Pinopsin mRNA levels are significantly elevated in the pineal glands of chickens carrying a null mutation in guanylate cyclase-1.

The purpose of this study was to determine if the absence of guanylate cyclase-1 (RetGC1, GC1), a key visual phototransduction cascade enzyme that is expressed in both retinal photoreceptors and pinealocytes, disrupts light regulation of pinopsin mRNA levels in the chicken pineal gland. In this series of experiments, we compared levels of pinopsin and tryptophan 5-hydroxylase mRNA in the pineal glands of GUCY1*B (*B) and normal chickens housed under either cyclic light or constant dark conditions. The *B chicken carries a null mutation in the gene encoding guanylate cyclase-1 that results in blindness in these animals at hatching. The results of our experiments show (1) that the amount of pinopsin mRNA in *B pineal is significantly higher than the amount in normal pineal in both light and dark conditions, (2) that light induces an increase in pinopsin mRNA levels in *B pineal, (3) that the relative magnitude of the light-induced increase in pinopsin mRNA in *B pineal is not significantly different from that observed in normal pineal, and (4) that the changes in the regulation of pinopsin mRNA levels in *B pineal gland are not accompanied by changes in the circadian expression of tryptophan 5-hydroxylase mRNA. These results show that the absence of guanylate cyclase-1 expression in the *B pineal gland leads to a significant increase in basal levels of pinopsin mRNA in this gland but does not alter the magnitude of the increase in pinopsin mRNA levels that is observed as a result of light stimulation.

A null mutation in guanylate cyclase-1 alters the temporal dynamics and light entrainment properties of the iodopsin rhythm in cone photoreceptor cells.

Guanylate cyclase-1 (GC1) plays a critical role in visual phototransduction and its absence severely compromises the ability of the photoreceptor cells to transduce light for vision. In this study we sought to determine if the absence of GC1 has any effect on light entrainment of the circadian oscillators located in these cells. We compared the rhythmic changes in transcript levels of iodopsin, a photoreceptor-specific gene whose expression is regulated by circadian oscillators, in retinas of normal chickens and GUCY1*B (*B) chickens that carry a null mutation in GC1. Our results show that iodopsin rhythms are present in *B retinas and that they can be entrained to light; however, the rise and fall of iodopsin transcript levels in *B retina under cyclic light conditions is significantly more rapid than that observed in normal retina, and under constant dark conditions, the phase of the iodopsin rhythm in *B retina is advanced by 6 h relative to that observed in normal retina. In addition, the rate of entrainment of the iodopsin rhythm in *B retina to a reversal of the light cycle is significantly slower than normal. The results of our study show that a functioning visual phototransduction cascade is not essential for light entrainment of the oscillators that drive the iodopsin rhythm in photoreceptor cells. We propose that the abnormal synthesis of cGMP in *B photoreceptors underlies the irregular iodopsin rhythms observed in post-hatch *B retina.

Comparative evaluation of cytokine profiles and reactive gliosis supports a critical role for interleukin-6 in neuron-glia signaling during regeneration.

Using reverse transcription polymerase chain reaction (RT-PCR), we have studied the temporal expression of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), transforming growth factor-beta 1 (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) mRNAs in three axotomy paradigms with distinct functional outcomes. Axotomy of adult rat facial motoneurons results in neuronal regeneration, axotomy of neonatal facial motoneurons results in neuronal apoptosis, and axotomy of rubrospinal neurons results in neuronal atrophy. Our RT-PCR findings show that a significant and sustained upregulation of IL-6 mRNA is associated uniquely with the regeneration of adult facial motoneurons. Histochemical studies using IL-6 immunohistochemistry show intense IL-6 immunoreactivity in axotomized adult facial motoneurons. Assessment of reactive glial changes with astroglial and microglial markers reveals that the reactive gliosis following adult facial nerve axotomy is more intense than that observed in either of the other two paradigms. Exposure of cultured microglial cells to IL-6 stimulates microglial proliferation in a dose-dependent manner. Cultured microglia also show expression of IL-6 receptor mRNA, as determined by RT-PCR. Our findings support the idea that reactive gliosis is required for neuron regeneration to occur, and more specifically, they suggest that neuron-derived IL-6 serves as a signalling molecule that induces microglial proliferation during motoneuron regeneration.

Characterization of the chicken GCAP gene array and analyses of GCAP1, GCAP2, and GC1 gene expression in normal and rd chicken pineal.

PURPOSE: This study had three objectives: (1) to characterize the structures of the chicken GCAP1 and GCAP2 genes; (2) to determine if GCAP1, GCAP2, and GC1 genes are expressed in chicken pineal gland; (3) if GC1 is expressed in chicken pineal, to determine if the GC1 null mutation carried by the retinal degeneration (rd) chicken is associated with degenerative changes within the pineal glands of these animals. METHODS: GCAP1 and GCAP2 gene structures were determined by analyses of chicken cosmid and cDNA clones. The putative transcription start points for these genes were determined using 5'-RACE. GCAP1, GCAP2 and GC1 transcripts were analyzed using Northern blot and RT-PCR. Routine light microscopy was used to examine pineal morphology. RESULTS: Chicken GCAP1 and GCAP2 genes are arranged in a tail-to-tail array. Each protein is encoded by 4 exons that are interrupted by 3 introns of variable length, the positions of which are identical within each gene. The putative transcription start points for GCAP1 and GCAP2 are 314 and 243 bases upstream of the translation start codons of these genes, respectively. As in retina, GCAP1, GCAP2 and GC1 genes are expressed in the chicken pineal. Although the GC1 null mutation is present in both the retina and pineal of the rd chicken, only the retina appears to undergo degeneration. CONCLUSIONS: The identical arrangement of chicken, human, and mouse GCAP1/2 genes suggests that these genes originated from an ancient gene duplication/inversion event that occurred during evolution prior to vertebrate diversification. The expression of GC1, GCAP1, and GCAP2 in chicken pineal is consistent with the hypothesis that chicken pineal contains a functional phototransduction cascade. The absence of cellular degeneration in the rd pineal gland suggests that GC1 is not critical for pineal cell survival.

Circadian regulation of iodopsin and clock is altered in the retinal degeneration chicken retina.

We are interested in determining if the visual phototransduction cascade plays a role in light entrainment of photoreceptor circadian oscillators. In this study, we compared mRNA levels of iodopsin and the chicken homolog of Clock (cClock) in the retinas of normal and rd (retinal degeneration) chickens that lack functional rod and cone phototransduction cascades. Iodopsin is a circadian-regulated, photoreceptor-specific gene expressed in chicken retina, and Clock is a transcription factor that has been shown to play a role in the circadian clock mechanism in mouse and Drosophila. The results of our analyses show that cClock and iodopsin transcript levels undergo daily oscillations in retinas of normal animals housed under 12 h light:12 h dark (12L:12D) conditions, and that these oscillations are maintained in the absence of light. Levels of these transcripts in the retinas of rd/rd chickens housed under cyclic light conditions did not change significantly over the course of a 12L:12D cycle; however, there was evidence that the photoreceptor oscillators were entrained in these animals. Comparisons of our normal and rd/rd data suggest that there are at least two light entrainment pathways that impinge on the oscillators found in photoreceptor cells, one of which is effectively disabled by the GC1 null mutation carried by the rd chicken.

Cytokine transcripts expressed by microglia in vitro are not expressed by ameboid microglia of the developing rat central nervous system.

Because of morphological similarities between ameboid microglia in the developing central nervous system (CNS), brain macrophages in the injured CNS, and cultured microglia in vitro, it is thought that these cell types are functionally equivalent. To investigate the validity of this assumption, we have compared mRNA levels of interleukin-1alpha and -1beta (IL-1alpha and IL-1beta), tumor necrosis factor-alpha and -beta (TNF-alpha and TNF-beta), transforming growth factor-beta1 (TGF-beta1), and macrophage colony-stimulating factor (M-CSF) in the postnatal day 4 (P4) supraventricular corpus callosum (SVCC) with those in unstimulated cultured microglia. Control tissues included spleen, cortex, hippocampus, and cerebellum. Our analyses have shown that while IL-1alpha, IL-1beta, TNF-alpha, TNF-beta, and TGF-beta1 transcripts are abundantly expressed by cultured microglia, they are very low to virtually undetectable in the SVCC. These data strongly suggest that ameboid microglia, which are concentrated in the SVCC, are unlikely to be a significant source of these cytokines. Our study, which shows clear differences in the functional status of cultured microglia vs. ameboid microglia in vivo, stresses the importance of using caution when interpreting in vitro findings in terms of the in vivo functions of microglia.

Relation of optical coherence tomography to microanatomy in normal and rd chickens.

PURPOSE: To elucidate the relation between optical coherence tomography (OCT) scans and retinal histology in normal and retinal degeneration (rd) chickens. METHODS: Retinas from adult normal and rd chickens were examined in vivo with OCT at 850 nm and compared quantitatively with stained cryosections of unfixed retinas from the same locations. RESULTS: The nerve fiber layer (NFL) and inner plexiform layer (IPL) show homogeneous backscatter throughout their thicknesses. NFL reflectivity is approximately 0.6 log units higher than that of the IPL. The inner nuclear layer shows a low reflectivity; the properties of reflections from ganglion cell and outer nuclear layers are indeterminate. The outer retina and choroid form a large reflective complex. Photoreceptor inner segments produce the highest of these reflections in normal chicken retinas, approximately 1.5 log units higher than that of the IPL. The retinal pigment epithelium also has a relatively large backscatter coefficient and is the dominant reflector in rd retinas that lack photoreceptors. Choroidal pigment produces an intermediate level of backscatter and is the largest attenuator of signal at 850 nm. CONCLUSIONS: Quantified OCT signals have a predictable relationship to histology and pathology in chicken retinas. The results from rd retinas represent a first step toward in vivo quantitation of retinal structure in retinal degenerative disease.

Cytokine mRNA profiles in contused spinal cord and axotomized facial nucleus suggest a beneficial role for inflammation and gliosis.

We have studied temporal mRNA expression patterns for interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), macrophage colony stimulating factor (M-CSF), and transforming growth factor-beta1 (TGF-beta1) in two rat injury paradigms with very different cellular inflammatory reactions: contussion of the spinal cord and axotomy of the facial nerve. Our comparative analyses using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) show an early and robust upregulation of IL-1beta, TNF-alpha, IL-6, and M-CSF mRNAs in spinal cord after contusion injury. Peak expression of these mRNAs was transient and returned to control levels by 24 h postinjury. In contrast, expression of IL-1beta and TNF-alpha mRNAs in the axotomized facial nucleus was minimal and delayed, and levels of M-CSF mRNA remained unaltered. Similar to injured spinal cord, the axotomized nucleus showed a dramatic and early upregulation of IL-6 mRNA, but unlike spinal cord, IL-6 mRNA levels subsided only gradually. Both injury paradigms showed gradually increasing levels of TGF-beta1 mRNA which were maximal at 7 days postinjury. RT-PCR analyses were also performed on isolated blood-borne mononuclear cells and neutrophils. The results showed that these cells contain high levels of IL-1beta and M-CSF mRNAs, moderate levels of TGF-beta and TNF-alpha mRNAs, and minimal levels of IL-6 mRNA. The RT-PCR analyses together with histological observations indicate that expression of the proinflammatory cytokines IL-1beta, TNF-alpha, and IL-6 is short-lived and self-limited after contusion injury, and that it occurs primarily within endogenous glial cells. Transient expression of these molecules likely triggers secondary events which may be beneficial to wound repair and regeneration.

A null mutation in the photoreceptor guanylate cyclase gene causes the retinal degeneration chicken phenotype.

The retinas of the retinal degeneration (rd) chicken are fully developed and possess normal morphology at hatching but fail to respond to light stimulation. Analyses of retinal cGMP, the internal messenger of phototransduction, show that the amount of cGMP in predegenerate, fully developed rd/rd photoreceptors is 5-10 times less than that seen in normal photoreceptor cells. We show that the low levels of cGMP in rd chicken retina are a consequence of a null mutation in the photoreceptor guanylate cyclase (GC1) gene. Thus, the rd chicken is a model for human Leber's congenital amaurosis. Absence of GC1 in rd retina prevents phototransduction and affects survival of rods and cones but does not interfere with normal photoreceptor development.

Effects on white Leghorn hens of constant exposure to ultraviolet light from insect traps.

Constant exposure of Hy-Line W-36 White Leghorn hens to ultraviolet light from insect traps resulted in no significant differences in egg production, fertility, hatchability of fertile eggs, or total hatchability. Also, there were no apparent effects on the eyes of the birds. Results were the same when either blacklight or blacklight blue tubes were used. The need for additional testing of light traps for nuisance fly control in commercial caged layer houses is discussed.

Effects of dexfenfluramine or 5,7-dihydroxytryptamine on tryptophan hydroxylase and serotonin transporter mRNAS in rat dorsal raphe.

Dexfenfluramine (DF), given in high doses, can produce long-lasting decreases in brain levels of serotonin (5-HT) and 5-HT transporter (5-HTT) protein. The purpose of this study was to determine if DF-induced decreases in 5-HT and 5-HTT in rat forebrain are correlated with compensatory changes in the expression of the genes for tryptophan hydroxylase (TPH) and 5-HTT in the dorsal raphe nucleus. Gene transcripts were measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Rats were treated with either one or eight injections of DF at either high (10 mg/kg) or low (2 mg/kg) doses. A positive control group for 5-HT cell loss received a single cerebroventricular injection of 5,7-dihydroxytryptamine (DHT). Rats were killed either 5, 15 or 30 days after their last treatment. Paroxetine binding to the 5-HTT protein in frontal cortex was, as expected, reduced in all of the treated groups relative to vehicle controls. TPH mRNA levels in the dorsal raphe of animals that received DHT were significantly higher than those measured in all other treatment groups 15 days following treatment. By 30 days, the amount of TPH mRNA in DHT-treated rats had fallen to well below control levels. None of the DF regimens significantly affected TPH mRNA levels. Unlike the TPH mRNA changes in DHT-treated rats, the 5-HTT mRNA levels in the dorsal raphe declined progressively throughout the 30 day survival period. None of the DF regimens significantly affected 5-HTT mRNA levels. The significance of these data are discussed in terms of whether loss of forebrain markers for 5-HT reflects either the loss of fine caliber 5-HT axon terminals or a decrease in the expression of these markers in the somata of these cells which are located in the dorsal raphe.

Expression of GCAP1 and GCAP2 in the retinal degeneration (rd) mutant chicken retina.

We cloned the guanylate cyclase activating proteins, GCAP1 and GCAP2, from chicken retina and examined their expression in normal and predegenerate rdlrd chicken retina. Northern analyses show that the amounts of the single transcripts encoding GCAP1 and GCAP2 are reduced to about 70% of normal levels in rdlrd retina. Western analyses reveal that GCAP2 levels appear normal in this retina, while GCAP1 levels are reduced by more than 90%. The specific downregulation of GCAP1 in rdlrd retina is consistent with a model for this disease in which activation of guanylate cyclase in the photoreceptors is abnormal, resulting in low levels of cGMP and an absence of phototransduction.

Analysis of TGF-beta 1 gene expression in contused rat spinal cord using quantitative RT-PCR.

We have used northern blot analysis and quantitative reverse transcription polymerase chain reaction (RT-PCR) to determine the postinjury expression profile of the transforming growth factor-beta 1 (TGF-beta 1) gene in the contused rat spinal cord. Spectrophotometric estimates of total sample RNA and quantitative analyses of cyclophilin mRNA using RT-PCR served as controls for comparisons between samples. No changes in cyclophilin gene expression were found at any postinjury survival times. The results of the TGF-beta 1 analyses, which were carried out on spinal cord samples taken at postinjury intervals ranging from 6 h to 10 days, show that the amount of TGF-beta 1 mRNA present in spinal cord increases rapidly following injury, reaching maximum levels 7 days postinjury. Unoperated control samples contained approximately 2 x 10(8) molecules of TGF-beta 1 mRNA/0.5 microgram total RNA. By 1 day postinjury, the amount of TGF-beta 1 mRNA in the cord had increased by a factor of 2.5 to 5 x 10(8) molecules/0.5 microgram total RNA. At 7 days postinjury, there were approximately 15 x 10(8) molecules of TGF-beta 1 mRNA/0.5 microgram total RNA. By 10 days postinjury the amount of TGF-beta 1 mRNA present in the spinal cord had declined to 8 x 10(8) molecules of TGF-beta 1 mRNA/0.5 microgram total RNA, a value similar to that observed at 3 days postinjury. The roles that TGF-beta 1 might play in modifying cellular responses in injured spinal cord are discussed.

Molecular and biochemical analyses of iodopsin in rd chick retina.

PURPOSE: The results of previous immunocytochemical and electrophysiological studies of retinas of rd (retinal degeneration) chicks suggest that the iodopsin cone visual pigment may be defective in this mutant. The goal of this study was to determine if the primary structure and synthesis of this protein is normal in this animal model of inherited retinal degeneration. METHODS: Northern cDNA sequence and western analyses were used to study rd/rd iodopsin. cDNAs encoding rd/rd iodopsin were obtained by screening an rd/rd cDNA retinal expression library and by reverse transcription PCR. Western blots were probed with either R4 or COS-1, two different monoclonal antibodies that have been shown to specifically recognize chicken iodopsin. RESULTS: Hybridization of the +/+, +/rd, and rd/rd poly(A)+ RNA with an iodopsin cDNA probe revealed the presence of a single 1.5 kb band in each of the samples, all of which were labeled with equal intensity. No significant differences were found between the published nucleic acid sequences for normal chicken iodopsin cDNA and that determined for rd/rd iodopsin cDNA. Antibody-dependent differences in the staining intensity of the 34 kDa band containing iodopsin were observed on western blots of +/+, +/rd, and rd/rd retinal protein. R4 stained the 34 kDa band in each sample with equal intensity. COS-1 labeling of the 34 kDa band in the rd/rd sample was less intense than that observed in the +/+ and +/rd samples. CONCLUSIONS: Based on the results of the cDNA sequence and northern blot experiments, the authors conclude that the gene encoding iodopsin and transcription of this gene are normal in the rd mutant. The results of the western blot analyses of rd/rd iodopsin suggest that post-translational processing of iodopsin may be abnormal in this mutant.

Oligodendrocytes produce low molecular weight glycoproteins containing N-acetyl-D-glucosamine in their Golgi apparatus.

Lectin histochemistry using the Griffonia simplicifolia II lectin (GSL II) has revealed a novel group of glycoproteins containing terminal N-acetyl-D-glucosamine (GlcNAc) residues in oligodendrocytes. The GlcNAc-containing glycoproteins were not present in other types of glial cells, but were expressed by some neuronal cell populations. Within oligodendrocytes their localization was confined to the Golgi apparatus, as determined ultrastructurally. Biochemical analyses using tricine/SDS-polyacrylamide gel electrophoresis and western blotting with GSL II showed the GlcNAc-containing glycoproteins to be insoluble, with molecular masses ranging from 15 to 30 kDa. Our study provides a first account of insoluble, GlcNAc-rich 15-30 kDa glycoproteins in oligodendroglia. The findings are discussed in the context of the functional significance of other known oligodendrocyte glycoproteins.

Loss of serotonin uptake sites and immunoreactivity in rat cortex after dexfenfluramine occur without parallel glial cell reactions.

The frontal cortices of rats which received either D,L- or D-fenfluramine (DFEN) for 4 days were examined 18 h to 2 weeks following treatment for changes in synaptosomal uptake of serotonin (5HT), paroxetine binding, 5HT-immunoreactivity (5HT-IR), and both astrocytic (GFAP) and microglial markers. Additional rats received intracerebroventricular injections of the neurotoxin 5,7-dihydroxytryptamine (DHT). Consistent with previous reports, D,L- and DFEN produced dose-dependent losses of both 5HT uptake and paroxetine binding, and loss of 5HT-IR which coincided with an abnormal or 'swollen' appearance of immunoreactive axon processes. Recovery of these serotonergic indices was greatest following the lowest doses of DFEN, but was absent after 5,7-DHT treatment. No evidence for an increase in GFAP synthesis or microglial activation was observed in frontal cortices of rats treated with either DFEN or 5,7-DHT. We conclude that the presence of swollen 5HT-IR axons in the cortices of both the 5,7-DHT and DFEN groups is insufficient to trigger the glial responses often associated with neuronal degeneration. Thus, it remains to be determined if swollen 5HT-IR axons are a prelude to neurodegeneration, or whether they represent reversible changes in axonal immunochemistry associated with decreases in 5HT levels. The implications of the data for the clinical safety of DFEN are briefly discussed.

Visinin: biochemical and molecular comparisons in normal and rd chick retina.

Western, northern and DNA sequence analyses were used to determine if the retinal protein, visinin, is defective in the chicken retinal degeneration mutant, rd. A 22kDa band, corresponding to purified visinin, was stained with equal intensity on Western blots of +/+, +/rd and rd/rd retinal protein probed with a visinin polyclonal antibody. Hybridization of a northern blot of +/+, +/rd and rd/rd poly(A)+ RNA with a random-primer labelled visinin cDNA probe showed a single, equally labelled 1-Kbp band in each of the samples. Finally, no differences were found between the nucleic acid sequences of the 579 bp cDNAs encoding +/+ and rd/rd visinin. We did, however, find one significant difference between our visinin DNA sequence and the previously published chick visinin DNA sequence. We consistently observed a C at position 118 rather than the published T which changes the amino acid residue at position 40 from serine to proline. Based on the results of this study, we conclude that visinin is not defective in the rd chick model of hereditary retinal degeneration.