Degeneration stage-specific response pattern of retinal ganglion cell spikes in rd10 mouse retina.

It is known that with retinal degeneration there is rewiring of retinal networks. Consequently, electrical stimulation of the degenerating retina produces responses that differ according to the stage of retinal degeneration. We sought to delineate a degeneration stage-specific parameter for the response pattern of retinal ganglion cell (RGC) spikes as a strategy for stage-specific electrical stimulation for perceptual efficiency of prosthetic vision devices. Electrically-evoked RGC spikes were recorded at different degeneration stages in the rd10 mouse model for human retinitis pigmentosa (RP). Retinal explants mounted on an 8×8 multi-electrode array were stimulated by applying 1 Hz cathodic-phase first biphasic current pulses. RGC firing rate during the first 100 ms post-stimulus was compared to that during the 100-1000 ms period and a response ratio of 100 ms (RR100 ms) was calculated through the different postnatal weeks. Our results show that during post-stimulus 100-1000 ms, the degree of correlation between pulse amplitude and evoked RGC spikes drastically decreases at PNW 4.5. This pattern was closely matched by the RR100 ms curve at this stage. We conclude that the RR100 ms might be a good indicator of the therapeutic potential of a retinal electrical prosthesis.

Are unconventional NMDA receptors involved in slowly adapting type I mechanoreceptor responses?

Specific immunohistochemical staining for NMDA receptor NR2A/B subunits was found in the outer root sheath layer of rat sinus hair (whisker) follicle. Co-localization with CK 20 confirmed that Merkel cells were stained. The NR2A/B staining seen on Merkel cells was pericellular. In addition it appeared that NF70-positive staining was in close proximity to, but did not colocalise with NR2A/B immunoreactivity, indicating that NR2A/B was only expressed by Merkel cells and not their adjacent nerve terminals. Merkel cells and the nerve terminals have previously been associated with electrophysiological recordings from slowly adapting type I (St I) mechanoreceptor unit activity. Pharmacological experiments with isolated sinus hairs using a wide range of ionotropic glutamate receptor antagonists found that only certain NMDA receptor blockers depressed St I unit responses to mechanical stimuli. AMPA/kainate receptor antagonists (CNQX and NBQX, 100 microM) had no effect, nor did classical competitive NMDA receptor antagonists, D-AP5 (600 microM) and R-CPP (100 microM), nor the NMDA glycine site antagonist 5,7-dichlorokynurenic acid (100 microM). The only effective NMDA receptor blockers were those selective for the polyamine site: ifenprodil (IC50 20 microM) and Ro 25-6981 (IC50 approximately 50 microM), and the associated ion channel: MK 801, ketamine and (+/-)-1-(1,2-diphenylethyl)piperidine (IC50 < 100 microM). The two enantiomers of MK 801 were equipotent. All effects were long lasting, consistent with their non-/uncompetitive actions. The most potent drug tested, ifenprodil, at an effective dose of 30 microM, had a mean recovery time of 74 min. A three-fold increase in drug concentration was required to depress St II units (associated with non-synaptic lanceolate endings). Changes in Zn2+ did not affect St I unit responses. These data suggest that unconventional NMDA receptors are involved in St I unit responses, but question the notion of a glutamatergic synapse between the Merkel cell and nerve terminal.

Evidence of collateralization of vagal efferents innervating subdiaphragmatic segments of the gastrointestinal tract in the rat using the double labelling fluorescent dyes technique

Collateralization of the abdominal vagal trunks was investigated in the rat using double labelling fluorescence dye technique. A total of 20 adult male and female rats were used for the study. The anterior and posterior walls of the corpus and fundus of the stomach were injected with 0.1 microliter (microliter) of 5 aqueous solution of diamidino yellow (Dy) in eight rats. The same quantity and percentage of fast blue (Fb) was injected into the walls of the duodenum and proximal jejunum in the same eight rats that were injected with Dy. The anterior and posterior walls of the stomach of four rats were injected with 0.1 microliter of 5 Dy only while four other rats had injections of 0.1 microliter of Fb only into the duodenum and proximal jejunum. Two control rats had stomach injections with normal saline, and two rats had saline injections into the intestine. Each rat was perfused with a preservative 14 days after injection and serial sections taken for examination with a fluorescence microscope. The results of the experiment revealed that in the eight rats injected simultaneously with Dy and Fb, some neurons of the dorsal motor nucleus of the vagus nerve (DMX) were labelled with Dy only, some with Fb only and some were doubly labelled with Dy and Fb. No double-labelled neurons were seen in the rat injected with one dye only and no labellings at all were seen in the controls. The pattern of labelling obtained in the study is suggestive of collateralization of axons of the abdominal vagal trunks

Evidence of collateralization of vagal efferents innervating subdiaphragmatic segments of the gastrointestinal tract in the rat using the double labelling fluorescent dyes technique.

Collateralization of the abdominal vagal trunks was investigated in the rat using double labelling fluorescence dye technique. A total of 20 adult male and female rats were used for the study. The anterior and posterior walls of the corpus and fundus of the stomach were injected with 0.1 microliter (microliter) of 5% aqueous solution of diamidino yellow (Dy) in eight rats. The same quantity and percentage of fast blue (Fb) was injected into the walls of the duodenum and proximal jejunum in the same eight rats that were injected with Dy. The anterior and posterior walls of the stomach of four rats were injected with 0.1 microliter of 5% Dy only while four other rats had injections of 0.1 microliter of Fb only into the duodenum and proximal jejunum. Two control rats had stomach injections with normal saline, and two rats had saline injections into the intestine. Each rat was perfused with a preservative 14 days after injection and serial sections taken for examination with a fluorescence microscope. The results of the experiment revealed that in the eight rats injected simultaneously with Dy and Fb, some neurons of the dorsal motor nucleus of the vagus nerve (DMX) were labelled with Dy only, some with Fb only and some were doubly labelled with Dy and Fb. No double-labelled neurons were seen in the rat injected with one dye only and no labellings at all were seen in the controls. The pattern of labelling obtained in the study is suggestive of collateralization of axons of the abdominal vagal trunks.

Demostration of collaterization of the abdominal vagus in the rat using the double flourescent dyes technique [abstract]

Since the earlier report of Mitchel (1935), the central origin of preganglionic parasympathetic fibres has been studied by various investigators using different techniques and animal species. While it is now generally accepted that the dorsal motor nucleus of the vagus nerve (DMNV) is the principal source of preganglionic parasympathetic fibres to several organs in the thorax and abdomen, there has been persistent controversy as regards topographic representation of these organs in the DMNV. In a previous study in the ferret using the Horseradish peroxidase technique, some degree of topographic representation of the subdiaphragmatic part of the gastrointestinal tract was observed. It was, however, noted that no part of this nucleus is exclusively responsible for innervation of any segment of the gut. The author went on to speculate that the pattern of representation of the gut demostrated in the study might supply more than one segment of the gut by collaterization. The present study was thus designed to test this hypothesis. A total of 16 male and female Sprague Dawley rats weight range from 350 to 500g were used for the study. Four of these rats were used as control while the remaining 12 were used as experimental rats. Eight rats were injected with 1æ1 of 5 percent Diamidino yellow (DY) by multiple penetrations into the walls of the stomach while the same quantity and percentage of Fast blue (FB) was injected in the same manner into the walls of the duodenum and upper jejunum in the eight rats. Two rats had multiple injections of 1æ1 of 5 percent DY into the walls of the stomach only and two other rats had multiple injections of 1æ1 of 5 percent FB into the walls of the duodenum and upper jejunum only. Four control rats were injected with 1æ1 of normal saline (2 in the stomach and 2 in the intestine) in the same manner in which the experimental rats were injected. Each rat was anaesthetized with pentobarbitone and then perfused transcardially 14 days after the injections. Serial sections of the medulla were cut at 20-micron thickness with the cryosat and the sections examined with a Nikon Apaphot flourescence microscope. The result of the experiment revealed that in 8 rats injected with DY and FB some cells of the DMNV were labelled with DY only, some with FB only and some were doubly labelled with FB and DY. The two rats injected with FB showed FB labelled cells only while the two injected with DY showed DY labelled cells only. (AU)

Methamphetamine-induced oxidative stress in cultured mouse astrocytes.

Methamphetamine (METH) is a monoaminergic toxin that destroys dopamine terminals and causes astrogliosis in vivo. Oxidative stress has been shown to play an important role in the toxic effects of METH. In the present study, we sought to determine whether astrocytes are involved in METH-induced oxidative stress. Reactive oxygen species (ROS), ATP, and change in mitochondria membrane potential (delta psi(m)) were examined in cultured striatal, mesencephalic, and cortical astrocytes after 4 to 48 h of 4 mM METH treatment. Results showed that only striatal and mesencephalic astrocytes showed a significant increase in ROS formation from 8 and 12 h, respectively. At 48 h treatment, there was a 55 and 53% increase in ROS content in striatal and mesencephalic astrocytes, respectively, whereas cortical astrocytes showed only a 25% (not significant) increase. JC-1, a delta psi(m)-sensitive dye, showed a decrease in delta psi(m) at 8 h treatment for striatal and mesencephalic astrocytes and at 12 h for cortical astrocytes. Astrocytes from all three regions showed a similar pattern of initial increase followed by a decrease in ATP content, with striatal astrocytes resulting in a maximum depletion (39% of control value) at 48 h treatment. These findings showed that METH treatment resulted in the formation of ROS in the order of striatal > mesencephalic > cortical astrocytes. Although the formation of ROS did not severely interfere with ATP production, a depolarization of mitochondria was observed. The present study suggested that astrocytes may be an important element governing the selective vulnerability to the striatum to METH-induced oxidative stress.

Functional evidence for calcium-induced calcium release in isolated rat vibrissal Merkel cell mechanoreceptors.

1. Single unit recordings were made from Merkel cell (sinus hair type I; St I) and sinus hair type II (St II) mechanoreceptors in isolated rat vibrissae. Responses were determined as the number of spikes evoked by controlled mechanical displacement of the hair shaft for 5 s every 30 s. 2. Superfusion of caffeine (10 mM) increased the responses of Merkel cell receptors by 50-180% of control (mean +/- S.E.M., 64 +/- 12.6%, n = 6, P < 0.001). Similar concentrations of caffeine inhibited St II receptor responses by 20-60% (mean +/- S.E.M., 35 +/- 8%, n = 5, P < 0.01). In both receptor types, caffeine induced a low-frequency increase in spontaneous firing. 3. When Merkel cell receptor responses were completely blocked by superfusion of high Mg2+-containing solution (to competitively block Ca2+ influx) caffeine had no effect when added after complete inhibition, but when added during partial inhibition of responses, the Mg2+-induced inhibition was transiently reversed or halted. This suggests that Ca2+ influx was a prerequisite for the action of caffeine. 4. Ryanodine (1 microM) increased the responses of Merkel cell receptors to mechanical stimulation by 7-60% (mean +/- S.E.M., 32 +/- 10.9 %, n = 5, P < 0.05) but had no effect on St II receptor responses. 5. The Ca2+-induced Ca2+ release (CICR) inhibitor procaine inhibited St I receptor responses in a concentration-dependent manner. Near-maximal inhibition was attained with 100 microM procaine. In four St I units, mean responses were depressed to 25% of control values. When both procaine (100 microM) and caffeine (10 mM) were introduced together, no net effect was seen. The responses of St II receptors were little affected by up to 100 microM procaine superfusion. 6. It is concluded that the mechano-electrical transduction process in St I receptors (but not St II) includes a CICR pathway. Taken with previous findings on the role of Merkel cells, it is likely that CICR is occurring in the Merkel cells.

Selective phototoxic destruction of quinacrine-loaded Merkel cells is neither selective nor complete.

Experiments were performed on slowly adapting type I mechanoreceptors in an isolated rat skin-nerve preparation (SA I receptors) and in an isolated rat sinus hair preparation (St I receptors). Merkel cells were stained in vitro with the fluorescent dye quinacrine and irradiated with ultraviolet (UV) light (2 mW for up to 1 h) while recording receptor responses to standard mechanical stimuli every 30 s. In addition, thresholds for electrically evoked action potentials were tested by applying electrical stimuli to the skin through the same stylus used for mechanical stimulation. UV irradiation resulted in abrupt failure to respond to mechanical stimuli in 73% of the SA I receptors examined (n = 37) within less than 1 h. This confirms previous reports of phototoxic destruction of Merkel cells. However, several minutes after the receptors failed to respond to mechanical stimulation, thresholds for electrical stimuli applied to the receptive field increased sharply. About 40% of the St I receptors (n = 13) irradiated with UV light following quinacrine staining stopped responding to bending of the hair within 1 h. In contrast, none of the seven St II receptors treated in the same way showed significant changes in the responses. Electron microscopic examination of sinus hairs after quinacrine staining alone showed slight changes in the appearance of Merkel cells, and in particular enlargement of the perinuclear space. These changes did not affect receptor responses. Electron microscopic studies of sinus hairs with receptors that had maintained normal responses to mechanical stimuli after quinacrine staining and 1 h of UV irradiation revealed that a substantial number of Merkel cells still had a normal ultrastructure while adjacent nerve terminals were severely swollen and partially compressing the Merkel cells. No changes were observed in lanceolate nerve terminals forming the morphological substrate of St II receptors. These results demonstrate that sensitivity to phototoxic destruction following quinacrine staining varies greatly among Merkel cells, with some maintaining normal function and ultrastructural appearance even after 1 h of UV irradiation. On the other hand there is clear evidence that the phototoxic damage affects the nerve terminals as well. Such experiments can therefore not provide conclusive proof about the role of Merkel cells in these mechanoreceptors.

Chloroquine specifically impairs Merkel cell mechanoreceptor function in isolated rat sinus hairs.

The function of Merkel cells in mechanotransduction has remained controversial Single unit recordings were made from Merkel cell receptors (sinus hair type I, St I) and another slowly adapting mechanoreceptor (sinus hair type II, St II) in isolated rat sinus hairs by applying controlled mechanical displacements to the hair shaft. Chloroquine (50-300 microM) caused a concentration dependent inhibition of Merkel cell receptor responses to mechanical stimulation. In contrast, both stimulated and spontaneous spike activity of St II receptors was increased by the same concentrations of chloroquine. Ultrastructural examination of chloroquine treated sinus hairs revealed swollen Merkel cells with multiple vacuoles and randomly distributed granules while other neural and surrounding structures showed no striking morphological changes. These results suggest that the Merkel cell plays a mechanotransducer role in Merkel cell receptors.

An isolated rat vibrissal preparation with stable responses of slowly adapting mechanoreceptors.

Sinus hairs were isolated from rats and examined in an isolated organ bath while superfused with oxygenated synthetic interstitial fluid. The distal end of the deep vibrissal nerve was teased for single unit recordings of responses from slowly adapting mechanoreceptors to standard bending of the hair. Sinus hair type I and type II receptors could be clearly identified by their respective characteristic firing pattern. Their responses were stable for at least 5 h even if the sinus hair had been stored at 4 degrees C for 24 h beforehand. Electron microscopic examination of these hairs at the end of experiments showed well preserved ultrastructure without abnormalities. The short diffusion distances in this preparation make it well suited for studying drug effects with the aim of investigating the mechanoelectric transduction process in these receptors.

Influence of the thyroid state on the intrinsic contractile properties of the bladder muscle.

The role of thyroid hormones on the contractile responses of the isolated rat urinary bladder strips to acetilcholine and potassium chloride (in depolarising Tyrode solution) was examined. Chronic administration of thyroxine (6-8 micrograms/100 g body wt/day) for 15 days caused stimulation of acetylcholine and potassium chloride-induced contractile responses of the rat urinary bladder strip. Thyroidectomy caused inhibition of acetylcholine and potassium chloride-induced contractile responses of the rat urinary bladder strip. These findings suggest that the thyroid state affects the intrinsic contractile state of bladder muscle.

The effect of thyroidectomy and thyroxine on the reactivity of rat diaphragm muscle to electrical stimulation in vitro.

The effect of thyroidectomy and thyroxine on the reactivity of diaphragm muscle to electrical stimulation was studied in adult albino Wistar rats. Thyroidectomy significantly affected the contractility of the diaphragm muscle. The result shows that thyroidectomy predisposes the muscle of the diaphragm to fatigue.