Dense core vesicles resemble active-zone transport vesicles and are diminished following synaptogenesis in mature hippocampal slices.

Large dense core vesicles (approximately 100 nm) contain neuroactive peptides and other co-transmitters. Smaller dense core vesicles (approximately 80 nm) are known to contain components of the presynaptic active zone and thought to transport and deliver these components during developmental synaptogenesis. It is not known whether excitatory axons in area CA1 contain such dense core vesicles, and whether they contribute to synaptic plasticity of mature hippocampus. Serial section electron microscopy was used to identify dense core vesicles in presynaptic axons in s. radiatum of area CA1 in adult rat hippocampus. Comparisons were made among perfusion-fixed hippocampus and hippocampal slices that undergo synaptogenesis during recovery in vitro. Dense core vesicles occurred in 26.1+/-3.6% of axonal boutons in perfusion fixed hippocampus, and in only 17.6+/-4.5% of axonal boutons in hippocampal slices (P<0.01). Most of the dense core vesicle positive boutons contained only one dense core vesicle, and no reconstructed axonal bouton had more than a total of 10 dense core vesicles in either condition. Overall the dense core vesicles had average diameters of 79+/-11 nm. These small dense core vesicles were usually located near nonsynaptic membranes and rarely occurred near the edge of a presynaptic active zone. Their size, low frequency, locations, and decrease following recuperative synaptogenesis in slices are novel findings that merit further study with respect to small dense core vesicle content and possible contributions to synapse assembly and plasticity in the mature hippocampus.

GeneKeyDB: a lightweight, gene-centric, relational database to support data mining environments.

BACKGROUND: The analysis of biological data is greatly enhanced by existing or emerging databases. Most existing databases, with few exceptions are not designed to easily support large scale computational analysis, but rather offer exclusively a web interface to the resource. We have recognized the growing need for a database which can be used successfully as a backend to computational analysis tools and pipelines. Such database should be sufficiently versatile to allow easy system integration. RESULTS: GeneKeyDB is a gene-centered relational database developed to enhance data mining in biological data sets. The system provides an underlying data layer for computational analysis tools and visualization tools. GeneKeyDB relies primarily on existing database identifiers derived from community databases (NCBI, GO, Ensembl, et al.) as well as the known relationships among those identifiers. It is a lightweight, portable, and extensible platform for integration with computational tools and analysis environments. CONCLUSION: GeneKeyDB can enable analysis tools and users to manipulate the intersections, unions, and differences among different data sets.

Dendritic spines disappear with chilling but proliferate excessively upon rewarming of mature hippocampus.

More dendritic spine synapses occur on mature neurons in hippocampal slices by 2 h of incubation in vitro, than in perfusion-fixed hippocampus. What conditions initiate this spinogenesis and how rapidly do the spines begin to proliferate on mature neurons? To address these questions, CA1 field of the hippocampus neurons expressing green fluorescent protein in living slices from mature mice were imaged with two-photon microscopy. Spines disappeared and dendrites were varicose immediately after slice preparation in ice-cold artificial cerebrospinal fluid (ACSF). Electron microscopy (EM) revealed disrupted dendritic cytoplasm, enlarged or free-floating postsynaptic densities, and excessive axonal endocytosis. Upon warming dendritic varicosities shrank and spines rapidly reappeared within a few minutes illustrating the remarkable resilience of mature hippocampal neurons in slices. When membrane impermeant sucrose was substituted for NaCl in ACSF dendrites remained spiny at ice-cold temperatures and EM revealed less disruption. Nevertheless, spine number and length increased within 30 min in warm ACSF even when the extracellular calcium concentration was zero and synaptic transmission was blocked. When slices were first recovered for several hours and then chilled in 6 degrees C ACSF many spines disappeared and the dendrites became varicose. Upon re-warming varicosities shrank and spines reemerged in the same position from which they disappeared. In addition, new spines formed and spines were longer suggesting that chilling, not the initial injury from slicing, caused the spines to disappear while re-warming triggered the spine proliferation on mature neurons. The new spines might be a substrate for neuronal recovery of function, when neurons have been chilled or exposed to other traumatic conditions that disrupt ionic homeostasis.

Detection of apoB-100 R3500Q mutation by competitive allele-specific polymerase chain reaction.

The apolipoprotein B-100 mutation R3500Q is one of the most common inherited defects causing abnormality of the lipid metabolism. We describe a one-step, single-tube PCR technique for detection of the mutation based on competition between allele-specific primers. Three oligonucleotides are used: two allele-specific primers differing in their 3' nucleotide (for the wild-type and the mutant allele) together with a common primer, resulting in simultaneous amplification of both alleles. This provided internal control of successful amplification and is expected to result in increased specificity. The allele-specific primers differ also in length, allowing us to distinguish both alleles by their size in a single electrophoretic run. For optimization of the protocol, DNAs genotyped before by oligonucleotide ligation assay were used. The individual genotypes obtained by CAS-PCR coincided fully with the ones from a referent OLA test: seven heterozygous individuals were found, 4 of them among 150 unrelated hypercholesterolemic individuals studied and other three in the pedigrees of heterozygous carriers. On the overall 160 genotypes were determined, neither false-positive (0 out of 153 non-carriers) nor false-negative (0 out of 7 carriers) results were obtained. No homozygous mutant genotypes were identified in this sample.

Dendrites are more spiny on mature hippocampal neurons when synapses are inactivated.

Dendrites of CA1 pyramidal neurons in mature rat hippocampal slices were exposed to different levels of synaptic activation. In some slices, synaptic transmission was blocked with glutamate receptor antagonists, sodium and calcium channel blockers and/or a nominally calcium-free medium with high magnesium. In other slices, synapses were activated with low-frequency control stimulation or repeated tetanic stimulation. In slices with blocked synaptic transmission, dendrites were spinier than in either of the activated states. Thus, mature neurons can increase their numbers of spines, possibly compensating for lost synaptic activity.

Slices have more synapses than perfusion-fixed hippocampus from both young and mature rats.

Hippocampal slices have long been used to investigate properties of synaptic transmission and plasticity. Here, for the first time, synapses in slices have been compared quantitatively with synapses occurring in perfusion-fixed hippocampus, which is presumed to represent the natural in vivo state. Relative to perfusion-fixed hippocampus, a remarkable 40-50% increase in spine number occurs in adult hippocampal slices, and a 90% increase occurs in slices from postnatal day 21 rats. Serial EM shows that all of the dendritic spines have normal synapses with presynaptic and postsynaptic elements; however, not all spine types are affected uniformly. Stubby and mushroom spines increase in the adult slices, and thin, mushroom, and branched spines increase in the immature slices. More axonal boutons with multiple synapses occur in the slices, suggesting that the new synapses form on preexisting axonal boutons. The increase in spine and synapse number is evident within a couple of hours after preparing the slices. Once the initial spine induction has occurred, no further change occurs for up to 13 hr in vitro, the longest time investigated. Thus, the spine increase is occurring during a period when there is little or no synaptic activity during the first hour, and the subsequent stabilization in spine synapse numbers is occurring after synaptic activity returns in the slice. These findings suggest that spines form in response to the loss of synaptic activity when slices are removed from the rest of the brain and during the subsequent 1 hr recovery period.

Evidence for metabotropic glutamate receptor activation in the induction of depolarization-induced suppression of inhibition in hippocampal CA1.

Depolarization-induced suppression of inhibition (DSI) is a transient reduction of GABAA receptor-mediated IPSCs that is mediated by a retrograde signal from principal cells to interneurons. Using whole-cell recordings, we tested the hypothesis that mGluRs are involved in the DSI process in hippocampal CA1, as has been proposed for cerebellar DSI. Group II mGluR agonists failed to affect either evoked monosynaptic IPSCs or DSI, and forskolin, which blocks cerebellar DSI, did not affect CA1 DSI. Group I and group III mGluR agonists reduced IPSCs, but only group I agonists occluded DSI. (S)-MCPG blocked (1S,3R)-ACPD-induced IPSC suppression and markedly reduced DSI, whereas group III antagonists had no effect on DSI. Many other similarities between DSI and the (1S,3R)-ACPD-induced suppression of IPSCs also were found. Our data suggest that a glutamate-like substance released from pyramidal cells could mediate CA1 DSI by reducing GABA release from interneurons via the activation of group I mGluRs.

N-ethylmaleimide blocks depolarization-induced suppression of inhibition and enhances GABA release in the rat hippocampal slice in vitro.

Regulation of synaptic, GABAA receptor-mediated inhibition is a process of critical importance to normal brain function. Recently, we have described a phenomenon in hippocampus of a transient, yet marked, decrease in spontaneous, GABAA receptor-mediated IPSCs after depolarization activated Ca2+ influx into a pyramidal cell. This process, depolarization-induced suppression of inhibition (DSI), is absent in hippocampal cells that previously had been exposed to pertussis toxin in vivo, implicating a G-protein in the DSI process. To circumvent the problem that a single cell cannot be studied before and after G-protein block using the pertussis toxin pretreatment method, we have used the sulfhydryl alkylating agent N-ethylmaleimide (NEM), which blocks pertussis toxin-sensitive G-proteins, to determine whether acute inhibition of G-proteins can eliminate DSI of spontaneous IPSCs (sIPSCs). In whole-cell recordings from CA1 pyramidal cells that were first determined to express DSI, we have found that NEM does block DSI of sIPSCs. We also report that DSI of monosynaptic, evoked IPSCs is blocked by NEM, suggesting that a similar mechanism underlies both forms of DSI. It was of interest that DSI was abolished at a time when NEM had increased, not decreased, GABA transmission. Indeed, NEM greatly increased quantal GABA release by a Ca(2+)-independent mechanism, an observation with potentially important implications for understanding synaptic GABA release.

Cold acclimation-associated changes in brown adipose tissue do not necessarily indicate an increase of nonshivering thermogenesis in C57BL/6J mice.

We have reported previously that a cold acclimation procedure (3-hr partial restraint at 6 degrees C, repeated 3 times at 2-week intervals) usually improves the cold tolerance of adult C57BL/6J mice. Those mice that did not improve their cold tolerance had lower cold-induced sympathetic nervous outflow to the interscapular brown adipose tissue (IBAT), suggesting a failure in the mechanisms of nonshivering thermogenesis. To understand the origin of this failure, this study was intended to measure nonshivering thermogenesis in mice that did not improve their cold tolerance after the cold acclimation procedure. After being subjected 3 times to a partial restraint at 6 degrees C, mice were anesthetized with urethane, immobilized with vecuronium bromide, and placed on artificial ventilation. The VO2 and VCO2 in expired air were measured and metabolic heat production (MHP) was calculated while body temperature was artificially lowered to 7.5 degrees C below control level. In a separate group of mice, the total amount and concentration of mitochondrial uncoupling protein, thermogenin (UCP), in IBAT was measured immediately after completion of the cold-acclimation procedure. The concentration and the amount of UCP in the mitochondria of IBAT was significantly higher in all mice that had been presented to the cold acclimation procedure, regardless of its outcome, than in mice that had never been exposed to an environment below room temperature (NAIVE). MHP increased significantly during body cooling in all mice. However, MHP before and during cold stimulation in mice that did not improve their cold tolerance as a result of the cold-acclimation procedure was significantly lower than the MHP of animals in which cold tolerance was improved, and was not different from MHP of the NAIVE group. Therefore, in mice in which cold tolerance did not improve after repeated cold exposure, the anatomical and biochemical changes in brown adipose tissue typical of cold acclimation were not associated with a cold-induced increase in MHP. We infer that the expression of UCP in brown adipose tissue is a necessary, but not sufficient, attribute of cold acclimation. Cold acclimation, measured as increased cold tolerance, occurs only if synthesis of UCP in BAT is associated with an increased cold-induced response of the sympathetic nervous system.

Nonshivering thermogenesis in adult and aged C57BL/6J mice housed at 22 degrees C and at 29 degrees C.

Twelve- and 28-month-old C57BL/6J male mice were housed either at room temperature of 22 degrees C or at thermoneutrality (29 degrees C) during the two months prior to experiments. Acute experiments were conducted under anesthesia, myorelaxation, and artificial ventilation. We recorded efferent electrical impulse activity in one of the sympathetic nerves innervating the interscapular brown adipose tissue in response to acute cold stimulation, when body temperature was lowered 7.5 degrees C below control level. In separate experiments we measured O2 consumption and CO2 production and calculated the nonshivering thermogenesis. We also measured the concentration of uncoupling protein in interscapular brown adipose tissue before and after three-hour cold stress. In aged mice, both sympathetic nervous activity and nonshivering thermogenesis were lower in animals housed at thermoneutrality (29 degrees C) than in mice housed at 22 degrees. Among mice maintained at 22 degrees C, but not at thermoneutrality, aged animals had greater nonshivering thermogenesis and greater cold induced concentration of uncoupling protein in the brown adipose tissue than adults. Sympathetic nervous outflow to brown adipose tissue was always greater in aged mice, regardless of the temperature of acclimation. We concluded that aged mice, housed at 22 degrees C, showed the changes in nonshivering thermogenesis associated with cold acclimation. However, an increased sympathetic outflow to brown adipose tissue in aged animals reflects an age-related elevation of the tone and responsiveness of the sympathetic nervous system.

Retrograde signalling in depolarization-induced suppression of inhibition in rat hippocampal CA1 cells.

1. We have investigated the phenomenon of 'depolarization-induced suppression of inhibition' (DSI) using whole-cell voltage-clamp techniques in Ca1 pyramidal cells of rat hippocampal slices. DSI was induced by eliciting voltage-dependent calcium (Ca2+) currents with 1 s voltage steps of +60 to +90 mV from the holding potential. DSI was apparent as a reduction in synaptic GABAA responses for a period of about 1 min following the voltage step. 2. TTX-sensitive spontaneous IPSCs (sIPSCs) were susceptible to DSI, while TTX-resistant miniature inhibitory postsynaptic current (mIPSCs) were not. Miniature IPSCs are ordinarily infrequent and independent of external Ca2+ in the CA1 region. To increase the frequency of mIPSCs and to induce a population of Ca(2+)-sensitive mIPSCs, we increased the bath K+ concentration to 15 mM. The increased mIPSCs were also insensitive to DSI, however. 3. T whole-cell pipette-filling solution contained 5 mM 2(triethylamino-N-(2,6-dimethyl-phenyl)acetamide (QX-314) to block voltage-dependent Na+ currents and caesium to block K+ currents. Nevertheless, bath application of 50 microM 4-aminopyridine (4-AP) or 250 nM veratridine both clearly reduced DSI, evidently by acting at presynaptic sites. 4. The amplitudes of monosynaptically evoked IPSCs (elicited in the presence of 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 50 microM 2-amino-5-phosphonovaleric acid (APV)) were dramatically reduced during the DSI period. Weak stimulation produced small IPSCs and occasional 'failures' of transmission during the control period. The percentage of failures increased markedly during the DSI period. Moderate-intensity stimulation produced larger IPSCs that were often composed of distinguishable multiquantal components. All-or-none failures of multiquantal IPSC components also occurred during DSI. 5. The degree of paired-pulse IPSC depression did not change during DSI, whereas it was decreased, as expected, by baclofen. 6. We conclude that the data represent novel evidence that DSI is mediated by a retrograde signalling process possibly involving presynaptic axonal conduction block.

Nonshivering thermogenesis during acute cold exposure in adult and aged C57BL/6J mice.

In C57BL/6J adult and aged mice, housed at room temperature (22.5 +/- 1 degrees C), we measured O2 consumption and CO2 production and calculated metabolic heat production under conditions of anesthesia and myorelaxation during acute cold stimulation when body temperature was lowered 7.5 degrees C below control level. An independent group of mice was subjected to a three hour partial physical restraint at 6 degrees C and concentration of uncoupling protein (thermogenin) was measured in interscapular brown adipose tissue mitochondria at different times after cold exposure. Heat production under anesthesia and myorelaxation was about 57-66% lower than in nonanesthetized conditions, but increased significantly during cold stimulation in both age groups. Under anesthesia and myorelaxation before and during cold stimulation aged mice produced about 20% more heat than adult mice. Because in these experiments all sources of facultative thermogenesis, except nonshivering, were suppressed by anesthesia and myorelaxation, and because brown adipose tissue is the major source of nonshivering thermoproduction, we concluded that aged mice housed at room temperature have an increased thermogenesis in brown adipose tissue. This conclusion was also supported by the finding that the concentration of uncoupling protein measured in the mitochondria of brown adipose tissue after single cold exposure was significantly higher in aged than in adult mice. Therefore, we propose that the lower, cold-induced, heat production typically observed in nonanesthetized aged mice may reflect reduced thermogenic capacity of skeletal muscles. While aged mice have less brown adipose tissue than adult animals, the remaining brown adipose tissue may compensate by increasing the concentration of uncoupling protein.

Sympathetic outflow to interscapular brown adipose tissue in cold acclimated mice.

C57BL/6J male mice were subjected to a cold acclimation procedure which consisted of three consecutive cold stress tests: 3-h partial restraint at 6 degrees C at 2-wk intervals. During the week following the last cold stress test, each animal previously subjected to the cold acclimation procedure, and an additional group of naive mice (animals that never had been exposed to an environment below room temperature) were anesthetized with urethan, paralyzed with vecuronium bromide, artificially ventilated, and subjected to cold stimulation for approximately 16 min. Electrical impulse activity from one of the fine sympathetic nerves entering the interscapular brown adipose tissue was recorded before and during cold stimulation, until body temperature dropped 8 degrees C below control level. Sympathetic outflow increased significantly during cold stimulation in all mice. Animals that did not achieve cold acclimation in three repeated cold stress tests (they demonstrated less cold tolerance in the last test) had lower sympathetic nervous outflow to brown adipose tissue at room temperature and during cold stimulation than mice that had achieved cold acclimation. In fact, sympathetic nervous outflow to brown adipose tissue in mice that had failed to show cold acclimation was similar to that of naive mice. These findings indicate that the sympathetic nervous system plays a primary role in cold acclimation.

[Benign neoplasms of the mediastinum]. / Dobrokachestvennye novoobrazovaniia sredosteniia.

The authors discuss the results of surgical treatment of 105 patients with benign neoplasms of the mediastinum. Cysts were encountered in 22.9% and benign tumors in 77.1% of cases. Two complications occurred during the operation: injury inflicted to the intercostal artery in removal of a neurofibroma and injury to the lung in removal of a mediastinal cyst. The authors conclude that surgery of mediastinal neoplasms is a radical method of treatment.

[Kinetics of the inactivation of tetrodotoxin-sensitive sodium channels in the spinal ganglion neurons of rats]. / Kinetika inaktivatsii tetrodotoksinchuvstvitel'nykh natrievykh kanalov neironov spinal'nykh gangliev krys.

The assessment of TTX-sensitive sodium transmembrane ionic current induced by depolarizing shifts in the MP performed in neurons of isolated dorsal root ganglion of adult rats revealed that the kinetics of the inactivation of TTX-sensitive sodium channels was more complicated than suggested by Hodgkin and Huxley for gating devices, the behaviour of the inactivation system following at least two exponential kinetics.