Inner retinal preservation in the photoinducible I307N rhodopsin mutant mouse,a model of autosomal dominant retinitis pigmentosa

Rod-cone degenerations, for example, retinitis pigmentosa are leading causes of blindness worldwide. Despite slow disease progression in humans, vision loss is inevitable; therefore, development of vision restoration strategies is crucial. Among others, promising approaches include optogenetics and prosthetic implants, which aim to bypass lost photoreceptors (PRs). Naturally, the efficacy of these therapeutic strategies will depend on inner retinal structural and functional preservation. The present study shows that in photoinducible I307N rhodopsin mice (Translational Vision Research Model 4 [Tvrm4]), a 12k lux light exposure eliminates PRs in the central retina in 1 week, but interneurons and their synapses are maintained for as long as 9 weeks postinduction. Despite bipolar cell dendritic retraction and moderate loss of horizontal cells, the survival rate of various cell types is very high. Significant preservation of conventional synapses and gap junctions in the inner plexiform layer is also observed. We found the number of synaptic ribbons to gradually decline and their ultrastructure to become transiently abnormal, although based on our findings intrinsic retinal architecture is maintained despite complete loss of PRs. Unlike common rodent models of PR degeneration, where the disease phenotype often interferes with retinal development, in Tvrm4 mice, the degenerative process can be induced after retinal development is complete. This time course more closely mimics the timing of disease onset in affected patients. Stability of the inner retina found in these mutants 2 months after PR degeneration suggests moderate, stereotyped remodeling in the early stages of the human disease and represents a promising finding for prompt approaches of vision restoration.     

Cytochrome oxidase staining marks dendritic zones of the rat olfactory bulb external plexiform layer

Cytochrome oxidase staining of the rat olfactory bulb external plexiform layer (EPL) produces a darkly stained intermediate zone bordered by lightly stained superficial and deep zones. Similar zonal staining was seen in cats, rabbits, and hamsters. These zones vary in relative thickness around the circumference of the olfactory bulb; the deep zone is proportionally thicker in the most dorsal and ventral parts of the bulb. Tufted cell somata are unevenly distributed within the EPL; the outer part of the EPL has more somata. The distribution of the cytochrome oxidase reaction product shows that the darkly stained intermediate zone is not produced by staining of tufted cell somata. Zones of cytochrome oxidase staining correspond to the sublaminar distribution of mitral and tufted cell basal dendrites. This was demonstrated by labeling mitral and tufted cells with small extracellular horseradish peroxidase injections and processing alternate sections for horseradish peroxidase and for cytochrome oxidase. Because there was cross-reaction of the cytochrome oxidase procedure with horseradish peroxidase, it was possible to trace many neurons through both series of sections. Middle tufted cells of the superficial EPL have basal dendrites confined to the superficial zone of light cytochrome oxidase staining. Internal tufted cells and middle tufted cells of the intermediate zone send their basal dendrites into the intermediate zone. One group of mitral cells (type I) has basal dendrites confined to the deep zone of lighter cytochrome oxidase staining. A second group of mitral cells (type II) and tufted cells of the intermediate EPL has basal dendrites primarily confined to the intermediate zone of dark cytochrome oxidase staining. The correlation of the enzyme staining with the dendritic laminar patterns supports the existence of three relatively distinct sublaminae in the EPL and supports the designation of two types of mitral cells. The staining pattern also provides an independent method for evaluating the sublaminae of the EPL without the necessity of labeling individual groups of cells. Finally, the staining pattern suggests that the intermediate zone of the EPL may be subjected to more tonic synaptic input, causing it to have an increased level of metabolic activity.     

DISTRIBUTION OF GABAERGIC SYNAPSES ONDENDRITIC BRANCHING POINTS AND STEMS OF DENDRITIC SPINES OF CA1 PYRAMIDAL NEURONS IN RAT HIPPOCAMPUS

目的：树突树的分支程度及其构筑对神经元的“计算”特性发挥着重要作用。为探讨树突计算的形态基础，本文研究γ氨基丁酸（ＧＡＢＡ）能突触在海马ＣＡ１区锥体细胞树突分支点和树突棘颈的分布。方法：实验用雄性Ｗｉｓｔａｒ大鼠，灌注固定取脑，按免疫电镜程序制备ＧＡＢＡ免疫电镜切片，电镜观察和拍照。结果：在大鼠海马ＣＡ１锥体细胞的大多数树突分支（６６７％）和树突棘棘颈处（６８３％）有ＧＡＢＡ能突触存在。它们位于树突分支发出处的下方和附近，带有抑制性突触的形态特征。在非分支处的树突干上很少见到ＧＡＢＡ突触。结论：海马ＣＡ１锥体细胞的树突分支和棘颈（也可视为分支处）有着高频度的ＧＡＢＡ能突触，这一特征可能是树突“计算”特性的形态学基础。分支处的ＧＡＢＡ突触不仅可以控制来自树突远端的突触电位向胞体的扩布，也可以控制发生在胞体的动作电位返回扩布到树突。     

Compartment of brain-type cretine kinase and ubiquitous mitochondrial cretine kinase in neurons: Evidence for a cretine phosphate energy shuttle in adult rat brain

Multiple isoforms of cretine kinase (CK) are expressed in specific cell types as part of an energy delivery or shuttle system. To test hypothesis that neurons utilize a cretine phosphate energy shuttle, we examined the pattern of CK isoform expression and localization in adult rat brain. Two isoforms of CK are present in brain extracts, “brain-type,” or BCK, and the ubiquitin form of the mitochondrial CK (uMtCK), as detected by enzyme activity following nondenaturing electrophoresis and by Western blotting following denaturing electrophorsis. In formalin-fixed and parafinn-embedded sections of rat brain, uMtCK immunostaining is detected in the somata of all Golgi type I neurons in the cerebellum, pontine reticular formation, red nucleus, hippocampus, and cerebral cortex. Immunostainig for uMtCK appears throughout the cell body but not in nuclei. BCK immunostaining is also present in somata of Golgi type I neurons in the cerebellum, red nucleus, and pons and is distributed throughout the cell body and within nuclei. BCK immunostaining also appeared in neuronal processes and is concentrated in the molecular layers of the cerebellum and the hippocampus and in cortical pyramidal cell dendrites. These results demonstrate a coordinate pattern of expression and compartmentation of BCK and MtCK isoform in neurons, which provides an anatomic basis for the transfer of metabolic energy via a cretine phosphate energy shuttle. © 1994 Wiley-Liss, Inc.     

Morphology and distribution of synapses onto a type of large field ganglion cell in the retina of the goldfish

The morphology and dendritic distribution of terminals that synapse onto a type of large-field ganglion cell in the retina of the goldfish are described. Electron microscopy was combined with retrograde labelling of cells with horseradish peroxidase (HRP). Synapses from both amacrine (four types) and bipolar cells contacted the dendrites (all orders) of these cells. In contrast to a recent report describing the synaptic organization of large-field ganglion cells in the catfish (Sakai et al., '86), the synapses were relatively evenly distributed throughout the dendritic arbor, not clustered at discrete sites, and no presynaptic specializations were seen in the dendrites of the ganglion cells.     

Protein synthesis in the neuropil of the rat dentate gyrus during synapse development

Previous studies have shown that there are dramatic accumulations of polyribosomes under developing synapses on dendrites of CNS neurons. The present study was designed to evaluate what types of proteins might be synthesized by the synapse-associated polyribosomes. Hippocampal slices from rat pups sacrificed at 4, 7, 10, 12, 14, and 21 days after birth as well as slices from adult animals were incubated in a modified Eagle's medium containing 3H-leucine. After a 30 min exposure to radiolabeled amino acids, the slices were microdissected, separating the dendritic enriched molecular layer from the cell bodies of the dentate gyrus and the hippocampus proper. The level of protein synthetic activity was assessed by comparing the incorporation in cell body and dendritic laminae. Polypeptides present in each dissected zone were separated electrophoretically on 1D SDS-polyacrylamide gels according to their molecular weight and the newly synthesized proteins were analyzed through gel fluorography. The overall level of 3H-amino acid incorporation into protein (measured as cpm/microgram protein) was higher than that of the adult at all postnatal ages. When the entire slice was analyzed, the maximum incorporation was at 12 days after birth. In the dissected subregions of the slice the peak protein synthetic activity in cell-body-enriched regions of dentate gyrus and hippocampus proper was at 4-7 days postnatal, declining between 7 and 21 days to values comparable to the adult. By contrast, protein synthesis in the molecular layer of the dentate gyrus did not peak until 12 days after birth, decreasing toward adult rates after 14 days. The overall pattern of Coomassie stained polypeptides present in the dentate molecular layer was comparable at all ages examined. Moreover, one-dimensional gel analysis showed no qualitative differences in the proteins that were synthesized in the three dissected zones across ages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Administration or deprivation of nerve growth factor during development permanently alters neuronal geometry.

We investigated whether the administration or deprivation of a neuronal growth factor during development can permanently alter the dendritic architecture of sensitive neurons. Nerve growth factor (NGF) or NGF antiserum treatment in the first 2-3 postnatal weeks markedly affected the survival, size, and dendritic arborization of mouse sympathetic ganglion cells acutely. Six months after the completion of treatment, the number of surviving neurons, cell body size, and higher order dendritic branching had changed considerably from their values at 3 weeks, suggesting that these parameters remain malleable throughout postnatal life. However, the number of primary dendrites, a fundamental determinant of organization within sympathetic ganglia, was permanently altered by the neonatal treatment protocol. The idea emerging from this study is that NGF influences the elaboration of primary dendrites by sympathetic ganglion cells only during a critical developmental period. In maturity, NGF acts as a "maintenance" factor necessary for normal neuronal function and survival, but neurons lose the capacity to respond with wholesale rearrangements of dendritic architecture.     

Synaptic Activation of GABAA Receptors Causes a Depolarizing Potential Under Physiological Conditions in Rat Hippocampal Pyramidal Cells

Intracellular recordings with K-acetate-filled microelectrodes were performed in slices of the adult rat hippocampus maintained in vitro at 35 - 36 degrees C to analyse the potentials associated with the orthodromic inhibitory sequence generated by CA1 pyramidal cells. In 43 of 72 cells, stimuli that were delivered in the stratum radiatum induced (i) an initial excitatory postsynaptic potential (EPSP), (ii) an early, hyperpolarizing inhibitory postsynaptic potential (IPSP) (peak latency from the stimulus artefact 20 ms), (iii) an intermediate depolarizing component (peak latency=60 - 120 ms; duration=60 - 150 ms, and (iv) a late, long-lasting hyperpolarizing IPSP (peak latency=120 - 160 ms, duration >400 ms). In the remaining cells the orthodromic inhibitory response lacked the intermediate depolarization. The depolarizing component was selectively blocked by local applications of bicuculline or picrotoxin on the apical dendrites of pyramidal cells. This pharmacological procedure induced an increase in the amplitude of the EPSP that was capable of triggering 2 - 3 action potentials, but no reduction of the recurrent IPSP which is caused by GABAA receptors located close to the soma. The amplitude and duration of the depolarizing component was enhanced by lowering the temperature in the tissue chamber to 29 - 31 degrees C or by application of the GABA uptake blocker nipecotic acid, further indicating that the depolarizing component represented an active phenomenon mediated through GABA. Application of the Cl- pump blocker furosemide reduced and eventually blocked the early IPSP and the depolarizing component. These data demonstrate that under physiological conditions rat hippocampal pyramidal cells generate a depolarization that is presumably caused by an outwardly directed Cl- movement due to the activation of GABAA receptors located on the apical dendrites. This novel mechanism might modulate hippocampal excitability in both physiological and pathophysiological conditions.     

Cerebrolysin ameliorates prefrontal cortex and hippocampus neural atrophy of spontaneous hypertensive rats with hyperglycemia

Hyperglycemia of diabetes mellitus causes damage at the vascular level, which at the renal level represents diabetic nephropathy. In this pathology, there is arterial hypertension. In addition, several reports suggest that hyperglycemia and arterial hypertension affect interneuronal communication at the level of dendritic morphology. We studied these changes in an animal model with streptozotocin-induced diabetes mellitus in the spontaneous hypertensive (SH) rat. Recent reports from our laboratory have demonstrated that cerebrolysin (CBL), a preparation of neuropeptides with protective and repairing properties, reduces dendritic deterioration in both pathologies, in separate studies. In the present study, we evaluated the effect of CBL using the animal model with hyperglycemia and arterial hypertension and assessed the dendritic morphology using a Golgi-Cox staining procedure. Our results suggest that CBL ameliorated the reduction in the number of dendritic spines in the PFC and hippocampus caused by hyperglycemia in the SH rat. In addition, CBL also increased distal dendritic length in the PFC and hippocampus in hyperglycemic SH rats. Consequently, the CBL could be a therapeutic tool used to reduce the damage at the level of dendritic communication present in both pathologies.