4-Aminopyridine Does Not Enhance Flocculus Function in Tottering,a Mouse Model of Vestibulocerebellar Dysfunction and Ataxia

The potassium channel antagonist 4-aminopyridine (4-AP) improves a variety of motor abnormalities associated with disorders of the cerebellum. The most rigorous quantitative data relate to 4-AP''s ability to improve eye movement deficits in humans referable to dysfunction of the cerebellar flocculus. Largely based on work in the ataxic mouse mutant tottering (which carries a mutation of the Cacna1a gene of the P/Q voltage-activated calcium channel), 4-AP is hypothesized to function by enhancing excitability or rhythmicity of floccular Purkinje cells. We tested this hypothesis by determining whether systemic or intrafloccular administration of 4-AP would ameliorate the eye movement deficits in tottering that are attributable to flocculus dysfunction, including the reductions in amplitude of the yaw-axis vestibulo-ocular reflex (VOR) and vision-enhanced vestibulo-ocular reflex (VVOR), and the optokinetic reflex (OKR) about yaw and roll axes. Because tottering''s deficits increase with age, both young and elderly mutants were tested to detect any age-dependent 4-AP effects. 4-AP failed to improve VOR, VVOR, and OKR gains during sinusoidal stimuli, although it may have reduced the tendency of the mutants'' responses to VOR and VVOR to decline over the course of a one-hour recording session. For constant-velocity optokinetic stimuli, 4-AP generated some enhancement of yaw OKR and upward-directed roll OKR, but the effects were also seen in normal C57BL/6 controls, and thus do not represent a specific reversal of the electrophysiological consequences of the tottering mutation. Data support a possible extra-floccular locus for the effects of 4-AP on habituation and roll OKR. Unilateral intrafloccular 4-AP injections did not affect ocular motility, except to generate mild eye elevations, consistent with reduced floccular output. Because 4-AP did not produce the effects expected if it normalized outputs of floccular Purkinje cells, there is a need for further studies to elucidate the drug''s mechanism of action on cerebellar motor dysfunction.     

Dopamine D1 Receptors Regulate the Light Dependent Development of Retinal Synaptic Responses

Retinal synaptic connections and function are developmentally regulated. Retinal synaptic activity plays critical roles in the development of retinal synaptic circuitry. Dopamine receptors have been thought to play important roles in the activity-dependent synaptic plasticity in central nervous system. The primary goal of this study is to determine whether dopamine D1 receptor regulates the activity-dependent development of retinal light responsiveness. Accordingly, we recorded electroretinogram from wild type mice and mice with genetic deletion of D1 dopamine receptor (D1−/− mice) raised under cyclic light conditions and constant darkness. Our results demonstrated that D1−/− mice have reduced amplitudes of all three major components of electroretinogram in adulthood. When the relative strength of the responses is considered, the D1−/− mice have selective reduction of the amplitudes of a-wave and oscillatory potentials evoked by low-intermediate intensities of lights. During postnatal development, D1−/− mice have increased amplitude of b-wave at the time of eye-opening but reduced developmental increase of the amplitude of b-wave after eye opening. Light deprivation from birth significantly reduced the amplitudes of b-wave and oscillatory potentials, increased the outer retinal light response gain and altered the light response kinetics of both a- and b-waves of wild type mice. In D1−/− mice, the effect of dark rearing on the amplitude of oscillatory potentials was diminished and dark rearing induced effects on the response gain of outer retina and the kinetics of a-wave were reversed. These results demonstrated roles of dopamine D1 receptor in the activity-dependent functional development of mouse retina.     

Depression of the vestibulo-ocular and optokinetic responses by intrafloccular microinjection of GABA-A and GABA-B agonists in the rabbit

The functional implication of the cerebellar flocculus in regulation of the VOR and OKR gain has mostly been studied by lesion experiments, and the hypotheses derived from these experiments are not always in line with one another. In the present study, a reversible method was used to inhibit floccular Purkinje cells. The GABA-A agonist muscimol or the GABA-B agonist baclofen were bilaterally injected into the flocculus of rabbits, and the effects of these injections on the gain of the VOR and OKR were studied. Both drugs induced a reduction by at least 50% of the gain of the VOR in light and darkness, and of the OKR. Although GABA-A and GABA-B receptors are known to have different cerebellar localizations, muscimol and baclofen injections resulted in quantitatively similar effects. It is suggested that these GABA-agonists cause either direct or indirect inhibition of floccular Purkinje cells, thus reducing modulation of the firing rate of these neurons by afferent mossy and climbing fibers. Because the flocular Purkinje cells act out of phase with the vestibular neurons which drive the oculomotor neurons, a reduced output of floccular Purkinje cells would result in a reduction of the VOR and OKR gain. These experiments provide strong evidence that the cerebellar flocculus has a positive influence on the basic VOR and OKR gain.     

In the absence of Sonic hedgehog, p53 induces apoptosis and inhibits retinal cell proliferation, cell-cycle exit and differentiation in zebrafish

Background

Sonic hedgehog (Shh) signaling regulates cell proliferation during vertebrate development via induction of cell-cycle regulator gene expression or activation of other signalling pathways, prevents cell death by an as yet unclear mechanism and is required for differentiation of retinal cell types. Thus, an unsolved question is how the same signalling molecule can regulate such distinct cell processes as proliferation, cell survival and differentiation.

Methodology/Principal Findings

Analysis of the zebrafish shh ^−/− mutant revealed that in this context p53 mediates elevated apoptosis during nervous system and retina development and interferes with retinal proliferation and differentiation. While in shh ^−/− mutants there is activation of p53 target genes and p53-mediated apoptosis, an increase in Hedgehog (Hh) signalling by over-expression of dominant-negative Protein Kinase A strongly decreased p53 target gene expression and apoptosis levels in shh ^−/− mutants. Using a novel p53 reporter transgene, I confirm that p53 is active in tissues that require Shh for cell survival. Proliferation assays revealed that loss of p53 can rescue normal cell-cycle exit and the mitotic indices in the shh ^−/− mutant retina at 24, 36 and 48 hpf. Moreover, generation of amacrine cells and photoreceptors was strongly enhanced in the double p53 ^−/− shh ^−/− mutant retina suggesting the effect of p53 on retinal differentiation.

Conclusions

Loss of Shh signalling leads to the p53-dependent apoptosis in the developing nervous system and retina. Moreover, Shh-mediated control of p53 activity is required for proliferation and cell cycle exit of retinal cells as well as differentiation of amacrine cells and photoreceptors.     

Stabilization of the gaze in chameleons: visual and vestibular reflexes

Some visual, vestibular and proprioceptive reflexes which contribute to gaze (head + eye) stabilization were quantified in the chameleon. All the reflexes were analysed in the horizontal plane, and the visual reflexes were also studied in the vertical plane. In restrained-head animals, both the optokinetic nystagmus (OKN) and the vestibulo-ocular reflex (VOR) had low gains. In free-head animals, the head (opto-collic or vestibulo-collic reflex) and eye (OKN or VOR) responses added their effects, thus improving gaze stabilization, especially during vestibular stimulation. Cervical stimulation provoked both a cervico-ocular reflex (COR) in the compensatory direction and a large number of saccades. The saccadic response was especially marked in the presence of patterned visual surroundings.     

Insulin receptor substrate 2 (IRS2)-deficient mice show sensorineural hearing loss that is delayed by concomitant protein tyrosine phosphatase 1B (PTP1B) loss of function

The insulin receptor substrate (IRS) proteins are key mediators of insulin and insulinlike growth factor 1 (IGF-1) signaling. Protein tyrosine phosphatase (PTP)-1B dephosphorylates and inactivates both insulin and IGF-1 receptors. IRS2-deficient mice present altered hepatic insulin signaling and β-cell failure and develop type 2–like diabetes. In addition, IRS2 deficiency leads to developmental defects in the nervous system. IGF1 gene mutations cause syndromic sensorineural hearing loss in humans and mice. However, the involvement of IRS2 and PTP1B, two IGF-1 downstream signaling mediators, in hearing onset and loss has not been studied. Our objective was to study the hearing function and cochlear morphology of Irs2-null mice and the impact of PTP1B deficiency. We have studied the auditory brainstem responses and the cochlear morphology of systemic Irs2^−/−Ptpn1^+/+, Irs2^+/+Ptpn1^−/−and Irs2^−/−Ptpn1^−/− mice at different postnatal ages. The results indicated that Irs2^−/−Ptpn1^+/+ mice present a profound congenital sensorineural deafness before the onset of diabetes and altered cochlear morphology with hypoinnervation of the cochlear ganglion and aberrant stria vascularis, compared with wild-type mice. Simultaneous PTP1B deficiency in Irs2^−/−Ptpn1^−/− mice delays the onset of deafness. We show for the first time that IRS2 is essential for hearing and that PTP1B inhibition may be useful for treating deafness associated with hyperglycemia and type 2 diabetes.     

Differential expression and function of ABCG1 and ABCG4 during development and aging

ABCG1 and ABCG4 are highly homologous members of the ATP binding cassette (ABC) transporter family that regulate cellular cholesterol homeostasis. In adult mice, ABCG1 is known to be expressed in numerous cell types and tissues, whereas ABCG4 expression is limited to the central nervous system (CNS). Here, we show significant differences in expression of these two transporters during development. Examination of β-galactosidase-stained tissue sections from Abcg1^−/−LacZ and Abcg4^−/−LacZ knockin mice shows that ABCG4 is highly but transiently expressed both in hematopoietic cells and in enterocytes during development. In contrast, ABCG1 is expressed in macrophages and in endothelial cells of both embryonic and adult liver. We also show that ABCG1 and ABCG4 are both expressed as early as E12.5 in the embryonic eye and developing CNS. Loss of both ABCG1 and ABCG4 results in accumulation in the retina and/or brain of oxysterols, in altered expression of liver X receptor and sterol-regulatory element binding protein-2 target genes, and in a stress response gene. Finally, behavioral tests show that Abcg4^−/− mice have a general deficit in associative fear memory. Together, these data indicate that loss of ABCG1 and/or ABCG4 from the CNS results in changes in metabolic pathways and in behavior.     

Interferon-Induced Ifit2/ISG54 Protects Mice from Lethal VSV Neuropathogenesis

Interferon protects mice from vesicular stomatitis virus (VSV) infection and pathogenesis; however, it is not known which of the numerous interferon-stimulated genes (ISG) mediate the antiviral effect. A prominent family of ISGs is the interferon-induced with tetratricopeptide repeats (Ifit) genes comprising three members in mice, Ifit1/ISG56, Ifit2/ISG54 and Ifit3/ISG49. Intranasal infection with a low dose of VSV is not lethal to wild-type mice and all three Ifit genes are induced in the central nervous system of the infected mice. We tested their potential contributions to the observed protection of wild-type mice from VSV pathogenesis, by taking advantage of the newly generated knockout mice lacking either Ifit2 or Ifit1. We observed that in Ifit2 knockout (Ifit2 ^−/−) mice, intranasal VSV infection was uniformly lethal and death was preceded by neurological signs, such as ataxia and hind limb paralysis. In contrast, wild-type and Ifit1 ^−/− mice were highly protected and survived without developing such disease. However, when VSV was injected intracranially, virus replication and survival were not significantly different between wild-type and Ifit2^−/− mice. When administered intranasally, VSV entered the central nervous system through the olfactory bulbs, where it replicated equivalently in wild-type and Ifit2 ^−/− mice and induced interferon-β. However, as the infection spread to other regions of the brain, VSV titers rose several hundred folds higher in Ifit2 ^−/− mice as compared to wild-type mice. This was not caused by a broadened cell tropism in the brains of Ifit2 ^−/− mice, where VSV still replicated selectively in neurons. Surprisingly, this advantage for VSV replication in the brains of Ifit2^−/− mice was not observed in other organs, such as lung and liver. Pathogenesis by another neurotropic RNA virus, encephalomyocarditis virus, was not enhanced in the brains of Ifit2 ^−/− mice. Our study provides a clear demonstration of tissue-, virus- and ISG-specific antiviral action of interferon.     

Differential Influences of the Aryl Hydrocarbon Receptor on Th17 Mediated Responses in vitro and in vivo

The aryl hydrocarbon receptor (AhR) has been attributed with anti-inflammatory effects in the development of pathological immune responses leading to experimental autoimmune encephalomyelitis (EAE) via the induction of regulatory T cells. In agreement with previously published findings, we find that TCDD administration confers protection from EAE, however, this immuno-modulatory effect was not the consequence of de novo Treg generation, but the inhibition of Th17 cell differentiation. Systemic application of FICZ at the time of immunization also reduced EAE pathology albeit to a lesser degree than TCDD. In vitro Th17 differentiation in the presence of AhR agonists, including TCDD, promoted IL-17 and IL-22 expression, but did not induce Treg differentiation. AhR affinity influenced the amounts of IL-17 and IL-22 protein that was secreted by Th17 cells, but did not seem to affect susceptibility to EAE in vivo. Making use of conditional AhR-deficient mice, we show that the anti-inflammatory effect of TCDD depends on AhR activation in both T cells and dendritic cells, further emphasising the ability of TCDD to interfere with T effector cell differentiation in vivo. The dichotomy between the in vivo and in vitro effects of AhR reveals the complexity of the AhR pathway, which has the capacity of affecting different AhR-expressing cell types involved in mounting immune responses, thus participating in defining their outcome.     

Aag DNA Glycosylase Promotes Alkylation-Induced Tissue Damage Mediated by Parp1

Alkylating agents comprise a major class of front-line cancer chemotherapeutic compounds, and while these agents effectively kill tumor cells, they also damage healthy tissues. Although base excision repair (BER) is essential in repairing DNA alkylation damage, under certain conditions, initiation of BER can be detrimental. Here we illustrate that the alkyladenine DNA glycosylase (AAG) mediates alkylation-induced tissue damage and whole-animal lethality following exposure to alkylating agents. Aag-dependent tissue damage, as observed in cerebellar granule cells, splenocytes, thymocytes, bone marrow cells, pancreatic β-cells, and retinal photoreceptor cells, was detected in wild-type mice, exacerbated in Aag transgenic mice, and completely suppressed in Aag ^−/− mice. Additional genetic experiments dissected the effects of modulating both BER and Parp1 on alkylation sensitivity in mice and determined that Aag acts upstream of Parp1 in alkylation-induced tissue damage; in fact, cytotoxicity in WT and Aag transgenic mice was abrogated in the absence of Parp1. These results provide in vivo evidence that Aag-initiated BER may play a critical role in determining the side-effects of alkylating agent chemotherapies and that Parp1 plays a crucial role in Aag-mediated tissue damage.     

Video-oculography in Mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals^1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system⁵. Second, the oculomotor system is relative simple⁶; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles⁷. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis⁸. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes⁹.Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels¹⁰. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice⁸.     

MFGE8 does not influence chorio-retinal homeostasis or choroidal neovascularization in vivo

Purpose

Milk fat globule-epidermal growth factor-factor VIII (MFGE8) is necessary for diurnal outer segment phagocytosis and promotes VEGF-dependent neovascularization. The prevalence of two single nucleotide polymorphisms (SNP) in MFGE8 was studied in two exsudative or “wet” Age-related Macular Degeneration (AMD) groups and two corresponding control groups. We studied the effect of MFGE8 deficiency on retinal homeostasis with age and on choroidal neovascularization (CNV) in mice.

Methods

The distribution of the SNP (rs4945 and rs1878326) of MFGE8 was analyzed in two groups of patients with “wet” AMD and their age-matched controls from Germany and France. MFGE8-expressing cells were identified in Mfge8 ^+/− mice expressing ß-galactosidase. Aged Mfge8 ^+/− and Mfge8 ^−/− mice were studied by funduscopy, histology, electron microscopy, scanning electron microscopy of vascular corrosion casts of the choroid, and after laser-induced CNV.

Results

rs1878326 was associated with AMD in the French and German group. The Mfge8 promoter is highly active in photoreceptors but not in retinal pigment epithelium cells. Mfge8^−/− mice did not differ from controls in terms of fundus appearance, photoreceptor cell layers, choroidal architecture or laser-induced CNV. In contrast, the Bruch''s membrane (BM) was slightly but significantly thicker in Mfge8^−/− mice as compared to controls.

Conclusions

Despite a reproducible minor increase of rs1878326 in AMD patients and a very modest increase in BM in Mfge8^−/− mice, our data suggests that MFGE8 dysfunction does not play a critical role in the pathogenesis of AMD.     

A non-linear model for visual-vestibular interaction during body rotation in man

A mathematical model for visual-vestibular interaction during body rotation in an illuminated visual surround is obtained by combining a previous model of the optokinetic reflex (OKR) with a simplified model of the vestibulo-ocular reflex (VOR). OKR is activated by the slip of the image of the external world on the retina, and represents a negative feedback loop around VOR. For large retinal slip velocities OKR behaves as a basically non-linear system. The validity of the model is proved via computer simulation by comparing predicted responses with the experimental results obtained in man by Koenig et al. (1978) in different situations of visual-vestibular interaction.Work supported by C.N.R. (Rome, Italy), Special Project on Piomedical Engineering, Grant No. 79.01255.86