Thioperamide delays vestibular compensation in goldfish

Unilateral lesion of the vestibular system induces posturo-locomotor deficits that are compensated for with time. Drug therapy is currently used to improve the recovery process and to facilitate vestibular compensation. We investigated the effects of thioperamide on functional recovery after unilateral labyrinthectomy in Carassius auratus. Approximately 24h after surgery, the animals were injected intraperitoneally with thioperamide (15 mg/kg) and saline (1.5 ml/kg). The injections were repeated daily for a total of 15 consecutive days. The substances were administered in a volume of 1.5 ml/kg body weight. Another group, which served as a non-lesion control, did not receive unilateral labyrinthectomy or system injections. Animals treated with saline presented a compensatory decrease in body tilt on the 7th day, while the animals treated with thioperamide presented a decrease in body tilt from the 13th day, suggesting a delay in the functional recovery process. These results suggest that an increase in cerebral histamine levels impairs vestibular compensation in goldfish.     

Neuronal gap junctions play a role in the secondary neuronal death following controlled cortical impact

Autosomal dominant cerebellar ataxias (ADCAs), genetically classified into spinocerebellar ataxias (SCAs), are a highly heterogeneous group of neurodegenerative disorders. Recently, mutations in the fibroblast growth factor 14 gene (FGF14) have been reported to cause SCA27 subtype. To evaluate the frequency of FGF14 mutations in mainland of China, we performed molecular genetic analysis in 67 unrelated familial ataxia cases and 500 normal controls by denaturing high-performance liquid chromatography (DHPLC) and DNA direct sequencing. Interestingly, we found a pair of siblings carried the same heterozygous variation (c.-10delC) characterized by different clinical features, which is probably a novel insertion/deletion (I/D) polymorphism in the 5'UTR region of the exon 1b. It suggests that SCA27 is a rare subtype in China.     

Cellular basis of vestibular compensation: analysis and modelling of the role of the commissural inhibitory system

In this study we used a cellular network model of the brainstem vestibulo-ocular reflex (VOR) pathways to investigate the role of the vestibular commissural system in "vestibular compensation", the behavioural recovery that takes place after unilateral labyrinthectomy (UL). The network was initialized on the basis of mathematical analysis and trial simulations to generate a VOR response with a physiologically realistic gain and time constant. The effects of a selective decrease in the strength of commissural inhibitory input to the ipsi-lesional medial vestibular nucleus (MVN) neurones, without changes in other parts of the network, were investigated. Thus we simulated the marked down-regulation of GABA receptor efficacy that our recent experimental results have demonstrated in these cells after UL. The main outcome of this study is the delineation, for the first time, of a specific region of parameter space within which an adaptive change in commissural inhibitory gain is appropriate and sufficient to bring about a re-balancing of bilateral vestibular nucleus activity after UL. For this to be achieved, the relative contribution of the intrinsic, pacemaker-like membrane properties of the ipsi-lesional MVN cells must be equal to or greater than the synaptic input from the primary vestibular afferents in determining the in vivo resting discharge rate of these cells. Recent experimental evidence supports the view that the intrinsic properties of the MVN cells do contribute substantially to their resting discharge in vivo. Previous modelling studies that have excluded a role for the commissural system in vestibular compensation have arrived at this conclusion, because their models operated outside this region of parameter space. A second finding of this study is that, in a network that compensates through a selective change in commissural gain, the time constant of the VOR response is significantly reduced, mimicking the loss of velocity storage after UL in vivo. By contrast, the time constant is unchanged in a network that compensates through changes involving other non-vestibular inputs. These findings indicate that adaptive changes in commissural gain, through the dynamic regulation of GABA receptor efficacy in the vestibular nucleus neurones, may play an important role in vestibular plasticity. Electronic Publication     

Dynamics of gap junctions between horizontal cells in the goldfish retina

Summary Experimental alterations of gap junctions between outer horizontal cells have been demonstrated in freeze-fracture replicas of goldfish retina. The alterations consisted predominantly of an increase of connexon densities and a decrease in the variability of the arrangement of connexons. They were observed i. in dark adapted retinae, ii. in animals with crushed optic nerves, iii. in picrotoxin-and bicuculline-treated animals. Since experiment i. is characterized by a depolarization of the horizontal cell, and experiment iii. was shown by others to result in uncoupling of horizontal cells, we conclude that the functional connectivity of horizontal cells might be correlated with the structure of gap junctions. An interesting detail is the differentiated reaction of axonal and perikaryal gap junctions on dark adaptation or blindness: whereas normally the axonal gap junctions are less densely packed, they increase their connexon density in darkness or blindness much more than the perikaryal gap junctions.     

Functional gap junctions in thymic epithelial cells are formed by connexin 43

A multiparametric study was carried out to investigate the presence and possible role of communicating junctions in the thymus, particularly in the thymic epithelium, the major component of the thymic microenvironment. The presence of direct cell-cell communication mediated by gap junctions was demonstrated in human and murine thymic epithelial cells (TEC) by means of in situ and in vitro immunohistochemical labeling as well as in vitro fluorochrome injection and double whole-cell patch clamp experiments. Moreover, both immuno- and Northern blot studies revealed that the gap junction protein connexin 43 and its mRNA were present in TEC. Importantly, we showed that thymic endocrine activity, as ascertained by thymulin production, could be specifically downmodulated in vitro by a gap junction inhibitor, octanol. We also investigated the existence of gap junctions between TEC and thymocytes. In thymic nurse cells we were able to detect cell-cell communication, although only a minor percentage of epithelial/thymocyte pairs were coupled in a given moment. In contrast, intercellular communication was not detected between cultured phagocytic cells of the thymic reticulum and the respective rosetting thymocytes. We suggest that gap junctions formed by connexin 43 may represent a novel (and rather cell type-specific) pathway for intrathymic cellular communication, including TEC/TEC as well as possible TEC/thymocyte interactions.     

Active response of a one-dimensional cardiac model with gap junctions to extracellular stimulation

To study the response of cardiac tissue to electrical stimulation, a one-dimensional model of cardiac tissue has been developed using linear core-conductor theory and the DiFrancesco-Noble model of Purkinje tissue. The cable lies in a restricted extracellular medium and includes a representation of the junctional resistances known to interconnect cardiac cells. Two electrode geometries are considered: (a) a semi-infinite cable with a monopolar electrode at the end of the cable and (b) a terminated cable with one electrode at each end of the cable. In a series of simulations, stimuli of varying magnitude and polarity are applied at three different times during the plateau of the action potential. The results at the stimulus site show that the action potential duration may either decrease or increase in response to the stimulus, depending on the polarity and application time of the stimulus. The spatial behaviour of the cable in response to the stimulus indicates that sites greater than 200 cells from the stimulating electrode are not affected by the stimulus.     

Formation of gap junctions by stem cells in the developing retina of the clawed frog (Xenopus laevis)

Summary To ascertain whether gap junctions are preferentially formed by proliferative retinal stem cells, an analysis by electron microscopic autoradiography was made on the retina of the Xenopus embryo at stages 26, 29 and 31, after pulse labelling with ³H-thymidine. When the administration of the isotope was carried out for 0.5 or 3 h, retinal cells in S phase or S to M phase in the cell cycle were marked with the isotope, respectively. In these specimens, most gap junctions were found on the isotope-labelled cells, and few on the unlabelled cells. Several cells in mitosis also formed gap junctions. These results would suggest that gap junctions are formed by retinal stem cells in S to M phases of the cell cycle, but not by cells in G₁ phase and post-mitotic cells.     

Effects of intra-vestibular nucleus injection of the Group I metabotropic glutamate receptor antagonist AIDA on vestibular compensation in guinea pigs

Removal of the peripheral vestibular receptor cells in one inner ear (unilateral vestibular deafferentation, UVD) results in a syndrome of ocular motor and postural disorders, many of which disappear over time in a process of behavioural recovery known as vestibular compensation. Excitatory amino acid receptors, in particular the N-methyl-D-aspartate (NMDA) receptor, have been implicated in vestibular compensation; however, the metabotropic glutamate receptors (mGluRs) have not been studied in this context. The aim of this study was to determine whether group I mGluRs in the brainstem vestibular nucleus complex (VNC) ipsilateral to the UVD are involved in vestibular compensation of the static symptoms of UVD in guinea pig. The selective group I mGluR antagonist (RS)-1-aminoindan-1,5,dicarboxylic acid (AIDA) was continuously infused into the ipsilateral VNC for 30-min pre-UVD and 30-min post-UVD by cannula, at a rate of 1 microl/h, using one of four doses: 0.1 fg, 0.1 pg, 0.1 ng or 0.1 microg (n=5 animals in each case). In control conditions, a 0.1-fg (n=4) or 0.1-microg (n=5) NaOH vehicle was infused into the ipsilateral VNC using the same protocol. In order to control for the possibility that AIDA disrupted spontaneous neuronal activity in the VNC in normal animals, 0.1 microg AIDA (n=4) or 0.1 microg NaOH (n=2) was infused into the VNC in labyrinthine-intact animals. In both groups, static symptoms of UVD (i.e. spontaneous nystagmus, SN, yaw head tilt, YHT and roll head tilt, RHT) were measured at 8, 10, 12, 15, 20, 25, 30, 35, 45 and 50 h post-UVD. In addition, the righting reflex latency (RRL) was measured in labyrinthine-intact animals in order to assess whether AIDA impaired motor coordination in labyrinthine-intact animals. In UVD animals, the highest dose of AIDA significantly reduced SN frequency and changed its rate of compensation (P<0.001 and P<0.0001, respectively). This dose of AIDA also caused a significant reduction in YHT (P<0.005) as well as a significant change in its rate of compensation (P<0.0001). However, RHT was not significantly affected. In the labyrinthine-intact animals, AIDA infusion did not induce a UVD syndrome, nor did it significantly affect RRL. These results suggest that group I mGluRs in the ipsilateral VNC may be involved in the expression of ocular motor and some postural symptoms following UVD. Furthermore, group I mGluRs may not contribute to the resting activity of vestibular nucleus neurons.     

Pericyte-endothelial gap junctions in developing rat cerebral capillaries: A fine structural study

Background: Fine structural study revealed the intercellular coupling between the pericyte and the endothelial cells via the gap junctions, in the capillaries of the basal forebrain of rat embryos. Results: Gap junctions were constructed by the adluminal plasmalemma of pericyte and the abluminal plasmalemma of endothelial cells. Conclusions: Gap junctions are membranous channels that directly join the cytoplasms of the pericyte and endothelial cell and imply some substantial role for the pericyte on the endothelial proliferation. It is postulated that the function of the pericyte in the prenatal mammals are assigned to the regulation of the development of cerebral microcirculation. © 1995 Wiley-Liss, Inc.     

Visual sensory substitution in vestibular compensation: neuronal substrates in the alert cat

The purpose of this study was to investigate adaptive changes in the activity of vestibular nuclei neurons unilaterally deprived of their primary afferent inputs when influenced by visual motion cues. These neuronal changes might account for the established role that vision plays in the compensation for posturo-kinetic deficits after the loss of vestibular inputs. Neuronal recordings were made in alert, non-paralysed cats that had undergone unilateral vestibular nerve sections. The unit responses collected in both Deiters' nuclei were compared to those previously recorded in intact cats. We analysed the extracellular activity of Deiters' nucleus neurons, as well as the optokinetic reflex (OKR) evoked during sinusoidal translation of a whole-field optokinetic stimulus in the vertical plane. In intact cats, we found the unit firing rate closely correlated with the visual surround translation velocity, and the relationship between the discharge rate and the motion frequency was tuned around an optimal frequency. The maximum firing rate modulation was generally below the 0.25 Hz stimulus frequency; unit responses were weak or even absent above 0.25 Hz. From the 4th day to the end of the 3rd week after ipsilateral deafferentation, a majority of cells was found to display maximum discharge modulation during vertical visual stimulation at 0.50 Hz, and even at 0.75 Hz, indicating that the frequency bandwidth of the visually induced responses of deafferented vestibular nuclei neurons had been extended. Consequently, the frequency-dependent attenuation in the sensitivity of vestibular neurons to visual inputs was much less pronounced. After the first 3 weeks postlesion, the unit response characteristics were very similar to those observed prior to the deafferentation. On the nucleus contralateral to the neurectomy, the maximum modulation of most cells was tuned to the low frequencies of optokinetic stimulation, as also seen prior to the lesion. We found, however, a subgroup of cells displaying well-developed responses above 0.50 Hz. Under all experimental conditions, the neuronal response phase still remained closely correlated with the motion velocity of the vertical sinusoidal visual pattern. We hypothesize that Deiters' neurons deprived of their primary afferents may transiently acquire the ability to code fast head movements on the basis of visual messages, thus compensating, at least partially, for the loss of dynamic vestibular inputs during the early stages of the recovery process. Since the overall vertical OKR gain was not significantly altered within the 0.0125 Hz–1 Hz range of stimulation after the unilateral neurectomy, it can be postulated that the increased sensitivity of deafferented vestibular neurons to visual motion cues was accounted for by plasticity mechanisms operating within the deafferented Deiters' nucleus. The neuroplasticity mechanisms underlying this rapid and temporary increase in neuronal sensitivity are discussed.     

GABA<Subscript>A</Subscript> receptor subunit expression in the guinea pig vestibular nucleus complex during the development of vestibular compensation

The aim of this experiment was to investigate whether vestibular compensation following unilateral vestibular deafferentation (UVD) is associated with changes in the expression of GABA_A receptor subunits in the guinea pig vestibular nuclear complex (VNC) at 2, 10, and 30 h post-surgery. Using Western blotting, the ₁ and ₂ subunits (but not the ₂ subunit) were detected in the VNC of labyrinthine-intact animals. However, there were no significant differences in the protein expression of the ₁ and ₂ subunits within the ipsilateral or contralateral VNC at any time post-UVD compared to sham and anesthetic controls. Furthermore, UVD did not induce the expression of the ₂ protein. These results suggest that vestibular compensation in guinea pig, as in the rat, is not associated with changes in the protein levels of the GABA_A receptor subunits ₁, ₂, and ₂ in the VNC. However, a limitation of this study is that the Western blotting technique can detect only changes that are larger than 30% and therefore small changes cannot be excluded.     

The incidence and size of gap junctions between the bone cells in rat calvaria

Polyclonal antisera to synthetic peptides matching sequences on the cytoplasmic regions of connexin-43, a gap junction protein first identified in rat heart, have been used to immunolabel gap junctions in the calvarial bone, maintained intact as in vivo, of 1- to 2-week-old rats. The specimens were examined in reflection and fluorescence modes by scanning laser confocal microscopy, and the numbers of gap junctions and their sizes estimated. The mean number of connexin-43 immunolabelled junctions per osteoblast (n=65) was 15.3 (SD ± 4.5). The mean length of 227 junctions, selected for the sharpness of the image of the fluorescent spot, was 0.67 m (SD ± 0.18; range 0.37–1.29 m) and their mean area 0.26 m² (SD ± 0.145; range 0.075–0.93 m²); these probably fell within the upper half of the total size range. Gap junctions were detected between preosteoblasts, osteoblasts, osteocytes and chondrocytes, and between these juxtaposed cell types. In addition, connexin-43 immuno-labelled junctions were found between some osteoclasts and overlying mononuclear cells at active sites of resorption.     

Neuronal gap junctions play a role in the secondary neuronal death following controlled cortical impact

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. Recent studies in mice showed a critical role for neuronal gap junctions in NMDA receptor-mediated excitotoxicity and ischemia-mediated neuronal death. Here, using controlled cortical impact (CCI) in adult mice, as a model of TBI, and Fluoro-Jade B staining for analysis of neuronal death, we set to determine whether neuronal gap junctions play a role in the CCI-mediated secondary neuronal death. We report that 24 h post-CCI, substantial neuronal death is detected in a number of brain regions outside the injury core, including the striatum. The striatal neuronal death is reduced both in wild-type mice by systemic administration of mefloquine (a relatively selective blocker of neuronal gap junctions) and in knockout mice lacking connexin 36 (neuronal gap junction protein). It is also reduced by inactivation of group II metabotropic glutamate receptors (with LY341495) which, as reported previously, control the rapid increase in neuronal gap junction coupling following different types of neuronal injury. The results suggest that neuronal gap junctions play a critical role in the CCI-induced secondary neuronal death.     

Compensation of horizontal canal related activity in the medial vestibular nucleus following unilateral labyrinth ablation in the decerebrate gerbil

The spontaneous activity and dynamic responses to two frequencies (1.3 and 0.13 Hz) of sinusoidal angular horizontal head acceleration of type I neurons in the medial vestibular nucleus were recorded bilaterally in decerebrate Mongolian gerbils (Meriones unguiculatus) under three experimental conditions; normal labyrinth intact, acutely following unilateral labyrinthine lesion, and four to seven weeks following labyrinthine lesion. The mean spontaneous activity and number of detected type I neurons decreased immediately ipsilateral to the lesion but recovered significantly with time. In contrast, spontaneous activity on the contralateral side increased during compensation following hemilabyrinthectomy. The mean response gains at both frequencies of head oscillation were depressed bilaterally and asymmetrically acutely following the lesion such that the response gain of cells on the intact side exceeded that of the neurons recorded on the injured side. After compensation the number of detected type I neurons on the side ipsilateral to the injury increased but remained below normal levels. The mean gains remained depressed but became symmetric with compensation as a result of improvement in the response of ipsilateral neurons. The phase of responses were significantly advanced in the compensated animals. Although response gain is not fully restored, the linearity of the dynamic modulation in compensated animals is improved as evidenced by a continuous modulation of the increased spontaneous activity of neurons contralateral to the hemilabyrinthectomy. It is proposed that this effect is related to the concurrent improvement in the linearity of the horizontal vestibulo-ocular response. Electrical cathodal polarization of the vestibular nerve ipsilateral to the ablated labyrinth was utilized to investigate the relationship between recovery of spontaneous activity and dynamic function. Acutely following hemilabyrinthectomy, cathodal polarization restored activity in second-order type I neurons to near normal levels but their response gain to head rotation remained depressed. Similar galvanic stimulation in compensated animals also elevated ipsilateral spontaneous activity. As in the acute preparation, such stimulation did not modify the response gain or phase. Thus, the improvement in response of type I neurons in the compensated gerbil was not a direct consequence of restoration of spontaneous activity on the side of the injury.     

Compensation of horizontal canal related activity in the medial vestibular nucleus following unilateral labyrinth ablation in the decerebrate gerbil

Summary The spontaneous activity and dynamic responses to sinusoidal horizontal head angular acceleration of type II horizontal semicircular canal related neurons in the medial vestibular nucleus (MVN) were recorded bilaterally in decerebrate Mongolian gerbils (Meriones unguiculatus) under three experimental conditions: normal labyrinths intact, acutely following unilateral labyrinthine lesion, and four to seven weeks following labyrinthine lesion. The number of type II neurons detected contralateral to the lesion was greatly reduced both in the acutely hemilabyrinthectomized animals and following compensation. The gain of the responses was depressed bilaterally acutely following the lesion. A greater reduction in response gain was noted in cells contralateral to the lesion. The gain of the contralateral type II responses increased with time such that in the compensated animal bilaterally symmetric gains were recorded. While the significant changes which occur in the gain of type II neurons with recovery from peripheral vestibular lesions can largely be attributed to type I neurons on the other side of the midline, changes in type I neurons were not entirely reflected in the type II population. The spontaneous activity of type II neurons did not undergo any significant changes following the labyrinthine lesion. We present a model utilizing the dynamic responses to estimate the functional recovery of commissural connections in compensated animals. The overall gain of the contralateral type I to ipsilateral type I commissural polysynaptic pathway appears to improve, while the efficacy in the reverse direction remains depressed, suggesting that modifications in commissural connections, particularly involving the type II to type I connections within the MVN on the injured side, mediate aspects of behavioral recovery.     

Evidence for glutamate as a neurotransmitter in the cat vestibular nerve: radioautographic and biochemical studies

Summary Evidence that glutamate acts as a neuro-transmitter in vestibular nerve fibers was sought (1) by electron microscope radioautographic identification of the uptake sites of [³H]-glutamic acid after incubation of slices of cat vestibular nuclei, and (2) by measuring changes in sodium-dependent high affinity glutamate uptake in nerve endings containing homogenates from normal and deafferented vestibular nuclei 8 to 11 days after unilateral vestibular nerve lesion. Electron microscopic radioautography revealed that glutamate had been taken up by numerous nerve endings projecting over the whole vestibular nuclear complex. The biochemical approach indicated that after section of the vestibular nerve, a significant decrease in high affinity glutamate uptake occurred in the vestibular nuclei, which lost their exclusively ipsilateral projection. This decrease varied from one area of the deafferented vestibular nuclei to another, reaching –58% in the lateral area of the central part corresponding to the ventral lateral vestibular nucleus and the rostral part of the descending vestibular nucleus. It is concluded that glutamate (or aspartate) is used by the vestibular nerve fibers as a neurotransmitter in the vestibular nuclei.     

戊四唑致痫大鼠脑内缝隙连接的作用

目的: 研究缝隙连接(gap junction, GJ)在癫痫发病中的作用及机制。方法: 以戊四唑 (pentylene-tetrazol, PTZ)致痫大鼠模型为研究对象,采用免疫组织化学和实时定量RT-PCR技术,分别检测缝隙连接蛋白Cx32和Cx43在癫痫发作后不同时点皮层和海马神经元的表达。加用卡马西平(carbamazepine, CBZ)和甘珀酸(carbenoxolone, CBX)干预,观察二者对Cx32/43表达以及大鼠癫痫发作的影响。结果: 免疫组织化学染色显示PTZ致痫2 h后大鼠脑内Cx32/43阳性细胞开始增多,8 h后增多更为明显。实时定量RT-PCR示致痫2 h Cx32 mRNA迅速升高,5 h达高峰。Cx43 mRNA表达水平较低,但明显高于对照组。CBX显著抑制了Cx32/43的表达,CBZ对Cx32和Cx43的表达无明显影响。二者均抑制了大鼠的痫样发作。结论: GJ参与癫痫的发病过程,具有促进癫痫发作的作用。CBZ不影响Cx32/43的表达,表明其抗癫痫作用机制与阻断GJ 无关。     

The crucial amino acid residue related to inactivation of Vibrio vulnificus hemolysin

Vibrio vulnificus, an opportunistic human pathogen causing fetal septicemia, produces a 50-kDa pore-forming toxin as a virulence factor. This toxin consists of 451 amino acid residues; however, there are two types of this toxin on the basis of the difference of some amino acid residues, type 1 (Leu(281), Ser(415), Asn(435)/Asp(435), Asn(438)) and type 2 (Ile(281), Asn(415), Asn(435), Thr(438)). In the present study, two characteristic properties of type 2 toxin that was elaborated by V. vulnificus cells or synthesized by the in vitro system were compared to those of type 1 toxin. Type 2 toxin was found to be more resistant to spontaneous inactivation at 37 degrees C and to specific inactivation by cholesterol. On the other hand, a variant of type 2 toxin (Asp(435), Asn(438)) showed the same properties as type 1 toxin. The replacement of the 438th Asn to Thr (N438T), but not the 435th Asp to Asn (D435N), resulted in reversion of the variant type 2 toxin to typical type 2 toxin. These findings indicate that a single amino acid residue, Thr(438), may be critical for higher stability of type 2 toxin.