Development of the rabbit retina

Summary Measures of rabbit eyes and retinal whole-mounts were used to evaluate the development of retinal area and shape. The retina is shown to have a horizontal axis about a third longer than the vertical axis just before birth, and to adopt an almost symmetrical shape during postnatal development to adulthood. In general, retinal thickness is shown to decrease after birth, but differently in particular retinal regions: the reduction is marked in the periphery, and less pronounced in the visual streak. As an exception, the myelinated region — after it becomes really myelinated, from 9 days p.p. — even increases in thickness. In all regions of the retina, the absolute and relative thickness of the nuclear layers decreases, whereas the relative thickness of plexiform and fibrous layers increases. Proliferation of cells within the rabbit retina was studied during the first three postnatal weeks. ³H-thymidine incorporation was used to demonstrate DNA synthesis autoradiographically in histological sections as well as in enzymatically isolated retinal cells. A first proliferation phase occurs in the neuroblastic cell layer and ceases shortly after birth in the retinal center, but lasts for about one week in the retinal periphery. We found, however, a few ³H-thymidine-labeled cells as late as in the third postnatal week.These late-labeled cells were found within the nerve fiber layer and in the inner plexiform layer. The latter cells were shown to express antigens detected by antibodies directed to the intermediate-sized filament protein vimentin, which are known to label Müller cells and neuroepithelial stem cells. This was confirmed in our preparation of enzymatically isolated cells; all cells with autoradiographically labeled nuclei revealed a characteristic elongated morphology typical for Müller radial glia (and also for early neuroepithelial stem cells). ³H-thymidine-labeled cells in the nerve fiber layer were most probably astrocytic. In analogy to the brain, we conclude that the mammalian retina undergoes a series of proliferation phases: first an early phase producing both neurons and glial cells, and then a late phase producing glial cells, e.g., in the nerve fiber layer. Most probably, the late phase within the inner nuclear layer is glial as well, i.e., consists of dividing Müller cells; it cannot be excluded, however, that there may remain some mitotically active stem cells.     

NOS-like immunoreactive neurons express GABA-like immunoreactivity in rabbit and rat retinae

In rabbit and rat retinae, wholemounted preparations and 40 μm thick vibratome sections were processed for nitric oxide synthase (NOS) immunoreactivity and consecutive semithin sections were immunostained with anti-NOS and anti-GABA antisera, respectively. Two types of NOS-labelled amacrine cells were identified: type 1 cells with larger somata were intensely stained, and type 2 cells with smaller somata were weakly stained. A few displaced amacrine cells also showed NOS-like immunoreactivity. All these NOS-like immunoreactive neurons also expressed GABA-like immunoreactivity. Thus, nitric-oxide-containing neurons might constitute a subpopulation of GABAergic neurons in rabbit and rat retinae. Received: 18 August 1997 / Accepted: 15 October 1997     

GABAergic circuitry in the opossum retina: a GABA release induced by l-aspartate

Glutamate and γ-amino butyric acid (GABA) are the major excitatory and inhibitory neurotransmitters, respectively, in the central nervous system (CNS), including the retina. Although in a number of studies the retinal source of GABA was identified, in several species, as horizontal, amacrine cells and cells in the ganglion cell layer, nothing was described for the opossum retina. Thus, the first goal of this study was to determine the pattern of GABAergic cell expression in the South America opossum retina by using an immunohistochemical approach for GABA and for its synthetic enzyme, glutamic acid decarboxylase (GAD). GABA and GAD immunoreactivity showed a similar cellular pattern by appearing in a few faint horizontal cells, topic and displaced amacrine cells. In an effort to extend the knowledge of the opossum retinal circuitry, the possible influence of glutamatergic inputs in GABAergic cells was also studied. Retinas were stimulated with different glutamatergic agonists and aspartate (Asp), and the GABA remaining in the tissue was detected by immunohistochemical procedures. The exposure of retinas to NMDA and kainate resulted the reduction of the number of GABA immunoreactive topic and displaced amacrine cells. The Asp treatment also resulted in reduction of the number of GABA immunoreactive amacrine cells but, in contrast, the displaced amacrine cells were not affected. Finally, the Asp effect was totally blocked by MK-801. This result suggests that Asp could be indeed a putative neurotransmitter in this non-placental animal by acting on an amacrine cell sub-population of GABA-positive NMDA-sensitive cells.     

Intracellular electrophysiology of CA1 pyramidal neurones in slices of the kainic acid lesioned hippocampus of the rat

Summary Intracellular recordings were made from hippocampal CA1 pyramidal cells in slices where the CA3/CA4 region had been lesioned using intracerebroventricular kainic acid. In 55% of the cells studied orthodromic excitation evoked bursts of action potentials. This bursting activity was associated with a decrease in or loss of the early phase to the hyperpolarisation which normally follows orthodromically evoked action potentials. The recurrent inhibitory post-synaptic potential produced by antidromic activation of pyramidal cells was also reduced or absent. A late phase to the orthodromic hyperpolarisation was reduced in cells from lesioned slices. However, in normal slices treated with bicuculline this potential showed an apparent increase. The afterhyperpolarisation which follows a short current evoked burst of action potentials was reduced in bursting cells from lesioned slices. In addition, a silent period in the firing pattern produced by long depolarising current pulses was reduced or absent in these cells. These results together with observations made with bicuculline suggest that the bursting activity in lesioned slices is largely due to a loss of inhibition mediated by -aminobutyric acid. It is proposed that the kainic acid-lesioned in vitro hippocampus may be a suitable preparation for studying the electrophysiology of temporal lobe epilepsy.     

The development of astrocytes in the albino rabbit retina and their relationship to retinal vasculature

We have examined the development of astrocytes in the albino rabbit retina, using antibodies to glial fibrillary acidic protein (GFAP) and vimentin. Vimentin immunoreactive (vimentin⁺) astrocyte-like cells first appear at the 24th postconceptional day (24 PCD), in a pattern similar to that of the adult. GFAP immunoreactivity was first detected in astrocytes at the 29 PCD, in a similar pattern. Vessels enter the retina from 29 PCD. The presence of astrocytes in a mature distribution prior to the ingrowth of vessels indicates that astrocytes are not dependent on the vessels for their early positioning and differentiation. In contrast with the rat and cat, we found no evidence of migration of astrocytes into the rabbit retina from the optic nerve.     

Age-related changes of benzodiazepine and GABA binding sites in the rat retina

The changes in the number and sensitivity of benzodiazepine and GABA binding sites in the rat retina during postnatal development, adulthood and ageing and their functional relationship at different ages have been studied. Data indicate an increase in the total number of both GABA and benzodiazepine binding sites with age. In contrast, the activation of retinal benzodiazepine receptor binding by GABA is significantly reduced in aged rats with respect to young adult and newborn rats. Moreover, the activation of retinal benzodiazepine receptor binding induced by dark exposure of the animals is present in young adult rats but is lost in aged rats. These results suggest that in the retina of aged rats there is an increase of GABA and benzodiazepine receptors which have lost their functional connection.     

红藻氨酸致(癎)大鼠神经元凋亡的动态研究

目的：探讨红藻氨酸（kainic acid，KA）诱导的癫痫大鼠脑内神经元凋亡在不同部位和致痫后不同时间的变化情况。方法：将50只SD大鼠随机分为对照组和KA组，KA组再按癫痫发作后1天、1周、2周、3周和4周不同时点分为5个亚组。KA注射后，观察大鼠癫痫发作后的行为学变化，采用原位细胞凋亡检测法观察不同部位、不同时点神经元凋亡情况。结果：KA注射后，大鼠出现严重的惊厥，在癫痫发作后1天、1周和4周凋亡细胞明显增多，以齿状回、丘脑和CA3区明显（P〈0．05）。结论：细胞凋亡贯穿KA致痫大鼠癫痫发作后神经元迟发性死亡的全过程。     

The doublet microtubules of rods of the rabbit retina

Summary The connecting cilium of the rabbit photoreceptor rod is composed of nine outer doublets, lacking dynein side arms. The central singlet microtubules are absent. In cross section, there is an inner dense ring situated between the doublets and the center core of the cilium. As the doublet microtubules progress from the connecting region into outer segments, the cylindrical array of the nine pairs of doublets spreads out as a brush-like arrangement into the incisure cavity of the outer segment. The microtubules continue as doublets for much of the length of the outer segment. The B-tubules terminate first; the A-tubules extend as single tubules into the apical region of the photoreceptor. Before the B-tubules end, they open up, forming hook-shaped projections from the A-tubules. The gradual reduction in length of these hook-shaped structures suggests that near their distal ends each B-tubule opens because of the separation of protofilament 1 of the B-tubule from protofilament 1 of the adjacent A-tubule. Subsequently, the B-tubule protofilaments terminate individually.     

Co-localization of serotonin and GABA in neurons of the Xenopus laevis retina

Serotonin-synthesizing neurons in the retina of Xenopus laevis have been identified using anti-phenylalanine hydroxylase (PH) antibody which recognizes tryptophan 5-hydroxylase, the rate-limiting enzyme for serotonin synthesis. Double-labelling experiments, using anti-PH antibody and anti-serotonin antibody/5,7-dihydroxytryptamine (5,7-DHT) uptake, have shown that some serotonin-like immunoreactive/5,7-DHT-labelled neurons exhibit PH-like immunoreactivity (PH-LI) (serotonin-synthesizing neurons), but the others do not (serotonin-accumulating neurons). In the present study, triple-labelling experiments were performed using 5,7-DHT uptake and antibodies raised against GABA and PH, to determine the possible co-localization of -aminobutyric acid (GABA) in serotonin-synthesizing and/or -accumulating neurons in the Xenopus retina. All 5,7-DHT-labelled bipolar cells lacked PH-LI; all of them were immunoreactive to GABA. In contrast, all 5,7-DHT-labelled large amacrine cells exhibited PH-LI, but none of them expressed GABA-LI. Small amacrine cells labelled with 5,7-DHT but not PH-LI exhibited GABA-LI, whilst the small amacrine cells with PH-LI lacked GABA-LI. These observations indicate that GABA is co-localized in serotonin-accumulating amacrine and bipolar cells, whereas serotonin-synthesizing large and small amacrine cells do not contain GABA-LI.     

Neuronal responses to iontophoretically applied dopamine,glutamate, and GABA of identified dopaminergic cells in the rat substantia nigra after kainic acid-induced destruction of the striatum

Summary Kainic acid (KA 1.2–1.5 g) was injected unilaterally into the rat striatum (ST). Fifteen to 30 days later neurons of the substantia nigra (SN) were identified by antidromic stimulation from the ST or medial forebrain bundle (MFB). The projecting axons had conduction velocity similar to that recorded in unlesioned animals.Responses to iontophoretically applied dopamine (DA), glutamate (GLU), and GABA (5–100 nA) were recorded from neurons of the dopaminergic pars compacta-striatal projection. Control experiments were performed in intact rats. GABA and DA inhibited neurons tested in controls while GLU had an excitatory effect. Changes in firing rate induced by GABA and GLU developed 1–5 s after the beginning of their ejection while the action of DA appeared after a delay of 20–40 s.Neurons in lesioned animals showed a net decrease in sensitivity to all three neurotransmitters. The highest current tested gave responses 50–70% lower than in controls.The data suggest that destruction of striatal efferents with KA does not induce hypersensitivity in the pars compacta of the SN.     

Decrease in GABA immunoreactivity and alteration of GABA metabolism after kindling in the rat hippocampus

Summary The kindling model of epilepsy, induced by tetanic stimulation of Schaffer collateral/commissural fibers, was studied in the rat hippocampus. Gamma-aminobutyric acid immunoreactivity was used to quantify the number of GABA-immunoreactive somata per mm² in CA1 region, 28 days after the last generalized seizure. Comparison of the numbers obtained from kindled animals with those from controls, showed a significant decrease (18%) on the ipsilateral stimulated side but none on the contralateral side. In control rats injection of the GABA-transaminase inhibitor, amino oxyacetic acid (AOAA), led to a 46% increase in the number of cell somata immunoreactive for GABA. This probably results from an accumulation of GABA, reflecting GABA synthesis by glutamate decarboxylase (GAD) activity, in somata of interneurons that had initially a GABA content below the immunocytochemical detection threshold. In kindled rats, 31 days after the last seizure, the number of GABA-immunoreactive cells that could be observed after AOAA-treatment was significantly lower (35% ipsilateral and 25% contralateral) when compared to AOAA-treated controls. This suggests that in kindled animals a GAD dependent increase in GABA content did not take place in a subpopulation of interneurons. The observations for kindled rats are interpreted as a long-term decrease in GABA content and as an alteration in GABA turnover in a subpopulation of interneuron somata, the latter possibly due to a decrease in GAD activity. The long-term enhanced seizure sensitivity, characteristic for kindled animals, may be due to a decreased GABAergic inhibitory control of the neuronal circuitry in the CA1 region of the hippocampus.     

Somatostatin-like immunoreactivity in amacrine cells of the chicken retina

Somatostatin-like immunoreactivity was detected in chicken retina by radioimmunoassay. The levels of somatostatin-like immunoreactivity decreased after intra-ocular injection of kainic acid, but were not affected by destruction of the ganglion cells. By immunohistochemistry, somatostatinimmunoreactive amacrine cells were found in the inner nuclear layer. These cells were destroyed by kainic acid. At least some of the cells projected to all three sub-layers of the inner plexiform layer in which there were diffuse bands of fluorescence. Specific immunofluorescence was also detected at the level of the outer limiting membrane and the optic nerve fibre layer, but the outer nuclear and plexiform layers, horizontal, bipolar and ganglion cells did not show specific immunofluorescence.It is suggested that other amacrine cell sub-classes, defined in terms of their putative transmitter, may show specific patterns of cell body location and size, and terminal arborisation.     

Dark threshold and steady-state reactions of the pupillary light reflex in the pigmented rabbit

Summary The light reflex of the pupil was used as an indicator of retinal activity in the awake, unanesthetized pigmented rabbit.The change of the area of the pupil in response to light flashes subtending a circular field of 22° and of 0.1 sec duration was measured continuously by reflecting infrared light from the iris to a photocell (dynamic response). The photopupillar contraction showed latencies between 0.136 sec (at 700 cd/m²) and 0.290 sec (at 0.12 cd/m²) and was maximal within 0.7 sec. The least stimulus luminance producing a just perceptible contraction of the iris muscle in the most sensitive animal was 4×10^–2 cd/m² which is nearly 10⁴ times the intensity threshold in the human eye. It is concluded that some interaction takes place in the rod-dominated rabbit's retina which results in a masking effect of the rod-mediated pupillomotor response to flashes. Evidence for this effect is the large change of threshold of the rabbit's pupillomotor response during illumination to steady lights of threshold luminance.The actual size of the pupil was measured photographically on infrared film first in the dark, then at increasingly higher levels of illumination. With illumination of white light the size of apparent pupillary diameter decreases from 6,0 mm (after dark adaptation) to 4,5 mm (during illumination with 500 cd/m²). Evidence is given that the steady state reactions of the rabbit's pupil are governed by the activity of the photopic system.     

Effect of azide on the ERG of the isolated mammalian retina

Summary Azide, which is known to affect the pigment epithelium strongly may be assumed to cause damage to the receptors, which are functionally connected to the pigment epithelium. To check this hypothesis the effect of azide on the ERG was investigated. An isolated retina preparation was used as in this preparation the P III component, which contains considerable receptor contribution, can be isolated.In 2 series of experiments the effects of azide on the P III and the complete ERG were investigated. Depending on the concentration azide was shown to abolish the b-wave, to cause delay and amplitude diminution of the P III and enhance a positive component in the off-effect.A number of plausible sites of origin of these azide effects on the ERG changes are discussed.Supported by a Netherlands Organization for the Advancement of Pure Research grant.     

Some behavioral effects of selective neuronal depletion by kainic acid in the dorsal hippocampus of rats

Iontophoretic injections of the neurotoxin kainic acid (KA) into the dorsal hippocampus resulted in an essentially complete attrition of neurons throughout the dorsal hippocampus but spared the fimbria/fornix system. The ventral hippocampus, subiculum, and associated aspects of the entorhinal area appeared completely intact. There was also no detectable damage to neocortex or thalamus adjacent to the dorsal hippocampus or to such KA-sensitive distant regions of the brain as the piriform cortex or striatum. Bilateral depletion of neurons from the dorsal hippocampus retarded the acquisition of a brightness discrimination (because the animals developed position habits during the early phases of acquisition) but had no deleterious effects on brightness discrimination reversal. The KA injections also sharply reduced spontaneous alternation in a T maze and increased locomotor activity in an open field. Food and water intake as well as saline and sucrose preferences were normal after the initial post-operative period but the animals continued to spill more of their food than controls.     

Distribution of enkephalin-like immunoreactivity in the rat main olfactory bulb

Enkephalin-like immunoreactivity was localized within the main olfactory bulb of the rat using immunohistochemical techniques. These studies utilized well characterized antisera directed to either leu⁵- or met⁵-enkephalin. Specificity was established by absorption of the antisera with either 10 μM synthetic leu⁵- or met⁵-enkephalin.Specific enkephalin-like immunoreactivity was observed within several different cell populations including (1) periglomerular cells, (2) granule cells and their processes within the external plexiform layer and (3) occasional short-axon (horizontal) cells within the granule and external plaxiform layers. The granule cell layer contained the greatest number of immunoreactive cells. Only a limited number of immunoreactive cells were found in both the periglomerular and granule cell layers, suggesting the enkephalin-containing neurons represent a sub-population within each layer.The absence of immunoreactive processes in the periventribular white matter, as well as the morphologies of immunoreactive bulbar neurons, indicates that enkephalin is found exclusively within intrinsic olfactory bulb neurons.     

Distribution of [H]kainic acid and binding sites in the rat brain: in vivo and in vitro receptor autoradiography

Brain sections incubated in vitro with a-[³H]kainic acid (KA; spec. act. 62.5 Ci/mmol), reveal a heterogenous distribution of low and high affinity KA binding sites in the brain. The highest density of KA binding sites was localised to the hippocampus CA₃ region and to superficial layers of the entorhinal cortex (3.8 6.0 μCi/g tissue). Intravenous injection of [³H]KA (1 μCi/g) reveals limited overall penetration of [³H]KA across the blood-brain barrier. However, a dense labelling of the hippocampus, entorhinal cortex and lateral septal regions (2.5–3.8 μCi/g tissue) was observed. Behaviourally, these rats exhibited mild limbic seizure activity possibly as a result of a direct action of KA in the hippocampus or entorhinal cortex.     

克罗米酚的抗癫癎作用

目的:了解雌激素受体调节剂克罗米酚(clomiphene citrate,CC)对海人藻酸(kainic acid, KA)致癎大鼠癫癎发作行为的影响。方法:健康雌性SD大鼠40只,均行双侧卵巢切除术,术后第八天将动物随机分4组:茶油对照组(OIL组)、KA组、雌二醇(E2) KA组、E2 CC KA组,每组10只。 OIL组、KA组连续5天腹腔注射茶油;E2 KA组连续5天腹腔注射E2(20mg／kg,10mg／ml);E2 CC KA组连续5天腹腔注射E2和CC(2mg／kg,2mg／ml),最后一次打药结束1 h后,致癎各组大鼠(KA 组、E2 KA组、E2 CC KA组)经腹腔注射KA(10mg／kg,2mg／ml),OIL组腹腔注射生理盐水后,连续观察大鼠2 h的行为改变。记录癎样发作的潜伏期、出现重型发作的时间以及发作程度。结果:E2 CC KA组的癫癎发作潜伏期为61．75±19．04 min,较E2 KA组的潜伏期23．8±6．03 min延长,且出现重型癫癎发作的时间晚,为50．20±20．37 min,而E2 KA组出现重型癫癎发作时间平均为30．70 ±13．58 min,两组比较差异有显著意义(P<0．05)。致癎2 h后,致癎各组大鼠的癫癎发作Racine分级比较差异显著意义(P>0．05)。结论:CC有拮抗雌激素的致癎作用,延缓癫癎的发作。     

Curcumin attenuates the kainic acid-induced hippocampal cell death in the mice

Kainic acid (KA) induced oxidative stress is associated with hippocampal cell death. Recent studies suggest that curcumin, a potent antioxidant, may provide protection for KA-induced oxidative stress. We investigated the effects of curcumin treatment on hippocampal reactive astrocytes in mice with KA-induced seizures. Eighteen hours after curcumin treatment, mice were treated with KA (30 mg/kg, i.p.), and then sacrificed after a further 48 h. Using cresyl violet staining and TUNEL analysis, histological evaluation revealed cell death in the KA-treated hippocampus. However, marked cell death was not observed in mice treated with curcumin. In addition, curcumin treatment reduced the KA-induced immunoreactivity of caspase-3. Similarly, immunoreactivity analyses indicated that KA causes upregulation of hippocampal GFAP, eNOS, and HO-1 levels, all of which were reduced in animals those received the curcumin treatment. Our findings indicate that curcumin is a potent inhibitor of reactive astrocyte expression and thus, prevents hippocampal cell death. These results also support its potential for use in the treatment of neurodegenerative diseases.