Comparison of central versus peripheral delivery of pregabalin in neuropathic pain states.

BACKGROUND: Although pregabalin therapy is beneficial for neuropathic pain (NeP) by targeting the CaVα2δ-1 subunit, its site of action is uncertain. Direct targeting of the central nervous system may be beneficial for the avoidance of systemic side effects. RESULTS: We used intranasal, intrathecal, and near-nerve chamber forms of delivery of varying concentrations of pregabalin or saline delivered over 14 days in rat models of experimental diabetic peripheral neuropathy and spinal nerve ligation. As well, radiolabelled pregabalin was administered to determine localization with different deliveries. We evaluated tactile allodynia and thermal hyperalgesia at multiple time points, and then analyzed harvested nervous system tissues for molecular and immunohistochemical changes in CaVα2δ-1 protein expression. Both intrathecal and intranasal pregabalin administration at high concentrations relieved NeP behaviors, while near-nerve pregabalin delivery had no effect. NeP was associated with upregulation of CACNA2D1 mRNA and CaVα2δ-1 protein within peripheral nerve, dorsal root ganglia (DRG), and dorsal spinal cord, but not brain. Pregabalin's effect was limited to suppression of CaVα2δ-1 protein (but not CACNA2D1 mRNA) expression at the spinal dorsal horn in neuropathic pain states. Dorsal root ligation prevented CaVα2δ-1 protein trafficking anterograde from the dorsal root ganglia to the dorsal horn after neuropathic pain initiation. CONCLUSIONS: Either intranasal or intrathecal pregabalin relieves neuropathic pain behaviours, perhaps due to pregabalin's effect upon anterograde CaVα2δ-1 protein trafficking from the DRG to the dorsal horn. Intranasal delivery of agents such as pregabalin may be an attractive alternative to systemic therapy for management of neuropathic pain states.

Melatonin modulates visual function and cell viability in the mouse retina via the MT1 melatonin receptor.

A clear demonstration of the role of melatonin and its receptors in specific retinal functions is lacking. The present study investigated the distribution of MT1 receptors within the retina, and the scotopic and photopic electroretinograms (ERG) and retinal morphology in wild-type (WT) and MT1 receptor-deficient mice. MT1 receptor transcripts were localized in photoreceptor cells and in some inner retinal neurons. A diurnal rhythm in the dark-adapted ERG responses was observed in WT mice, with higher a- and b-wave amplitudes at night, but this rhythm was absent in mice lacking MT1 receptors. Injection of melatonin during the day decreased the scotopic response threshold and the amplitude of the a- and b-waves in the WT mice, but not in the MT1(-/-) mice. The effects of MT1 receptor deficiency on retinal morphology was investigated at three different ages (3, 12, and 18 months). No differences between MT1(-/-) and WT mice were observed at 3 months of age, whereas at 12 months MT1(-/-) mice have a significant reduction in the number of photoreceptor nuclei in the outer nuclear layer compared with WT controls. No differences were observed in the number of cells in inner nuclear layer or in ganglion cells at 12 months of age. At 18 months, the loss of photoreceptor nuclei in the outer nuclear layer was further accentuated and the number of ganglion cells was also significantly lower than that of controls. These data demonstrate the functional significance of melatonin and MT1 receptors in the mammalian retina and create the basis for future studies on the therapeutic use of melatonin in retinal degeneration.

Circadian variations in coagulation and fibrinolytic factors among four different strains of mice.

This study examined circadian variation in coagulation and fibrinolytic parameters among Jcl:ICR, C3H/HeN, BALB/cA, and C57BL/6J strains of mice. Plasma plasminogen activator inhibitor 1 (PAI-1) levels fluctuated in a circadian manner and peaked in accordance with the mRNA levels at the start of the active phase in all strains. Fibrinogen mRNA levels peaked at the start of rest periods in all strains, although plasma fibrinogen levels remained constant. Strain differences in plasma antithrombin (AT) activity and protein C (PC) levels were then identified. Plasma AT activity was circadian rhythmic only in Jcl:ICR, but not in other strains, although the mRNA levels remained constant in all strains. Levels of plasma PC and its mRNA fluctuated in a circadian manner only in Jcl:ICR mice, whereas those of plasma prothrombin, factor X, factor VII, prothrombin time (PT), and activated partial thrombin time (APTT) remained constant in all strains. These results suggest that genetic heterogeneity underlies phenotypic variations in the circadian rhythmicity of blood coagulation and fibrinolysis. The circadian onset of thrombotic events might be due in part to the rhythmic gene expression of coagulation and fibrinolytic factors. The present study provides fundamental information about mouse strains that will help to understand the circadian variation in blood coagulation and fibrinolysis.

Localization of a circadian clock in mammalian photoreceptors.

Several studies have demonstrated that the mammalian retina contains an autonomous circadian clock. Dopaminergic and other inner retinal neurons express many of the clock genes, whereas some of these genes seem to be absent from the photoreceptors. This observation has led to the suggestion that in mammalian retina the circadian pacemaker driving retinal rhythms is located in the inner nuclear layer. However, other evidence points to the photoreceptor layer as the site of the mammalian retinal clock. The goal of the present study was to demonstrate the presence of a functional circadian clock in photoreceptors. First, using laser capture microdissection and reverse transcriptase-polymerase chain reaction, we investigated which of the clock genes are expressed in rat photoreceptors. We then prepared photoreceptor layer cultures from the retina to test whether these isolated cultures were viable and could drive circadian rhythms. Our data indicated that Per1, Per3, Cry1, Cry2, Clock, Bmal1, Rev-erb alpha, and Rora RNAs were present in the photoreceptors, whereas we were unable to amplify mRNA for Per2 and Npas2. Photoreceptor layers obtained from Period1-luciferase rats expressed a robust circadian rhythm in bioluminescence and melatonin synthesis. These results demonstrate that mammalian photoreceptors contain the circadian pacemaker driving rhythmic melatonin synthesis.

Clock gene expression in the rat retina: effects of lighting conditions and photoreceptor degeneration.

Previous studies have shown that, in the Royal College of Surgeon rat, circadian rhythms in the retinal dopaminergic and melatonergic systems are still present after the photoreceptors have degenerated, thus demonstrating that circadian rhythmicity in the mammalian retina can be generated independently from the photoreceptors. The aim of the present study was to investigate the pattern of expression of the clock genes in the retina of the Royal College of Surgeons rat under different lighting conditions. Expression of clock genes was investigated in the retina of normal and dystrophic Royal College of Surgeons rats under 12 h of light/12 h of dark (LD), constant darkness (DD) and constant light (LL) using Real Time Quantitative RT-PCR. Our data indicate that, in control animals, Period1, Period2, Cryptochrome1, Cryptochrome2, Clock, Rora, Rev-Erb alpha and Npas2 mRNA levels showed a significant variation over the sampling period in LD cycles and in DD, whereas Bmal1 mRNA did not show any significant variation. In LL, the transcripts for Per1, Per2, Clock and Rev-Erb alpha showed significant temporal variations. In the dystrophic retina, only Per1 and Per2 mRNA levels showed a temporal variation over the 20-h period. Our work indicates that degeneration of the photoreceptor cells dramatically affected the expression levels and patterns of many clock genes. Finally, the present study suggests that investigating the expression pattern of clock genes using the whole retina or animals with photoreceptor degeneration may not provide any definitive answers about the working of the retinal circadian clock system.

Effect of photoreceptor degeneration on circadian photoreception and free-running period in the Royal College of Surgeons rat.

The study of how the retina processes the photic information required for the entrainment of the circadian system is an exciting new topic in retinal neurobiology. We have recently shown that in RCS/N-rdy rats melanopsin mRNA levels are dramatically reduced (about 90%) and melanopsin immunoreactivity cannot be detected in the retina of these rats at 60 days of age. Although RCS/N-rdy rats are a widely used model to investigate mechanisms of photoreceptor degeneration, no study has investigated circadian photoreception in these animals. The aim of this study was to examine circadian photoreception in RCS/N-rdy(+) (rdy(+)) rats homozygous for the normal rdy allele and age-matched RCS/N-rdy (rdy) homozygotes with retinal dystrophy. No differences between RCS/N-rdy and rdy(+) were observed in light-induced phase shift of locomotor activity at the three light intensities used (1 x 10(-3), 1 x 10(-1), and 1 x 10(1) microW cm(-2)). Surprisingly, we observed that in RCS/N-rdy the free-running period of the circadian rhythm of locomotor activity was shorter (P<0.01) than in rdy(+), thus suggesting that photoreceptor degeneration may affect the free-running period of the locomotor activity rhythm.

Clock mutation affects circadian regulation of circulating blood cells.

BACKGROUND: Although the number of circulating immune cells is subject to high-amplitude circadian rhythms, the underlying mechanisms are not fully understood. METHODS: To determine whether intact CLOCK protein is required for the circadian changes in peripheral blood cells, we examined circulating white (WBC) and red (RBC) blood cells in homozygous Clock mutant mice. RESULTS: Daytime increases in total WBC and lymphocytes were suppressed and slightly phase-delayed along with plasma corticosterone levels in Clock mutant mice. The peak RBC rhythm was significantly reduced and phase-advanced in the Clock mutants. Anatomical examination revealed hemoglobin-rich, swollen red spleens in Clock mutant mice, suggesting RBC accumulation. CONCLUSION: Our results suggest that endogenous clock-regulated circadian corticosterone secretion from the adrenal gland is involved in the effect of a Clock mutation on daily profiles of circulating WBC. However, intact CLOCK seems unnecessary for generating the rhythm of corticosterone secretion in mice. Our results also suggest that CLOCK is involved in discharge of RBC from the spleen.

Intraocular injection of kainic acid does not abolish the circadian rhythm of arylalkylamine N-acetyltransferase mRNA in rat photoreceptors.

PURPOSE: Melatonin synthesis in mammalian retinal photoreceptors is under photic and circadian control and regulated by changes in the activity of arylalkylamine N-acetyltransferase (AANAT). Recent studies have suggested that retinal dopaminergic neurons contain a circadian pacemaker, and dopamine is the neurotransmitter that drives circadian rhythmicity in the mammalian retina. METHODS: To investigate the role of inner retinal neurons, including dopamine neurons, in generating the rhythm of melatonin synthesis, rat retinas were lesioned with kainic acid (KA), which was shown previously to induce degeneration of neurons in the inner nuclear layer and to eliminate rhythmicity in the dopaminergic system. Aanat, rhodopsin, medium wavelength (mwl) opsin, short wavelength (swl) opsin, and period1 (Per1), and period2 (Per2) mRNA levels were measured using real-time quantitative RT-PCR in KA injected and control eyes. RESULTS: Our data show that intraocular injections of KA did not abolish the daily and circadian rhythms of Aanat mRNA in the photoreceptors, but it did shift the phase of the Aanat transcript rhythm in constant darkness. Surprisingly, KA injections reduced the levels and eliminated daily rhythms of mwl and swl opsin transcripts, but not of rhodopsin mRNA. Per1 and Per2 mRNA levels were rhythmic in saline injected and in KA-treated retinas, and Per2 mRNA levels were significantly reduced (20-50%) in KA-treated retinas. CONCLUSIONS: These findings demonstrate that the circadian clock generating melatonin rhythmicity is largely KA insensitive and likely to be located in the rod photoreceptors, although KA-sensitive neurons do influence its timing. More important, our data demonstrate that dopamine rhythmicity is not necessary for generating the circadian rhythm of Aanat mRNA in the photoreceptors. Our data also indicate that Per1 and Per2 are rhythmically transcribed in the rat retina and KA treatment has a dramatic effect on the overall levels of Per2 mRNA.

Disrupted fat absorption attenuates obesity induced by a high-fat diet in Clock mutant mice.

The Clock gene is a core component of the circadian clock in mammals. We show here that serum levels of triglyceride and free fatty acid were significantly lower in circadian Clock mutant ICR than in wild-type control mice, whereas total cholesterol and glucose levels did not differ. Moreover, an increase in body weight induced by a high-fat diet was attenuated in homozygous Clock mutant mice. We also found that dietary fat absorption was extremely impaired in Clock mutant mice. Circadian expressions of cholecystokinin-A (CCK-A) receptor and lipase mRNAs were damped in the pancreas of Clock mutant mice. We therefore showed that a Clock mutation attenuates obesity induced by a high-fat diet in mice with an ICR background through impaired dietary fat absorption. Our results suggest that circadian clock molecules play an important role in lipid homeostasis in mammals.

Dopamine regulates melanopsin mRNA expression in intrinsically photosensitive retinal ganglion cells.

In mammals a subpopulation of retinal ganglion cells are intrinsically photosensitive (ipRGCs), express the photopigment melanopsin, and play an important role in the regulation of the nonimage-forming visual system. We have recently reported that melanopsin mRNA and protein levels in the rat retina are under photic and circadian control. The aim of the present work was to investigate the mechanisms that control melanopsin expression in the rat retina. We discovered that dopamine (DA) is involved in the regulation of melanopsin mRNA, possibly via dopamine D2 receptors that are located on these ipRGCs. Interestingly, we also discovered that pituitary adenylate cyclase-activating peptide (PACAP) mRNA levels are affected by DA. Dopamine synthesis and release in the retina are regulated by the rod and the cone photoreceptors via retinal circuitry; our new data indicate that DA controls melanopsin expression, indicating that classical photoreceptors may modulate the transcription of this new photopigment. Our study also suggests that DA may have an important role in mediating the light signals that are used for circadian entrainment and for other responses that are mediated by the nonimage-forming visual system.

Tissue-specific augmentation of circadian PAI-1 expression in mice with streptozotocin-induced diabetes.

Diabetes is associated with an excess risk of cardiac events, and the risk for infarction is partly determined by plasminogen activator inhibitor-1 (PAI-1). We found that plasma total and active PAI-1 levels increased in a circadian manner in mice with streptozotocin (STZ)-induced diabetes. Circadian expression of PAI-1 mRNA in the lung, heart, liver, and kidney increased in a tissue-specific manner. Peak to peak comparisons revealed that the mRNA expression levels increased by 1.7, 1.7, 1.2, and 1.6-fold in the heart, lung, liver, and kidney, respectively. In contrast, the circadian expression of the clock gene, mPer2, was preserved in the diabetic mice, suggesting that the altered expression of PAI-1 mRNA did not arise due to impaired circadian clocks. Our results suggest that impairment of the coagulation and fibrinolytic systems induced by diabetes is partly due to impaired circadian PAI-1 fluctuation at the level of mRNA expression.

Gene- and tissue-specific alterations of circadian clock gene expression in streptozotocin-induced diabetic mice under restricted feeding.

Circadian clocks are located in the suprachiasmatic nucleus (SCN) of the hypothalamus as well as in the peripheral tissues of mammals. Recent molecular studies have revealed that the phase of circadian gene expressions in peripheral clocks could entrain to time-imposed restricted feeding (RF) independently of the SCN. To elucidate whether endogenous insulin is involved in the entraining mechanisms of peripheral clocks to RF, we examined the expression profiles of clock genes in peripheral tissues of mice with diabetes induced by streptozotocin (STZ). The circadian expressing genes (mPer1, mPer2, and BMAL1) underwent a phase-shift induced by RF in the heart, liver, and kidney of both diabetic and normal animals. However, the expression phase of mPer1 in these tissues of the diabetic mice significantly differed from those in the normal animals under RF. The expression phase of the circadian output gene, albumin D-site binding protein (DBP), was completely shifted by RF both in normal and in diabetic mice, suggesting that the endogenous insulin is not essential for the entrainment of peripheral clocks to feeding cycles in mice.

Feeding is not a more potent Zeitgeber than the light-dark cycle in Drosophila.

Daily scheduled feeding is a potent Zeitgeber that elicits anticipatory activity in mammals. Recent studies have revealed that daytime feeding of nocturnal laboratory rodents completely inverts the phase of circadian gene expression in peripheral tissues such as heart, liver and kidney, independently of environmental light cycles. To investigate whether feeding is a potent time cue for Drosophila, we examined the behavioral activity rhythm and peripheral expression profile of clock genes in Drosophila under 12 h of night-time restricted feeding. We found that flies could not exhibit food-anticipatory activity rhythms under restricted feeding. Expression profiles of the clock genes period and timeless were not affected by either the phase or the amplitude in the periphery. These results suggest that feeding is not a more potent Zeitgeber than the light/dark cycle at either the individual behavioral level or at the peripheral molecular clock levels in Drosophila.