An enriched environment restores normal behavior while providing cytoskeletal restoration and synaptic changes in the hippocampus of rats exposed to an experimental model of depression.

The exposure of rats to an enriched environment (EE) has several effects in common with the administration of antidepressants. However, there is still little information about the molecular underpinnings of these effects on rats subjected to experimental models of depression. The aim of this research was to evaluate the effects of EE on rats exposed to the learned helplessness paradigm (LH), a well-known model of the disease. We found that a 21 day exposure to EE reverts helplessness behavior to normal in LH animals. Inmunohistochemical labeling showed that this effect was accompanied by normalization of two structural proteins of hippocampal neurons to control values: the light neurofilament subunit (NFL) and the postsynaptic density 95 (PSD-95) protein, which were decreased in LH animals housed in standard cages. The decrease in the presynaptic protein synaptophysin (SYN) observed in LH animals remained unchanged after exposure to EE. There was no increase in neurogenesis as measured by quantification of double-labeled cells with 5-bromo-2'-deoxyuridine (BrdU) and the neuronal marker beta-tubulin class III. These results show that EE may have behavioral and synaptic effects on animals exposed to an experimental model of depression, and that such actions seem to be independent from neurogenesis.

Endobain E, a brain endogenous factor, is present and modulates NMDA receptor in ischemic conditions.

We have isolated from rat cerebral cortex an endogenous Na(+), K(+)-ATPase inhibitor, termed endobain E, which modulates glutamatergic N-methyl-d-aspartate (NMDA) receptor. This endogenous factor allosterically decreases [(3)H]dizocilpine binding to NMDA receptor, most likely acting as a weak channel blocker. In the present study we investigated whether endobain E is present in the cerebral cortex of rats subjected to ischemia and modulates NMDA receptor exposed to the same conditions. Ischemia-reperfusion was carried out by bilateral occlusion of common carotid arteries followed by a 15-min reperfusion period. Elution profile of brain soluble fraction showed that endobain E is present in cerebral cortex of ischemia-reperfusion rats. On assaying its effect on synaptosomal membrane Na(+), K(+)-ATPase activity and [(3)H]dizocilpine binding to cerebral cortex membranes prepared from animals without treatment, it was found that the endogenous modulator isolated from ischemia-reperfusion rats was able to inhibit both enzyme activity and ligand binding. On the other hand, endobain E prepared from rats without treatment also decreased binding to cerebral cortex or hippocampal membranes obtained from animals exposed to ischemia-reperfusion. Since ischemia decreases tissue pH and NMDA receptor activity varies according to proton concentration, pH influence on endobain E effect was tested. Endobain E ( approximately 80 mg original tissue) decreased [(3)H]dizocilpine binding 25% at pH 7.4 or 8.0 but 90% at pH 6.5. These results demonstrate that endobain E is present and also able to modulate NMDA receptor in the short-term period that follows cerebral ischemia and that its effect depends on proton concentration, suggesting greater NMDA receptor modulation by endobain E at low pH, typical of ischemic tissues.

Neuronal cytoskeletal alterations in an experimental model of depression.

It has been proposed that depression is associated with hippocampal morphological changes. The apical dendrite atrophy of hippocampal CA3 pyramidal neurons has been described in experimental models of depression. The aim of the present study was to determine which cytoskeletal components are involved in the morphological changes previously described in the hippocampus of depressed animals. The expression of different neuronal cytoskeletal markers was analyzed by immunohistochemistry in rats exposed to a learned helplessness paradigm, an experimental model of depression. Rats were trained with 60 inescapable foot shocks (0.6 mA/15 s) and escape latencies and failures were tested 4 days after training. Animals in which learned helplessness behavior persisted for 21 days were included in the depressed group. No foot shocks were delivered to control rats. Microtubule-associated protein 2 (MAP-2) and light (NFL; 68 kDa), medium (NFM; 160 kDa) and heavy (NFH; 200 kDa) neurofilament subunit immunostainings were analyzed employing morphometric parameters. In the depressed group, NFL immunostaining decreased 55% (P<0.05) and 60% (P<0.001) in CA3 and dentate gyrus, respectively. In the same areas, MAP-2, NFM and NFH immunostainings did not differ between depressed and control animals. Since NFL is present in the core of mature neurofilament, it is proposed that hippocampal depression-associated plastic alterations may be due to changes in the dynamics of the neurofilament assembly.

The effect of endogenous modulator endobain E on NMDA receptor is interfered by Zn2+ but is independent of modulation by spermidine.

A brain endogenous factor, termed endobain E, allosterically decreases [3H]dizocilpine binding to NMDA receptor. Such effect depends on receptor activation by the coagonists glutamate and glycine and is interfered by channel blockers, suggesting its interaction with the inner surface of the associated channel. To further analyze endobain E effect on NMDA receptor, in the current study competitive [3H]dizocilpine binding assays to brain membranes were performed with Zn2+ to block the associated channel, as well as with spermidine (SPD), which exerts positive allosteric modulation of NMDA receptor. Partially or nonadditive effects on [3H]dizocilpine binding were recorded, respectively, in the presence of endobain E at a concentration that inhibits binding 25% plus IC25 Zn2+ or endobain E at a concentration that inhibits binding 50% plus IC50 Zn2+. With an endobain E concentration that decreases 25% ligand binding, SPD potentiated binding over a wide concentration range but failed to modify endobain E effect. Similarly, [3H]dizocilpine binding reduction over a wide endobain E concentration range remained unaltered by high SPD concentrations. Additive effects were observed with endobain E at a concentration that decreases binding 25% plus IC25 SPD site antagonists arcaine or ifenprodil. Zn2+ experiments indicated that endobain E effect is interfered by channel blockade produced by this ion. Although endobain E effect is dependent on NMDA receptor activation by glutamate and glycine, it proves independent of the positive modulation exerted by SPD. Thus the endogenous modulator seems not to interact at NMDA receptor polyamine site, favoring the hypothesis that endobain E binds inside the associated channel.

[3H]dizocilpine binding to N-methyl-D-aspartate (NMDA) receptor is modulated by an endogenous Na+, K+-ATPase inhibitor. Comparison with ouabain.

An endogenous Na+, K+-ATPase inhibitor termed endobain E has been isolated from rat brain which shares several biological properties with ouabain. This cardiac glycoside possesses neurotoxic properties attributable to Na+, K+-ATPase inhibition, which leads to NMDA receptor activation, thus supporting the concept that Na+/K+ gradient impairment has a critical impact on such receptor function. To evaluate potential direct effects of endobain E and ouabain on NMDA receptors, we assayed [3H]dizocilpine binding employing a system which excludes ionic gradient participation. Brain membranes thoroughly washed and stored as pellets ('non-resuspended' membranes) or after resuspension in sucrose ('resuspended' membranes) were employed. Membrane samples were incubated with 4 or 10 nM ligand with or without added endobain E or ouabain, in the presence of different glutamate plus glycine combinations, with or without spermidine. [3H]dizocilpine basal binding and Na+, K+- and Mg2+-ATPase activities proved very similar in 'non-resuspended' or 'resuspended' membranes. Endobain E decreased [3H]dizocilpine binding to 'resuspended' membranes in a concentration-dependent manner, attaining roughly 50% binding inhibition with the highest endobain E concentration assayed. Among tested conditions, only in 'resuspended' membranes, with 4 nM ligand and with 1x10(-8) M glutamate plus 1x10(-5) M glycine, was [3H]dizocilpine binding enhanced roughly +24% by ouabain (1 mM). After Triton X-100 membrane treatment, which drastically reduces Na+, K+-ATPase activity, the effect of ouabain on binding was lost whereas that of endobain E remained unaltered. Results indicate that not only membrane preparation but also treatment and storage are crucial to observe direct endobain E and ouabain effects on NMDA receptor, which are not attributable to changes in Na+, K+-ATPase activity or to Na+/K+ equilibrium alteration.

Kinetics of Na+, K+-ATPase inhibition by an endogenous modulator (II-A).

We have previously reported the isolation by gel filtration and anionic exchange HPLC of two brain Na+, K+-ATPase inhibitors, II-A and II-E, and kinetics of enzyme interaction with the latter. In the present study we evaluated the kinetics of synaptosomal membrane Na+, K+-ATPase with II-A and found that inhibitory activity was independent of ATP (2-8 mM), Na+ (3.1-100 mM), or K+ (2.5-40 mM) concentration. Hanes-Woolf plots showed that II-A decreases Vmax in all cases; KM value decreased for ATP but remained unaltered for Na+ and K+, indicating respectively uncompetitive and noncompetitive interaction. However, II-A became a stimulator at 0.3 mM K+ concentration. It is postulated that brain endogenous factor II-A may behave as a sodium pump modulator at the synaptic region, an action which depends on K+ concentration.

Na+,K+-ATPase interaction with a brain endogenous inhibitor (endobain E).

Na+,K+-ATPase activity of rat brain synaptosomal membranes was evaluated in the presence of an inhibitory fraction II-E (termed endobain E), isolated by gel filtration and anionic exchange HPLC of a rat brain soluble fraction. We studied endobain E aging, analyzed its inhibitory potency in the absence or presence of ouabain as well as its ability to block high affinity [3H]ouabain binding to cerebral cortex membranes. Similar loss of endobain E activity was observed when samples were stored either dried or in solution. Endobain E fraction inhibited synaptosomal membrane Na+,K+-ATPase activity in a concentration-dependent manner and the slope of the corresponding curve strongly resembled that of ouabain. Assays performed in the presence of endobain E and ouabain indicated that the inhibitory effect was additive or less than additive, depending on their respective concentrations during preincubation and/or incubation. High affinity [3H]ouabain binding to cerebral cortex membranes proved concentration-dependent from 0.10 to 0.50 mg protein per ml; binding inhibition by endobain E was independent of protein concentration within the above range. [3H]ouabain binding inhibition by endobain E was concentration-dependent over a 10-fold range, an effect similar to that found for Na+,K+-ATPase inhibition. The extent of endobain E effect on Na+,K+-ATPase inhibition was much higher (90-100%) than that on [3H]ouabain binding blockade (50%). Findings suggest some type of interaction between endobain E and ouabain inhibitory mechanisms and favour the view that the former behaves as an endogenous ouabain.