Gurprit S. Lall

Melanopsin cells are the principal conduits for rod-cone input to non-image-forming vision

Rod and cone photoreceptors detect light and relay this information through a multisynaptic pathway to the brain by means of retinal ganglion cells (RGCs). These retinal outputs support not only pattern vision but also non-image-forming (NIF) functions, which include circadian photoentrainment and pupillary light reflex (PLR). In mammals, NIF functions are mediated by rods, cones and the melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs). Rod–cone photoreceptors and ipRGCs are complementary in signalling light intensity for NIF functions. The ipRGCs, in addition to being directly photosensitive, also receive synaptic input from rod–cone networks. To determine how the ipRGCs relay rod–cone light information for both image-forming and non-image-forming functions, we genetically ablated ipRGCs in mice. Here we show that animals lacking ipRGCs retain pattern vision but have deficits in both PLR and circadian photoentrainment that are more extensive than those observed in melanopsin knockouts. The defects in PLR and photoentrainment resemble those observed in animals that lack phototransduction in all three photoreceptor classes. These results indicate that light signals for irradiance detection are dissociated from pattern vision at the retinal ganglion cell level, and animals that cannot detect light for NIF functions are still capable of image formation.

Distinct contributions of rod, cone, and melanopsin photoreceptors to encoding irradiance.

Summary Photoreceptive, melanopsin-expressing retinal ganglion cells (mRGCs) encode ambient light (irradiance) for the circadian clock, the pupillomotor system, and other influential behavioral/physiological responses. mRGCs are activated both by their intrinsic phototransduction cascade and by the rods and cones. However, the individual contribution of each photoreceptor class to irradiance responses remains unclear. We address this deficit using mice expressing human red cone opsin, in which rod-, cone-, and melanopsin-dependent responses can be identified by their distinct spectral sensitivity. Our data reveal an unexpectedly important role for rods. These photoreceptors define circadian responses at very dim “scotopic” light levels but also at irradiances at which pattern vision relies heavily on cones. By contrast, cone input to irradiance responses dissipates following light adaptation to the extent that these receptors make a very limited contribution to circadian and pupillary light responses under these conditions. Our data provide new insight into retinal circuitry upstream of mRGCs and optimal stimuli for eliciting irradiance responses.

2-Aminoethoxydiphenylborane is an acute inhibitor of directly photosensitive retinal ganglion cell activity in vitro and in vivo.

The mammalian retina contains directly photosensitive retinal ganglion cells (RGCs), which use the photopigment melanopsin. The generation of mice lacking melanopsin has been invaluable in elucidating the function of these cells. These animals display deficiencies in circadian photoentrainment, the pupil light reflex, and the circadian regulation of the cone pathway. Interpreting the results from such gene knock-out models is always complicated by neuronal plasticity and the potential for restructuring of neuronal networks. Until now, the study of photosensitive RGCs has lacked an acute inhibitor. 2-Aminoethoxydiphenylborane (2-APB) is an antagonist at IP3 receptors and an inhibitor of canonical transient receptor potential ion channels (TRPCs). Here, we show that 2-APB is an extremely potent in vitro inhibitor of the photosensitive RGCs and that its effect is independent of store-dependent Ca2+ release. The identification of canonical TRPC6 and TRPC7 ion channels in melanopsin-expressing ganglion cells suggests that 2-APB may act directly on a TRPC ion channel. Importantly, using the pupil light reflex as a functional assay, we show that 2-APB inhibits photosensitive RGC activity in vivo. Collectively, our data further elucidate the phototransduction pathway in the photosensitive RGCs and demonstrate that 2-APB can be used to silence activity in these cells both in vitro and in vivo.

How rod, cone, and melanopsin photoreceptors come together to enlighten the mammalian circadian clock

In mammals, a small number of retinal ganglion cells express melanopsin, an opsin photopigment, allowing them to be directly photoreceptive. A major function of these so-called intrinsically photosensitive retinal ganglion cells (ipRGCs) is to synchronize (entrain) endogenous circadian clocks to the external light:dark cycle. Thanks to their intrinsic light response, ipRGCs can support photoentrainment even when the other retinal photoreceptors (rods and cones) are absent or inactive. However, in the intact retina the ipRGC light response is a composite of extrinsic (rod/cone) and intrinsic (melanopsin) influences. As a result all three photoreceptor classes contribute to the retinal pathways providing light information to the clock. Here, we consider what each photoreceptor type contributes to the clock light response. We review electrophysiological and behavioral data pertinent to this question, primarily from laboratory rodents, drawing them together to provide a conceptual model in which each photoreceptor class plays a distinct role in encoding the light environment. We finally use this model to highlight some of the important outstanding questions in this field.

Gold Nanoparticles Downregulate Interleukin‐1β‐Induced Pro‐Inflammatory Responses

Interleukin 1 beta (IL-1β)-dependent inflammatory disorders, such as rheumatoid arthritis and psoriasis, pose a serious medical burden worldwide, where patients face a lifetime of illness and treatment. Organogold compounds have been used since the 1930s to treat rheumatic and other IL-1β-dependent diseases and, though their mechanisms of action are still unclear, there is evidence that gold interferes with the transmission of inflammatory signalling. Here we show for the first time that citrate-stabilized gold nanoparticles, in a size dependent manner, specifically downregulate cellular responses induced by IL-1β both in vitro and in vivo. Our results indicate that the anti-inflammatory activity of gold nanoparticles is associated with an extracellular interaction with IL-1β, thus opening potentially novel options for further therapeutic applications.

Attenuation of circadian light induced phase advances and delays by neuropeptide Y and a neuropeptide Y Y1/Y5 receptor agonist

Circadian rhythms can be synchronised to photic and non-photic stimuli. The circadian clock, anatomically defined as the suprachiasmatic nucleus in mammals, can be phase shifted by light during the night. Non-photic stimuli reset the circadian rhythm during the day. Photic and non-photic stimuli have been shown to interact during the day and night. Precise mechanisms for these complex interactions are unknown. A possible pathway for non-photic resetting of the clock is thought to generate from the intergeniculate leaflet, which conveys information to the suprachiasmatic nucleus (SCN) through the geniculohypothalamic tract and utilises neuropeptide Y (NPY) as its primary neurotransmitter. Interactions between light and NPY were investigated during the early (2 h after activity onset) and late (6 h after activity onset) night in male Syrian hamsters. NPY microinjections into the region of the SCN significantly attenuated light-induced phase delay, during the early subjective night. Phase advances to light were completely inhibited by the administration of NPY during the late night. The precise mechanism by which NPY attenuates or blocks photic phase shifts is unclear, but the NPY Y5 receptor has been implicated in the mediation of this inhibitory effect. The NPY Y1/Y5 receptor agonist, [Leu(31),Pro(34)]NPY, was administered via cannula microinjections following light exposure during the early and late night. [Leu(31),Pro(34)]NPY significantly attenuated phase delays to light during the early night and blocked phase advances during the late night, in a manner similar to NPY. These results show the ability of NPY to attenuate phase shifts to light during the early night and block light-induced phase advances during the late night. Furthermore, this is the first in vivo study implicating the involvement of the NPY Y1/Y5 receptors in the complex interaction of photic and non-photic stimuli during the night. The alteration of photic phase shifts by NPY may influence photic entrainment within the circadian system.

Attenuation of phase shifts to light by activity or neuropeptide Y: a time course study

Circadian rhythms in mammals can be synchronised to photic and non-photic stimuli. Interactions between photic and behavioural stimuli were investigated during the late subjective night, 6 h after activity onset in Syrian hamsters (CT18). Light pulses of 130 lx for 15 min at this time resulted in phase advance shifts. Novel wheel exposure, for a period of 3 h, following photic stimulation was able to attenuate the phase advancing effects of light. A time delay of up to 60 min between photic and behavioural stimuli also resulted in significant attenuation of light-induced phase shifts (P<0.05). A 90-min interval between stimuli resulted in no significant attenuation. Novel wheel exposure mediates its effects via the intergeniculate leaflet, which conveys information to the SCN and utilises neuropeptide Y (NPY) as its primary neurotransmitter. Phase shifts to light pulses given at CT18 were attenuated by NPY administration. Neuropeptide Y injections up to 60 min post-light exposure significantly attenuated phase shifts by 50% on average. However a 90-min interval between light and NPY microinjection did not significantly affect light-induced phase shifts. These results confirm previous work indicating that novel wheel exposure and NPY administration can modulate light-induced phase shifts during the late night. Further, they show for the first time that the time course for this interaction is similar between wheel running and NPY. Most significantly, our work indicates that the time course in vivo in the late night is similar to that shown previously in vitro during the early night.

Neuropeptide Y, GABA and circadian phase shifts to photic stimuli.

Circadian rhythms can be phase shifted by photic and non-photic stimuli. The circadian clock, anatomically defined as the suprachiasmatic nucleus (SCN), can be phase delayed by light during the early subjective night and phase advanced during the late subjective night. Non-photic stimuli reset the clock when presented during the subjective day. A possible pathway for the non-photic resetting of the clock is thought to originate from the intergeniculate leaflet, which conveys information to the SCN through the geniculohypothalamic tract and utilizes among others neuropeptide Y (NPY) and GABA as neurotransmitters. Photic and non-photic stimuli have been shown to interact during the early and late subjective night. Microinjections of NPY or muscimol, a GABA(A) receptor agonist, into the region of the SCN can attenuate light-induced phase shifts during the early and late subjective night. The precise mechanism for these interactions is unknown. In the current study we investigate the involvement of a GABAergic mechanism in the interaction between NPY and light during the early and late subjective night. Microinjections of NPY significantly attenuated light-induced phase delays and inhibited phase advances (P<0.05). The administration of bicuculline during light exposure, before NPY microinjection did not alter the ability of NPY to attenuate light-induced phase delays and block photic phase advances. These results indicate that NPY attenuates photic phase shifts via a mechanism independent of GABA(A) receptor activation. Furthermore it is evident that NPY influences circadian clock function via differing cellular pathways over the course of a circadian cycle.

Crucial involvement of xanthine oxidase in the intracellular signalling networks associated with human myeloid cell function

Xanthine oxidase (XOD) is an enzyme which plays a central role in purine catabolism by converting hypoxanthine into xanthine and then further into uric acid. Here we report that XOD is activated in THP-1 human myeloid cells in response to pro-inflammatory and growth factor stimulation. This effect occurred following stimulation of THP-1 cells with ligands of plasma membrane associated TLRs 2 and 4, endosomal TLRs 7 and 8 as well as stem cell growth factor (SCF). Hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) transcription complexes were found to be responsible for XOD upregulation. Importantly, the mammalian target of rapamycin (mTOR), a major myeloid cell translation regulator, was also found to be essential for XOD activation. Specific inhibition of XOD by allopurinol and sodium tungstate led to an increase in intracellular AMP levels triggering downregulation of mTOR activation by phosphorylation of its T2446 residue. Taken together, our results demonstrate for the first time that XOD is not only activated by pro-inflammatory stimuli or SCF but also plays an important role in maintaining mTOR-dependent translational control during the biological responses of human myeloid cells.

Dysfunctional mitochondria contain endogenous high-affinity human Toll-like receptor 4 (TLR4) ligands and induce TLR4-mediated inflammatory reactions.

Mitochondria, known to share many common features with prokaryotic cells, accumulate several endogenous ligands of the pattern-recognition Toll-like receptor 4 (TLR4), such as the heat shock proteins (Hsp) 70 and 60. TLR4 specifically recognises and responds to LPS of Gram-negative bacteria and participates in both autoimmune reactions and tissue regeneration due to its ability to recognise endogenous ligands. In the present study we show that mitochondria extracts obtained from hydrogen peroxide-dysfunctionalised cells induce a pro-inflammatory response in human THP-1 myeloid leukaemia cells. This inflammatory response was similar to that caused by LPS and much stronger than that induced by the extracts of normal mitochondria. Such reactions include activation of stress-adaptation hypoxia-inducible factor 1 alpha (HIF-1α) and expression/release of the pro-inflammatory cytokines IL-6 and TNF-α. Pre-treatment of THP-1 myeloid macrophages with TLR4-neutralising antibody before exposure to mitochondria extracts or LPS attenuated the inflammatory responses. Signalling pathways recruited by TLR4 in response to LPS and mitochondria-derived ligands were found to be the same. An in vitro ELISA-based TLR4-ligand binding assay, in which the ligand-binding domain of human TLR4 was immobilised, showed that mitochondria extracts contain endogenous TLR4 ligands. These results were verified in surface plasmon resonance experiments in which the affinity of the ligands derived from dysfunctional mitochondria was comparable with that of LPS and was much higher than that observed for normal mitochondria.