Pigmented Rats Research Articles

Alleviation of mortality induced by salicylate and stress.

Protection from the deleterious effects of the interaction of environmental stress and salicylate by calcium supplement was investigated in 96 pigmented rats. Within a 2 x 2 x 4 factorial design, rats were assigned to groups defined by: A) ad lib access to 1) plain tap water, or 2) 50 mM calcium chloride solution; B) exposure to stressors consisting of daily 10 h periods of 1) 98 dB SPL noise, or 2) confinement precluding movements; C) daily injections of 233, 350, or 410 mg/kg of sodium salicylate or the saline vehicle. For subjects maintained on tap water, weight loss and mortality increased with salicylate levels, with all subjects dying in the group drinking water and injected with 410 mg/kg. Calcium protected all of the subjects in the noise stress group but not in the confined group.

Impact of lipofuscin on the retinal pigment epithelium: electroretinographic evaluation of a protease inhibition model

With aging, the retinal pigment epithelium (RPE) becomes increasingly congested with residual debris called lipofuscin. Little is known about the impact of lipofuscin on retinal function, and this was addressed in the present study by examining the influence of RPE debris on electroretinographic (ERG) parameters utilizing an experimental model of lipofuscin accumulation. Pigmented rats were injected intravitreally with the protease inhibitor leupeptin, and examined 1 week later by electroretinogram (ERG) recording and light and electron microscopy. Relative to vehicle-injected controls, leupeptin-treated retinas showed abundant accumulation throughout the RPE cytoplasm of inclusions that resembled lipofuscin. RPE cells filled with this debris showed a marked increase in height and a displacement of melanin from their apical border. Morphological changes in the RPE had no influence on retinal function since ERG threshold, a- and b-wave maximum amplitude, latency and implicit time were not significantly different between leupeptin-treated eyes and controls. Furthermore, leupeptin-induced RPE inclusions did not alter either the rate or extent of ERG dark adaptation. These findings suggest that filling of the RPE cytoplasm with residual debris is not in itself likely to be the cause of functional alterations in the aging eye.

Circularly polarized 50-Hz magnetic field exposure reduces pineal gland and blood melatonin concentrations of Long-Evans rats

In order to determine if pigmented rats also exhibit melatonin suppression like that described for albino rats exposed to circularly polarized, 50-Hz, 1-μT magnetic fields for 6 weeks, two experiments were conducted with male Long-Evans rats. The field-exposed experimental group received circularly polarized, 50-Hz, 1-μT magnetic fields for 6 weeks, the concurrent sham-exposed control group was exposed to the stray field of 0.02 μT. In addition, prior to the exposure experiment, two cage-control groups were placed in the facility for 6 weeks without activation of the 50-Hz magnetic field generation apparatus. Rats were sacrificed at 12.00 and at 24.00 h for collection of plasma and pineal gland: melatonin was determined by radioimmunoassay. Significant reductions of plasma and pineal gland melatonin contents were observed at 0.02 μT as compared to the control values, and a further reduction was observed at 1 μT. As do albino rats, pigmented rats also exhibit melatonin suppression when exposed to time-varying magnetic fields.

A new procedure for fundus photography and fluorescein angiography in small laboratory animal eyes.

Increasing interest in retinal research demands continuous improvement of experimental techniques and interpretation. Thus, the purpose of our research was to devise a new method for funduscopic photography and fluorescein angiography in the normal or diseased retina of the small laboratory animal that would produce results comparable in optical quality and field coverage to those obtained in human clinical practice. To enhance the view of the small eye, a 2.2 Volk Panretinal lens was held in apposition to the lens of a clinical fundus camera, the Topcon TRC 50FT, by means of a custom made metal sleeve. Albino mice, albino rats, and pigmented rats were photographed. Fluorescein angiography was performed on pigmented rats. Fluorescein was administered intravenously via the jugular vein at a dose of 5 mg/kg. Various speeds of film and flash settings were used depending on the light source and the pigmentation of the animal. Attachment of the 2.2 Panretinal lens to the clinical fundus camera allowed for more clearly defined fundus photographs of the small laboratory animal, as well as an enlarged field of observation over conventional techniques. Consequently, angiography fields and stages documented in the small laboratory animal approximated those obtained in human clinical practice. This technique facilitates the visualization of small fundi and it allows for a fuller documentation of experimental retinal models.

Corticostriatal and corticotectal projections from visual cortical areas 17, 18 and 18a in the pigmented rat.

Regions of the visual cortex and the deep layers of the superior colliculus (SC) have been suggested to be functionally linked via an 'indirect' pathway through the basal ganglia. The present report demonstrates projections from the striate (area 17) and extrastriate (areas 18 and 18a) visual cortex in Long-Evans hooded rats to the striatum (ST) and SC with anterograde (biocytin) and retrograde (WGA-HRP and fluorescent dyes) tracers. Biocytin injections into each cortical area produced markedly different patterns of labeling in ST and SC. Injections into area 17 resulted in a dense labeling in the superficial layers of SC, with little labeling present in the deep layers of SC or ST. In contrast, injections into area 18a, which produced marked labeling in the deep layers of SC with moderate in the superficial layers, resulted in dense labeling in the caudal two-thirds of the dorsal region of ST; injections into area 18 produced numerous terminals mainly in the deep layers of SC and in the rostral two-thirds of the dorsal aspect of ST. WGA-HRP injections into ST resulted in numerous retrogradely labeled pyramidal neurons in areas 18 and 18a, but only a few labeled neurons were observed in area 17. Corticostriatal neurons in extrastriate visual cortex were distributed predominantly in layer V, with smaller numbers in layers II and III, whereas corticotectal neurons were located only in layer V of both the striate and extrastriate visual cortex. Although corticostriatal and corticotectal neurons were intermingled in layer V, no double-labeled neurons were observed following injections of different fluorescent dyes into ST and SC. It appears, therefore, that: 1) the major source of visual input to both ST and the deep layers of SC in the rat arises not from the primary visual cortex but from extrastriate visual areas; and that 2) the projections from the extrastriate visual area to ST and SC originate from different populations of corticofugal neurons.

Effect of a phase advance of the light/dark cycle on pineal function and orcadian running activity in individual rats

Circadian rhythms of urinary 6-sulphatoxy-melatonin excretion and spontaneous wheel-running activity were monitored in individual male Wistar albino and hooded rats entrained to a 12 h L:12 h D photoperiod, before and after an 8 h phase advance of the light cycle. The pigmented hooded strain commenced melatonin metabolite excretion 2 h after darkness, whereas the albino rats did not excrete detectable amounts until 4 h of darkness had elapsed ( p < 0.05). There was no correlation between the time of onset of spontaneous wheel-running activity and the onset of excretion of 6-sulphatoxy-melatonin under entrained conditions. When the photoperiod was advanced by 8 h, the albino rats took a median of 4 days (range 2–10 days) to establish a normal phase angle for the onset of 6-sulphatoxymelatonin excretion in contrast to the pigmented rats, which took in excess of 10 days to entrain. Albino rats reentrained running activity significantly earlier than pigmented rats (8 days: range 6–9 days), compared to 10 days (range 8 to >12 days). There was no consistent relationship between the pineal and running rhythms with respect to the time taken to entrain following a lighting phase shift. All rats entrained the 6-sulphatoxy-melatonin rhythm by advancing the onset of excretion in contrast to the running rhythm where three rats reentrained by advancing, three by delaying, and in four, the direction of entrainment could not be accurately determined. In two of the animals (both albino), there was unequivocal evidence that the 6-sulphatoxymelatonin rhythm and running reentrained in different directions. The results obtained during entrainment suggest that the pineal and spontaneous running rhythms can be dissociated and may even be controlled by different rhythm centres.

Significance of Bruch's membrane in the creation of iatrogenic chorioretinal venous anastomosis.

Iatrogenic retinal to choroidal vein anastomoses, as a method of bypassing retinal venous occlusions has been reported in dogs in which Bruch's membrane is poorly formed. In order to determine whether chorioretinal venous anastomoses can be induced in an animal with a Bruch's membrane that is well developed as in humans, pigmented rats were chosen. A high intensity, small spot size argon green laser beam of 514 nm was used to induce the anastomosis. Three out of 5 rat eyes developed a retinal vein to choroidal vein anastomosis. The success rate of iatrogenic retinal to choroidal vein anastomoses in the rat was comparable to that obtained in the dog. This study suggests that Bruch's membrane was not significant in the creation of an iatrogenic chorioretinal venous anastomosis.

The effects of visual deprivation on adrenergic and glutamatergic receptors in the visual structures of the pigmented rat,: An autoradiographic study

The effects of visual deprivation on adrenergic and glutamatergic receptors in the visual structures of the pigmented rat,: An autoradiographic study

Failure to induce rapid eye movement sleep by dark pulses in pigmented inbred rat strains

Studies of albino Lewis rats, pigmented Brown Norway rats, and their F2 backcross progeny have demonstrated that the ability to trigger rapid eye movement (REM) sleep by turning off cage lights (dark pulses) is associated with albinism in these rat strains. Other studies have shown that pigmented inbred rats show REM sleep induction in the dark portion of short light:dark cycles or skin temperature changes. In the present study, these same pigmented breeds, Dark Agouti and hooded Long-Evans rats, were subjected to 5-min dark pulses and failed to show any evidence of REM sleep triggering. In fact, they showed trends towards REM sleep suppression during dark pulses. These results extend the finding that dark pulse triggering of REM sleep, readily evoked in albino rats, does not appear in pigmented rat strains.

Parabigeminal, pretectal and hypothalamic afferents to rat's dorsal lateral geniculate nucleus. Comparison between albino and pigmented strains

In adult pigmented and albino rats different fluorescent dyes were injected into the dorsal lateral geniculate nuclei of opposite sides. Differences between the strains occur mainly in parabigemino-geniculate and pretecto-geniculate projections. Both the major contralateral and the minor ipsilateral parabigemino-geniculate projections in albinos were clearly smaller then those in pigmented rats. In pigmented rats but not in albinos the parabigemino-geniculate projections originated mainly from the region where the vertical meridian is represented and contained a small number of neurones projecting bilaterally. In each strain, a small number of retrogradely labelled neurones was found in the ipsilateral and contralateral lateral hypothalami.

Effect of exposure to 2,5-hexanediol in light or darkness on the retina of albino and pigmented rats. II. Electrophysiology.

Albino (Sprague-Dawley) and pigmented (Norwegian Brown) male rats were exposed to 2,5-hexanediol (H; 1%) in their drinking water for 5 or 8 weeks, respectively. Half of the rats of each strain were housed in light (average 30 cd/cm2 inside cage, 12 h/day); the other half was kept in constant darkness. Control groups were studied in parallel under identical conditions but without H. Electrophysiological recordings were made 2-5 days and 13 weeks after the end of the exposure to H. Alterations in the visual system, as measured by electroretinography and visual evoked response, were found in groups of albino rats exposed to H and/or light. The pupillary diameter was enlarged in the albino group exposed to both H and light. Among the pigmented rats, alterations were recorded only in the visual evoked response of the H exposed groups. The results demonstrate that simultaneous exposure to H and light can lead to alterations in visual function that are more severe than those induced by each agent alone, and may exceed a simple summation.

Effect of exposure to 2,5-hexanediol in light or darkness on the retina of albino and pigmented rats. I. Morphology

Effect of exposure to 2,5-hexanediol in light or darkness on the retina of albino and pigmented rats. I. Morphology

Glutamatergic and GABAergic synaptic currents in ganglion cells from isolated retinae of pigmented rats during postnatal development

This study was aimed at characterizing the earliest phases of synaptogenesis in the mammalian retina. Spontaneous activity of ganglion cells in the isolated superfused retina was used as an indicator for the functionality of synaptic connections. Retinal ganglion neurons (RGNs) were identified by location of their somata in the ganglion cell layer (GCL) and by their ability to generate large (> 500 pA) voltage-activated sodium currents. Spontaneous spiking was found in many RGNs prior to cell perfusion. Between postnatal day (P) 1 and 18, a total of 195 RGNs was tested for light-induced currents, conductance changes in response to exogenous glutamate (Glu) and γ-aminobutyric acid (GABA), and depolarizing or hyperpolarizing synaptic activity. The vast majority of the material was derived from RGNs at day P5. Whole-cell ion currents were always sampled at somatic sites, using either conventional or perforated patch whole-cell recordings. On day P5, 5% of tested RGNs ( n = 73) were already responsive to light stimulation. A higher percentage of cells (23%, n = 187) generated spontaneous depolarizing currents that were regarded as glutamatergic excitatory postsynaptic currents (EPSCs), since (1) they were blocked by Glu antagonists, (2) they conformed to the Na +/Cs + equilibrium potential, (3) and they displayed a time course characteristic of glutamatergic EPSCs. The mean EPSC amplitude was 19.0 pA (S.D. 11.83 pA). Amplitude distributions were fitted by multiple Gaussian equations rendering a quantal size of 6.6 to 9.1 pA at a holding voltage ( V h) of −70 mV (driving force about 70 mV). Spontaneous EPSCs were never observed under condition of Ca 2+-free solutions, but they persisted in the presence of tetrodotoxin. Bath application of quisqualate (500 μM) consistently increased EPSC frequencies. In contrast to the relatively high percentage of RGNs generating spontaneous EPSCs, very few RGNs at P5 (3%, n = 187) displayed inhibitory postsynaptic currents (IPSCs), although by that time all tested RGNs ( n = 14) were responsive to both exogenous Glu and GABA. These results indicate that in the postnatal rat retina development of excitatory synapses precedes the maturation of inhibitory afferents. Excitatory inputs to RGNs were to some extent functional before the animals opened their eyes. Glutamatergic synaptic activity may, thus, play an important role in shaping visual connections in the absence of visual experience.

Appearance of cGMP-phosphodiesterase immunoreactivity parallels the morphological differentiation of photoreceptor outer segments in the rat retina.

We have investigated by immunofluorescence the appearance of immunoreactive guanosine 3'-5' cyclic monophosphate phosphodiesterase (cGMP-PDE) during the postnatal development of the retina of the pigmented rat. We show that a sudden increase in immunoreactivity takes place during postnatal day five (P5), when rod outer segments begin to form; immunoreactivity develops rapidly in the following days. Labeling is restricted to the developing photoreceptor outer segments, sparing other retinal cells, as confirmed by electron microscopy immunocytochemistry. In addition, cGMP-PDE immunoreactivity follows a center-to-periphery gradient paralleling photoreceptor differentiation. It appears that cGMP-PDE is expressed when the photoreceptor subcellular compartments are already formed, and represents a specific marker of late photoreceptor differentiation. The appearance of cGMP-PDE during development is temporally correlated with the appearance of other proteins of the phototransduction machinery.

Ontogeny of 2f1-f2 acoustic and cochlear microphonic distortion products

The development of the acoustic distortion product (ADP) and cochlear microphonic distortion product (CMDP) 2f1-f2 was studied in pigmented rats, beginning 10 days after birth (p10). Both the ADP and CMDP were measured as a function of stimulus frequency (1.04 to 10.24 kHz with an f2/f1 ratio of 1.25) and level (20 to 80 dB SPL). The onset of the ADP occurred first at f2=5.12 kHz (p11–13), then at higher and lower frequencies. The amplitude of the ADP also matured first at f2=5.12 kHz (p17–18). This result is similar to the earlier work [S. J. Norton etal., Hear. Res. 51, 73–92 (1991); C. M. Henley etal., Hear. Res. 43, 141–148 (1989); M. Lenoir and J.-L. Puel, Hear Res. 29, 265–271 (1987)]. The onset and maturation of the DMDP was similar to ADP’s, except at lower frequencies where the CMDP developed somewhat earlier. There is a strong correlation in amplitudes of the ADPs and CMDPs across ages, consistent with the hypothesis that the ADP and CM DP may be linked to the same process [D. T. Kemp and A. M. Brown, Hear. Res. 13, 39–46 (1984)].

Rat retinal ganglion cells express Ca 2+-permeable non-NMDA glutamate receptors during the period of histogenetic cell death

Local application of glutamate agonists to retinal ganglion cells (RGNs) was performed in retinae isolated from pigmented rats aged between 3 and 8 days postnatally. A vast majority of RGNs displayed current responses to glutamate (Glu), N-methyl- d-aspartate (NMDA), quisqualate (QA), α-amino-2,3-dihydro-5-methyl-3-oxo-4-isoazolepropanoic acid (AMPA), kainate (KA) and domoate (DA). In Na +-free extracellular solution with elevated Ca 2+, non-NMDA agonists elicited large (up to 200 pA) inward currents that were completely blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) and Cd 2+. MK-801 also induced a partial block of cationic currents in Na +-free saline. In standard salt solutions, current-voltage relationships of Glu-R-mediated currents were often inwardly rectifying in the presence of d-aminophosphonovalerat (D-APV), as is typical of Ca 2+-permeable non-NMDA receptors. The presence of inward rectification in the current voltage relationship was always associated with a high value of the cationic permeability ratioP Ca 2+/ P Cs + (>0.8). However, in about half of the investigated RGNs no inward rectification was observed under standard recording conditions. Our results lead to the suggestion that expression of Ca 2+-permeable Glu receptor subunits may contribute to regulation of cell numbers in the postnatal retina.

Salicylate-induced phantom auditory effects on reinforced behavior in pre- and postweanling rats

In two behavioral experiments, normal hearing or salicylate-induced phantom auditory perception were investigated in young pigmented rats. Twenty-four hours prior to training in Experiment 1, 45 preweanling pups (14–21 days of age) were exposed to continuous background noise. They were injected with sodium salicylate or saline either before or after training to suppress approach responses to a lactating dam during noise offset periods. The sequence of injection initiation affected acquisition and extinction rates, and the oldest subjects behaved similarly to adults reported in studies of salicylate-induced auditory effects. Adapting the task in Experiment 2 for 18 postweanling pups 32 days of age produced greater suppression and prolonged extinction when salicylate was administered prior to both acquisition and extinction sessions, whereas subjects injected with saline before training and salicylate before extinction showed rapid recovery from suppression. The experiments support the notion that the auditory system undergoes functionally important change at around 15–16 days of age.

Dendritic competition in the developing retina: ganglion cell density gradients and laterally displaced dendrites.

Dendrites of retinal ganglion cells (RGCs) tend to be distributed preferentially toward areas of reduced RGC density. This, however, does not occur in the retina of normal pigmented rats, in which it has been suggested that the centro-peripheral gradient of RGC density is too shallow to provide directional guidance to growing dendrites. In this study, laterally displaced dendrites of RGCs retrogradely labeled with horseradish peroxidase were related to cell density gradients induced experimentally in the rat retina. Neonatal unilateral lesions of the optic tract produced retrograde degeneration of contralaterally projecting RGCs, but spared ipsilaterally projecting neurons in the same retina. These lesions created an anomalous temporal to nasal gradient of cell density across the decussation line, opposite to the nasal to temporal gradient found along the same axis in either normal rats or rats that had the contralateral eye removed at birth. RGCs in rats that received optic tract lesions had their dendrites displaced laterally toward the depleted nasal retina, while in either normal or enucleated rats there was no naso-temporal asymmetry. The lateral displacement affected both primary dendrites and higher-order branches. However, the gradient of cell density after optic tract lesions was less steep than the gradient in either normal or enucleated rats. To test for the presence of steeper gradients at early stages of development, RGC density gradients were also examined at postnatal day 5 (P5). In normal rats, the RGCs were homogeneously distributed throughout the retina, while rats given optic tract lesions at birth already showed a temporo-nasal density gradient at P5. Still, this anomalous gradient was less steep than that found in normal adults. It is concluded that the time course, rather than the steepness of the RGC density gradient, is the major determinant of the lateral displacement of dendritic arbors with respect to the soma in developing RGCs. The data are consistent with the idea that the overall shape of dendritic arbors depends in part on dendritic competition during retinal development.

Expression of GAP-43 in growing efferent fibers during cochlear development.

The development of olivocochlear efferent fibers has been studied by means of the immunocytochemical detection of the growth-associated protein GAP-43. This study has been carried out in pre- and postnatal, developing, pigmented rats. Results indicate that olivocochlear efferents reach the developing auditory epithelium from embryonic day 18 on. GAP-43-like immunoreactivity persists in the organ of Corti until the beginning of the second postnatal month. The relationships between the expression of GAP-43 in olivocochlear efferent fibers and other plasticity-related phenomena are discussed.

Diltiazem protects against neurotoxicity induced by excitotoxic amino acids on cochlear afferent fibers.

The neurotoxic effects of two glutamate agonists, kainic acid (KA) and monosodium glutamate (MSG), were analyzed in the pigmented rat cochlea. Neural damage occurring after KA or MSG treatments were evidenced by the presence of a majority of swollen fibers in the inner spiral bundle. The use of diltiazem (DI), a Ca2+ L-channel antagonist, to prevent cochlear neural damage induced by glutamate agonists resulted in a significant increase of unswollen afferent nerve fibers. MSG-treated animals, after DI pretreatment, recovered the normal latency of the compound action potential, and only unswollen nerve fibers were observed.