Functional specialization in rat occipital and temporal visual cortex.

Recent studies have revealed a surprising degree of functional specialization in rodent visual cortex. Anatomically, suggestions have been made about the existence of hierarchical pathways with similarities to the ventral and dorsal pathways in primates. Here we aimed to characterize some important functional properties in part of the supposed "ventral" pathway in rats. We investigated the functional properties along a progression of five visual areas in awake rats, from primary visual cortex (V1) over lateromedial (LM), latero-intermediate (LI), and laterolateral (LL) areas up to the newly found lateral occipito-temporal cortex (TO). Response latency increased >20 ms from areas V1/LM/LI to areas LL and TO. Orientation and direction selectivity for the used grating patterns increased gradually from V1 to TO. Overall responsiveness and selectivity to shape stimuli decreased from V1 to TO and was increasingly dependent upon shape motion. Neural similarity for shapes could be accounted for by a simple computational model in V1, but not in the other areas. Across areas, we find a gradual change in which stimulus pairs are most discriminable. Finally, tolerance to position changes increased toward TO. These findings provide unique information about possible commonalities and differences between rodents and primates in hierarchical cortical processing.

Dynamic, semi-quantitative imaging of intracellular ROS levels and redox status in rat hippocampal neurons.

The cellular redox status is determined by various extra- and intracellular factors, and contributes to cytosolic signaling and oxidative stress. Especially mitochondria modulate the cytosolic redox status by oxidizing NADH and FADH(2) and generating reactive oxygen species (ROS). Whereas cellular NADH and FAD levels are reliably detectable as autofluorescence, quantifying cellular ROS production is more demanding, because the various redox-sensitive dyes share major disadvantages including irreversible oxidation, autooxidation and photosensitivity. As an alternative, we took advantage of a genetically engineered redox-sensitive green fluorescent protein (roGFP1), carefully evaluated its response properties, and succeeded to monitor ROS dynamics in cultured rat hippocampal neurons and organotypic slices. The ratiometric properties and reversible oxidation/reduction of roGFP1 enable reliable, semi-quantitative analyses of cytosolic ROS levels and redox status. Cytosolically expressed roGFP1 readily responded to hydrogen peroxide, superoxide and hydroxyl radicals, and was only negligibly affected by intracellular pH or Cl(-) content. Furthermore, roGFP1 was well suited for two-photon excitation, reliably detected changes in endogenous ROS production during impaired mitochondrial respiration or neuronal stimulation, and was even capable of visualizing perimitochondrial ROS microdomains. Modulation of cellular scavenging systems confirmed the functional integration of roGFP1 into the cellular ROS and redox balance. We conclude that roGFP1 is well suited for dynamic, compartment specific, subcellular analyses even in complex neuronal networks. The ability to correlate dynamic changes in cellular ROS levels with mitochondrial metabolism and neuronal network activity is a promising step towards a detailed mechanistic understanding of redox- and ROS-mediated signaling in normal and diseased brain function.

H(2)O(2)-mediated modulation of cytosolic signaling and organelle function in rat hippocampus.

Reactive oxygen species (ROS) released from (dys-)functioning mitochondria contribute to normal and pathophysiological cellular signaling by modulating cytosolic redox state and redox-sensitive proteins. To identify putative redox targets involved in such signaling, we exposed hippocampal neurons to hydrogen peroxide (H(2)O(2)). Redox-sensitive dyes indicated that externally applied H(2)O(2) may oxidize intracellular targets in cell cultures and acute tissue slices. In cultured neurons, H(2)O(2) (EC(50) 118 microM) induced an intracellular Ca(2+) rise which could still be evoked upon Ca(2+) withdrawal and mitochondrial uncoupling. It was, however, antagonized by thapsigargin, dantrolene, 2-aminoethoxydiphenyl borate, and high levels of ryanodine, which identifies the endoplasmic reticulum (ER) as the intracellular Ca(2+) store involved. Intracellular accumulation of endogenously generated H(2)O(2)-provoked by inhibiting glutathione peroxidase-also released Ca(2+) from the ER, as did extracellular generation of superoxide. Phospholipase C (PLC)-mediated metabotropic signaling was depressed in the presence of H(2)O(2), but cytosolic cyclic adenosine-5'-monophosphate (cAMP) levels were not affected. H(2)O(2) (0.2-5 mM) moderately depolarized mitochondria, halted their intracellular trafficking in a Ca(2+)- and cAMP-independent manner, and directly oxidized cellular nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH(2)). In part, the mitochondrial depolarization reflects uptake of Ca(2+) previously released from the ER. We conclude that H(2)O(2) releases Ca(2+) from the ER via both ryanodine and inositol trisphosphate receptors. Mitochondrial function is not markedly impaired even by millimolar concentrations of H(2)O(2). Such modulation of Ca(2+) signaling and organelle interaction by ROS affects the efficacy of PLC-mediated metabotropic signaling and may contribute to the adjustment of neuronal function to redox conditions and metabolic supply.

Enhanced hypoxia susceptibility in hippocampal slices from a mouse model of rett syndrome.

Rett syndrome is a neurodevelopmental disorder caused by mutations in the X-chromosomal MECP2 gene encoding for the transcriptional regulator methyl CpG binding protein 2 (MeCP2). Rett patients suffer from episodic respiratory irregularities and reduced arterial oxygen levels. To elucidate whether such intermittent hypoxic episodes induce adaptation/preconditioning of the hypoxia-vulnerable hippocampal network, we analyzed its responses to severe hypoxia in adult Rett mice. The occurrence of hypoxia-induced spreading depression (HSD)--an experimental model for ischemic stroke--was hastened in Mecp2-/y males. The extracellular K+ rise during HSD was attenuated in Mecp2-/y males and the input resistance of CA1 pyramidal neurons decreased less before HSD onset. CA1 pyramidal neurons were smaller and more densely packed, but the cell swelling during HSD was unaffected. The intrinsic optical signal and the propagation of HSD were similar among the different genotypes. Basal synaptic function was intact, but Mecp2-/y males showed reduced paired-pulse facilitation and higher field potential/fiber volley ratios, but no increased seizure susceptibility. Synaptic failure during hypoxia was complete in all genotypes and the final degree of posthypoxic synaptic recovery indistinguishable. Cellular ATP content was normal in Mecp2-/y males, but their hematocrit was increased as was HIF-1alpha expression throughout the brain. This is the first study showing that in Rett syndrome, the susceptibility of telencephalic neuronal networks to hypoxia is increased; the underlying molecular mechanisms apparently involve disturbed K+ channel function. Such an increase in hypoxia susceptibility may potentially contribute to the vulnerability of male Rett patients who are either not viable or severely disabled.

Optical and pharmacological tools to investigate the role of mitochondria during oxidative stress and neurodegeneration.

Mitochondria are critical for cellular adenosine triphosphate (ATP) production; however, recent studies suggest that these organelles fulfill a much broader range of tasks. For example, they are involved in the regulation of cytosolic Ca(2+) levels, intracellular pH and apoptosis, and are the major source of reactive oxygen species (ROS). Various reactive molecules that originate from mitochondria, such as ROS, are critical in pathological events, such as ischemia, as well as in physiological events such as long-term potentiation, neuronal-vascular coupling and neuronal-glial interactions. Due to their key roles in the regulation of several cellular functions, the dysfunction of mitochondria may be critical in various brain disorders. There has been increasing interest in the development of tools that modulate mitochondrial function, and the refinement of techniques that allow for real time monitoring of mitochondria, particularly within their intact cellular environment. Innovative imaging techniques are especially powerful since they allow for mitochondrial visualization at high resolution, tracking of mitochondrial structures and optical real time monitoring of parameters of mitochondrial function. The techniques discussed include classic imaging techniques, such as rhodamine-123, the highly advanced semi-conductor nanoparticles (quantum dots), and wide field microscopy as well as high-resolution multiphoton imaging. We have highlighted the use of these techniques to study mitochondrial function in brain tissue and have included studies from our laboratories in which these techniques have been successfully applied.

Mitochondrial inhibition prior to oxygen-withdrawal facilitates the occurrence of hypoxia-induced spreading depression in rat hippocampal slices.

Oxygen withdrawal blocks mitochondrial respiration. In rat hippocampal slices, this triggers a massive depolarization of CA1 neurons and a negative shift of the extracellular DC potential, the characteristic sign of hypoxia-induced spreading depression (HSD). To unveil the contribution of mitochondria to the sensing of hypoxia and the ignition of HSD, we modified mitochondrial function. Mitochondrial uncoupling by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 microM) prior to hypoxia hastened the onset and shortened the duration of HSD. Blocking mitochondrial ATP synthesis by oligomycin (10 microg/ml) was without effect. Inhibition of mitochondrial respiration by rotenone (20 microM), diphenyleneiodonium (25 microM), or antimycin A (20 microM) also hastened HSD onset and shortened HSD duration. 3-nitropropionic acid (1 mM) increased HSD duration. Cyanide (100 microM) hastened HSD onset and increased HSD duration. At higher concentrations, cyanide (1 mM), azide (2 mM), and FCCP (10 microM) triggered SD episodes on their own. Compared with control HSD, the spatial extent of the intrinsic optical signals of cyanide- and azide-induced SDs was more pronounced. Monitoring NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) autofluorescence and mitochondrial membrane potential verified the mitochondrial targeting by the drugs used. Except 1 mM cyanide, no treatment reduced cellular ATP levels severely and no correlation was found between ATP, NADH, or FAD levels and the time to HSD onset. Therefore ATP depletion or a cytosolic reducing shift due to NADH/FADH2 accumulation cannot serve as a general explanation for the hastening of HSD onset on mitochondrial inhibition. Additional redox couples (glutathione) or events downstream of the mitochondrial depolarization need to be considered.

Sulfhydryl oxidation reduces hippocampal susceptibility to hypoxia-induced spreading depression by activating BK channels.

The cytosolic redox status modulates ion channels and receptors by oxidizing/reducing their sulfhydryl (SH) groups. We therefore analyzed to what degree SH modulation affects hippocampal susceptibility to hypoxia. In rat hippocampal slices, severe hypoxia caused a massive depolarization of CA1 neurons and a negative shift of the extracellular DC potential, the characteristic sign of hypoxia-induced spreading depression (HSD). Oxidizing SH groups by 5,5'-dithiobis 2-nitrobenzoic acid (DTNB, 2 mM) postponed HSD by 30%, whereas their reduction by 1,4-dithio-dl-threitol (DTT, 2 mM) or alkylation by N-ethylmaleimide (500 microM) hastened HSD onset. The DTNB-induced postponement of HSD was not affected by tolbutamide (200 microM), dl-2-amino-5-phosphonovaleric acid (150 microM), or 6-cyano-7-nitroquinoxaline-2,3-dione (25 microM). It was abolished, however, by Ni2+ (2 mM), withdrawal of extracellular Ca2+, charybdotoxin (25 nM), and iberiotoxin (50 nM). In CA1 neurons DTNB induced a moderate hyperpolarization, blocked spontaneous spike discharges and postponed the massive hypoxic depolarization. DTT induced burst firing, depolarized glial cells, and hastened the onset of the massive hypoxic depolarization. Schaffer-collateral/CA1 synapses were blocked by DTT but not by DTNB; axonal conduction remained intact. Mitochondria did not markedly respond to DTNB or DTT. While the targets of DTT are less clear, the postponement of HSD by DTNB indicates that sulfhydryl oxidation increases the tolerance of hippocampal tissue slices against hypoxia. We identified as the underlying mechanism the activation of BK channels in a Ca(2+)-sensitive manner. Accordingly, ionic disregulation and the loss of membrane potential occur later or might even be prevented during short-term insults. Therefore well-directed oxidation of SH groups could mediate neuroprotection.