Jeremy A. Murphy

Differential c-Fos immunoreactivity in arousal-promoting cell groups following systemic administration of caffeine in rats.

Despite the widespread use of caffeine, the neuronal mechanisms underlying its stimulatory effects are not completely understood. By using c‐Fos immunohistochemistry as a marker of neuronal activation, we recently showed that stimulant doses of caffeine activate arousal‐promoting hypothalamic orexin (hypocretin) neurons. In the present study, we investigated whether other key neurons of the arousal system are also activated by caffeine, via dual immunostaining for c‐Fos and transmitter markers. Rats were administered three doses of caffeine or saline vehicle during the light phase. Caffeine at 10 and 30 mg/kg, i.p., increased motor activities, including locomotion, compared with after saline or a higher dose, 75 mg/kg. The three doses of caffeine induced distinct dose‐related patterns of c‐Fos immunoreactivity in several arousal‐promoting areas, including orexin neurons and adjacent neurons containing neither orexin nor melanin‐concentrating hormone; tuberomammillary histaminergic neurons; locus coeruleus noradrenergic neurons; noncholinergic basal forebrain neurons that do not contain parvalbumin; and nondopaminergic neurons in the ventral tegmental area. At any dose used, caffeine induced little or no c‐Fos expression in cholinergic neurons of the basal forebrain and mesopontine tegmentum; dopaminergic neurons of the ventral tegmental area, central gray, and substantia nigra pars compacta; and serotonergic neurons in the dorsal raphe nucleus. Saline controls exhibited only few c‐Fos‐positive cells in most of the cell groups examined. These results indicate that motor‐stimulatory doses of caffeine induce a remarkably restricted pattern of c‐Fos expression in the arousal‐promoting system and suggest that this specific neuronal activation may be involved in the behavioral arousal by caffeine. J. Comp. Neurol. 498:667–689, 2006.

Stimulant doses of caffeine induce c-FOS activation in orexin/hypocretin-containing neurons in rat.

Although caffeine is a commonly used CNS stimulant, neuronal mechanisms underlying its stimulatory effect are not fully understood. Orexin (hypocretin)-containing neurons play a critical role in arousal and might be activated by acute administration of caffeine. We examined this possibility by using dual-immunostaining for orexin B and c-Fos protein as a marker for neuronal activation. Rats were administered intraperitoneally with 10, 30 or 75 mg/kg caffeine, or saline. As previously reported, caffeine increased locomotion at 10 and 30 mg/kg, but not at 75 mg/kg. The numbers of orexin-immunoreactive and non-orexin-immunoreactive neurons expressing c-Fos were analysed using three counting boxes within the orexin field in the posterior hypothalamus. Compared with saline, all doses of caffeine increased the number of cells immunoreactive for both orexin and c-Fos. The average magnitude of this increase across doses in orexin neurons differed amongst regions; c-Fos expression increased by 343% in the perifornical area and by 158% in the more medial, dorsomedial nucleus. In the lateral hypothalamic area, c-Fos expression increased by 226% at 10 and 30 mg/kg but no change was seen at 75 mg/kg. In contrast, caffeine significantly increased the number of non-orexin-immunoreactive neurons expressing c-Fos only in the dorsomedial nucleus. These results indicate that systemically administered caffeine preferentially activates orexin neurons over non-orexin neurons in the same field, and that this activation is most pronounced in the perifornical region where orexin neurons are most concentrated. The activation of orexin neurons might play a role in the behavioural activation by caffeine.

Enriched environment during adolescence changes brain-derived neurotrophic factor and TrkB levels in the rat visual system but does not offer neuroprotection to retinal ganglion cells following axotomy

The purpose of the present experiment was to characterize changes in TrkB signaling in the rat visual system resulting from exposure to enriched environment. Female Sprague-Dawley rats were placed in enriched or impoverished conditions for 1, 7 or 28 days. Levels of BDNF protein and its predominant receptor TrkB were examined in the retina, superior colliculus and visual cortex. In the retina, 1 day of enrichment increased full-length TrkB and after 28 days increased BDNF. In the superior colliculus, enrichment for 7 days reduced full-length TrkB and after 28 days increased BDNF and full-length TrkB. One day of enrichment significantly increased BDNF, reduced full-length TrkB and increased truncated TrkB in the visual cortex. Consequently, we further investigated whether exposure to enriched environment and the subsequent changes in BDNF and TrkB translates into a neuroprotective effect on retinal ganglion cells (RGCs) following transection of the optic nerve. Although exogenous intraocular application of BDNF provides neuroprotection to RGCs after axotomy, the endogenous increase in BDNF in the retina after 28 days of enrichment had no effect on RGC survival. While enriched housing conditions offer a model of non-invasive rehabilitation treatment for injury and modulates changes in BDNF and TrkB levels, these molecular changes did not translate into a neuroprotective effect on RGCs following transection of the optic nerve.

The neural cell adhesion molecule is necessary for normal adult retinal ganglion cell number and survival

Retinal ganglion cells (RGCs) undergo apoptotic death in predictable time-dependant manners during development and as a consequence of injury. Recently, synthetic neural cell adhesion molecule (NCAM) agonists have been shown to provide neuroprotective support. Within the adult mouse retina, NCAM has been localized on all neurons and glia; however, no functional role has been determined. Using adult NCAM-/- mice, we directly tested the potential influence of NCAM on neuron survival in vivo and observed that, in NCAM-/- retinas, RGC densities are greater, RGC loss after injury is earlier and target tissue significantly influences adult RGC survival, all in contrast to wild-type retinas. Collectively, our results indicate that NCAM may play a vital role in regulating the developmental change in the effectiveness of local versus target-derived RGC trophic support and that, in the adult, endogenous NCAM influences the total number of CNS neurons and their survival following injury.

The role of endothelin-1 and its receptors in optic nerve head astrocyte proliferation

Aim To characterise the influence of endothelin-1 (ET-1), a vasoactive peptide, and its receptors (endothelin B (ETB) and endothelin A (ETA)) on rat optic nerve head astrocyte (ONHA) proliferation. Methods ONHAs were isolated from adult Brown Norway rats. ONHA specificity was determined with immunohistochemistry for: glial fibrillary acidic protein (GFAP); A2B5, a marker of type II astrocytes located outside the ONH; and myelin basic protein (MBP). ONHA proliferation was quantified following treatment with ET-1 (1×10−6, 1×10−7, 1×10−9 or 1×10−11 M) or vehicle for 24, 48 or 72 h. ETB and ETA antagonists were used to assess the role of each receptor in ONHA proliferation. Results ONHA specificity was confirmed with positive labelling for GFAP, and negative labelling for A2B5 and MBP. ONHAs also expressed ETB and ETA. Cell percentages increased significantly beginning 48 h after ET-1 exposure with 1×10−7 (20%) and 1×10−9 M (15%). After 72 h, ONHA percentages increased significantly at all ET-1 concentrations (25%, 21%, 29%, 28% increases relative to vehicle, for 1×10−6, 1×10−7, 1×10−9 and 1×10−11 M, respectively). No significant proliferation occurred in the presence of either antagonist. Conclusion ONHAs proliferated following 48 h or more of exposure to ET-1. The proliferation required both ETB and ETA receptors.

Endogenous polysialylated neural cell adhesion molecule enhances the survival of retinal ganglion cells.

PURPOSE During development, all retinal cells express polysialylated neural cell adhesion molecule (PSA-NCAM). PSA is localized only on glia in the adult retina, but as Müller glial processes ensheathe most retinal neurons, PSA remains in the extracellular environment of adult neurons. The authors sought to investigate the influence of endogenous PSA on the survival of neonatal as well as adult normal and injured retinal ganglion cells (RGCs). METHODS Endogenous retinal PSA was selectively degraded by application of endoneuraminidase. PSA presence and removal were confirmed by immunohistochemistry and levels were assessed by Western Blot analysis. Neonatal RGC survival after PSA removal was assessed in vitro in RGCs immunopanned from rat pups. Adult RGC survival was assessed in vivo in mice by investigating RGC densities after removal of PSA in normal retinas and after optic nerve transection. RESULTS Virtually all neonatal RGCs express PSA-NCAM and survive well in vitro; however, removal of PSA resulted in a 42% loss of these cells 3 days after the treatment. Similarly, removal of PSA in the adult retina in vivo induced a loss of 25% of RGCs at 14 days, and significantly reduced RGC densities after optic nerve transection by an additional 27% (relative to injured retinas with a vehicle injection) at 7 days. CONCLUSIONS Together, these findings demonstrate that endogenous PSA supports the survival of neonatal as well as injured and normal adult RGCs and provide the first functional evidence of a role for PSA in the adult retina.

Injury to retinal ganglion cell axons increases polysialylated neural cell adhesion molecule (PSA-NCAM) in the adult rodent superior colliculus.

The adult mammalian central nervous system (CNS) exhibits a limited regenerative response to injury. It is well established that polysialylated neural cell adhesion molecule (PSA-NCAM) contributes to nervous system plasticity. In the visual system, PSA-NCAM participates in retinal ganglion cell (RGC) axon growth during development and specifically influences RGC innervation of its principle target tissue, the superior colliculus (SC). The goals of this study were to determine whether PSA-NCAM is expressed in the normal adult mouse SC and to evaluate PSA-NCAM expression following RGC injury. In the normal rostral, but not caudal, SC we find that PSA-NCAM is present in the retinorecipient layers; however, PSA-NCAM and RGC axons do not co-localize. In the deeper collicular layers, PSA-NCAM is observed as several distinct patches that occur at the same depth along the medial-lateral axis throughout the colliculus. RGC axotomy denervates predominantly the contralateral colliculus, where increased PSA-NCAM levels are seen at 7 and 10 days after the injury. Further evaluation of the retinorecipient layers of the partially denervated SC reveals that some intact CTB-traced RGC axons (less than 5%) labeled from the ipsilateral eye do co-localize with PSA-NCAM. This study is the first characterization of PSA-NCAM expression in the normal and partially denervated adult SC and may indicate that PSA-NCAM is involved in attempted visual system remodeling after injury.

Endothelin-1–Induced Proliferation Is Reduced and Ca2+ Signaling Is Enhanced in Endothelin B–Deficient Optic Nerve Head Astrocytes

PURPOSE To characterize the influence of endothelin-1 (ET-1) on optic nerve head astrocyte (ONHA) proliferation and Ca²⁺ signaling in ONHAs lacking functional endothelin B (ETB) receptors. METHODS ONHAs were isolated from adult wild type (WT) and transgenic spotting lethal (TSL) rats, lacking functional ETB receptors. ONHA specificity was confirmed by positive glial fibrillary acidic protein (GFAP), negative A2B5 (a marker for type II astrocytes located outside the optic nerve head) and myelin basic protein (MBP) labeling. The mitogenic effects of 10⁻⁷ or 10⁻⁹ M ET-1, or vehicle were investigated for 48 or 72 hours in WT and TSL ONHAs. Intracellular calcium levels ([Ca²⁺](i)) were assessed in ONHAs loaded with fura-2 calcium indicator dye. RESULTS ET-1-induced proliferation of TSL ONHAs was blunted at 48 hours (by 37% at 10⁻⁷ M and by 33% at 10⁻⁹ M) and 72 hours (by 117% at 10⁻⁷ M and by 100% at 10⁻⁹ M) compared with WT cells. ET-1-induced ONHA fura-2 ratio increases were significantly greater in TSL ONHAs (by 20% at 10⁻⁷ M and by 48% at 10⁻⁹ M) compared with WT ONHAs. ET-1-induced fura-2 ratio increases were blocked after pretreatment with BQ-610 (ETA antagonist) in WT and TSL ONHAs, but not by BQ-788 (ETB antagonist) in WT ONHAs. CONCLUSIONS ET-1-induced ONHA proliferation is reduced in cells lacking functional ETB receptors, ET-1-induced [Ca²⁺](i) increases are enhanced in the absence of functional ETB receptors, and ETA, but not ETB, is required for ET-1-induced [Ca²⁺](i) elevation.