| Diabetes mellitus (DM) is a common disease induced by hyperglycemia. The morbidity of DM has been elevated in both developed and developing countries. Diabetic retinopathy (DR) is one of the common complicants of diabetes, which may cause defects in visual function or even blindness. Within the retina, the vascular system as well as the neural circuit is susceptible to the development of DR. The damage and degeneration of retinal neurons induced by diabetes have been repeatedly reported in both diabetic candidates and animal models. However, the functional changes of retinal neurons and underlying physiological mechanisms remain to be elucidated. Consequently, this situation contributes fundamentally to the shortage of effective methods in the clinical treatment and prevention of DR.Retinal ganglion cells (GCs) are the third-order neurons as well as the only type of output neurons in the retina, which receive and integrate visual signals from preceding neurons, including the excitatory inputs from bipolar cells and inhibitory inputs from amacrine cells. The visual signals are then primarily processed and conveyed by GCs to higher vision center in manner of action potentials. As a result, GCs play a critical role during the signal process within the retina, and the defects in GC function often lead to severe visual diseases. The GCs are on the surface of the retina, facilitating the observation and recording on this type of neurons. The studies into the diabetic GCs are advancing in the last decade. However, few work has reveal how diabetes affects GC function on a single neuron level. In the present work, we analyzed the electrophysiological activities of a large number of GCs in diabetic animals on a single cell level, in order to explore the pathological changes within the retinal neural circuits induced by diabetes.In the present work, STZ-induced type-Ⅰ diabetic mice were used as the animal model. During 3 to 4 month after the onset of diabetes, the physiological activities of GCs were observed, including light induced and spontaneous activites, on dark-adapted whole mount retinas. Meanwhile, TUNEL staining and counting techniques were adopted to study the neuronal apoptosis in the retina.After the intraperitoneal injection of STZ, the blood glucose levels of the diabetic mice were elevated and subsequently maintained. The changes in gain of body weight, the polyuria and polydipsia also strongly suggest the onset of diabetes and an effective and reliable type-Ⅰ diabetic animal model. During 3 to 4 month after the onset of hyperglycemia, GC activity was recorded in dark-adapted mouse retina by using loose-patch clamp recording techniques.When exposed to light stimuli, most GCs in normal retina produce ON-or OFF-light response. GCs are classified into ON-GC, OFF-GC, and ON-OFF-GCs according to their response to light. It is observed that, some GCs failed to show light responsive capacity during diabetes. This result suggests that the fundamental physiological function of some GC was damaged by diabetes, which may, in turn, injure visual function.The main body of the study focused on the spontaneous firing of GCs in dark adapted mouse retina. We make continuous loose-patch recordings to different GC subtypes. After we achieved stable recordings, the last minute of the recording was selected for further analysis. It is observed that the firing rate varies greatly among different neurons. By analysing the three subtypes, we found that the distribution pattern of sponataneous firing rate in ON-and OFF-GCs turn out to be non-normal. After 3 month diabetic induction, the spontaneous activity of ON-GCs was significantly enhanced in dark. Furthermore, the bursting pattern, including bursting frequency and time spent bursting (%) in ON-GCs were also elevated by diabetes. The sample size of OFF-and ON-OFF-GCs are far less than that of ON-GCs. The analysis based on the present sample size reveals no difference in the spontaneous firing rate between daibetic and control groups.The potential mechanism underlying the increase of the spontaneous firing rate of ON-GCs was studies by pharmacologic methods. In dark, the spontaneous activity of ON-GCs in normal animals was eliminated by synaptic blocker cocktail, indicating that this activity is synaptic input dependent. Similar condition was also witnessed in most, but not all ON-GCs from diabetic mice. And some diabetic ON-GCs exhibit input independent activities, suggesting that their intrinsic physiological properties of some ON-GCs were possiblly altered by diabetes.The inhibitory signal transmission in the retina was mainly mediated by GABA and glycine receptors. ON-GCs of both diabetic and control groups showed an elevated firing rate in the presence of inhibitory receptor antagonists. Under this circumstance, no difference was detected between two groups. Combined with the fact that ON-GCs showed an significant high firing rate than control group in normal Ringer’s, we propose that the inhibitory inputs to ON-GC from higher level neurons were suppressed in diabetes, which contributed, if not abolutely, to the increased spontaneous activity. This result also presents a potential evidence that the excitatory inputs to ON-GCs was less affected by diabetes.The whole-mount retina was marked by TUNEL and conterstained by DAPI to confirm the location of retinal neurons while setting aside the false positive TUNEL signals. Neurolucida system was used to count the total number of TUNEL signals. Compared with control group, the total number of TUNEL positive signals per unit area was not changed in 3-month diabetic mice, indicating that the survival of retinal neurons were not affected by diabetes at this stage, which should be an early stage of diabetes.In conclusion, after 3 months of STZ-induced diabetes, the neurons in mice retina has not undergone massive apoptosis. At this time, however, the physiological properties of GCs were fundamentally affected, including the loss of light responsiveness, enhanced spontaneous activities in ON-GCs, possible alterations of intrinsic physiological properties and suppressed inhibitory synaptic inputs. These findings may not only provide important information to the understanding of funcitonal changes of retinal neurons during the early diabetes, but propose useful reference to find potential targets to treat and curb the development of DR. |
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