Maternal nutrient restriction alters thyroid hormone dynamics in placentae of sheep having small for gestational age fetuses.

Thyroid hormones regulate a multitude of metabolic and cellular processes involved in placental and fetal growth, while maternal nutrient restriction (NR) has the potential to influence these processes. Those fetuses most impacted by NR, as categorized by weight, are termed small for gestational age (SGA), but the role of thyroid hormones in these pregnancies is not fully understood. Therefore, the aims of the present study were to determine effects of NR during pregnancy on maternal and fetal thyroid hormone concentrations, as well as temporal and cell-specific expression of mRNAs and proteins for placental thyroid hormone transporters, thyroid hormone receptors, and deiodinases in ewes having either SGA or normal weight fetuses. Ewes with singleton pregnancies were fed either a 100% NRC (n = 8) or 50% NRC (NR; n = 28) diet from Days 35 to 135 of pregnancy with a single placentome surgically collected on Day 70. Fetal weight at necropsy on Day 135 was used to designate the fetuses as NR NonSGA (n = 7; heaviest NR fetuses) or NR SGA (n = 7; lightest NR fetuses). Thyroid hormone levels were lower in NR SGA compared to NR NonSGA ewes, while all NR fetuses had lower concentrations of thyroxine at Day 135. Expression of mRNAs for thyroid hormone transporters SLC16A2, SLC16A10, SLCO1C1, and SLCO4A1 were altered by day, but not nutrient restriction. Expression of THRA mRNA and protein was dysregulated in NR SGA fetuses with protein localized to syncytial and stromal cells in placentomes in all groups. The ratio of deiodinases DIO2 and DIO3 was greater for NR SGA placentae at Day 70, while DIO3 protein was less abundant in placentae from NR SGA than 100% NRC ewes. These results identify mid-gestational modifications in thyroid hormone-associated proteins in placentomes of ewes having SGA fetuses, as well as a potential for placentomes from NonSGA pregnancies to adapt to, and overcome, nutritional restrictions during pregnancy.

Expression of progesterone receptor in the porcine uterus and placenta throughout gestation: correlation with expression of uteroferrin and osteopontin.

Progesterone (P4) stimulates production and secretion of histotroph, a mixture of hormones, growth factors, nutrients, and other substances required for growth and development of the conceptus (embryo or fetus and placental membranes). Progesterone acts through the progesterone receptor (PGR); however, there is a gap in our understanding of P4 during pregnancy because PGR have not been localized in the uteri and placentae of pigs beyond day 18. Therefore, we determined endometrial expression of PGR messenger RNA (mRNA) and localized PGR protein in uterine and placental tissues throughout the estrous cycle and through day 85 of pregnancy in pigs. Further, 2 components of histotroph, tartrate-resistant acid phosphatase 5 (ACP5; uteroferrin) and secreted phosphoprotein 1 (SPP1; osteopontin) proteins, were localized in relation to PGR during pregnancy. Endometrial expression of PGR mRNA was highest at day 5 of the estrous cycle, decreased between days 5 and 11 of both the estrous cycle and pregnancy, and then increased between days 11 and 17 of the estrous cycle (P < 0.01), but decreased from days 13 to 40 of pregnancy (P < 0.01). Progesterone receptor protein localized to uterine stroma and myometrium throughout all days of the estrous cycle and pregnancy. PGR were expressed by uterine luminal epithelium (LE) between days 5 and 11 of the estrous cycle and pregnancy, then PGR became undetectable in LE through day 85 of pregnancy. During the estrous cycle, PGR were downregulated in LE between days 11 and 15, but expression returned to LE on day 17. All uterine glandular epithelial (GE) cells expressed PGR from days 5 to 11 of the estrous cycle and pregnancy, but expression decreased in the superficial GE by day 12. Expression of PGR in GE continued to decrease between days 25 and 85 of pregnancy; however, a few glands near the myometrium and in close proximity to areolae maintained expression of PGR protein. Acid phosphatase 5 protein was detected in the GE from days 12 to 85 of gestation and in areolae. Secreted phosphoprotein 1 protein was detected in uterine LE in apposition to interareolar, but not areolar areas of the chorioallantois on all days examined, and in uterine GE between days 35 and 85 of gestation. Interestingly, uterine GE cells adjacent to areolae expressed PGR, but not ACP5 or SPP1, suggesting these are excretory ducts involved in the passage, but not secretion, of histotroph into the areolar lumen and highlighting that P4 does not stimulate histotroph production in epithelial cells that express PGR.

Removing seminal plasma improves bovine sperm sex-sorting.

Bull ejaculates with sperm concentrations of less than 1 billion sperm sort poorly for sex chromosomes, but whether this is because of the sperm concentration or the concomitant seminal plasma content has not been elucidated. Experiments were conducted to determine why ejaculates with lower sperm concentrations sort poorly and develop a protocol to increase sorting efficiency. In Experiment I, spermatozoa at 160 or 240 × 106 sperm/mL were stained at 49, 65 or 81 µm Hoechst 33342 with 0 or 10% seminal plasma and then sex-sorted. In Experiment II, seminal plasma was adjusted to create samples with sperm concentrations of 0.7, 1.4 and 2.1 × 109 sperm/mL, prior to sex-sorting. In Experiment III, spermatozoa were diluted to 0.7, 1.4 and 2.1 × 109 sperm/mL using TALP containing 0 or 10% seminal plasma prior to sex-sorting and cryopreservation. In Experiment I, the optimal staining combination was 160 × 106 sperm/mL stained with 65 µm Hoechst 33342 and no seminal plasma. In Experiment II, the percentages of membrane-impaired sperm were lower for sample concentrations of 2.1 × 109 sperm/mL (15%) than for samples at 1.4 × 109 (17%) or 0.7 × 109 sperm/mL (18%; p < 0.01). The X sort rate was slower for samples stored at 0.7 × 109 sperm/mL (3.45 × 103 sperm/sec) than for samples stored at 1.4 × 109 and 2.1 × 109 sperm/mL (3.85 and 3.94 × 103 sperm/sec, respectively; p < 0.05). In Experiment III, samples containing 0% seminal plasma had higher percentages of live-oriented cells (54 vs. 50%; p < 0.05), fewer dead sperm (19 vs. 22%; p < 0.01) and higher post-thaw motility (41 vs. 35%; p < 0.05) than samples containing 10% seminal plasma. Ejaculates with high sperm concentrations result in superior sorting because these samples have less seminal plasma during staining than ejaculates with lower initial sperm concentrations as all samples are diluted to 160 × 106 sperm/mL for staining. Therefore, sorting efficiency appears to be affected by seminal plasma concentration, not by the original sperm concentration.

Crucial role of astrocytes in temporal lobe epilepsy.

Astrocytes sense and respond to synaptic activity through activation of different neurotransmitter receptors and transporters. Astrocytes are also coupled by gap junctions, which allow these cells to redistribute through the glial network the K(+) ions excessively accumulated at sites of intense neuronal activity. Work over the past two decades has revealed important roles for astrocytes in brain physiology, and it is therefore not surprising that recent studies unveiled their involvement in the etiology of neurological disorders such as epilepsy. Investigation of specimens from patients with pharmacoresistant temporal lobe epilepsy and epilepsy models revealed alterations in expression, localization and function of astrocytic connexins, K(+) and water channels. In addition, disturbed gliotransmission as well as malfunction of glutamate transporters and of the astrocytic glutamate- and adenosine-converting enzymes - glutamine synthetase and adenosine kinase, respectively - have been observed in epileptic tissues. Accordingly, increasing evidence indicates that dysfunctional astrocytes are crucially involved in processes leading to epilepsy. These new insights might foster the search for new targets for the development of new, more efficient anti-epileptogenic therapies.

Histochemical screening, metabolite profiling and expression analysis reveal Rosaceae roots as the site of flavan-3-ol biosynthesis.

Histochemical screening of 30 Rosaceae genera representing all classic subfamilies demonstrated flavan-3-ols (catechins) as general secondary metabolites in roots of Rosaceae. Semi-quantitative LC-MS analyses confirmed the presence of catechin, epicatechin and various dimeric flavan-3-ols (also representing higher polymeric proanthocyanidins) as prominent polyphenols in root tips of Fragaria (strawberry), Malus (apple), Rosa (rose), Pyrus (pear) and Prunus (plum). Distinct patterns of flavan-3-ol distribution at the cellular level were found in strawberry (Fragaria × ananassa) and apple (Malus × domestica) root tips. The calyptras (root caps) showed the most prominent flavan-3-ol staining for these two genera. Border cells of Fragaria and Malus, as first demonstrated here for Rosaceae, were also found to contain flavan-3-ols. Transcript analyses with cDNA demonstrated root expression of known flavonoid genes expressed in the respective fruits and leaves. Primarily, this proves in situ biosynthesis of flavan-3-ols in these roots. Knowledge of the distinct cellular distribution patterns and their in situ biosynthesis in roots provides a basis for analysis of the functional roles of Rosaceae root flavan-3-ols.

Laminar-specific and developmental expression of aquaporin-4 in the mouse hippocampus.

Mice deficient in the water channel aquaporin-4 (AQP4) demonstrate increased seizure duration in response to hippocampal stimulation as well as impaired extracellular K+ clearance. However, the expression of AQP4 in the hippocampus is not well described. In this study, we investigated (i) the developmental, laminar and cell-type specificity of AQP4 expression in the hippocampus; (ii) the effect of Kir4.1 deletion on AQP4 expression; and (iii) performed Western blot and RT-PCR analyses. AQP4 immunohistochemistry on coronal sections from wild-type (WT) or Kir4.1-/- mice revealed a developmentally-regulated and laminar-specific pattern, with highest expression in the CA1 stratum lacunosum-moleculare (SLM) and the molecular layer (ML) of the dentate gyrus (DG). AQP4 was colocalized with the glial markers glial fibrillary acidic protein (GFAP) and S100ß in the hippocampus, and was also ubiquitously expressed on astrocytic endfeet around blood vessels. No difference in AQP4 immunoreactivity was observed in Kir4.1-/- mice. Electrophysiological and postrecording RT-PCR analyses of individual cells revealed that AQP4 and Kir4.1 were co-expressed in nearly all CA1 astrocytes. In NG2 cells, AQP4 was also expressed at the transcript level. This study is the first to examine subregional AQP4 expression during development of the hippocampus. The strikingly high expression of AQP4 in the CA1 SLM and DG ML identifies these regions as potential sites of astrocytic K+ and H2O regulation. These results begin to delineate the functional capabilities of hippocampal subregions and cell types for K+ and H2O homeostasis, which is critical to excitability and serves as a potential target for modulation in diverse diseases.

Analysis of phosphorylation-dependent modulation of Kv1.1 potassium channels.

The voltage-gated potassium channel Kv1.1 contains phosphorylation sites for protein kinase A (PKA) and protein kinase C (PKC). To study Kv1.1 protein expression and cellular distribution in regard to its level of phosphorylation, the effects of PKA and PKC activation on Kv1.1 were investigated in HEK 293 cells stably transfected with Kv1.1 (HEK 293/1). Without kinase activation, HEK 293/1 cells carry unphosphorylated Kv1.1 protein in the plasma membranes, whereas large amounts of phosphorylated and unphosphorylated Kv1.1 protein were located intracellularly. Activation of PKA resulted in phosphorylation of intracellular Kv1.1 protein, followed by a rapid translocation of Kv1.1 into the plasma membrane. Patch-clamp analysis revealed an increase in current amplitude upon PKA activation and demonstrated differences in the voltage dependence of current activation between unphosphorylated and phosphorylated Kv1.1 channels. In contrast to PKA, even prolonged activation of PKC did not lead to direct phosphorylation of Kv1.1, but induced Kv1.1 protein synthesis. Thus, protein kinases have direct and indirect effects on the functional expression of voltage-gated potassium channels. Our data suggest that the synergistic action of protein kinases may play an important role in the fine-tuning of Kv channel function.

Modulation of voltage-gated K(+) channels Kv11 and Kv1 4 by forskolin.

Forskolin (FSK) affects voltage-gated K(+) (Kv) currents in different cell types, but it is not known which of the various subunits form FSK-sensitive Kv channels. We compared the effect of the compound at Kv1.1 and Kv1.4 channels ectopically expressed in HEK 293 cells. Low FSK concentrations induced a phosphorylation-dependent potentiation of Kv1.1 currents. At higher concentrations, this effect was superimposed by a fast, cAMP-independent channel block. Kv1.4 currents were inhibited with lower potency by FSK but were not modified by phosphorylation. The variable effect of the compound might help to distinguish between Kv subunits expressed by native cells.

Distribution of P2X receptors on astrocytes in juvenile rat hippocampus.

Recent evidence suggested that ATP acting via ionotropic (P2X) and metabotropic (P2Y) purinergic receptors might be involved in signaling between glial cells and within glial-neuronal networks. In contrast to their neuronal counterpart, the identity of P2X receptors in CNS glial cells is largely unknown. In the present study, antibodies recognizing the subunits P2X1-P2X7 were applied together with the astroglial marker S100beta and nuclear labeling with Hoechst 33342 to investigate semiquantitatively the distribution of the whole set of P2X receptors in astrocytes of the juvenile rat hippocampus. Expression of P2X1-P2X4, P2X6, and P2X7 subunits was observed in astrocytes of various hippocampal subregions, but the cells were completely devoid of P2X5 protein. S100beta-positive cells expressing subunits P2X3-P2X7 occurred evenly in the different subfields, while P2X1- and P2X2-positive astrocytes were distributed more heterogeneously. The staining pattern of P2X subunits also differed at the subcellular level. Antibodies against P2X2 and P2X4 labeled both astroglial cell bodies and processes. Immunoreactivity for P2X1 and P2X6 was mainly confined to somatic areas of S100beta-positive cells, whereas the subunit P2X3 was primarily localized along astroglial processes. Knowledge of the distribution of P2X receptors might provide a basis for a better understanding of their specific role in cell-cell signaling.

6-Hydroxykynurenic acid and kynurenic acid differently antagonise AMPA and NMDA receptors in hippocampal neurones.

6-Hydroxykynurenic acid (6-HKA), a derivative of kynurenic acid (KYNA) extracted from Ginkgo biloba leaves, was tested for its putative glutamate receptor (GluR) antagonism in comparison to the scaffold substance. The patch-clamp method together with fast-application techniques were used to estimate inhibition by 6-HKA and KYNA of agonist binding at NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (NMDARs and AMPARs) of CA1 pyramidal neurones. 6-Hydroxykynurenic acid proved to be a low-affinity antagonist. When comparing with KYNA, 6-HKA was less potent at NMDARs (IC(50) = 136 versus 59 microM), but showed a higher affinity to AMPARs (K(B) = 22 versus 172 microM). The replacement of 6-HKA and KYNA by glutamate was investigated on outside-out patches. Both antagonists competitively inhibited AMPAR responses and displayed fast unbinding kinetics, but the derivative was significantly slower displaced than KYNA (tau = 1.63 versus 1.22 ms). Our findings demonstrate that 6-hydroxylation considerably changes the pharmacological profile of KYNA. Among the 6-derivatives of KYNA, 6-HKA shows the highest affinity to AMPARS: Despite its relatively low lipophily, these properties might be of clinical relevance under conditions that compromise the integrity of the blood-brain barrier. Furthermore, 6-HKA should be a useful tool to analyse glutamate-mediated synaptic responses.

Characterization of the anticonvulsant and neuroprotectant BIIR 561 CL in vitro: effects on native and recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors.

BIIR 561 CL is a novel blocker of AMPA receptors and voltage-dependent sodium channels. In this study we further describe the effects of BIIR 561 CL on AMPA receptor-mediated membrane currents in rodent neurons, as well as in cells expressing recombinant human GluR1/2 receptors in more detail. BIIR 561 CL suppressed responses to kainate in neuronal cultures from rat cortex with an IC50 of 9.8 microM. Similar effects were observed using acutely dissociated neurons from the CA1 region of rat hippocampus (IC50 = 9.5 microM). Inhibition of kainate responses by BIIR 561 CL was prevented by preapplication of GYKI 53655, suggesting that both non-competitive inhibitors bind to a common site of the receptor. The effect of 10 microM BIIR 561 CL on kainate-induced currents was dependent on extracellular pH, with more pronounced block (84.1%) under acidic conditions (pHextern=6.4), compared to only 30.1% at a pHextern of 8.4. Thus, it can be hypothesized that BIIR 561 CL inhibits AMPA receptors in ischaemic brain regions more effectively than in healthy tissue. BIIR 561 CL inhibited responses to 1 mM glutamate in cells expressing recombinant human GluR1/2 receptors with similar potency, as compared to kainate responses in rat neurons (IC50=17.3 microM). The reference compound NBQX had an IC50 of 25.2 nM. None of the two compounds affected the glutamate-induced receptor desensitization at any tested concentration. The block by BIIR 561 CL was not use-dependent and had fast on- and off-kinetics (tauon=6.8 s; tauoff=1.3 s in hGluR1/2 receptors with 30 microM BIIR 561 CL). Thus, BIIR 561 CL can be anticipated to have a promising profile for the treatment of neurological disorders like brain ischaemia and head trauma.

Modulation of calcium channels by group I and group II metabotropic glutamate receptors in dentate gyrus neurons from patients with temporal lobe epilepsy.

PURPOSE: Metabotropic glutamate receptors (mGluRs) might be promising new drug targets for the treatment of epilepsy because the expression of certain mGluRs is regulated in epilepsy and because activation of mGluRs results in distinctive anti- and proconvulsant effects. Therefore, we examined how mGluR activation modulates high-voltage-activated (HVA) Ca2+ channels. METHODS: Whole-cell patch-clamp recordings were obtained from granule cells and interneuron-like cells acutely isolated from the dentate gyrus of patients with pharmacoresistent temporal lobe epilepsy. RESULTS: Agonists selective for either group I or group II mGluRs rapidly and reversibly reduced HVA currents in most dentate gyrus cells. These modulatory effects were inhibited by the respective group I and group II mGluR antagonists. The specific Ca2+ channel antagonists nifedipine and omega-conotoxin GVIA potently occluded the effects of group I and II mGluR agonists, respectively, indicating that group I mGluRs acted on L-type channels and group II mGluRs affected N-type channels. About two thirds of the responsive neurons were sensitive either to group I or group II mGluRs, whereas a minority of cells showed effects to agonists of both groups, indicating a variable mGluR expression pattern. CONCLUSIONS: Group I and group II mGluRs are expressed in human dentate gyrus neurons and modulate L- and N-type HVA channels, respectively. The data shed light on the possible cellular sequelae of the mGluR1 upregulation observed in human epileptic dentate gyrus as well as on possible mGluR-mediated anticonvulsant mechanisms.

Functional and molecular properties of human astrocytes in acute hippocampal slices obtained from patients with temporal lobe epilepsy.

PURPOSE: The specific role of glial cells in epilepsy is still elusive. In this study, functional properties of astrocytes were investigated in acute hippocampal brain slices obtained from surgical specimens of patients with drug-resistant temporal lobe epilepsy (TLE). METHODS: The patch-clamp technique together with a single-cell reverse transcription-polymerase chain reaction approach were used to combine functional and molecular analysis in the same individual cell in situ. RESULTS: In patients with Ammon's horn sclerosis, the glial current patterns resembled properties of immature astrocytes in rodent hippocampus. Depolarizing voltage steps activated delayed rectifier and transient K+ currents as well as tetrodotoxin-sensitive Na+ currents. Hyperpolarizing voltages elicited inward rectifier K+ currents. Comparative recordings were made in astrocytes from patients with lesion-associated TLE that lacked significant histopathological hippocampal alterations. The inward rectifier K+ current density was significantly smaller in astrocytes from the sclerotic group compared with lesion-associated TLE patients. CONCLUSIONS: During normal development of rodent brain, astroglial inward rectification gradually increases. It thus appears that astrocytes in human sclerotic tissue reexpress an immature current pattern. Reduced astroglial inward rectification in conjunction with seizure-induced shrinkage of the extracellular space may lead to impaired spatial K+ buffering. This will result in stronger and prolonged depolarization of glial cells and neurons in response to activity-dependent K+ release and may thus contribute to seizure generation and spread in this particular condition of human TLE.

Astrocytes in the hippocampus of patients with temporal lobe epilepsy display changes in potassium conductances.

Functional properties of astrocytes were investigated with the patch-clamp technique in acute hippocampal brain slices obtained from surgical specimens of patients suffering from pharmaco-resistant temporal lobe epilepsy (TLE). In patients with significant neuronal cell loss, i.e. Ammon's horn sclerosis, the glial current patterns resembled properties characteristic of immature astrocytes in the murine or rat hippocampus. Depolarizing voltage steps activated delayed rectifier and transient K+ currents as well as tetrodotoxin-sensitive Na+ currents in all astrocytes analysed in the sclerotic human tissue. Hyperpolarizing voltages elicited inward rectifier currents that inactivated at membrane potentials negative to -130 mV. Comparative recordings were performed in astrocytes from patients with lesion-associated TLE that lacked significant histopathological hippocampal alterations. These cells displayed stronger inward rectification. To obtain a quantitative measure, current densities were calculated and the ratio of inward to outward K+ conductances was determined. Both values were significantly smaller in astrocytes from the sclerotic group compared with lesion-associated TLE. During normal development of rodent brain, astroglial inward rectification gradually increases. It thus appears reasonable to suggest that astrocytes in human sclerotic tissue return to an immature current pattern. Reduced astroglial inward rectification in conjunction with seizure-induced shrinkage of the extracellular space may lead to impaired spatial K+ buffering. This will result in stronger and prolonged depolarization of glial cells and neurons in response to activity-dependent K+ release, and may thus contribute to seizure generation in this particular condition of human TLE.

Voltage- and gamma-aminobutyric acid-activated membrane currents in the human medulloblastoma cell line MHH-MED-3.

The whole-cell patch clamp technique was used to characterize voltage- and neurotransmitter-activated currents in the medulloblastoma cell line MHH-MED-3 and cells from tissue slices and primary cultures of two medulloblastoma biopsies. These preparations revealed similar electrophysiological properties. All tested cells displayed 4-aminopyridine-sensitive delayed rectifying K(+) currents, gamma-aminobutyric acid(A) receptor-mediated Cl(-) currents and most of them inward rectifier K(+) currents. Transient inward currents were mainly carried by low-voltage activated T-type Ca(2+) channels in MHH-MED-3 cells, and tetrodotoxin-sensitive Na(+) channels in cells from the primary culture. From these characteristics we conclude that medulloblastoma cells share physiological features with developing cerebellar granule cells at an immature stage.

Ion channels in glial cells.

Functional and molecular analysis of glial voltage- and ligand-gated ion channels underwent tremendous boost over the last 15 years. The traditional image of the glial cell as a passive, structural element of the nervous system was transformed into the concept of a plastic cell, capable of expressing a large variety of ion channels and neurotransmitter receptors. These molecules might enable glial cells to sense neuronal activity and to integrate it within glial networks, e.g., by means of spreading calcium waves. In this review we shall give a comprehensive summary of the main functional properties of ion channels and ionotropic receptors expressed by macroglial cells, i.e., by astrocytes, oligodendrocytes and Schwann cells. In particular we will discuss in detail glial sodium, potassium and anion channels, as well as glutamate, GABA and ATP activated ionotropic receptors. A majority of available data was obtained from primary cell culture, these results have been compared with corresponding studies that used acute tissue slices or freshly isolated cells. In view of these data, an active glial participation in information processing seems increasingly likely and a physiological role for some of the glial channels and receptors is gradually emerging.

Developmental regulation of AMPA-receptor properties in CA1 pyramidal neurons of rat hippocampus.

AMPA-receptor (AMPA-R) currents were recorded from CA1 pyramidal neurons in situ and after acute isolation from the hippocampus of 3- to 45-day-old rats. Membrane currents were analyzed by combining the patch clamp method with fast application techniques. The complete block of receptor currents by GYKI 53655 and the absence of modulation by Concanavalin A indicated that the cells exclusively expressed non-NMDA glutamate receptors of the AMPA subtype while functional kainate receptors could not be detected. The lowest sensitivity to kainate and NBQX was observed at postnatal day (p) 18. These changes might reflect a lower abundance of GluR1 at that developmental stage. A decrease of potentiation of receptor currents by cyclothiazide (CTZ), an acceleration of the recovery from CTZ potentiation and a faster and more complete desensitization of glutamate-evoked currents suggest an up-regulation of flop splice variants with increasing age. These functional data indicate that AMPA-R expression in CA1 pyramidal neurons varies during postnatal development which can be expected to influence the kinetics of synaptic transmission and the excitotoxic vulnerability as well.

Lesion-induced changes of electrophysiological properties in astrocytes of the rat dentate gyrus.

Reorganization of the adult dentate gyrus following unilateral entorhinal cortex lesion (ECL) is a well-established model for studying mechanisms of trauma-induced neuronal plasticity. The lesion induces deafferentiation of the outer molecular layer, which is accompanied by a strong astroglial reaction. This glial response is thought to contribute to subsequent repair processes, but the underlying mechanisms are poorly understood. In this study we addressed the question whether denervation leads to modifications in the electrophysiological properties of astrocytes, assuming that such changes might be involved in the remodeling of neural circuitry. Patch-clamp recordings were obtained from astrocytes in the dentate gyrus of adult rats that underwent ECL and compared to corresponding data from control animals. We observed a significant reduction of inward rectifier K(+) current densities, a positive shift of resting potentials, and an increase in input resistance in astrocytes of the denervated molecular layer. Current densities were reduced between 6 and 19 days postlesion (dpl), reaching a minimum at 10 dpl. Voltage-gated outward K(+) currents were not affected by the lesion. Inward rectifier K(+) currents increase with maturation in astrocytes. Thus, our results provide evidence that, following ECL, mature astrocytes dedifferentiated and readapted an immature current pattern. Presumably, these changes lead to stronger and prolonged depolarization of glial cells and neurons in response to activity-dependent K(+) release, which in turn might enhance the synthesis of neurotrophic factors and contribute to a permissive environment for neuronal reorganization.

Two electrophysiologically distinct types of granule cells in epileptic human hippocampus.

We investigated the electrophysiology of morphologically identified human granule cells with conventional current-clamp recordings. Slices were prepared from 14 human epileptic sclerotic hippocampi. Granule cells appeared to have a diverse electrophysiology. Each cell was distinguished by the shape of the afterhyperpolarization following single action potentials. Two types could be discerned: type I afterhyperpolarizations were monophasic and brief (typically 10-40 ms), whilst type II afterhyperpolarizations were biphasic and long (typically 50-100 ms). The two types also differed in their repetitive firing behaviour and action potential morphology: type I cells had significantly weaker spike frequency adaptation, lower action potential amplitude and smaller action potential upstroke/downstroke ratio. Thus, the firing pattern of type I cells resembled that of rodent dentate interneurons. In contrast, the corresponding parameters of type II cells were comparable to rodent dentate granule cells. Despite the distinct firing patterns, membrane properties were not different. The two types of cells also differed in their synaptic responses to stimulation of the perforant path. At strong suprathreshold stimulation intensity, type I cells always generated multiple action potentials, whereas type II cells usually spiked once only. Slow inhibitory postsynaptic potentials were not detected in type I neurons, but were easily identified in type II neurons. Extracellular recordings of perforant path-evoked field potentials in the cell layer confirmed that the majority of granule cells showed multiple discharges even when we recorded simultaneously from a type II cell that generated one action potential only. The morphology of both types of cells was characteristic of what has been described for primate dentate granule cells. Based on comparisons with previous studies on rodent and human granule cells, we tentatively hypothesize that: (i) the majority of granule cells from sclerotic hippocampus display an hyperexcitable epileptogenic electrophysiology; (ii) there is a subset of granule cells whose electrophysiology is preserved and is more comparable to granule cells from non-epileptic hippocampus.