Intrinsic photosensitive retinal ganglion cells in the diurnal rodent, Arvicanthis ansorgei.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) represent a new class of photoreceptors which support a variety of non-image forming physiological functions, such as circadian photoentrainment, pupillary light reflex and masking responses to light. In view of the recently proposed role of retinal inputs for the regulation of diurnal and nocturnal behavior, we performed the first deep analysis of the ipRGC system in a diurnal rodent model, Arvicanthisansorgei, and compared the anatomical and physiological properties of ipRGCs with those of nocturnal mice. Based on somata location, stratification pattern and melanopsin expression, we identified two main ipRGC types in the retina of Arvicanthis: M1, constituting 74% of all ipRGCs and non-M1 (consisting mainly of the M2 type) constituting the following 25%. The displaced ipRGCs were rarely encountered. Phenotypical staining patterns of ganglion cell markers showed a preferential expression of Brn3 and neurofilaments in non-M1 ipRGCs. In general, the anatomical properties and molecular phenotyping of ipRGCs in Arvicanthis resemble ipRGCs of the mouse retina, however the percentage of M1 cells is considerably higher in the diurnal animal. Multi-electrode array recordings (MEA) identified in newborn retinas of Arvicanthis three response types of ipRGCs (type I, II and III) which are distinguished by their light sensitivity, response strength, latency and duration. Type I ipRGCs exhibited a high sensitivity to short light flashes and showed, contrary to mouse type I ipRGCs, robust light responses to 10 ms flashes. The morphological, molecular and physiological analysis reveals very few differences between mouse and Arvicanthis ipRGCs. These data imply that the influence of retinal inputs in defining the temporal niche could be related to a stronger cone input into ipRGCs in the cone-rich Arvicanthis retina, and to the higher sensitivity of type I ipRGCs and elevated proportion of M1 cells.

The output signal of Purkinje cells of the cerebellum and circadian rhythmicity.

Measurement of clock gene expression has recently provided evidence that the cerebellum, like the master clock in the SCN, contains a circadian oscillator. The cerebellar oscillator is involved in anticipation of mealtime and possibly resides in Purkinje cells. However, the rhythmic gene expression is likely transduced into a circadian cerebellar output signal to exert an effective control of neuronal brain circuits that are responsible for feeding behavior. Using electrophysiological recordings from acute and organotypic cerebellar slices, we tested the hypothesis whether Purkinje cells transmit a circadian modulated signal to their targets in the brain. Extracellular recordings from brain slices revealed the typical discharge pattern previously described in vivo in single cell recordings showing basically a tonic or a trimodal-like firing pattern. However, in acute sagittal cerebellar slices the average spike rate of randomly selected Purkinje cells did not exhibit significant circadian variations, irrespective of their specific firing pattern. Also, frequency and amplitude of spontaneous inhibitory postsynaptic currents and the amplitude of GABA- and glutamate-evoked currents did not vary with circadian time. Long-term recordings using multielectrode arrays (MEA) allowed to monitor neuronal activity at multiple sites in organotypic cerebellar slices for several days to weeks. With this recording technique we observed oscillations of the firing rate of cerebellar neurons, presumably of Purkinje cells, with a period of about 24 hours which were stable for periods up to three days. The daily renewal of culture medium could induce circadian oscillations of the firing rate of Purkinje cells, a feature that is compatible with the behavior of slave oscillators. However, from the present results it appears that the circadian expression of cerebellar clock genes exerts only a weak influence on the electrical output of cerebellar neurons.

The insulin-like growth factor 1 receptor is essential for axonal regeneration in adult central nervous system neurons.

Axonal regeneration is an essential condition to re-establish functional neuronal connections in the injured adult central nervous system (CNS), but efficient regrowth of severed axons has proven to be very difficult to achieve. Although significant progress has been made in identifying the intrinsic and extrinsic mechanisms involved, many aspects remain unresolved. Axonal development in embryonic CNS (hippocampus) requires the obligate activation of the insulin-like growth factor 1 receptor (IGF-1R). Based on known similarities between axonal growth in fetal compared to mature CNS, we decided to examine the expression of the IGF-1R, using an antibody to the ßgc subunit or a polyclonal anti-peptide antibody directed to the IGF-R (C20), in an in vitro model of adult CNS axonal regeneration, namely retinal ganglion cells (RGC) derived from adult rat retinas. Expression of both ßgc and the ß subunit recognized by C20 antibody were low in freshly isolated adult RGC, but increased significantly after 4 days in vitro. As in embryonic axons, ßgc was localised to distal regions and leading growth cones in RGC. IGF-1R-ßgc co-localised with activated p85 involved in the phosphatidylinositol-3 kinase (PI3K) signaling pathway, upon stimulation with IGF-1. Blocking experiments using either an antibody which neutralises IGF-1R activation, shRNA designed against the IGF-1R sequence, or the PI3K pathway inhibitor LY294002, all significantly reduced axon regeneration from adult RGC in vitro (∼40% RGC possessed axons in controls vs 2-8% in the different blocking studies). Finally, co-transfection of RGC with shRNA to silence IGF-1R together with a vector containing a constitutively active form of downstream PI3K (p110), fully restored axonal outgrowth in vitro. Hence these data demonstrate that axonal regeneration in adult CNS neurons requires re-expression and activation of IGF-1R, and targeting this system may offer new therapeutic approaches to enhancing axonal regeneration following trauma.

Heterogeneity of intrinsically photosensitive retinal ganglion cells in the mouse revealed by molecular phenotyping.

Intrinsically photosensitive retinal ganglion cell (ipRGC) types can be distinguished by their dendritic tree stratification and intensity of melanopsin staining. We identified heavily stained melanopsin-positive M1 cells branching in the outermost part of the inner plexiform layer (IPL) and weakly melanopsin-positive M2 cells branching in the innermost layer of the IPL. A third type can be distinguished by the displacement of the soma to the inner nuclear layer and has morphological similarities with either M1 cells or M2 cells, and is termed here displaced or M-d cells. The aim of the present study was to examine the phenotypic traits of ipRGC types. Using whole retinae from adult mice, we performed immunohistochemistry using melanopsin immunostaining and a number of antibodies directed against proteins typically expressed in retinal ganglion cells. The majority of M1 and M2 ipRGCs expressed Isl-1, microtubule associated protein-2 (MAP2), γ-synuclein, and NeuN, whereas Brn3 transcription factor and the different neurofilaments (NF68, NF160, NF200) were able to discriminate between ipRGC subtypes. Brn3 was expressed preferentially in M2 cells and in a small subpopulation of weakly melanopsin-positive M-d cells with similarities to M2 cells. All three neurofilaments were primarily expressed in large M2 cells with similarities to the recently described alpha-like M4 cells, but not in M1 cells. Expression of NF68 and NF160 was also observed in a few large M-d ipRGCs. These findings show that ipRGCs are not a phenotypically homogenous population and that specific neuronal markers (Brn3 and neurofilament) can partly distinguish between different ipRGC subtypes.

Activation of glycine receptor phase-shifts the circadian rhythm in neuronal activity in the mouse suprachiasmatic nucleus.

In mammals, the master clock in the suprachiasmatic nucleus (SCN) of the hypothalamus is composed of numerous synchronized oscillating cells that drive daily behavioural and physiological processes. Several entrainment pathways, afferent inputs to the SCN with their neurotransmitter and neuromodulator systems, can reset the circadian system regularly and also modulate neuronal activity within the SCN. In the present study, we investigated the function of the inhibitory neurotransmitter glycine on neuronal activity in the mouse SCN and on resetting of the circadian clock. The effects of glycine on the electrical activity of SCN cells from C57Bl/6 mice were studied either by patch-clamp recordings from acute brain slices or by long-term recordings from organotypic brain slices using multi-microelectrode arrays(MEA). Voltage-clamp recordings confirmed the existence of glycine-induced, chloride-selective currents in SCN neurons. These currents were reversibly suppressed by strychnine, phenylbenzeneω-phosphono-α-amino acid (PMBA) or ginkgolide B, selective blockers of glycine receptors(GlyRs). Long-term recordings of the spontaneous activity of SCN neurons revealed that glycine application induces a phase advance during the subjective day and a phase delay during the early subjective night. Both effects were suppressed by strychnine or by PMBA. These results suggest that glycine is able to modulate circadian activity by acting directly on its specific receptors in SCN neurons.