CaV3.2 KO mice have altered retinal waves but normal direction selectivity.

Early in development, before the onset of vision, the retina establishes direction-selective responses. During this time period, the retina spontaneously generates bursts of action potentials that propagate across its extent. The precise spatial and temporal properties of these "retinal waves" have been implicated in the formation of retinal projections to the brain. However, their role in the development of direction selective circuits within the retina has not yet been determined. We addressed this issue by combining multielectrode array and cell-attached recordings to examine mice that lack the CaV3.2 subunit of T-type Ca2+ channels (CaV3.2 KO) because these mice exhibit disrupted waves during the period that direction selective circuits are established. We found that the spontaneous activity of these mice displays wave-associated bursts of action potentials that are altered from that of control mice: the frequency of these bursts is significantly decreased and the firing rate within each burst is reduced. Moreover, the projection patterns of the retina demonstrate decreased eye-specific segregation in the dorsal lateral geniculate nucleus (dLGN). However, after eye-opening, the direction selective responses of CaV3.2 KO direction selective ganglion cells (DSGCs) are indistinguishable from those of wild-type DSGCs. Our data indicate that although the temporal properties of the action potential bursts associated with retinal waves are important for activity-dependent refining of retinal projections to central targets, they are not critical for establishing direction selectivity in the retina.

The role of neuronal connexins 36 and 45 in shaping spontaneous firing patterns in the developing retina.

Gap junction coupling synchronizes activity among neurons in adult neural circuits, but its role in coordinating activity during development is less known. The developing retina exhibits retinal waves--spontaneous depolarizations that propagate among retinal interneurons and drive retinal ganglion cells (RGCs) to fire correlated bursts of action potentials. During development, two connexin isoforms, connexin 36 (Cx36) and Cx45, are expressed in bipolar cells and RGCs, and therefore provide a potential substrate for coordinating network activity. To determine whether gap junctions contribute to retinal waves, we compared spontaneous activity patterns using calcium imaging, whole-cell recording, and multielectrode array recording in control, single-knock-out (ko) mice lacking Cx45 and double-knock-out (dko) mice lacking both isoforms. Wave frequency, propagation speed, and bias in propagation direction were similar in control, Cx36ko, Cx45ko, and Cx36/45dko retinas. However, the spontaneous firing rate of individual retinal ganglion cells was elevated in Cx45ko retinas, similar to Cx36ko retinas (Hansen et al., 2005; Torborg and Feller, 2005), a phenotype that was more pronounced in Cx36/45dko retinas. As a result, spatial correlations, as assayed by nearest-neighbor correlation and functional connectivity maps, were significantly altered. In addition, Cx36/45dko mice had reduced eye-specific segregation of retinogeniculate afferents. Together, these findings suggest that although Cx36 and Cx45 do not play a role in gross spatial and temporal propagation properties of retinal waves, they strongly modulate the firing pattern of individual RGCs, ensuring strongly correlated firing between nearby RGCs and normal patterning of retinogeniculate projections.

Development of asymmetric inhibition underlying direction selectivity in the retina.

Establishing precise synaptic connections is crucial to the development of functional neural circuits. The direction-selective circuit in the retina relies upon highly selective wiring of inhibitory inputs from starburst amacrine cells (SACs) onto four subtypes of ON-OFF direction-selective ganglion cells (DSGCs), each preferring motion in one of four cardinal directions. It has been reported in rabbit that the SACs on the 'null' sides of DSGCs form functional GABA (γ-aminobutyric acid)-mediated synapses, whereas those on the preferred sides do not. However, it is not known how the asymmetric wiring between SACs and DSGCs is established during development. Here we report that in transgenic mice with cell-type-specific labelling, the synaptic connections from SACs to DSGCs were of equal strength during the first postnatal week, regardless of whether the SAC was located on the preferred or null side of the DSGC. However, by the end of the second postnatal week, the strength of the synapses made from SACs on the null side of a DSGC significantly increased whereas those made from SACs located on the preferred side remained constant. Blocking retinal activity by intraocular injections of muscimol or gabazine during this period did not alter the development of direction selectivity. Hence, the asymmetric inhibition between the SACs and DSGCs is achieved by a developmental program that specifically strengthens the GABA-mediated inputs from SACs located on the null side, in a manner not dependent on neural activity.

Decreased brain zinc availability reduces hippocampal neurogenesis in mice and rats.

In the adult brain, neurogenesis occurs in the subgranular zone of the dentate gyrus (DG), where high levels of vesicular zinc are localized in the presynaptic terminals. To determine whether zinc has a role in modulating hippocampal neurogenesis under normal or pathologic conditions, we manipulated the level of vesicular zinc experimentally. To reduce hippocampal vesicular zinc, rats were either fed a zinc-deficient diet or treated with a zinc chelator, clioquinol (CQ). The number of progenitor cells and immature neurons was decreased significantly in the DG after 6 weeks of dietary zinc deprivation. Conversely, the number of progenitor cells and immature neurons was restored after a 2-week reversal to a normal zinc-containing diet. Similarly, a 1-week treatment with the zinc chelator, CQ, reduced the number of progenitor cells. The results of our previous study showed that hypoglycemia increased hippocampal neurogenesis. This study shows that zinc chelation reduced hypoglycemia-induced progenitor cell proliferation and neurogenesis. Finally, the role of vesicular zinc on neurogenesis was further assessed in zinc transporter 3 (ZnT3) gene deleted mice. Zinc transporter 3 knockout (KO) mice had significantly fewer proliferating progenitor cells and immature neurons after hypoglycemia. Our data provide converging evidence in support of the essential role zinc has in modulating hippocampal neurogenesis.

Inhibition of poly(ADP-ribose) polymerase suppresses inflammation and promotes recovery after ischemic injury.

The brain inflammatory response induced by stroke contributes to cell death and impairs neurogenesis. Poly(ADP-ribose) polymerase-1 (PARP-1) is a coactivator of the transcription factor NF-kappaB and required for NF-kappaB-mediated inflammatory responses. Here we evaluated PARP inhibition as a means of suppressing post-stroke inflammation and improving outcome after stroke. Rats were subjected to bilateral carotid occlusion-reperfusion, and treatment with the PARP inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide (PJ34) was begun 48 h later. PJ34 was found to rapidly suppress the ischemia-induced microglial activation and astrogliosis. Behavioral tests performed 6 to 8 weeks after ischemia showed deficits in spatial memory and learning that were lessened by the PJ34 treatment. Immunohistochemical evaluation of hippocampus at 8 weeks after ischemia showed increased neuronal density in CA1 layer of PJ34-treated animals relative to vehicle-treated animals. Bromodeoxyuridine labeling showed formation of new neurons in hippocampal CA1 area in PJ34-treated animals, but not in vehicle-treated animals. Together, these results suggest that treatment with a PARP inhibitor for several days after ischemia enhances long-term neuronal survival and neurogenesis by reducing inflammation.

Sequential release of nitric oxide, zinc, and superoxide in hypoglycemic neuronal death.

Oxidative stress and zinc release are both known to contribute to neuronal death after hypoglycemia; however, the cause-effect relationships between these events are not established. Here we found, using a rat model of profound hypoglycemia, that the neuronal zinc release and translocation that occur immediately after hypoglycemia are prevented by the nitric oxide synthase inhibitor 7-nitroindazole but not by overexpression of superoxide dismutase-1 (SOD-1). However, overexpression of SOD-1 prevented activation of poly(ADP-ribose) polymerase-1 (PARP-1) and neuronal death, suggesting that zinc release is upstream of superoxide production. Accordingly, zinc-induced superoxide production was blocked in neuronal cultures by the NADPH oxidase inhibitor apocynin and by genetic deficiency in the p47(phox) subunit of NADPH oxidase. A key role for the vesicular zinc pool in this process was suggested by reduced superoxide formation and neuronal death in mice deficient in zinc transporter 3. Together, these findings suggest a series of events in which nitric oxide production triggers vesicular zinc release, which in turn activates NADPH oxidase and PARP-1. This sequence may also occur in other central nervous system disorders in which zinc, nitric oxide, and oxidative stress have been linked.

Zinc inhibits astrocyte glutamate uptake by activation of poly(ADP-ribose) polymerase-1.

Several processes by which astrocytes protect neurons during ischemia are now well established. However, less is known about how neurons themselves may influence these processes. Neurons release zinc (Zn2+) from presynaptic terminals during ischemia, seizure, head trauma, and hypoglycemia, and modulate postsynaptic neuronal function. Peak extracellular zinc may reach concentrations as high as 400 microM. Excessive levels of free, ionic zinc can initiate DNA damage and the subsequent activation of poly(ADP-ribose) polymerase 1 (PARP-1), which in turn lead to NAD+ and ATP depletion when DNA damage is extensive. In this study, cultured cortical astrocytes were used to explore the effects of zinc on astrocyte glutamate uptake, an energy-dependent process that is critical for neuron survival. Astrocytes incubated with 100 or 400 microM of zinc for 30 min showed significant decreases in ATP levels and glutamate uptake capacity. These changes were prevented by the PARP inhibitors benzamide or DPQ (3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone) or PARP-1 gene deletion (PARP-1 KO). These findings suggest that release of Zn2+ from neurons during brain insults could induce PARP-1 activation in astrocytes, leading to impaired glutamate uptake and exacerbation of neuronal injury.

Hypoglycemia, brain energetics, and hypoglycemic neuronal death.

Hypoglycemia is a common and serious problem among diabetic patients receiving treatment with insulin or other glucose-lowering drugs. Moderate hypoglycemia impairs neurological function, and severe hypoglycemia leads to death of selectively vulnerable neurons. Recent advances have shed new light on the underlying processes that cause neuronal death in hypoglycemia and the factors that may render specific neuronal populations especially vulnerable to hypoglycemia. In addition to its clinical importance, the pathophysiology of hypoglycemia is an indicator of the unique bioenergetic properties of the central nervous system, in particular the metabolic coupling of neuronal and astrocyte metabolism. This review will focus on relationships between bioenergetics and brain dysfunction in hypoglycemia, the neuronal cell death program triggered by hypoglycemia, and the role of astrocytes in these processes.

Hypoglycemic neuronal death is triggered by glucose reperfusion and activation of neuronal NADPH oxidase.

Hypoglycemic coma and brain injury are potential complications of insulin therapy. Certain neurons in the hippocampus and cerebral cortex are uniquely vulnerable to hypoglycemic cell death, and oxidative stress is a key event in this cell death process. Here we show that hypoglycemia-induced oxidative stress and neuronal death are attributable primarily to the activation of neuronal NADPH oxidase during glucose reperfusion. Superoxide production and neuronal death were blocked by the NADPH oxidase inhibitor apocynin in both cell culture and in vivo models of insulin-induced hypoglycemia. Superoxide production and neuronal death were also blocked in studies using mice or cultured neurons deficient in the p47(phox) subunit of NADPH oxidase. Chelation of zinc with calcium disodium EDTA blocked both the assembly of the neuronal NADPH oxidase complex and superoxide production. Inhibition of the hexose monophosphate shunt, which utilizes glucose to regenerate NADPH, also prevented superoxide formation and neuronal death, suggesting a mechanism linking glucose reperfusion to superoxide formation. Moreover, the degree of superoxide production and neuronal death increased with increasing glucose concentrations during the reperfusion period. These results suggest that high blood glucose concentrations following hypoglycemic coma can initiate neuronal death by a mechanism involving extracellular zinc release and activation of neuronal NADPH oxidase.

Use of a poly(ADP-ribose) polymerase inhibitor to suppress inflammation and neuronal death after cerebral ischemia-reperfusion.

BACKGROUND AND PURPOSE: Most stroke patients do not present for medical treatment until several hours after onset of brain ischemia. Consequently, neuroprotective strategies are required with comparably long therapeutic windows. Poly(ADP-ribose) polymerase inhibitors such as PJ34 are known to suppress microglial activation, a postischemic event that may contribute to neuronal death. We evaluated the effects of PJ34 administered 8 hours after transient forebrain ischemia. METHODS: Rats were subjected to 10 minutes of forebrain ischemia and treated with PJ34 for 7 days beginning 8 hours after reperfusion. Activated microglia and infiltrating macrophages were evaluated at serial time points between zero and 14 days after ischemia by immunostaining for CD11b. CA1 neuronal survival was evaluated 7 days after ischemia. RESULTS: Rats treated with PJ34 showed a near-complete inhibition of microglia/macrophage activation (evaluated on day 5) and an 84% reduction in CA1 neuronal death. CONCLUSIONS: Administration of PJ34 as late as 8 hours after transient ischemia-reperfusion has a large protective effect on CA1 survival. This effect may be mediated by suppression of the postischemic brain inflammatory response.

Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient mouse.

Uptake of the neurotransmitter glutamate is effected primarily by transporters expressed on astrocytes, and downregulation of these transporters leads to seizures and neuronal death. Neurons also express a glutamate transporter, termed excitatory amino acid carrier-1 (EAAC1), but the physiological function of this transporter remains uncertain. Here we report that genetically EAAC1-null (Slc1a1(-/-)) mice have reduced neuronal glutathione levels and, with aging, develop brain atrophy and behavioral changes. EAAC1 can also rapidly transport cysteine, an obligate precursor for neuronal glutathione synthesis. Neurons in the hippocampal slices of EAAC1(-/-) mice were found to have reduced glutathione content, increased oxidant levels and increased susceptibility to oxidant injury. These changes were reversed by treating the EAAC1(-/-) mice with N-acetylcysteine, a membrane-permeable cysteine precursor. These findings suggest that EAAC1 is the primary route for neuronal cysteine uptake and that EAAC1 deficiency thereby leads to impaired neuronal glutathione metabolism, oxidative stress and age-dependent neurodegeneration.