6 channels were found to be expressed in the ganglion cell layer (GCL), most of them already at P1 or P3. Their expression upregulates postnatally and the pattern and distribution change in an isoform-specific manner. Additionally Kv1 channels are found in the outer and inner plexiform layer (OPL, IPL) and in the inner nuclear layer (INL) at different postnatal stages. In adult retina the Kv 1.3 channel localizes to the inner and outer segments of cones. In contrast, Kv1.4 is highly expressed in the outer retina at P8. In adult retina Kv1.4 occurs in rod inner segments (RIS) near the connecting cilium where it colocalizes with synapse associated protein SAP 97. By using confocal laser scanning microscopy we showed a differential localization of Kv1.1-1.6 to cholinergic amacrine and rod bipolar cells of the INL of the adult retina.

Spermidine synthase is prominently expressed in the striatal patch compartment and in putative interneurones of the matrix compartment.

The ubiquitous polyamines spermidine and spermine are known as modulators of glutamate receptors and inwardly rectifying potassium channels. They are synthesized by a set of specific enzymes in which spermidine synthase is the rate-limiting step catalysing the formation of the spermine precursor spermidine from putrescine. Spermidine and spermine were previously localized to astrocytes, probably reflecting storage rather than synthesis in these cells. In order to identify the cellular origin of spermidine and spermine synthesis in the brain, antibodies were raised against recombinant mouse spermidine synthase. As expected, strong spermidine synthase-like immunoreactivity was obtained in regions known to express high levels of spermidine and spermine, such as the hypothalamic paraventricular and supraoptic nuclei. In the striatum, spermidine synthase was found in neurones and the neuropil of the patch compartment (striosome) as defined by expression of the micro opiate receptor. The distinct expression pattern of spermidine synthase, however, only partially overlapped with the distribution of the products spermidine and spermine in the striatum. In addition, spermidine synthase-like immunoreactivity was seen in patch compartment-apposed putative interneurones. These spermidine synthase-positive neurones did not express any marker characteristic of the major striatal interneurone classes. The neuropil labelling in the patch compartment and in adjacent putative interneurones may indicate a role for polyamines in intercompartmental signalling in the striatum.

Tandem-pore domain potassium channels are functionally expressed in retinal (Müller) glial cells.

Tandem-pore domain (2P-domain) K+-channels regulate neuronal excitability, but their function in glia, particularly, in retinal glial cells, is unclear. We have previously demonstrated the immunocytochemical localization of the 2P-domain K+ channels TASK-1 and TASK-2 in retinal Müller glial cells of amphibians. The purpose of the present study was to determine whether these channels were functional, by employing whole-cell recording from frog and mammalian (guinea pig, rat and mouse) Müller cells and confocal microscopy to monitor swelling in rat Müller cells. TASK-like immunolabel was localized in these cells. The currents mediated by 2P-domain channels were studied in isolation after blocking Kir, K(A), K(D), and BK channels. The remaining cell conductance was mostly outward and was depressed by acid pH, bupivacaine, methanandamide, quinine, and clofilium, and activated by alkaline pH in a manner consistent with that described for TASK channels. Arachidonic acid (an activator of TREK channels) had no effect on this conductance. Blockade of the conductance with bupivacaine depolarized the Müller cell membrane potential by about 50%. In slices of the rat retina, adenosine inhibited osmotic glial cell swelling via activation of A1 receptors and subsequent opening of 2P-domain K+ channels. The swelling was strongly increased by clofilium and quinine (inhibitors of 2P-domain K+ channels). These data suggest that 2P-domain K+ channels are involved in homeostasis of glial cell volume, in activity-dependent spatial K+ buffering and may play a role in maintenance of a hyperpolarized membrane potential especially in conditions where Kir channels are blocked or downregulated.

Polyamines increase in sympathetic neurons and non-neuronal cells after axotomy and enhance neurite outgrowth in nerve growth factor-primed PC12 cells.

Following axonal damage, sympathetic neurons are capable of regenerating and reinnervating their target tissues. Some years ago exogenous administration of polyamines was shown to enhance this regeneration. Recently, it was found that axonal injury leads to a dramatic up-regulation of the expression of arginase I in sympathetic neurons. This enzyme catalyzes the conversion of arginine to ornithine, which can subsequently be converted to the diamine putrescine and, ultimately, to the polyamines spermidine and spermine. In the present study, using an antiserum that reacts with both spermidine and spermine, we have found an increase in polyamine levels in both neurons and non-neuronal cells in the superior cervical ganglion 2 and 5 days following transection of the ganglion's postganglionic trunks. Using PC12 cells primed with nerve growth factor and then stripped off the culture dish and replated as a model system for axotomized sympathetic neurons, we found that spermidine treatment, with or without nerve growth factor, resulted in an increased percentage of cells with a neurite whose length was at least twice the diameter of the neuron's cell body. These increases could be seen within 48 h and were still evident after 8 days. Together, these data support the possibility that endogenous polyamines are involved in the normal regeneration which occurs following sympathetic axonal damage.

Kir6.1 is the principal pore-forming subunit of astrocyte but not neuronal plasma membrane K-ATP channels.

ATP-sensitive potassium channels (K-ATP channels) directly couple the energy state of a cell to its excitability, are activated by hypoxia, and have been suggested to protect neurons during disturbances of energy metabolism such as transient ischemic attacks or stroke. Molecular studies have demonstrated that functional K-ATP channels are octameric protein complexes, consisting of four sulfonylurea receptor proteins and four pore-forming subunits which are members of the Kir6 family of inwardly rectifying potassium channels. Here we show, using specific antibodies against the two known pore-forming subunits (Kir6.1 and Kir6.2) of K-ATP channels, that only Kir6.1 and not Kir6.2 subunits are expressed in astrocytes. In addition to a minority of neurons, Kir6.1 protein is present on hippocampal, cortical, and cerebellar astrocytes, tanycytes, and Bergmann glial cells. We also provide ultrastructural evidence that Kir6.1 immunoreactivity is primarily localized to distal perisynaptic and peridendritic astrocyte plasma membrane processes, and we confirm the presence of functional K-ATP channels in Bergmann glial cells by slice-patch-clamp experiments. The identification of Kir6.1 as the principal pore-forming subunit of plasma membrane K-ATP channels in astrocytes suggests that these glial K-ATP channels act in synergy with neuronal Kir6.2-mediated K-ATP channels during metabolic challenges in the brain.

Regulation of apolipoprotein E secretion in rat primary hippocampal astrocyte cultures.

Apolipoprotein E isoforms may have differential effects on a number of pathological processes underlying Alzheimer's disease. Recent studies suggest that the amount, rather than the type, of apolipoprotein E may also be an important determinant for Alzheimer's disease. Therefore, understanding the regulated synthesis of apolipoprotein E is important for determining its role in Alzheimer's disease. We show here that in rat primary hippocampal astrocyte cultures, dibutyryl-cAMP increased apolipoprotein E secretion with time in a dose-dependent manner (to 177% at 48 h) and that retinoic acid potentiated this effect (to 298% at 48 h). Dibutyryl-cAMP also gave a rapid, albeit transient, increase of apolipoprotein E mRNA expression (to 200% at 1 h). In contrast, the protein kinase C activator phorbol 12-myristate 13-acetate decreased both apolipoprotein E secretion (to 59% at 48 h) and mRNA expression (to 22% at 1 h). Phorbol 12-myristate 13-acetate also reversed the effects of dibutyryl-cAMP. Apolipoprotein E secretion was also modulated by receptor agonists for the adenylyl cyclase/cAMP pathway. Isoproterenol (50 nM, a beta-adrenoceptor agonist) enhanced, while clonidine (250 nM, an alpha2-adrenoceptor agonist) decreased, secreted apolipoprotein E. We also analysed the effects of agonists for the phospholipase C/protein kinase C pathway. Arterenol (1 microM, an alpha1-adrenoceptor agonist) and serotonin (2.5 microM) enhanced, whereas carbachol (10 microM, an acetylcholine muscarinic receptor agonist) decreased secreted apolipoprotein E. The effects of these non-selective receptor agonists were modest, probably due to effects on different signalling pathways. Arterenol also potentiated the isoproterenol-mediated increase. We also show that phorbol 12-myristate 13-acetate and dibutyryl-cAMP have opposite effects on nerve growth factor, as compared to apolipoprotein E, secretion, suggesting that the results obtained were unlikely to be due to a general effect on protein synthesis. We conclude that astrocyte apolipoprotein E production can be regulated by factors that affect cAMP intracellular concentration or activate protein kinase C. Alterations in these signalling pathways in Alzheimer's disease brain may have consequences for apolipoprotein E secretion in this disorder.

Kir subfamily in frog retina: specific spatial distribution of Kir 6.1 in glial (Müller) cells.

We show by immunocytochemistry in frog retina that most members of the Kir subfamily are expressed in specific neuronal compartments. However, Kir 6.1, the pore-forming subunit of K(ATP) channels, is expressed exclusively in glial Müller cells. Müller cell endfeet display strong Kir 6.1 immunolabel throughout the retina, whereas the somata are labeled only in the retinal periphery. This spatial pattern is similar to that of Kir 4.1, of the ratio of inward to outward K+ currents, and of spermine/spermidine immunoreactivity. We suggest that the co-expression of Kir 4.1 and Kir 6.1 subunits may enable the cells to maintain their high K+ conductance and hyperpolarized membrane potentials both at high ATP levels (Kir 4.1) and during ATP deficiency (Kir 6.1).

Cellular localization of the potassium channel Kir7.1 in guinea pig and human kidney.

BACKGROUND: K(+) channels have important functions in the kidney, such as maintenance of the membrane potential, volume regulation, recirculation, and secretion of potassium ions. The aim of this study was to obtain more information on the localization and possible functional role of the inwardly rectifying K(+) channel, Kir7.1. METHODS: Kir7.1 cDNA (1114 bp) was isolated from guinea pig kidney (gpKir7.1), and its tissue distribution was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, a genomic DNA fragment (6153 bp) was isolated from a genomic library. cRNA was expressed in Xenopus laevis oocytes for functional studies. Immunohistochemistry and RT-PCR were used to localize Kir7.1 in guinea pig and human kidney. RESULTS: The expression of gpKir7.1 in Xenopus laevis oocytes revealed inwardly rectifying K(+) currents. The reversal potential was strongly dependent on the extracellular K(+) concentration, shifting from -14 mV at 96 mmol/L K(+) to -90 mV at 1 mmol/L K(+). gpKir7.1 showed a low affinity for Ba(2+). Significant expression of gpKir7.1 was found in brain, kidney, and lung, but not in heart, skeletal muscle, liver, or spleen. Immunocytochemical detection in guinea pig identified the gpKir7.1 protein in the basolateral membrane of epithelial cells of the proximal tubule. RT-PCR analysis identified strong gpKir7.1 expression in the proximal tubule and weak expression in glomeruli and thick ascending limb. In isolated human tubule fragments, RT-PCR showed expression in proximal tubule and thick ascending limb. CONCLUSION: Our results suggest that Kir7.1 may contribute to basolateral K(+) recycling in the proximal tubule and in the thick ascending limb.

Comparison of cloned Kir2 channels with native inward rectifier K+ channels from guinea-pig cardiomyocytes.

The aim of the study was to compare the properties of cloned Kir2 channels with the properties of native rectifier channels in guinea-pig (gp) cardiac muscle. The cDNAs of gpKir2.1, gpKir2.2, gpKir2.3 and gpKir2.4 were obtained by screening a cDNA library from guinea-pig cardiac ventricle. A partial genomic structure of all gpKir2 genes was deduced by comparison of the cDNAs with the nucleotide sequences derived from a guinea-pig genomic library. The cell-specific expression of Kir2 channel subunits was studied in isolated cardiomyocytes using a multi-cell RT-PCR approach. It was found that gpKir2.1, gpKir2.2 and gpKir2.3, but not gpKir2.4, are expressed in cardiomyocytes. Immunocytochemical analysis with polyclonal antibodies showed that expression of Kir2.4 is restricted to neuronal cells in the heart. After transfection in human embryonic kidney cells (HEK293) the mean single-channel conductance with symmetrical K+ was found to be 30.6 pS for gpKir2.1, 40.0 pS for gpKir2.2 and 14.2 pS for Kir2.3. Cell-attached measurements in isolated guinea-pig cardiomyocytes (n = 351) revealed three populations of inwardly rectifying K+ channels with mean conductances of 34.0, 23.8 and 10.7 pS. Expression of the gpKir2 subunits in Xenopus oocytes showed inwardly rectifying currents. The Ba2+ concentrations required for half-maximum block at -100 mV were 3.24 M for gpKir2.1, 0.51 M for gpKir2.2, 10.26 M for gpKir2.3 and 235 M for gpKir2.4. Ba2+ block of inward rectifier channels of cardiomyocytes was studied in cell-attached recordings. The concentration and voltage dependence of Ba2+ block of the large-conductance inward rectifier channels was virtually identical to that of gpKir2.2 expressed in Xenopus oocytes. Our results suggest that the large-conductance inward rectifier channels found in guinea-pig cardiomyocytes (34.0 pS) correspond to gpKir2.2. The intermediate-conductance (23.8 pS) and low-conductance (10.7 pS) channels described here may correspond to gpKir2.1 and gpKir2.3, respectively.

Serotonin uptake and release mechanisms in developing cultures of rat embryonic raphe neurons: age- and region-specific differences.

The development of serotonergic neurons of the rat raphe was followed in primary neuronal cell cultures taken at embryonic days embryonic day 13 and embryonic day 14 from three different raphe sub-groups, topographically defined with respect to their position to the isthmus as rostral (R1), intermediate (R2) and caudal (R3). In neurons cultivated from embryonic day 13 raphe serotonin, immunoreactivity was detected after only two days in vitro in the rostral R1 and the intermediate R2 sub-groups. Within two weeks of cultivation the number of serotonergic neurons as well as the dendritic branching continuously increased in all three sub-groups. In cultures obtained from embryonic day 13 raphe a specific uptake of [3H]serotonin could not be detected during the first days in vitro. Specific uptake as well as regulated serotonin release, however, was clearly discernible in these cultures after nine days in vitro, indicating developmental differentiation of the initially immature serotonergic neurons in culture. In contrast, serotonergic neurons obtained from the three raphe sub-groups at embryonic day 14 took up and released [3H]serotonin, as early as after two days in culture. Basal as well as stimulated serotonin release was diminished when preincubating the cells with tetanus toxin, which cleaves synaptobrevin thereby blocking exocytosis. Our data indicate that the differential development of serotonergic neurons in the various raphe sub-groups in vivo is also sustained in culture. The differences observed when comparing neurons from embryonic days 13 and 14 suggest that a short time-period of about 24h appears to be crucial for the developmental upregulation of serotonin uptake, storage and release.