Influence of parallel fiber–Purkinje cell synapse formation on postnatal development of climbing fiber–Purkinje cell synapses in the cerebellum

The climbing fiber (CF) to Purkinje cell (PC) synapse in the cerebellum provides an ideal model for the study of developmental rearrangements of neural circuits. At birth, each PC is innervated by multiple CFs. These surplus CFs are eliminated during postnatal development, and mono innervation is attained by postnatal day 20 (P20) in mice. Earlier studies on spontaneous mutant mice and animals with “hypogranular” cerebella indicate that regression of surplus CFs requires normal generation of granule cells and their axons, parallel fibers (PFs), and normal formation of PF–PC synapses. Our understanding of how PF–PC synapse formation affects development of CF–PC synapse has been greatly advanced by analyses of mutant mice deficient in glutamate receptor δ2 subunit (GluRδ2), an orphan receptor expressed selectively in PCs. Deletion of GluRδ2 results in impairment of PF–PC synapse formation, which leads to defects in development of CF–PC synapses. In this article, we review how impaired PF–PC synapse formation affects wiring of CFs to PCs based mostly on our data on GluRδ2 knockout mice. We propose a new scheme that CF–PC synapses are shaped by the three consecutive events, namely functional differentiation of multiple CFs into one strong and a few weak inputs from P3 to P7, “early phase” of CF synapse elimination from P7 to around P11, and “late phase” of CF synapse elimination from around P12. Normal PF–PC synapse formation is required for the “late phase” of CF synapse elimination.     

G-protein-independent modulation of P-type calcium channels by μ-opioids in Purkinje neurons of rat

P-type calcium channels play a key role in the synaptic transmission between mammalian central neurons since a major part of calcium entering pre-synaptic terminals is delivered via these channels. Using conventional whole-cell patch clamp techniques we have studied the effect of μ-opioids on P-type calcium channels in acutely isolated Purkinje neurons from rat cerebellum. The selective μ-opioid agonist DAMGO (10 nM) produced a small, but consistent facilitation of current through P-type calcium channels (10 ± 1%, n = 27, p < 0.001). The effect of DAMGO was rapid (less than 10 s) and fully reversible. This effect was both concentration and voltage-dependent. The EC₅₀ for the effect of DAMGO was 1.3 ± 0.4 nM and the saturating concentration was 100 nM. The endogenous selective agonist of μ-opioid receptors, endomorphin-1 demonstrated similar action. Intracellular perfusion of Purkinje neurons with GTPγS (0.5 mM) or GDPβS (0.5 mM), as well as strong depolarizing pre-pulses (+50 mV), did not eliminate facilitatory action of DAMGO on P-channels indicating that this effect is not mediated by G-proteins. Furthermore, the effect of DAMGO was preserved in the presence of a non-specific inhibitor of PKA and PKC (H7, 10 μM) inside the cell. DAMGO-induced facilitation of P-current was almost completely abolished by the selective μ-opioid antagonist CTOP (100 nM). These observations indicate that μ-type opioid receptors modulate P-type calcium channels in Purkinje neurons via G-protein-independent mechanism.     

Activity-dependent plasticity of developing climbing fiber–Purkinje cell synapses

Elimination of redundant synapses and strengthening of the surviving ones are crucial steps in the development of the nervous system. Both processes can be readily followed at the climbing fiber to Purkinje cell synapse in the cerebellum. Shortly after birth, around five equally strong climbing fiber synapses are established. Subsequently, one of these five synaptic connections starts to grow in size and synaptic strength, while the others degenerate and eventually disappear. Both the elimination of the redundant climbing fiber synapses and the strengthening of the surviving one depend on a combination of a genetically coded blueprint and synaptic activity. Recently, it has been shown that synaptic activity affects the synaptic strength of developing climbing fibers. Remarkably, the same pattern of paired activity of the presynaptic climbing fiber and the postsynaptic Purkinje cell resulted in strengthening of already “large” climbing fibers and weakening of already “weak” climbing fibers. In this review, we will integrate the current knowledge of synaptic plasticity of climbing fibers with that of other processes affecting climbing fiber development.     

Expression of TGF-β1 in the blood during fracture repair in an estrogen-deficient rat model

OBJECTIVES:

Previous studies have reported that osteoporosis due to estrogen deficiency influences fracture healing. Transforming growth factor (TGF-β) has been found to be involved in fracture healing via the regulation of the differentiation and activation of osteoblasts and osteoclasts. The current study aimed to determine the effects of estrogen on the expression of TGF-β1 during fracture healing in ovariectomized rats.

METHODS:

Thirty female Sprague-Dawley rats weighing 200–250 g were assigned to: (i) a sham-operated group that was given a normal saline; (ii) an ovariectomized control group that was given a normal saline; or (iii) an ovariectomized + estrogen (100 µg/kg/day) group that was treated with conjugated equine estrogen. The right femur of all rats was fractured, and a Kirschner wire was inserted six weeks post-ovariectomy. Treatment with estrogen was given for another six weeks post-fracture. At the end of the study, blood samples were taken, and the right femur was harvested and subjected to biomechanical strength testing.

RESULTS:

The percentage change in the plasma TGF-β1 level before treatment was significantly lower in the ovariectomized control and estrogen groups when compared with the sham group (p<0.001). After six weeks of treatment, the percentage change in the plasma TGF-β1 level in the estrogen group was significantly higher compared with the level in the ovariectomized control group (p = 0.001). The mean ultimate force was significantly increased in the ovariectomized rats treated with estrogen when compared with the ovariectomized control group (p = 0.02).

CONCLUSION:

These data suggest that treatment with conjugated equine estrogen enhanced the strength of the healed bone in estrogen-deficient rats by most likely inducing the expression of TGF-β1.     

Does the vestibular system contribute to head direction cell activity in the rat?

Head direction cells (HDC) located in several regions of the brain, including the anterior dorsal nucleus of the thalamus (ADN), postsubiculum (PoS), and lateral mammillary nuclei (LMN), provide the neural substrate for the determination of head direction. Although activity of HDC is influenced by various sensory signals and internally generated cues, lesion studies and some anatomical and physiological evidence suggest that vestibular inputs are critical for the maintenance of directional sensitivity of these cells. However, vestibular inputs must be transformed considerably in order to signal head direction, and the neuronal circuitry that accomplishes this signal processing has not been fully established. Furthermore, it is unclear why the removal of vestibular inputs abolishes the directional sensitivity of HDC, as visual and other sensory inputs and motor feedback signals strongly affect the firing of these neurons and would be expected to maintain their directional-related activity. Further physiological studies will be required to establish the role of vestibular system in producing HDC responses, and anatomical studies are needed to determine the neural circuitry that mediates vestibular influences on determination of head direction.     

Effect of nigrostriatal damage induced by 6-hydroxydopamine on the expression of glial cell line–derived neurotrophic factor in the striatum of the rat

Several types of brain injuries have been associated with alterations in the striatal expression of neurotrophic factors, including glial cell line–derived neurotrophic factor (GDNF). However, contradictory results on the striatal expression of GDNF have been reported in different animal models of Parkinson's disease. For this reason, we examined the effect of nigrostriatal damage on the mRNA and protein expression levels of GDNF in the striatum as a function of time following a striatal or medial forebrain bundle 6-hydroxydopamine lesion. At different time points after the administration of 6-hydroxydopamine, striatal expression levels of GDNF were analyzed with semi-quantitative Western blotting. No significant changes in GDNF expression levels were observed within the 35-day observation period, either between the denervated and the intact striatum of medial forebrain bundle and striatally lesioned rats or between the striata of lesioned animals and those of control animals. In order to reinforce these results, striata of lesioned rats, sacrificed 18 days after lesioning, were analyzed with enzyme-linked immunosorbent assay and real-time polymerase chain reaction. At this time point, both techniques confirmed the results of the Western blot analysis, detecting no changes in striatal expression of GDNF, either at the protein level, or at the mRNA level. These data show that nigrostriatal damage induced by 6-hydroxydopamine has no effect on the striatal expression of GDNF.     

Quantitative determination of cell surface β-adrenoceptors in different rat skeletal muscles

In the present study, the density of cell surface beta-adrenergic receptors was determined in different skeletal muscles using the hydrophilic ligand [3H]CGP 12177. The density of beta-adrenergic receptors was highest in the slow-twitch soleus muscle (32.8+/-0.9 fmol mg dw(-1)) and lowest in the fast-twitch glycolytic white gastrocnemius (10.4+/-0.5 fmol mg dw(-1)) beta-Adrenoceptor density correlated closely with the percentage of type-I fibres (r=0.979; P<0.0001) and inversely with the percentage of type-IIB fibres (r=696; P<0.03). Incubation with isoprenaline (10 microM) for 30 min decreased the density of beta-adrenergic receptors in the cell surface from 32.9+/-0.8 to 19.3+/-0.7 fmol mg dw(-1) in the soleus and from 16.8+/-1.0 to 12.0+/-0.7 fmol mg dw(-1) in the epitrochlearis. Internalisation appeared rapid (half-time less than 5 min). To study externalisation of beta-adrenergic receptors, soleus strips were incubated 30 min with 10 microM isoprenaline and then transferred to buffer without agonist. The first incubation reduced the density to approximately 50%, the subsequent incubation without agonist increased cell surface receptor density to approximately 80% of the initial density after 1 h. No further increase was observed over the next 2 h, suggesting that some of the receptors had been degraded. Insulin or contractile activity did not influence rate of externalisation.     

Glial cell line-derived neurotrophic factor in Purkinje cells of adult zebrafish: An autocrine mode of action?

Glial cell line-derived neurotrophic factor (GDNF) has a neuroprotective role in Purkinje cells of cerebellum, promoting the survival and the differentiation of these cells. Its signalling is mediated by a receptorial complex GFRalpha1/RET. In the brain of adult zebrafish (Danio rerio) we previously investigated GDNF expression and localization, but no data exist regarding GFRalpha1 and RET presence. Thus, the present study was designed to clarify the morphological relation between GDNF and its receptorial complex GFRalpha1/RET immunoreactivity in the cerebellum of adult zebrafish. The expression of gdnf, GFRalpha1 and ret genes was demonstrated in adult zebrafish cerebellum by a standard RT-PCR. The distribution of GDNF and its receptorial complex GFRalpha1/RET was examined by single and double immunocytochemical stainings. In the valvula and corpus cerebelli GDNF, GFRalpha1 and RET immunoreactivity was seen co-localized in Purkinje cells, identified morphologically and by using an antiserum against a specific marker for these cells, aldolase C enzyme. In the vestibulolateralis lobe, Purkinje neurons were lacking in both the eminentiae granulares and medial caudal lobe. These results demonstrated the expression of the GDNF receptorial complex in adult zebrafish cerebellum and suggest an autocrine mode of action of GDNF in Purkinje cells.     

β-Galactofuranose-containing structures present in the cell wall of the saprophytic fungus Cladosporium (Hormoconis) resinae

A peptidogalactomannan was isolated from mycelia of Cladosporium (Hormoconis) resinae and characterized using methylation–fragmentation analysis, partial acid hydrolysis and ¹H and ¹³C-NMR spectroscopy. The galactomannan component was a branched structure and consisted of a main chain containing (1→6)-linked α-d-Manp residues substituted at O-2 by side chains containing (1→2)-linked α-d-Manp residues. β-d-Galf residues were present as side chains of 3–4 units that are (1→5)-interlinked. This structure is very similar to a pGM isolated from Aspergillus fumigatus and differs from that of Cladosporium werneckii (currently named Hortaea werneckii), with this pGM and other fungal galactomannans having single terminal (1→6)-linked β-Galf residues. The importance of the carbohydrate moiety of Cladosporium resinae pGM in immunoassays was also demonstrated. On FACS examination, a decrease (60%) in rabbit serum anti- C. resinae binding to C. resinae conidia occurred when this serum had been previously incubated with pGMs from C. resinae and A. fumigatus or with mannoprotein from Candida parapsilosis, suggesting the presence of cross-reactive determinants in these fungi.     

Effects of transient global ischemia on the neurons in the marginal division of the rat striatum

<正> Transient global ischemia frequently occurs following cardiac arrest and causes ischemic brain injury and vascular dementia.It wassuppised that the injury of neurons in the hippocampus formation sponsors the cognitive defictis.But recently this view has been challenged bya number of experiments.A memory process,objective recognition,found to be largely intact following selective hippocampal formation lesionsboth in monkeys and rats.Some of the extra-hippocampal damages are involved in the process of learning and memory.The marginal division(MrD)is a new subdivision in the striatum firstly discovered by Si Yun Shu in 1988.It is consisted of a band of fusiform neurons at the caudalmargin of the striatum.Depression of learning and memory occurred following injury of MrD.Global cerebral ischemia models were conducted by4-vessel occlusion.Ischemia induced hydrocephalus in hippocapus,cortex and striatum almost at same level.MrD was the injured by ischemia.Y-maze test demonstrated distinct and stable impairmen     

β1,4-galactosyltransferase-I in synovial tissue of collagen-induced rat model of rheumatoid arthritis

β1,4-galactosyltransferase-I (β1,4-GalT-I), which is one of the best-studied glycosyltransferases, plays a key role in the synthesis of selectin ligands such as sialyl Lewis (sLe^x) and sulfated sLe^x. Previous studies showed that inflammatory responses of β1,4-GalT-I-deficient mice were impaired because of the defect in selectin-ligand biosynthesis. However, the expression of β1,4-GalT-I and its biological function in rheumatoid arthritis (RA) remain to be elucidated. The mRNA and protein expression of β1,4-GalT-I increased in synovial tissue of the RA group compared with the Normal group at 10d and 15d after collagen-induced. Double staining indicated β1,4-GalT-I overlapped with macrophage-like synoviocytes (MLSs), fibroblast-like synoviocytes (FLSs), neutrophils and tumor necrosis factor-α (TNF-α). Moreover, β1,4-GalT-I mRNA in FLSs in vitro was affected in a dose- and time-dependent manner in response to TNF-α stimulation. ELISA revealed that expression of TNF-α was attenuated in FLSs in vitro treated with β1,4-GalT-I-Ab. We therefore suggest that β1,4-GalT-I may play an important role in the inflammation process of RA synovial tissue, which would provide the foundation for further researching into the concrete mechanism of β1,4-GalT-I in RA.     

Optimization of Multiplate® whole blood platelet aggregometry in the Beagle dog and Wistar rat for ex vivo drug toxicity testing

This study was performed to optimize and standardize the use of the Multiplate^® whole blood impedance aggregometer in the Beagle dog and Wistar rat for use in a research laboratory environment. The anticoagulants citrate, heparin and hirudin were compared and platelet aggregation responses to ADP, collagen, arachidonic acid and Par-4 agonist were evaluated to determine their half maximal effective concentrations (EC₅₀) in blood containing low concentrations of a drug solvent (0.1% DMSO). The results indicate that citrate anticoagulation is not suitable for Multiplate^® whole blood aggregometry because of the presence of spontaneous aggregation. ADP and collagen were found to be appropriate agonists for both species, whereas in the Beagle dog Par-4 agonist failed to induce aggregation and arachidonic acid induced platelet aggregation showed a high interindividual variability. The agonists EC₅₀ calculated in hirudin blood were 2.70 μM ADP, 0.85 μg/ml collagen, 0.03 mM arachidonic acid and 165.7 μM Par-4 agonist in the Wistar rat, and 0.95 μM ADP and 0.23 μg/ml collagen in the Beagle dog.     

Inhibition of rat pleural mesothelial cell nitric oxide synthesis by transforming growth factor-β1

Pleuritis is a common initial clinical manifestation of tuberculosis. It is associated with an accumulation of a variety of cytokines in the pleura and pleural fluid. We have recently shown that these proinflammatory cytokines induce the pleural mesothelial cell to produce large amounts of nitric oxide, a nitrogen intermediate that has been shown to have a tuberculocidal effect. TGF- has also been found in situ in tuberculous effusions and pleural tissues and is thought to suppress the immune response and promote tissue repair. This study examined the effects of TGF- on cytokine-induced NO synthesis by rat pleural mesothelial cells in vitro. Results demonstrated that TGF- significantly inhibited NO synthesis and that this inhibition was associated with a proportionate decrease in iNOS mRNA and iNOS protein. Suppression of pleural mesothelial cell NO synthesis by TGF- may be important in the pathogenesis of tuberculous pleuritis.