Dendritic spikes mediate negative synaptic gain control in cerebellar Purkinje cells

Dendritic spikes appear to be a ubiquitous feature of dendritic excitability. In cortical pyramidal neurons, dendritic spikes increase the efficacy of distal synapses, providing additional inward current to enhance axonal action potential (AP) output, thus increasing synaptic gain. In cerebellar Purkinje cells, dendritic spikes can trigger synaptic plasticity, but their influence on axonal output is not well understood. We have used simultaneous somatic and dendritic patch-clamp recordings to directly assess the impact of dendritic calcium spikes on axonal AP output of Purkinje cells. Dendritic spikes evoked by parallel fiber input triggered brief bursts of somatic APs, followed by pauses in spiking, which cancelled out the extra spikes in the burst. As a result, average output firing rates during trains of input remained independent of the input strength, thus flattening synaptic gain. We demonstrate that this "clamping" of AP output by the pause following dendritic spikes is due to activation of high conductance calcium-dependent potassium channels by dendritic spikes. Dendritic spikes in Purkinje cells, in contrast to pyramidal cells, thus have differential effects on temporally coded and rate coded information: increasing the impact of transient parallel fiber input, while depressing synaptic gain for sustained parallel fiber inputs.     

The relationship between birth timing,circuit wiring,and physiological response properties of cerebellar granule cells

Cerebellar granule cells (GrCs) are usually regarded as a uniform cell type that collectively expands the coding space of the cerebellum by integrating diverse combinations of mossy fiber inputs. Accordingly, stable molecularly or physiologically defined GrC subtypes within a single cerebellar region have not been reported. The only known cellular property that distinguishes otherwise homogeneous GrCs is the correspondence between GrC birth timing and the depth of the molecular layer to which their axons project. To determine the role birth timing plays in GrC wiring and function, we developed genetic strategies to access early- and late-born GrCs. We initiated retrograde monosynaptic rabies virus tracing from control (birth timing unrestricted), early-born, and late-born GrCs, revealing the different patterns of mossy fiber input to GrCs in vermis lobule 6 and simplex, as well as to early- and late-born GrCs of vermis lobule 6: sensory and motor nuclei provide more input to early-born GrCs, while basal pontine and cerebellar nuclei provide more input to late-born GrCs. In vivo multidepth two-photon Ca²⁺ imaging of axons of early- and late-born GrCs revealed representations of diverse task variables and stimuli by both populations, with modest differences in the proportions encoding movement, reward anticipation, and reward consumption. Our results suggest neither organized parallel processing nor completely random organization of mossy fiber→GrC circuitry but instead a moderate influence of birth timing on GrC wiring and encoding. Our imaging data also provide evidence that GrCs can represent generalized responses to aversive stimuli, in addition to recently described reward representations.

Cerebellar granule cells (GrCs) comprise the majority of neurons in the mammalian brain (1, 2). Each GrC receives only four excitatory inputs from mossy fibers, which originate in a variety of brainstem nuclei and the spinal cord, and the vast number of GrCs permits diverse combinations of mossy fiber inputs. Classical theories of cerebellar function have therefore proposed that GrCs integrate diverse, multimodal mossy fiber inputs and thus collectively expand coding space in the cerebellum (3–5). Until recently, studies have focused on the role of GrCs in implementing sparse coding of sensorimotor variables and stimuli (6–9). However, recent physiological studies of GrCs in awake, behaving animals highlight GrC encoding of cognitive signals in addition to sensorimotor signals (10–13). GrCs have also been recently shown to encode denser representations than expected by classical theory (10–12, 14–18), including a lack of dimensionality expansion under certain conditions (18).Despite the vast number of GrCs, stable molecularly or physiologically defined GrC subtypes within a single cerebellar region or lobule have not been described (19–22), although variation in gene expression across different regions has been reported (22, 23). The only known axis along which spatially intermingled GrCs can be distinguished from each other is the depth of the molecular layer to which their parallel fiber axons (PFs) project, which is dictated by GrC lineage and birth timing (24, 25). Birth timing predicts the wiring and functional properties of diverse neuron types in many neural systems (26), including the neocortex (27, 28), other forebrain regions (29, 30), olfactory bulb (31–33), and ventral spinal cord (34, 35). Furthermore, classic studies utilizing γ-irradiation at different times during rat postnatal development to ablate different cerebellar GrC and interneuron populations suggested that GrCs born at different times could contribute differentially to motor vs. action coordination (36). These observations also led to an as-of-yet untested hypothesis that mossy fibers arriving at different times during development could connect with different GrC populations. Could GrC birth timing be an organizing principle for information processing in the cerebellum?Recent evidence and modeling point to the possibility of spatial clusters of coactivated PFs (15, 37), suggesting that GrCs born around the same time may disproportionally receive coactive mossy fiber inputs. However, another study using different methods and stimuli did not find differences in the physiological responses of early- and late-born GrCs to various sensorimotor stimuli (38). Here, we address the role of birth timing in GrC wiring and function. We developed strategies to gain genetic access to early- and late-born GrCs, as well as control GrCs not restricted by birth timing. We report the first monosynaptic input tracing to GrCs, finding differential mossy fiber inputs to GrCs in vermis lobule 6 and simplex, as well as different patterns of input to early- and late-born GrCs in vermis lobule 6. Finally, we performed in vivo multidepth two-photon Ca²⁺ imaging of PFs of early- and late-born GrCs during an operant task and presentation of a panel of sensory, appetitive, and aversive stimuli. We found modest differences in the proportions of early- and late-born GrCs encoding of a subset of movement and reward parameters. Together, these results reveal a contribution of GrC birth timing to their input wiring and diverse encoding properties.     

A stable proportion of Purkinje cell inputs from parallel fibers are silent during cerebellar maturation

Cerebellar Purkinje neurons integrate information transmitted at excitatory synapses formed by granule cells. Although these synapses are considered essential sites for learning, most of them appear not to transmit any detectable electrical information and have been defined as silent. It has been proposed that silent synapses are required to maximize information storage capacity and ensure its reliability, and hence to optimize cerebellar operation. Such optimization is expected to occur once the cerebellar circuitry is in place, during its maturation and the natural and steady improvement of animal agility. We therefore investigated whether the proportion of silent synapses varies over this period, from the third to the sixth postnatal week in mice. Selective expression of a calcium indicator in granule cells enabled quantitative mapping of presynaptic activity, while postsynaptic responses were recorded by patch clamp in acute slices. Through this approach and the assessment of two anatomical features (the distance that separates adjacent planar Purkinje dendritic trees and the synapse density), we determined the average excitatory postsynaptic potential per synapse. Its value was four to eight times smaller than responses from paired recorded detectable connections, consistent with over 70% of synapses being silent. These figures remained remarkably stable across maturation stages. According to the proposed role for silent synapses, our results suggest that information storage capacity and reliability are optimized early during cerebellar maturation. Alternatively, silent synapses may have roles other than adjusting the information storage capacity and reliability.

Typical central excitatory synapses are formed onto dendritic spines, the distinctive morphology of which enables their unambiguous identification (1–3). It has generally been assumed that the presence of a dendritic spine equates to the existence of a functional excitatory transmission (4). Based on this assumption, the observation of spine motility in several brain areas (motor cortex and somatosensory cortex) has been considered to reflect synaptic plasticity (5). Indeed, a number of studies have established a correlation between learning and spine formation (6–8) or pruning (9, 10). However, in some conditions, morphological and synaptic plasticity have been shown to be dissociated (11), in line with the view that morphology does not provide all the information necessary to infer synaptic function.The cerebellum contains the majority of brain neurons (12, 13) and the predominant excitatory synapses found in this structure connect granule cells (GC) to Purkinje cells (PC). These synapses are formed on typical spines borne by PC dendrites (14), the majestic shape of which is likely related to the huge amount of independent inputs they receive. The GC-to-PC synapse is generally acknowledged to be an essential site for plasticity (15–19). However, in sharp contrast to other parts of the brain such as motor and somatosensory cortices, PC spines appear to be constitutive (20, 21), i.e., they appear to be an inherent property of PCs, independent of external factors. Indeed, pruning of these synapses has not been reported. Novel spine formation has been reported, but likely as a result of dendritic tree expansion (22, 23). The high density of spines along PC dendritic branchlets (5 to 17 per linear micrometer in rat; refs. 14 and 24–27) and their regular ordering in a helical pattern (28) support the idea that they optimize space occupancy with little room for spine addition, in accordance with their constitutive nature.The apparent morphological homogeneity of PC spines is in sharp contrast with the spectacular heterogeneity observed in the strength of GC-to-PC synapses. An in vivo study has reported that the receptive field of a PC was much smaller than that of the GCs putatively connecting to it (29), suggesting that most GC-to-PC synapses are electrically silent. This has been confirmed by an in vitro report (30) showing that synaptic transmission between paired-recorded GC and PC was detected nearly 10 times less frequently than expected from the occurrence of morphologically defined synaptic connections predicted by anatomical data (14, 31–33). Taken together, these two studies conducted in adult rats suggest that most (85% according to ref. 30) morphologically and molecularly defined GC-to-PC synapses are silent, i.e., they do not transmit any detectable electrical signal.If silent synapses do not transmit information, what is their role? Are they a reserve for additional information storage? Or do they result from information storage optimization (34)? According to this latter proposal (17, 34), since the requirement for optimized information storage is more and more critical as the amount of learned information increases, one might expect that the proportion of silent synapses increases with the amount learning. As previously suggested (34), this hypothesis could be tested by comparing the proportion of silent synapses in young versus adult animals. Indeed, the mouse cerebellar circuitry is not fully in place until the third postnatal week. Then, for at least 3 wk, the mouse acquires basic skills (eating, walking, and social interactions), adapts to changes in muscle strengths and sensitivity to stimuli, and improves its agility (35). Although the amount of cerebellar learning occurring over this maturation period is unclear, it can be reasonably assumed that cerebellar operation continuously optimizes. Here, we investigate how the proportion of silent synapses changes over this period of maturation.We determine the proportion of silent GC-to-PC synapses by a method based on the determination of the average postsynaptic response per activated synapse (average synaptic weight, w¯) in superfused acute slices. Thanks to the geometrical and repetitive architecture of the cerebellar cortex, calcium imaging is used to quantitatively map GC inputs. This mapping, combined with postsynaptic recording of transmission, and the determination of two cerebellar anatomical features (the average synapse density and spacing between PC planar dendritic trees) enables the determination of w¯. By comparison with the properties of synapses that produce an electrical postsynaptic response (investigated by paired recording and quantal analysis), we show that the proportion of silent synapses is higher than 70% and stable between the postnatal stages of interest. This suggests that cerebellar maturation has insignificant impact on the proportion of silent synapses.     

Melatonin inhibits nicotinic currents in cultured rat cerebellar granule neurons

There is limited data regarding the effects of melatonin on the activity of neuronal acetylcholine receptors (nAChRs) themselves. This study analyzes the effects of low concentrations of melatonin on nicotine-evoked currents from cerebellar granule neurons (CGNs) in culture. Using electrophysiological and Ca(2+)-imaging techniques, it was found a subset of rat CGNs to which nicotine application elicited both intracellular Ca(2+) transients and inward whole-cell currents. These responses were mediated by heteromeric nAChRs, as assessed by their sensitivity to nicotine and time constant of current decay. Preincubating the cells with low melatonin concentrations (down to 1 pm) significantly reduced the current amplitude in a dose-dependent manner, without affecting the receptor's apparent affinity and voltage-dependency, nor the current's rise and decay time course. The inhibitory effect of melatonin was significantly reduced by luzindole, a competitive antagonist of both MT(1) and MT(2) melatonin receptors. In conclusion, melatonin inhibits nicotinic currents through non-alpha7 heteromeric nAChRs expressed by CGNs in culture, an effect that appears to be at least partially mediated by melatonin membrane receptors. Direct modulation of nicotinic receptors is accomplished at doses that are likely to be physiologically relevant, thus providing a mechanism through which melatonin circadian rhythmic levels could modulate cholinergic activity.     

Learning-related long-term potentiation of inhibitory synapses in the cerebellar cortex

Despite the widespread distribution of inhibitory synapses throughout the central nervous system, plasticity of inhibitory synapses related to associative learning has never been reported. In the cerebellum, the neural correlate of fear memory is provided by a long-term potentiation (LTP) of the excitatory synapse between the parallel fibers (PFs) and the Purkinje cell (PC). In this article, we provide evidence that inhibitory synapses in the cerebellar cortex also are affected by fear conditioning. Whole-cell patch-clamp recordings of spontaneous and miniature GABAergic events onto the PC show that the frequency but not the amplitude of these events is significantly greater up to 24 h after the conditioning. Adequate levels of excitation and inhibition are required to maintain the temporal fidelity of a neuronal network. Such fidelity can be evaluated by determining the time window for multiple input coincidence detection. We found that, after fear learning, PCs are able to integrate excitatory inputs with greater probability within short delays, but the width of the whole window is unchanged. Therefore, excitatory LTP provides a more effective detection, and inhibitory potentiation serves to maintain the time resolution of the system.     

Conditioned eyeblink learning is formed and stored without cerebellar granule cell transmission

Classical conditioning of the eyeblink reflex is elicited by paired presentation of a conditioned stimulus and an unconditioned stimulus and represents a basic form of cerebellum-dependent motor learning. Purkinje cells and the deep nuclei receive convergent information of conditioned stimulus and unconditioned stimulus through the mossy fiber and climbing fiber projections, respectively. To explore the relative importance of these neural circuits and the underlying mechanism in associative eyeblink learning, we adopted a novel gene-manipulating technique, termed reversible neurotransmission blocking (RNB). In this technology, cerebellar granule cells specifically expressed neurotransmission-blocking tetanus toxin in a doxycycline (DOX)-dependent manner. Extracellular recording of Purkinje cells in awake RNB mice revealed that DOX treatment and withdrawal reversibly turned off and on simple spikes elicited by granule cell inputs, respectively, without interference with complex spikes evoked by climbing fiber inputs. Blockade of granule cell inputs to Purkinje cells abolished eyeblink conditioned responses (CRs) in a DOX-dependent manner. Importantly, when granule cell inputs recovered by removal of DOX, normal CRs were immediately produced in the DOX-treated, CR-negative RNB mice from the beginning of reconditioning. This learning process in RNB mice during DOX treatment was completely abolished by bilateral lesion of the interpositus nucleus before eyeblink conditioning. These results indicate that the convergent information at the interpositus nucleus is critical for acquisition and storage of learning in intimate association with the Purkinje cell circuit for expression of CRs in eyeblink conditioning.     

Melatonin receptor agonist 2-iodomelatonin prevents apoptosis of cerebellar granule neurons via K(+) current inhibition

Activation of K(+) current plays a critical role in the control of programmed cell death. In the present study, whole-cell patch-clamp recording, a caspase-3 activity assay, and flow cytometric analysis were used to examine the effects of the MT2 melatonin receptor agonist 2-iodomelatonin on the delayed-rectifier K(+) current (IK) and the prevention of apoptosis. It was found that apoptosis of cerebellar granular neurons induced by low-K(+) (5 mm) incubation was associated with an increase in IK amplitude and caspase-3 activity. After 6 hr of low-K(+) treatment, IK was increased by 45% (n = 86). Flow cytometry showed that the apoptosis rate increased by 333% compared with the control neurons. In addition, exposure of cultured granule cells to low K(+) also resulted in a significant activation of caspase-3, by 466%. 2-Iodomelatonin (10 microm in injection pipette) inhibited the IK amplitude recorded from control cells and from cells undergoing apoptosis. However, 2-iodomelatonin only modified the IK-channel activation kinetics of cells under both conditions. Furthermore, 2-iodomelatonin reduced the rate of apoptosis and caspase-3 activation, by 66 and 64%, respectively. The melatonin receptor antagonist, 4P-PDOT, abrogated the effect of 2-iodomelatonin on the IK augmentation, caspase-3 activity, and apoptosis. These results suggest that the neuroprotective effects of melatonin are not only because of its function as a powerful antioxidant, but also to its interactions with specific receptors. The effect of 2-iodomelatonin against apoptosis may be mediated by activating a melatonin receptor, which modulates IK channels and reduces K(+) efflux.     

2-iodomelatonin prevents apoptosis of cerebellar granule neurons via inhibition of A-type transient outward K+ currents

Compelling evidence indicates that excessive K+ efflux and intracellular K+ depletion are key early steps in apoptosis. Previously, we reported that apoptosis of cerebellar granular neurons induced by incubation under low K+ (5 mM) conditions was associated with an increase in delayed rectifier outward K+ current (IK) amplitude and caspase-3 activity. Moreover, the melatonin receptor antagonist 4P-PDOT abrogated the effects of 2-iodomelatonin on IK augmentation, caspase-3 activity and apoptosis. Here, we show that incubation under low K+/serum-free conditions for 6 hr led to a dramatic increase in the A-type transient outward K+ current (IA) (a 27% increase; n=31); in addition, fluorescence staining showed that under these conditions, cell viability decreased by 30% compared with the control. Treatment with 2-iodomelatonin inhibited the IA amplitude recorded from control and apoptotic cells in a concentration-dependent manner and modified the IA channel activation kinetics of cells under control conditions. Moreover, 2-iodomelatonin increased the viability of cell undergoing apoptosis. Interestingly, 4P-PDOT did not abrogate the effect of 2-iodomelatonin on IA augmentation under these conditions; in the presence of 4P-PDOT (100 microm), 2-iodomelatonin reduced the average IA by 41+/-4%, which was similar to the effect of 2-iodomelatonin alone. These results suggest that the neuroprotective effects of 2-idomelatonin are not only because of its antioxidant or receptor-activating properties, but rather that 2-iodomelatonin may inhibit IA channels by acting as a channel blocker.     

Dual regulation of NR2B and NR2C expression by NMDA receptor activation in mouse cerebellar granule cell cultures

In the developing cerebellum, switching of the subunit composition of NMDA receptors occurs in granule cells from NR2B-containing receptors to NR2C-containing ones. We investigated the mechanisms underlying switching of NR2B and NR2C subunit composition in primary cultures of mouse granule cells at the physiological KCl concentration (5 mM). Granule cells extensively extended their neuritic processes 48 h after having been cultured in serum-free medium containing 5 mM KCl. Consistent with this morphological change, NR2B mRNA and NR2C mRNA were down- and up-regulated, respectively, in the granule cells. This dual regulation of the two mRNAs was abrogated by blocking excitation of granule cells with TTX. This neuronal activity–dependent regulation of NR2B and NR2C mRNAs was abolished by the addition of selective antagonists of AMPA receptors and NMDA receptors. Furthermore, the dual regulation of NR2B and NR2C mRNAs in TTX-treated cells was restored by the addition of NMDA in the presence of the AMPA receptor antagonist, but not by that of AMPA in the presence of the NMDA receptor antagonist. Importantly, the NMDA receptor activation drove the NR2B/NR2C switching of NMDA receptors in the cell-surface membrane of granule cells. This investigation demonstrates that stimulation of NMDA receptors in conjunction with the AMPA receptor–mediated excitation of granule cells plays a key role in functional subunit switching of NMDA receptors in maturing granule cells at the physiological KCl concentration.     

大鼠小脑颗粒神经元体外原代培养

目的 体外原代培养大鼠小脑颗粒神经元,为研究慢性砷暴露对小脑神经元的毒性作用提供实验方法.方法 取生后5～7天Wistar仔鼠,体式显微镜下分离小脑皮层,0.25％胰蛋白酶消化、DNA Ⅰ酶洗涤制成单细胞悬液,两次差速贴壁后接种在多聚赖氨酸包被的培养板内,相差镜下观察大鼠小脑颗粒神经元成长、发育变化及突触形成.采用神经元特异性烯醇化酶(NSE)免疫荧光技术鉴定神经元.结果 培养后第24小时,相差显微镜下可见大鼠小脑颗粒神经元贴壁,呈网状排列;第2～3天,神经元胞体由椭圆形变成圆形,轮廓逐渐清晰,细胞伸出突起,突起逐渐延长,细胞间通过突起连接,形成了稀疏的神经元突触网络;第4～6天,细胞体积进一步增大,细胞间通过广泛的突触联系,神经元清晰饱满,形成了复杂的神经元网络.共聚焦显微镜下,可见大量含NSE的神经元.结论 成功地进行了大鼠小脑颗粒神经元的原代培养,该方法可为今后研究慢性砷暴露对小脑细胞的毒性作用提供实验依据.     

Dendrites of cerebellar granule cells correctly recognize their target axons for synaptogenesis in vitro

Neural circuits are generated by precisely ordered synaptic connections among neurons, and this process is thought to rely on the ability of neurons to recognize specific partners. However, it is also known that neurons promiscuously form synapses with nonspecific partners, in particular when cultured in vitro, causing controversies about neural recognition mechanisms. Here we reexamined whether neurons can or cannot select particular partners in vitro. In the cerebellum, granule cell (GC) dendrites form synaptic connections specifically with mossy fibers, but not with climbing fibers. We cocultured GC neurons with pontine or inferior olivary axons, the major sources for mossy and climbing fibers, respectively, as well as with hippocampal axons as a control. The GC neurons formed synapses with pontine axons predominantly at the distal ends of their dendrites, reproducing the characteristic morphology of their synapses observed in vivo, whereas they failed to do so when combined with other axons. In the latter case, synaptic proteins could accumulate between axons and dendrites, but these synapses were randomly distributed throughout the contact sites, and also their synaptic vesicle recycling was anomalous. These observations suggest that GC dendrites can select their authentic partners for synaptogenesis even in vitro, forming the synapses with a GC-specific nature only with them.     

NMDARs in granule cells contribute to parallel fiber–Purkinje cell synaptic plasticity and motor learning

Long-term synaptic plasticity is believed to be the cellular substrate of learning and memory. Synaptic plasticity rules are defined by the specific complement of receptors at the synapse and the associated downstream signaling mechanisms. In young rodents, at the cerebellar synapse between granule cells (GC) and Purkinje cells (PC), bidirectional plasticity is shaped by the balance between transcellular nitric oxide (NO) driven by presynaptic N-methyl-D-aspartate receptor (NMDAR) activation and postsynaptic calcium dynamics. However, the role and the location of NMDAR activation in these pathways is still debated in mature animals. Here, we show in adult rodents that NMDARs are present and functional in presynaptic terminals where their activation triggers NO signaling. In addition, we find that selective genetic deletion of presynaptic, but not postsynaptic, NMDARs prevents synaptic plasticity at parallel fiber-PC (PF-PC) synapses. Consistent with this finding, the selective deletion of GC NMDARs affects adaptation of the vestibulo-ocular reflex. Thus, NMDARs presynaptic to PCs are required for bidirectional synaptic plasticity and cerebellar motor learning.

The ability of an organism to adjust its behavior to environmental demands depends on its capacity to learn and execute coordinated movements. The cerebellum plays a central role in this process by optimizing motor programs through trial-and-error learning (1). Within the cerebellum, the synaptic output from granule cells (GCs) to Purkinje cells (PCs) shapes computational operations during basal motor function and serves as a substrate for motor learning (2). Several forms of motor learning depend on changes in the strength of the parallel fiber (PF), the axon of GCs, to the PC synapse (3, 4).In the mammalian forebrain, synaptic plasticity typically relies on postsynaptic N-methyl-D-aspartate receptor (NMDAR) activation, which alters AMPA receptor (AMPAR) turnover at the postsynaptic site (5). However, this may not extend to the cerebellar synapse between GCs and PCs, since no functional postsynaptic NMDARs have been identified in young or adult rodents (6, 7). Pharmacological approaches, however, have shown that both long-term depression (LTD) and long-term potentiation (LTP) induction depend on NMDAR activation at the PF-PC synapse in young rodents (8–12). Hence, the alternative mechanisms for NMDAR-dependent synaptic modulation may involve presynaptic NMDARs activation [(12–15); for review: refs. 16 and 17]. Indeed, cell-specific deletion of NMDARs in GCs abolishes LTP in young rodents (12). In addition to NMDARs, PF-PC synaptic plasticity also requires nitric-oxide (NO) signaling (18–20). As nitric-oxide synthase (NOS) is expressed in GCs, but not in PCs (21), the activation of presynaptic NMDARs might allow Ca²⁺ influx that activates NO synthesis, which in turn may act upon the PCs. However, in the mature cerebellum, the existence of presynaptic NMDARs on PFs and the role of NO in PF-PC plasticity remains a matter of debate. Previously, we have proposed that the activation of putatively presynaptic NMDARs in young rodents is necessary for inducing PF-PC synaptic plasticity without affecting transmitter release (8, 9, 11, 12). More recently, it has been shown that a subset of PFs express presynaptic NMDARs containing GluN2A subunits and that these receptors are functional (11, 12). Thus, in contrast to their role at other synapses, at least in young rodent, presynaptic NMDARs as part of the PF-PC synapses might act via the production of NO to induce postsynaptic plasticity, without altering neurotransmitter release (9, 11, 12, 18–22). However, a causal link between NMDARs activation in PFs, NO synthesis, and synaptic plasticity induction is still missing.In the cerebral cortex, the expression of presynaptic NMDARs is developmentally regulated (23, 24). However, little is known about the presence and function of presynaptic NMDARs in adult tissue. In the adult cerebellum, PCs only express postsynaptic NMDARs at their synapse with climbing fibers (CFs) (25). It has been proposed that the activation of these receptors could have heterosynaptic effects during PF-PC LTD. This mechanism would explain why LTD in adults depends on NMDARs. According to this model, presynaptic NMDARs would be a transient feature of developing tissue and not necessary for induction of synaptic plasticity and motor learning in adult animals (25).Here, we combine electron microscopy, two-photon calcium imaging, synaptic plasticity experiments, and behavioral measurements to show that presynaptic NMDARs are not developmentally regulated but are required for cerebellar motor learning in adults. We demonstrate that presynaptic NMDARs are present and functional in PFs of mature rodents. By specifically deleting the NMDAR subunit GluN1 either in the post- (PC) or the presynaptic cells (GCs), we demonstrate that NMDAR activation in GCs plays a key role in bidirectional synaptic plasticity and in vestibulo-ocular reflex (VOR) adaptation, an important paradigm for testing cerebellar motor learning (26–28). In contrast, NMDARs in PCs are neither involved in PF-PC synaptic plasticity nor required for cerebellar motor learning.     

CD95 (Fas) expression is regulated by sequestration in the Golgi complex in B-cell lymphoma

The CD95 (Fas) molecule transmits apoptotic signals important in B-cell development and the genesis of B-cell lymphoma. We have investigated the surface and intracellular expression of CD95 in Burkitt's lymphoma (BL) cells, an important non-Hodgkin's lymphoma of B-cell origin. Group I BL cells did not express CD95 at the cell surface, but contained high levels of this receptor in the cytoplasm. In contrast, group III BL cells expressed CD95 intracellularly and at the cell surface. In group I and group III BL cells, cytoplasmic CD95 was localized to the Golgi complex, as assessed by confocal immunofluorescence microscopy and subcellular fractionation followed by immunoblotting. Trafficking through the Golgi complex is regulated by elements within the target protein and cellular sorting mechanisms. CD95 contains candidate signals for interaction with trafficking machinery. Group I BL cells can be induced to upregulate surface expression of CD95 following CD40 ligation and certain group I BL cell lines drift invitro to a group III phenotype, with consequent surface expression of CD95. Taken together, these observations show that CD95 can either be retained in the Golgi complex or exported to the cell surface, and suggest that membrane trafficking has an important and previously unrecognized role in regulating CD95 expression in B lymphocytes.     

Dual personality of GABA/glycine-mediated depolarizations in immature spinal cord

The inhibitory action of glycine and GABA in adult neurons consists of both shunting incoming excitations and moving the membrane potential away from the action potential (AP) threshold. By contrast, in immature neurons, inhibitory postsynaptic potentials (IPSPs) are depolarizing; it is generally accepted that, despite their depolarizing action, these IPSPs are inhibitory because of the shunting action of the Cl(-) conductance increase. Here we investigated the integration of depolarizing IPSPs (dIPSPs) with excitatory inputs in the neonatal rodent spinal cord by means of both intracellular recordings from lumbar motoneurons and a simulation using the compartment model program "Neuron." We show that the ability of IPSPs to suppress suprathreshold excitatory events depends on E(Cl) and the location of inhibitory synapses. The depolarization outlasts the conductance changes and spreads electrotonically in the somatodendritic tree, whereas the shunting effect is restricted and local. As a consequence, dIPSPs facilitated AP generation by subthreshold excitatory events in the late phase of the response. The window of facilitation became wider as E(Cl) was more depolarized and started earlier as inhibitory synapses were moved away from the excitatory input. GAD65/67 immunohistochemistry demonstrated the existence of distal inhibitory synapses on motoneurons in the neonatal rodent spinal cord. This study demonstrates that small dIPSPs can either inhibit or facilitate excitatory inputs depending on timing and location. Our results raise the possibility that inhibitory synapses exert a facilitatory action on distant excitatory inputs and slight changes of E(Cl) may have important consequences for network processing.     

Growth inhibition of human gastrointestinal cancer cells by cyclosporin A

We have studied the ability of cyclosporin A (CsA) to inhibit the growth of human AGS gastric and HT29 colon carcinoma cells in vitro. Using continuous drug exposure in growth assays of cultured tumour cells we found that CsA produced a dose-dependent growth inhibition in gastric and colon cancer cells with a half-maximal effect at 5 M and 6 M CsA respectively. The growth inhibition of CsA was reversible in AGS cells, when the tumour cells were incubated in normal growth medium following CsA treatment. Trypan blue dye exclusion in AGS cells indicated a cytostatic rather than a cytotoxic effect in the concentration range used. Coincubation of CsA-treated cells with 10–400 U/ml interleukin-2 (IL-2) could not abrogate this growth inhibition, suggesting an IL-2 independent mechanism of action. Flow-cytometric analysis did not reveal a phase arrest of the gastric cancer cells within the cell cycle. We conclude from our experiments that CsA cytostatically and reversibly inhibits the growth of human gastric cancer cells in a dose-dependent manner. In contrast to its mechanism of action in lymphocytes, this direct antiproliferative effect of CsA seems not to be mediated by an IL-2-dependent pathway or a cell-cycle-phase arrest of the tumour cells.Abbreviations CsA cyclosporin A - IL-2 interleukin-2     

软肝冲剂对肝纤维化大鼠组织金属蛋白酶抑制剂的影响

目的：探讨软肝冲剂治疗肝纤维化和肝硬化的机制是否与抑制TIMP-1的表达有关。方法：用CCl4诱导大鼠的肝纤维化模型（病理分期为0-Ⅴ期），将大鼠随机分成两组，分别用软肝冲剂和生理盐水治疗2个月后。对肝组织TIMP-1mRNA的表达进行分析，并检测羟脯氨酸（Hyp）的量，同时检测血清中TIMP-1和TGF-β1的浓度。结果：软肝冲剂能够降低大鼠肝组织中TIMP-1mRNA的表达和胶原蛋白的含量，同时血清中TIMP-1和TGF-β1较CCl4诱导的模型组和生理盐水治疗组显著降低（P〈0．05）。结论：软肝冲剂治疗肝纤维化可能通过阻止TIMP-1的表达起作用。     

抗纤软肝颗粒对大鼠肝星状细胞增殖的影响

目的:探讨抗纤软肝颗粒(KXRG)对大鼠肝星状细胞(HSC)增殖的影响。方法:传代培养的HSC-T6(肝星状细胞系)与KXRG(1.25mg/ml、2.5mg/ml、5mg/ml)及其药物血清(5%、10%、20%)共同培养72小时后,应用MTT法测定细胞增殖、流式细胞仪测定HSC细胞周期各时相的DNA含量。结果:KXRG及药物血清均能显著抑制HSC的增殖,使细胞周期阻滞于S期,且2.5mg/ml药物和10%药物血清作用最强(P0.01)。结论:KXRG通过抑制HSC的活化,阻滞其细胞周期的正常进行,进而抑制HSC增殖,从而起到抗肝纤维化的作用。     

Degeneration of the Golgi and neuronal loss in dorsal root ganglia in diabetic BioBreeding/Worcester rats

Aims/hypothesis The aim of this study was to evaluate the nature and extent of neuronal loss in dorsal root ganglia (DRG) in diabetic polyneuropathy.Materials and methods We examined 10-month diabetic BioBreeding/Worcester (BB/Wor) rats with respect to DRG ultrastructure and morphometry, sural nerve morphometry, pro- and anti-apoptotic proteins, the expression of neurotrophic factors and their receptors, and sensory nerve functions.Results In diabetic rats, DRG neurons decreased to 73% of normal, owing to loss of substance P and calcitonin gene-related peptide-positive neurons. Levels of pro-apoptotic active caspase-3, Bax and low-affinity nerve growth factor (NGF) were increased in DRG. The concentration of anti-apoptotic heat shock protein (HSP) 70 in DRG was decreased, whereas concentrations of Bcl-xl and HSP27 were unaltered. Levels of poly(ADP-ribose) polymerase (PARP) and cleaved PARP were unaltered. Levels of NGF in sciatic nerve and concentrations of the high-affinity NGF receptor, insulin receptor and IGF-I receptor in DRG were significantly decreased. Sensory nerve conduction velocity decreased to 78% of normal. Hyperalgesia increased up to 6 months. Myelinated and unmyelinated fibre numbers of the sural nerve were significantly decreased in diabetic rats. DRG examinations revealed no evidence of apoptosis, mitochondrial changes or abnormalities of the endoplasmic reticulum. Instead, neurons demonstrated progressive vacuolar degenerative changes of the Golgi apparatus, with fragmentation and formation of large cytoplasmic vacuoles. These data show that sustained apoptotic stress is present in DRG of chronically diabetic BB/Wor rats, but fails to proceed to apoptotic cell death.Conclusions/interpretation Progressive DRG neuronal loss, particularly of small neurons, occurs in the type 1 diabetic BB/Wor rat. This is associated with neurotrophic withdrawal and progressive degeneration of the Golgi apparatus.Electronic supplementary material Supplementary material is available for this article at and is accessible to authorised users.