Transgenic ablation of rod photoreceptors alters the circadian phenotype of mice

The impact of photoreceptor loss on the circadian system was examined by utilizing a transgenic mouse model (rdta) in which rod photoreceptors were specifically ablated. These mice were able to phase-shift their circadian locomotor behaviour in response to light, but features of this circadian behaviour were markedly altered. The amplitude of circadian responses to light were approximately 2.5 greater, the circadian period (tau) was reduced (c. 20 min) and the total duration of activity (alpha) was increased (c. 50 min) when compared to wild type (+/+) and rd/rd mice (retinal degeneration, mice which also lack rod photoreceptors) of the same genetic background. The pattern of Fos expression in the suprachiasmatic nuclei (the site of the primary circadian clock in mammals) was indistinguishable between +/+ and rdta mice. However, Fos expression in the retina suggested that rod loss in rdta mice resulted in a functional reorganization of the retina and the constitutive activation of a population of retinal ganglion cells. Although it has been known for several years that the entraining photoreceptors of mammals are ocular, and that rod photoreceptors are not required for light regulation of the clock, these are the first data to show that features of the circadian phenotype (amplitude of the phase response curve, alpha, tau) can be influenced by photoreceptor ablation. These data support the hypothesis that the circadian phenotype of mammals is the product of an interaction between the suprachiasmatic nuclei and the retina. Thus, mammals which show an altered circadian behaviour can no longer be assumed to have defects associated only with specific clock genes; genes that affect photoreceptor survival may also modify circadian behaviour.     

Emergence of sustained spontaneous hyperactivity and temporary preservation of OFF responses in ganglion cells of the retinal degeneration (rd1) mouse

Complex alterations in the anatomy of outer retinal pathways accompany photoreceptor degeneration in the rd1 mouse model of retinitis pigmentosa, whereas inner retinal neurons appear relatively preserved. However, the progressive loss of photoreceptor input likely alters the neural circuitry of the inner retina. This study investigated resulting changes in the activity of surviving ganglion cells. Multielectrode recording monitored spontaneous and light-evoked extracellular action potentials simultaneously from 30 to 90 retinal ganglion cells of wild-type (wt) or rd1 mice. In rd1 mice, this activity evolves through three phases. First, normal spontaneous "waves" of correlated firing are seen at postnatal day 7 (P7) and last until shortly after eye opening. Second, at P14, full-field light flashes evoke reliable responses in many cells, with preferential preservation of off responses. These diminish as photoreceptor degeneration progresses. Third, once light-evoked responses have disappeared in early adulthood, surviving rd1 ganglion cells fire at a much higher spontaneous frequency than normal, sometimes in rhythmic bursts that are distinct from the developmental "waves." This hyperactivity is sustained well into adulthood, for weeks after photoreceptors have disappeared. Thus striking alterations occur in inner retinal physiology as retinal degeneration progresses in the rd1 mouse. Blindness occurs in the face of sustained hyperactivity among ganglion cells, which remain viable for months despite this activity. On and off responses are differentially affected in early stages of degeneration. While the source of these changes remains to be learned, such features should be considered in designing more effective treatments for these disorders.     

Characterization of an ocular photopigment capable of driving pupillary constriction in mice

This work demonstrates that transgenic mice lacking both rod and cone photoreceptors (rd/rd cl) retain a pupillary light reflex (PLR) that does not rely on local iris photoreceptors. These data, combined with previous reports that rodless and coneless mice show circadian and pineal responses to light, suggest that multiple non-image-forming light responses use non-rod, non-cone ocular photoreceptors in mice. An action spectrum for the PLR in rd/rd cl mice demonstrates that over the range 420-625 nm, this response is driven by a single opsin/vitamin A-based photopigment with peak sensitivity around 479 nm (opsin photopigment/OP479). These data represent the first functional characterization of a non-rod, non-cone photoreceptive system in the mammalian CNS.     

睫状神经营养因子对视网膜色素变性rd小鼠感光细胞作用的电镜观察

目的观察玻璃体腔注射睫状神经营养因子(ciliary neurotrophic factor,CNTF)对视网膜色素变性小鼠模型(retinal degeneration,rd)视网膜感光细胞的作用。方法将12只出生10d(P10)rd小鼠随机分成3组:CNTF治疗组、PBS(磷酸缓冲液)注射组、空白对照组;CNTF治疗组和PBS注射组的玻璃体腔分别注射鼠CNTF和磷酸缓冲液PBS,在出生后14d过量麻醉处死小鼠,取眼球行光、电镜观察。结果透射电镜结果显示凋亡是rd小鼠视网膜色素变性的主要病理表现,同时有炎症的参与,CNTF治疗组的rd小鼠视网膜光感受器细胞的超微结构好于对照组。结论CNTF玻璃体腔注射对视网膜色素变性rd小鼠视网膜感光细胞凋亡有抑制作用。     

Characterization of receptors for glutamate and GABA in retinal neurons

Glutamate and gamma-aminobutyric acid (GABA) are major excitatory and inhibitory neurotransmitters in the vertebrate retina, "a genuine neural center" (Ramón y Cajal, 1964, Recollections of My Life, C.E. Horne (Translater) MIT Press, Cambridge, MA). Photoreceptors, generating visual signals, and bipolar cells, mediating signal transfer from photoreceptors to ganglion cells, both release glutamate, which induces and/or changes the activity of the post-synaptic neurons (horizontal and bipolar cells for photoreceptors; amacrine and ganglion cells for bipolar cells). Horizontal and amacrine cells, which mediate lateral interaction in the outer and inner retina respectively, use GABA as a principal neurotransmitter. In recent years, glutamate receptors and GABA receptors in the retina have been extensively studied, using multi-disciplinary approaches. In this article some important advances in this field are reviewed, with special reference to retinal information processing. Photoreceptors possess metabotropic glutamate receptors and several subtypes of GABA receptors. Most horizontal cells express AMPA receptors, which may be predominantly assembled from flop slice variants. In addition, these cells also express GABAA and GABAC receptors. Signal transfer from photoreceptors to bipolar cells is rather complicated. Whereas AMPA/KA receptors mediate transmission for OFF type bipolar cells, several subtypes of glutamate receptors, both ionotropic and metabotropic, are involved in the generation of light responses of ON type bipolar cells. GABAA and GABAC receptors with distinct kinetics are differentially expressed on dendrites and axon terminals of both ON and OFF bipolar cells, mediating inhibition from horizontal cells and amacrine cells. Amacrine cells possess ionotropic glutamate receptors, whereas ganglion cells express both ionotropic and metabotropic glutamate receptors. GABAA receptors exist in amacrine and ganglion cells. Physiological data further suggest that GABAC receptors may be involved in the activity of these neurons. Moreover, responses of these retinal third order neurons are modulated by GABAB receptors, and in ganglion cells there exist several subtypes of GABAB receptors. A variety of glutamate receptor and GABA receptor subtypes found in the retina perform distinct functions, thus providing a wide range of neural integration and versatility of synaptic transmission. Perspectives in this research field are presented.     

Static,but not optokinetic visual stimuli induce Fos expression in the retina and brain of retinal degeneration mice

The stimulated expression of the c-fos gene was used in this study to characterize the residual vision of retinal degeneration (rd) mice. Conventional immunohistochemistry was employed to detect Fos-positive nuclei in the rd mouse retina and brain after monocular application of static and dynamic photic stimuli. Diffuse light induced Fos-positive nuclei in the inner nuclear and ganglion cell layers of the stimulated retina and in the contralateral pretectal olivary nucleus. On the other hand, Fos-positive nuclei were rarely detected in the rd mouse retina and brain after stimulation with a slowly displaced random-dot pattern. These results suggest that the residual vision of the rd mouse involves mainly the circuits that are related to the processing of static photic stimuli.     

rd1小鼠视网膜退变中期节细胞功能的变化

 目的:探讨视网膜色素变性模型rd1小鼠视网膜退变中期各类视网膜节细胞(retinal ganglion cells, RGCs)功能的变化情况。方法:运用多电极阵列(multi-electrode arrays,MEA)记录方法,记录视网膜退变中期(出生后20 d,P20)的rd1小鼠或正常对照小鼠视网膜中多个节细胞动作电位的发放,并比较自发发放和光反应特征等指标,评价幸存的节细胞功能变化。另外,采用免疫组化染色方法验证视网膜感光细胞的退化情况。结果:免疫组化的结果表明rd1小鼠视网膜感光层的厚度显著低于正常小鼠。根据节细胞光反应特性的不同,可以将其分成6类:ON sustained、ON transient、ON-OFF sustained、ON-OFF transient、OFF sustained和OFF transient RGCs,但OFF sustained RGCs所占比重极小(1.0%~3.1%)。rd1小鼠视网膜中保持光反应的节细胞比例显著低于正常小鼠。rd1小鼠节细胞的自发发放显著高于正常小鼠,而不同类型的节细胞变化情况有所不同。rd1小鼠视网膜各类节细胞的光反应强度及光敏感度均显著低于正常小鼠。结论:在rd1小鼠退变的中期,视网膜感光层明显退变;rd1小鼠退变中期的视网膜节细胞发生明显的功能退变,而且不同类型的节细胞变化情况有所不同。     

Neurotrophin receptors expression in the developing mouse retina: an immunohistochemical study

 Neurotrophins and their receptors (p75 and Trk family of receptors) play an important role in the survival of different populations of neurons in the central and peripheral nervous system. Expression of p75, TrkA, TrkB and TrkC was examined in mouse retinas by means immunohistochemistry in the postnatal development of normal and rd/rd mice (C57BL/6J). The rd/rd mice suffer a degeneration that causes a massive lost of photoreceptor cells. Results showed immunoreactivity to all three Trk proteins in both normal and rd/rd mice during the first 21 postnatal days, but some variations in intensity and localization were found. p75 immunoreaction was only present in rd/rd mice at the end of the degeneration process. These results could indicate a role of neurotrophins and their receptors in both the postnatal development of mouse retina and the degeneration process of rd/rd mice. Accepted: 30 October 1996     

Resting CD8 T cells recognize beta-galactosidase expressed in the immune-privileged retina and mediate autoimmune disease when activated

Although the expression of class II major histocompatibility complex (MHC) in retina is extremely low, it is an established fact that activated CD4 T cells, specific for retinal antigens (Ags), mediate experimental autoimmune uveoretinitis (EAU). Conversely, CD8 T cells have not been shown to recognize Ag in the retina. This study investigated whether retinal-specific Ags are detected by class I MHC-restricted CD8 T cells. Using a CD8 T-cell clone (beta3) specific for an immunodominant epitope of beta-galactosidase (beta-gal), local Ag recognition was shown by transfer of activated beta3 cells into beta-gal transgenic (Tg) mice expressing beta-gal in the retina (hi-arr-beta-gal mice), or in the brain and eye (GFAP-beta-gal mice). Beta-gal-positive photoreceptor cells were damaged in the retina of hi-arr-beta-gal mice, and anterior segment disease was found in the eyes of GFAP-beta-gal mice. Ag recognition by resting CD8 T cells was also evaluated. Recovery of 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE)-labelled beta3 cells from hi-arr-beta-gal mice was slightly decreased compared to recovery from B10.A mice, while recovery from GFAP-beta-gal mice was transiently increased. Conversely, recovery of CFSE- cells increased in hi-arr-beta-gal mice, consistent with an Ag-dependent response. The CFSE content of the CFSE+ population was unchanged relative to beta3 cells recovered from controls. Intracellular cytokine responses of beta3 cells recovered from hi-arr-beta-gal and GFAP-beta-gal mice correlated with the number of cells recovered, regardless of CFSE content. Even though their production of interferon-gamma and tumour necrosis factor-alpha was affected little by transfer into hi-arr-beta-gal recipients, the ability of beta3 cells to mediate delayed-type hypersensitivity was inhibited in hi-arr-beta-gal mice. These results show that resting CD8 T cells are affected by the presence of Ag that originates in retina and, when activated prior to transfer, mediate pathogenic autoimmunity against retinal and other ocular targets.     

Light aversion in mice depends on nonimage-forming irradiance detection

Detection of light in the eye underlies image-forming vision, but also regulates adaptive responses in physiology and behavior. Typically these adaptive responses do not involve image-forming vision, but depend on a relatively absolute measure of brightness (nonimage-forming irradiance detection). The goal of this study was to further understand how image-forming vision and nonimage-forming irradiance detection contribute to the effects of light on behavior. Three light dependent behaviors were assessed in wild-type, Rpe65-/- and rd1 mice. In Rpe65-/- mice, nonimage-forming irradiance detection is severely attenuated, but rod based visual acuity is relatively preserved. In rd1 mice visual acuity is nonrecordable, but nonimage-forming responses are less severely attenuated than Rpe65-/-. Positive masking, an image-forming vision dependent increase in wheel running, was absent in rd1 and restricted to higher irradiances in Rpe65-/-. Negative masking, a suppression of wheel running sensitivity with nonimage-forming irradiance detection input, was increased in rd1, but reduced in Rpe65-/- mice. By contrast, light aversion, an avoidance of brightly lit areas, was abolished in both Rpe65-/- and rd1. This shows that image-forming vision is not sufficient for light aversion, suggesting nonimage-forming irradiance detection motivates this behavior. Further, the differing effects of disease suggest that negative masking and light aversion are distinct responses with specialized nonimage-forming irradiance detection pathways.     

Terbium simulates the action of calcium on electrophysiological activity in the isolated fish retina

It has been suggested that terbium (Tb) could be substituted for calcium (Ca) in biological tissue and, because of its useful spectroscopic properties, provide an assessment of calcium distribution in the tissue. In order to assess their physiological effects, we have examined the action of free terbium ions on the membrane potential and light-evoked responses of horizontal cells, and on the massed light evoked response (ERG) of the neural retina. Measurements were performed by intracellular and extracellular recording from the isolated fish retina, superfused with Ringers of various ionic compositions, and certain technical difficulties which occurred in the use of terbium are discussed. We show that Tb ions mimic Ca ions in suppression of the ERG and in hyperpolarization of the horizontal cell membrane potential. The effects of Tb persist after its removal from the superfusate for a longer period than do those of calcium, which indicates that terbium binds more firmly at its site(s) activity. Tb and Ca both suppress the light-evoked S-potentials from the horizontal cells, although some differences were observed in the waveform of the S-potentials during the onset of suppression. We conclude that the physiological effects of Tb on the retina are essentially similar to those of Ca, and that Tb could, therefore, prove a useful marker of Ca distribution in neural tissue.     

遗传性视网膜变性rd小鼠及其感光细胞凋亡研究

目的 研究遗传性视网膜变性rd小鼠感光细胞层的发育变化及细胞凋亡。方法 对出生后5d到40d的rd小鼠及对照小鼠视网膜感光细胞层进行光镜及超微结构观察、TUNEL法检测及形态计量学分析。结果 与同龄对照鼠相比,rd小鼠出生后第10d视网膜开始变性,尔后1周内感光细胞迅速减少,第18d时只残留一层视椎细胞。rd小鼠出生后第10d感光细胞层开始出现TUNEL染色阳性细胞,第14d及16d达到高峰。电镜下变性高峰期rd小鼠视网膜感光细胞层可见大量浓缩核、染色质边聚及凋亡小体。结论 rd小鼠视网膜感光细胞在发育过程中变性,并通过凋亡的方式死亡。     

Retinal Prosthesis： A Potential Benefit for the Blind

Retinal degenerative diseases may induce the degeneration of outer retina and in turn, blindness. Nevertheless, due to the maintenance of inner retina, the coding and processing of visual neurons responses are still able to be executed naturally. Therefore, an effective retinal prosthesis device may be developed by mimicking the function of outer retina： transferring the visual light into artificial stimulus and delivering the stimulus to the retina aiming to evoke the neural responses. As two main developing directions for current retinal prosthesis,epiretinal （ER） and subretinal （SR） prosthesis are both undergoing experimental stage and possessing advantages and limitations. Further investigations in power supply, biocompatibility, etc. are still required. Additionally, suprachoroidal transretinal stimulation （STS） and neurotransmitter-induced stimulation as some other alternatives in retinal prosthesis are also considered as promising research directions, although they are not mature enough to be applied commercially, either.     

Primate horizontal cell dynamics: an analysis of sensitivity regulation in the outer retina

The human cone visual system maintains sensitivity over a broad range of illumination, from below 1 troland to 1,000,000 trolands. While the cone photoreceptors themselves are an important locus for sensitivity regulation-or light adaptation-the degree to which they contribute in primates remains unclear. To determine the range of sensitivity regulation in the outer retina, the temporal dynamics, neural gain control, and response range compression were measured in second-order neurons, the H1 horizontal cells, of the macaque retina. Situated at the first synapse in the retina, H1 cells receive input from a large population of cones. Lee et al. have previously shown that sensitivity regulation in H1 cells is both cone type-specific and spatially restricted. The sensitivity regulation seen in H1 cells at moderate illuminances thus takes place before the summation of cone signals in these cells, and the data establish the H1 cell as a convenient locus for analyzing cone signals. In the present study, cone-driven responses of primate H1 cells to temporally modulated sine-wave stimuli and to increment pulses were measured at steady levels of 1-1,000 trolands. The H1 cell gave a modulated response to sine-wave stimuli and hyperpolarized to increment pulses with overshoots at stimulus onset and offset. The temporal amplitude sensitivity function was primarily low-pass in shape, with a small degree of low-frequency roll off and a resonance shoulder near 40 Hz. A model incorporating a cascade of first-order filters together with an underdamped second-order filter could describe both temporal sinusoidal and pulse hyperpolarizations. Amplitude sensitivity was estimated from both pulse and sine-wave data as a function of the steady adaptation level. Sensitivity at low light levels (1 troland) showed a slowing in temporal dynamics, indicating time-dependent sensitivity regulation. Sensitivity was reduced at light levels above approximately 10 trolands, reflecting both response range compression and neural gain control. Thus the outer retina is a major locus for sensitivity regulation in primates.     

Functional and structural modifications during retinal degeneration in the rd10 mouse

Mouse models of retinal degeneration are useful tools to study therapeutic approaches for patients affected by hereditary retinal dystrophies. We have studied degeneration in the rd10 mice both by immunocytochemistry and TUNEL-labeling of retinal cells, and through electrophysiological recordings. The cell degeneration in the retina of rd10 mice produced appreciable morphological changes in rod and cone cells by P20. Retinal cell death is clearly observed in the central retina and it peaked at P25 when there were 800 TUNEL-positive cells per mm(2). In the central retina, only one row of photoreceptors remained in the outer nuclear layer by P40 and there was a remarkable deterioration of bipolar cell dendrites postsynaptic to photoreceptors. The axon terminals of bipolar cells also underwent atrophy and the inner retina was subject to further changes, including a reduction and disorganization of AII amacrine cell population. Glutamate sensitivity was tested in rod bipolar cells with the single cell patch-clamp technique in slice preparations, although at P60 no significant differences were observed with age-matched controls. Thus, we conclude that rod and cone degeneration in the rd10 mouse model is followed by deterioration of their postsynaptic cells and the cells in the inner retina. However, the functional preservation of receptors for photoreceptor transmission in bipolar cells may open new therapeutic possibilities.     

Excitatory amino acid receptors of rod- and cone-driven horizontal cells in the rabbit retina

Intracellular recordings were obtained from horizontal cells in the superfused retina-eyecup preparation of the rabbit. Rod- and cone-dominated horizontal cells were studied using bath-applied excitatory amino acid analogues. Cone-dominated horizontal cell somas were depolarized by kainate (KA) or quisqualate (QQ) and their light responses were reduced or abolished. They were not affected by N-methyl-DL-aspartate (NMDLA) at concentrations up to 2 mM or by 2-amino-4-phosphonobutyrate (APB), a selective agonist for the ON bipolar cell. When synaptic transmission was blocked with cobalt, horizontal cell somas were hyperpolarized. Under these conditions, KA and QQ caused large depolarizations suggesting that these agents have a direct action on horizontal cell somas. Excitatory amino acid antagonists such as cis-2,3-piperidine dicarboxylic acid (PDA) and kynurenic acid (Kyn) hyperpolarized horizontal cell somas to the level of the light-driven membrane potential. These antagonists blocked both the light-driven responses and the depolarizing action of KA. The specific NMDA antagonist 2-amino-7-phosphonoheptanoate (AP-7) had no effect on the membrane potential or light-driven responses of horizontal cell somas. In contrast to a previous report, we found no evidence that low concentrations of NMDLA could hyperpolarize horizontal cells or act as a KA antagonist in the rabbit retina. Rod-dominated axon terminals were identified by waveform, threshold, and the presence of a large rod after-potential evoked by high light intensity. These cells were depolarized by KA and their light responses were attenuated. NMDLA and APB had no effect on these cells. The general antagonists, PDA and Kyn, hyperpolarized axon terminals and blocked their light-evoked responses. The specific NMDA antagonist, AP-7, had no effect on these cells. These results suggest that the synaptic receptors that mediate light input to both rod- and cone-dominated horizontal cells are kainate or quisqualate receptors. This implies that the rod and cone transmitters of the rabbit retina are similar, with the characteristics of an excitatory amino acid, such as glutamate.     

Decreased glutathione transferase levels in rd1/rd1 mouse retina: replenishment protects photoreceptors in retinal explants

Currently much attention is focused on glutathione S transferase (GST)-induced suppression of apoptosis. The objective of our studies was therefore to see if GST isoenzymes rescue photoreceptors in retinal explants from rd1/rd1 mice, in which photoreceptors degenerate rapidly. Eyes from C3H rd1/rd1 and +/+ mice were collected at various time points between postnatal day (PN) 2 and PN28. Localization and content of alpha-GST and mu-GST was investigated by immunofluorescence and semi-quantitative Western blot analysis, respectively. In addition, PN2 and PN7 retinal explants were cultured till PN28, during which they were treated with 10 ng/ml alpha-GST or mu-GST. The spatiotemporal expression of both GST isoforms was closely similar: early presence in ganglion cell layer after which staining became restricted to Muller cells (particularly in the endfeet) and horizontal cell fibers in both rd1/rd1 and +/+. Doublets of alpha-GST and mu-GST were detected by Western blot analysis. Densitometry of these bands indicated steady reduction of alpha-GST content in rd1/rd1 retina starting from the second postnatal week. When alpha-GST and mu-GST were added exogenously to rd1/rd1 explants, photoreceptor rescue was produced that was more prominent in PN2 than in PN7 explants and more effective by alpha-GST than mu-GST. We propose that alpha-GST neuroprotection is mediated by reduction of tissue oxidative stress.     

Developmental time course distinguishes changes in spontaneous and light-evoked retinal ganglion cell activity in rd1 and rd10 mice

In a subset of hereditary retinal diseases, early photoreceptor degeneration causes rapidly progressive blindness in children. To better understand how retinal development may interact with degenerative processes, we compared spontaneous and light-evoked activity among retinal ganglion cells in rd1 and rd10 mice, strains with closely related retinal disease. In each, a mutation in the Pde6b gene causes photoreceptor dysfunction and death, but in rd10 mice degeneration starts after a peak in developmental plasticity of retinal circuitry and thereafter progresses more slowly. In vitro multielectrode action potential recordings revealed that spontaneous waves of correlated ganglion cell activity comparable to those in wild-type mice were present in rd1 and rd10 retinas before eye opening [postnatal day (P) 7 to P8]. In both strains, spontaneous firing rates increased by P14-P15 and were many times higher by 4-6 wk of age. Among rd1 ganglion cells, all responses to light had disappeared by ~P28, yet in rd10 retinas vigorous ON and OFF responses were maintained well beyond this age and were not completely lost until after P60. This difference in developmental time course separates mechanisms underlying the hyperactivity from those that alter light-driven responses in rd10 retinas. Moreover, several broad physiological groups of cells remained identifiable according to response polarity and time course as late as P60. This raises hope that visual function might be preserved or restored despite ganglion cell hyperactivity seen in inherited retinal degenerations, particularly if treatment or manipulation of early developmental plasticity were to be timed appropriately.     

Role of ubiquitin carboxy terminal hydrolase-L1 in neural cell apoptosis induced by ischemic retinal injury in vivo

Ubiquitin is thought to be a stress protein that plays an important role in protecting cells under stress conditions; however, its precise role is unclear. Ubiquitin expression level is controlled by the balance of ubiquitinating and deubiquitinating enzymes. To investigate the function of deubiquitinating enzymes on ischemia-induced neural cell apoptosis in vivo, we analyzed gracile axonal dystrophy (gad) mice with an exon deletion for ubiquitin carboxy terminal hydrolase-L1 (UCH-L1), a neuron-specific deubiquitinating enzyme. In wild-type mouse retina, light stimuli and ischemic retinal injury induced strong ubiquitin expression in the inner retina, and its expression pattern was similar to that of UCH-L1. On the other hand, gad mice showed reduced ubiquitin induction after light stimuli and ischemia, whereas expression levels of antiapoptotic (Bcl-2 and XIAP) and prosurvival (brain-derived neurotrophic factor) proteins that are normally degraded by an ubiquitin-proteasome pathway were significantly higher. Consistently, ischemia-induced caspase activity and neural cell apoptosis were suppressed approximately 70% in gad mice. These results demonstrate that UCH-L1 is involved in ubiquitin expression after stress stimuli, but excessive ubiquitin induction following ischemic injury may rather lead to neural cell apoptosis in vivo.