Tubulin expression in the developing and adult gerbil organ of Corti.

In the late stages of inner ear development, the relatively undifferentiated cells of Kollicker's organ are transformed into the elaborately specialized cell types of the organ of Corti. Microtubules are prominent features of adult cells in the organ of Corti, particularly supporting cells. To test the possible role of microtubules in organ of Corti development, the microtubule organization in the organ of Corti has been examined using indirect immunofluorescence to beta-tubulin in the developing gerbil cochlea. Tubulin first appears at post-natal day 0 (P0) as filamentous asters in inner hair cells and by P2, asters are also seen in outer hair cells. Tubulin appears at P3 in inner pillar cells in a tooth crown-like figure. By P6, tubulin expression is also evident in outer pillar cells and by P9, it is seen in Deiters cells. Elaboration of microtubules in pillar cells was observed to proceed from the reticular lamina towards the basilar membrane. The pattern of tubulin expression in the apical organ of Corti lags the base by about 3 days until P6, but by P9, apical and basal organ of Corti appear substantially the same.

Structural and segregational stability of various replicons in Actinobacillus actinomycetemcomitans.

Few vectors suitable for cloning in Actinobacillus actinomycetemcomitans, a periodontal pathogen, have been described. These plasmids were based either on the native A. actinomycetemcomitans replicon, pVT736-1, or derivatives of the IncP and IncQ family of plasmids. Their usefulness as cloning vectors is limited because of instability or size. Therefore, the ability of additional replicons to function in A. actinomycetemcomitans was evaluated. Derivatives of p15A, ColE1/f1, and pWV01 transformed A. actinomycetemcomitans efficiently and exhibited no structural instability in the new host. The stable maintenance of A. actinomycetemcomitans-specific DNA fragments inserted into the p15A derivative, pDMG4, demonstrated the ability of this plasmid to function as a cloning vector. In addition, pVT736-1 was used to clone selectable markers directly into A. actinomycetemcomitans. These constructs were structurally and segregationally stable.

Dose and time dependency of angiotensin II inhibition of hippocampal long-term potentiation.

We previously reported that injection of 1.0 microliter of 4.78 microM angiotensin II (AII) above the hippocampus in rats inhibits long-term potentiation (LTP) induction in medial perforant path-stimulated dentate granule cells. The present experiments were conducted in urethane-anesthetized Sprague-Dawley rats. LTP was measured in terms of the relative change in slope of the population EPSP compared to baseline. Effects of 0.48, 0.956, 1.195, 2.39, and 4.78 microM AII and time delays of 30, 60, 90, and 150 min with the 4.78 microM dose were determined. Results were significant and demonstrate that AII inhibition of LTP in dentate granule cells is both dose and time dependent. The threshold is approximately 1.0 pmol of peptide. Inhibition due to the 4.78 microM AII begins slowly after 1 h and is complete over the next 30 min, continues for another 30 min, and then fully recovers by the end of the next 30 min. This time dependency could be due to the internalization of the AII, interaction with a cytosolic receptor, and eventual degradation.

Losartan reduces ethanol intoxication in the rat.

Results of a previous study showed that ethanol inhibition of hippocampal long-term potentiation (LTP) induction was mediated by angiotensin II (AII) and the AT1 subtype receptor because it was blocked by losartan, a specific AT1 antagonist. Because LTP is an important hippocampal function involved in the memory process and other behaviors, it is possible that losartan might block some of the directly observable ethanol-induced changes in rat behavior. Results demonstrate that losartan can effectively block some of the intoxicating effects of low doses of ethanol, 2 g/kg PO or IP. However, even a high dose of losartan 20 mg/kg IP, did not reduce significantly any of the intoxicating effects of the higher dose of 4 g/kg administered by gavage. Higher doses of ethanol might be more difficult to block because of a direct effect on the post synaptic membrane.

Angiotensin AII AT1 receptor mediates ethanol-diazepam inhibition of hippocampal LTP.

Within a limited range of doses, co-administration of ethanol (EtOH) and diazepam (DZ) produce enhanced anxiolytic effects. These combined effects on long-term potentiation (LTP) in the hippocampus of rats anesthetized with urethane were studied in an attempt to provide an explanation at a more fundamental neuronal level. Male Sprague-Dawley rats received 0.1, 0.5, 0.75 and 1.0 mg/kg DZ i.p. in combination with 0.1, 0.5, 0.75 g and 1.0 g/kg EtOH by gavage, respectively. Drugs were administered 20 min (EtOH) and 15 min (DZ) prior to tetanic stimulation of the medial perforant path which resulted in LTP induction measured in terms of the relative change in amplitude of the population EPSP as compared to baseline. Effects of both drugs alone and in combination on LTP are presented. Both drugs depressed LTP induction and significant enhanced interactive effects were observed. We have previously shown that angiotensin II (AII) inhibits LTP induction and that the inhibition can be blocked by losartan, an AII AT1 receptor antagonist. Our present results demonstrate that the combined inhibitory effects of EtOH and DZ on LTP induction are also blocked by losartan.

Role of angiotensin II and AT1 receptors in hippocampal LTP.

Results of a previous study showed that angiotensin II (AII) inhibited the induction of long-term potentiation (LTP) in hippocampal granule cells in response to dorsomedial perforant path stimulation in urethane-anesthetized rats. The results of present experiments demonstrate a dose-dependent inhibition of LTP induction under the same conditions due to ethanol (EtOH) administered by stomach tube and diazepam (DZ) injected IP. The inhibition of LTP induction by EtOH and DZ can be blocked by saralasin (SAR) applied directly to the dorsal hippocampus and by lorsartan (DuP 753) administered IP. Lorsartan or a metabolite crosses the blood-brain barrier because it also blocks the inhibition of LTP induction due to AII administration directly into the dorsal hippocampus. Lorsartan is a competitive antagonist of the AT1 subtype AII receptor. Therefore, the AII and the EtOH and DZ inhibition of LTP induction are mediated by the AII subtype receptor AT1. AIII and the AT2 antagonist PD123319 did not produce any significant effects. These in vivo effects can be reproduced in brain slices and therefore cannot be attributed to other factors, such as the urethane. In addition, electrical stimulation of the lateral hypothalamus (LH) inhibits LTP induction, and the inhibition can be blocked by SAR. These data on LH stimulation indicate that LH AII-containing neurons send axons into the hippocampus that inhibit the induction of LTP. These results not only provide new information on a neurotransmitter involved in the amnesic effects of benzodiazepines and ethanol-induced memory blackouts, but also testable hypotheses concerning recent observations that angiotensin converting enzyme (ACE) inhibitors elevate mood and improve certain cognitive processes in the elderly.

Chronic exposure to environmental levels of lead impairs in vivo induction of long-term potentiation in rat hippocampal dentate.

This study examined the effects of chronic developmental lead (Pb) exposure in rats on hippocampal long-term potentiation (LTP). Male offspring were exposed to 0.2% Pb acetate continuously from birth until testing at 85-105 days. Excitatory postsynaptic potential (EPSP) and population spike amplitudes were measured in the dentate hilar region in response to stimulation applied to the lateral perforant path. LTP was induced in control animals with an average maximal EPSP potentiation of 41%, which was significantly greater than the increase in EPSP amplitudes (2%) in exposed animals after tetanizing stimulation. Current-voltage curves in controls demonstrated significant increases in EPSPs and population spikes after application of pulse trains to induce LTP, while exposed rats exhibited no discernible change in responses. These findings suggest that induction or development of LTP in the dentate hilar region in vivo is impaired by chronic developmental exposure to environmentally relevant levels of Pb.

Ethanol and diazepam inhibition of hippocampal LTP is mediated by angiotensin II and AT1 receptors.

Angiotensin II (AII) inhibits the induction of hippocampal long-term potentiation (LTP), a frequency-dependent model of learning and memory. These results demonstrate that the dose-dependent inhibition of LTP due to ethanol (EtOH) and diazepam (DZ) involves AII. Inhibition of LTP induction by AII, EtOH, and DZ can be blocked by AII receptor antagonists saralasin and lorsartan (DuP 753). Lorsartan is a competitive antagonist of the AT1 subtype AII receptor. Therefore, the EtOH and DZ inhibition of LTP induction is mediated by AT1 receptors. These results indicate a new role for AII in the brain in the possible mediation of memory deficits associated with alcohol and the benzodiazepines.

Angiotensin II blocks hippocampal long-term potentiation.

We have found that injection of angiotensin II (AII) above the hippocampus in the intact rat blocks the induction of long-term potentiation (LTP) in perforant path-stimulated dentate granule cells. A minimum dose of 4.78 pmol AII was required for the complete blockade of LTP and this blockade was entirely prevented if the AII-specific antagonist saralasin was co-injected at a 50-fold molar excess. AII thus appears to act via AII receptors and does not cause non-specific inhibition. The injection of saralasin alone yielded LTP comparable to that obtained when vehicle was injected. Angiotensin III was found to be 40-50 fold less potent than AII in blocking LTP. Both AII and AII receptors of unknown function occur in the hippocampal formation. The results reported here suggest a role for these molecules in the control of hippocampal LTP.

Evidence that protein kinase M does not maintain long-term potentiation.

We have shown that the induction but not maintenance of long-term potentiation (LTP) in the Schaffer collateral-CA1 synaptic zone of the rat hippocampus is blocked by the extracellular application of the protein kinase inhibitor staurosporine. This compound was also found to block the induction of LTP in the perforant path-granule cell synaptic zone of the intact hippocampus. We have determined that staurosporine is membrane-permeable and can be detected inside cells by fluorescence microscopy. When cultured fetal hippocampal neurons were treated with staurosporine, fluorescence was observed throughout the cytoplasm and in neurites. Other cell types gave similar results. It has been proposed that constitutively active cytosolic protein kinase M or other protein kinases maintain long-term potentiation. Since staurosporine has access to the cytosol and inhibits protein kinase M in vitro, our results suggest that this enzyme is not responsible for the maintenance of LTP. This conclusion may extend to other protein kinases as well, since staurosporine has been shown to inhibit a variety of these enzymes.

Calpain inhibitors block long-term potentiation.

Long-term potentiation (LTP) is a form of synaptic plasticity that serves as a model for certain types of learning and memory. The role of the calcium-activated thiol proteases or calpains in the biochemical mechanism of LTP has been explored. We show that the extracellular application of two newly developed, highly potent calpain inhibitors, N-acetyl-Leu-Leu-norleucinal and N-acetyl-Leu-Leu-methioninal, block LTP in both the Schaffer collateral-CA1 synaptic zone of the rat hippocampal slice and in perforant path-stimulated dentate granule cells in the intact hippocampus. The inhibitors do not affect baseline synaptic transmission and block LTP in the slice preparation if applied before but not after tetanic stimulation. The calpain inhibitor leupeptin is less potent than the above peptides but also blocks LTP if applied at a sufficient concentration.