The Extent of the Dendritic Tree and the Number of Synapses in the Frog Motoneuron

Frog motoneurons were intracellularly labelled with cobaltic lysine in the brachial and the lumbar segments of the spinal cord, and the material was processed for light microscopy in serial sections. With the aid of the neuron reconstruction system NEUTRACE, the dendritic tree of neurons was reconstructed and the length and surface area of dendrites measured. The surface of somata was determined with the prolate - oblate average ellipsoid calculation. Corrections were made for shrinkage and for optical distortion. The mean surface area of somata was 6710 microm2; lumbar motoneurons were slightly larger than brachial motoneurons. The mean length of the combined dendritic tree of brachial neurons was 29 408 microm and that of lumbar neurons 46 806 microm. The mean surface area was 127 335 microm2 in brachial neurons, and 168 063 microm2 in lumbar neurons. The soma - dendrite surface area ratio was 3 - 5% in most cases. Dendrites with a diameter of 600 microm from the soma. This suggests that about two-thirds of the synapses impinged upon distant dendrites >600 microm from the soma. The efficacy of synapses at these large distances is investigated on model neurons in the accompanying paper (Wolf et al., Eur. J. Neurosci., 4 1013 - 1021, 1992).     

Increase in ω3 (Peripheral-Type Benzodiazepine) binding site densities in different types of human brain tumours

Summary The density of ₃ (peripheral type benzodiazepine) binding sites, a marker of reactive and tumoural cells, has been measured in different types of human brain tumours; ₃ sites were quantified autoradiographically in sections from biopsy or autopsy specimens labelled with the specific radioligand³H-PK 11195. Compared to normal brain parenchyma, up to 12-fold increase in ₃ site densities were found in appparently viable areas of high grade astrocytoma and glioblastoma specimens, whereas more limited increases (2 to 3-fold) in this marker were observed in areas of necrosis. Low grade gliomas (astrocytomas) and meningiomas exhibited only moderate increases (2 to 3-fold) in this autoradiographic marker. Metastases of lung or kidney origin were characterized by greatly elevated (up to 20-fold) ₃ site densities as compared to normal brain parenchyma. In every case, there was a good spatial correspondence between the histopathological limits of the tumour and the anatomical location of the increase in ₃ site densities. These results suggest that ₃ site densities in human brain tumours reflect their proliferative activity and point to a possible future usefulness of positron or gamma-ray emitting ₃ site ligands for the clinical investigation and detection of human brain proliferative diseases.     

Effects of interleukin-1, tumor necrosis factor-β, and forskolin on tissue plasminogen activator activity in human osteoblastic osteosarcoma cells

Summary The effects of interleukin-1 (IL-1), forskolin, and tumor necrosis factor (TNF-) on tissue plasminogen activator (t-PA) activity were studied in the human osteoblastic osteosarcoma cell line, G292. t-PA activity was measured in the cell media using the chromogenic substrate, S-2251. After a 24 hour incubation period, IL-1 increased t-PA in a dose-dependent manner. The effect of IL-1 at 10.0 U/ml was partially inhibited in the presence of indomethacin. Forskolin (1.0 M) increased t-PA activity after 24 hours with the effects of combined treatment of IL-1 (1.0 U/ml, 10.0 U/ml) and forskolin being apparently additive in nature. TNF- (10^-8–10^-7 M) also produced increased t-PA activity in the cell medial after a 24 hour incubation period. These results suggest that the cytokines, IL-1 and TNF-, can increase t-PA activity in G292 cells and that there is both a cAMP-dependent as well as a cAMP-independent pathway involved in the regulation of this osteoblastic cell function.     

Spatial and Temporal Patterns of Neurogenesis in the Chick Retina

Chick embryo retinas were labelled in ovo by single injections of [3H]thymidine at selected times between days 2 and 12 of incubation. Embryos were later removed, at different stages of development, and the retinas processed for autoradiography of either serial sections or dissociated cell preparations. Analysis of unlabelled cells shows that neurogenesis starts, on day 2 of incubation, in a dorsotemporal area of the central retina, close to the posterior pole and to the optic nerve head. A gradient of neurogenesis spreads from this central area to the periphery, where neurogenesis ends, shortly after day 12, when the last few bipolar cells withdraw from the cell cycle. Additional dorsal-to-ventral and temporal-to-nasal gradients can be discerned in our autoradiographs. In all retinal sectors, ganglion cells start first to withdraw from the cell cycle, followed, with substantial overlapping, by amacrine, horizontal, photoreceptor plus Müller, and bipolar neuroblasts. Ganglion cells are also the first to reach the 50% level of unlabelled cells, followed this time by horizontal, photoreceptor, amacrine, Müller and bipolar cells. Finally, 100% levels of unlabelled cell populations are attained simultaneously by ganglion, horizontal and photoreceptor cells, followed by amacrine, then by Müller, and last by bipolar cells. Although all classes of neurons, in varying proportions, are being produced most of the time, our results also demonstrate that, in any given retinal area, the first cells leaving the cycle are determined to become ganglion cells, and the last ones bipolar cells, and not other types.     

Characterization and distribution of [3H]ohmefentanyl binding sites in the human brain

Binding properties and localization of [3H]ohmefentanyl, a new ligand for mu opioid receptors, were investigated on normal human brain sections. Binding assays performed at the level of the basal ganglia revealed: (1) a steady-state binding reached after 60 min incubation at room temperature, (2) the presence, in saturation experiments, of an apparent single class of binding sites with a Kd = 1.68 +/- 0.45 nM and a Bmax = 162 +/- 9 fmol/mg protein, (3) an order of potency to inhibit [3H]ohmefentanyl binding as follows: ohmefentanyl greater than [D-Ala2, MePhe4, Gly-ol5] enkephalin (DAGO) greater than ethylketocyclazocine (EKC) much greater than Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu) (BUBU) and U-50,488H. Quantitative autoradiography showed an heterogeneous distribution of [3H]ohmefentanyl binding sites with the highest densities in amygdala, medical geniculate body, thalamus, and caudate nucleus. Binding characteristics and anatomical distribution also show that [3H]ohmefentanyl may bind to a small proportion of additional sites called "DAGO-inaccessible [3H]ohmefentanyl specific binding sites." [3H]Ohmefentanyl binding to these sites can be partly inhibited by sigma ligands such as 1,3-di-o-tolylguanidine (DTG) and haloperidol. However, unlabeled DAGO inhibited more than 80% of [3H]ohmefentanyl specific binding in most of the human brain regions studied, suggesting that the major population of sites labeled by [3H]ohmefentanyl represented mu opioid receptors.     

Developmentally Regulated Expression of HDNF/NT-3 mRNA in Rat Spinal Cord Motoneurons and Expression of BDNF mRNA in Embryonic Dorsal Root Ganglion

Northern blot analysis was used to demonstrate high levels of hippocampus-derived neurotrophic factor/neurotrophin-3 (HDNF/NT-3) mRNA in the embryonic day (E) 13 - 14 and 15 - 16 spinal cord. The level decreased at E18 - 19 and remained the same until postnatal day (P) 1, after which it decreased further to a level below the detection limit in the adult. In situ hybridization revealed that the NT-3 mRNA detected in the developing spinal cord was derived from motoneurons and the decrease seen at E18 - 19 was caused by a reduction in the number of motoneurons expressing NT-3 mRNA. The distribution of NT-3 mRNA-expressing cells in the E15 spinal cord was very similar to the distribution of cells expressing choline acetyltransferase or nerve growth factor receptor (NGFR) mRNA. Moreover, a striking similarity between the developmentally regulated expression of NT-3 and NGFR mRNA was noted in spinal cord motoneurons. A subpopulation of all neurons in the dorsal root ganglia expressed brain-derived neurotrophic factor (BDNF) mRNA from E13, the earliest time examined, to adulthood. These results are consistent with a trophic role of NT-3 for proprioceptive sensory neurons innervating the ventral horn, and imply a local action of BDNF for developing sensory neurons within the dorsal root ganglia.     

Pulmonary mechanics and outcome of neonates on ECMO.

Deciding when to wean neonates from extracorporal membrane oxygenation (ECMO) can be difficult. The usefulness of simple measurements of pulmonary mechanics e.g., dynamic compliance (Cdyn) has been questioned. We investigated the pulmonary mechanics of eight neonates using the interrupter technique, which allows the partitioning of pulmonary mechanics into compartments representing the conducting airways and more peripheral phenomena (viscoelastic properties and "pendelluft"). Three neonates required ECMO for a congenital diaphragmatic hernia (CDH), two for hyaline membrane disease (HMO), two for meconium aspiration syndrome (MAS), and one for pneumonia. All neonates with MAS, HMD, and pneumonia were successfully weaned from ECMO when their Cdyn was 0.3 mL/cmH2O/kg or greater [mean 0.34 +/- 0.06 (SEM)]. All three neonates with CDH died and their highest Cdyn was 0.21, 0.19, and 0.09 mL/cmH2O/kg respectively (mean, 0.16 +/- 0.037). The airway resistance (Raw) and the slower component of pressure change after interruption (delta Pdiff), a measure of the more peripheral phenomena of the lung, were not significantly different in those neonates who survived and those who did not. The values for delta Pdiff in all patients were higher than those in healthy neonates. However, the Raw was not different. This suggests that the major disturbance in pulmonary mechanics was distal to the conducting airways. Those neonates who were successfully weaned from ECMO had a significantly higher Cdyn 24-48 hours prior to decannulation. Considering the lung as a two-compartment model offers no advantages when compared to the one-compartment model for the prediction of the outcome of a neonate on ECMO.     

Experimental vascularized total joint autografts--a primate study

Autogenous vascularized and nonvascularized total joint transfers were studied in the hands of Macaca fascicularis monkeys. Nine second toe proximal interphalangeal joints were transferred as a vascularized free graft to the hand, and the excised finger joints were transferred to the foot as a nonvascularized free graft. The grafts were examined clinically and histopathologically at 16 weeks to 10 months. Two of the nonvascularized free grafts were amputated because of infection and necrosis. Three had chronic infections. The four surviving nonvascularized grafts demonstrated necrosis of the hyaline cartilage and degenerative changes. Of the nine vascularized joints, one developed a wound infection that required amputation, another failed at 2 weeks because of wound dehiscence, and the remaining seven survived with preservation of the hyaline articular surfaces. The experimental technique was designed to be easily applied to clinical use. A skin island is provided as a "patency/viability monitor." The extensor mechanism is included in the graft for early function.     

Presence of functionally active β-adrenoceptors in rat mast cells

Summary The correlation between the binding of a -adrenoceptor antagonist, (–)[³H]-dihydroalprenolol (DHAP), and the adrenergic inhibition of histamine release by acetylcholine and by compound 48/80 was studied with isolated purified rat mast cells and in rat mast cell crude membrane fractions.Acetylcholine-evoked histamine release was inhibited by catecholamines, in the order isoprenaline > adrenaline > noradrenaline. Pretreatment of cells with (–)alprenolol antagonized the inhibitory effect of isoprenaline on acetylcholine-induced histamine release.40/80-evoked histamine release was bocked by isoprenaline at significantly higher concentrations than those required to inhibit cholinergic histamine release. The inhibitory effect of isoprenaline was equally antagonized by preincubating mast cells with (–)alprenolol.Specific binding sites for DHAP have been demonstrated in rat mast cell membranes. The specific binding of DHAP was inhibited by adrenoceptor agonists and antagonists according to the stereospecificity of these compounds.A close correlation between the binding-inhibitory potency of various adrenergic compounds and the data obtained in the pharmacological experiments was found, thus indicating the presence of -adrenoceptors in rat mast cells.     

In vitro and in vivo down-regulation of human plateletα 2-adrenoceptors by clonidine

Summary The effect of clonidine on the number of ₂-adrenoceptors in human platelet membranes, determined by³H-yohimbine binding, was investigatedin vitro andin vivo. Incubation of platelet membranes with clonidine (1–100 µM) for 16 h at 25 °C led to a concentration-dependent decrease in the number of³H-yohimbine binding sites of 10–25%; the affinity of³H-yohimbine to the sites was not changed (K_D approximately 3–4 nM). In such desensitized membranes, inhibition of³H-yohimbine binding by clonidine resulted in steep, monophasic displacement curves, which in comparison to the curves from control membranes (IC₅₀ for clonidine 90 nM), were shifted to the right (IC₅₀: 321 nM) and were not affected by 10^–4M guanosine-5-triphosphate (GTP).Treatment of 3 hypertensive patients with clonidine (3×150 µg/d for 7 days) reduced blood pressure and heart rate. Simultaneously, both³H-yohimbine binding sites on platelet membranes and plasma catecholamine levels decreased within three days and remained at a reduced level during treatment. After abrupt cessation of clonidine treatment, blood pressure, heart rate and plasma catecholamines rapidly increased, reaching values after two days similar to or higher than those before treatment.³H-yohimbine binding sites, however, initially decreased further before returning to control values. In platelet membranes derived from hypertensive patients treated with clonidine for at least three weeks, GTP (10^–4M) had no influence on inhibition of³H-yohimbine binding by (—)-adrenaline and clonidine. It is concluded that clonidine desensitizes ₂-adrenoceptors in human platelet membranesin vitro andin vivo. An important step in the desensitization process is the uncoupling of receptor occupancy by agonists and adenylate cyclase activity, as indicated by loss of the regulatory activity of GTP on desensitized membranes. The clonidine withdrawal syndrome may be caused by enhanced release of endogenous catecholamines not adequately regulated by presynaptic ₂-adrenoceptors, which have become subsensitive after chronic clonidine treatment.