Signatures of hippocampal oxidative stress in aged spatial learning-impaired rodents.

Neurons and glia within the hippocampus of aged, spatial learning-impaired Long-Evans rats exhibit uniquely altered gene expression profiles, and we have postulated oxidative stress as the basis for this. To test this hypothesis we quantitated the extent of protein and nucleic acid oxidative damage, evaluated the status of mitochondrial DNA integrity, and examined several signaling entities and molecular indicators frequently associated with oxidative stress and gliosis. Immunoblotting demonstrated elevated heme oxygenase-1 in the aged-impaired hippocampus and immunocytochemistry suggested that heme oxygenase-1 is largely cytosolic and at least partly neuronal in nature. In the aged-impaired group, immunoreactivity to 8-hydroxy-2'-deoxyguanosine, an oxidative nucleic acid adduct, was found to be elevated in the dentate gyrus and in area CA1 of the hippocampal formation. Isolated mitochondrial DNA was found to be significantly damaged in the aged-impaired group. In the aged learning-impaired rats only, proteins in a 65-kDa band were found to contain excessive levels of carbonyl residues. Glial activation was examined by in situ hybridization histochemistry to tumor necrosis factor alpha and by immunocytochemistry with OX-6, which detects activated microglia. White matter in aged brains exhibited a modest up-regulation of tumor necrosis factor alpha mRNA and OX-6 immunoreactivity, but the hippocampal formation expressed tumor necrosis factor alpha mRNA equivalent to young animals and few OX-6-positive microglia. The mRNA for manganese-dependent superoxide dismutase, which is elevated in the aged hippocampus, was found preferentially expressed in neurons. We conclude that aged hippocampal neurons appear to be under oxidative stress and this is more severe in the learning-impaired subjects, suggesting a possible basis for age-induced cognitive decline.

Distribution of neurons immunoreactive for parvalbumin and calbindin in the somatosensory thalamus of the raccoon.

The aim of this study was to assess the distribution of neurons immunoreactive for parvalbumin (PV), calbindin (CaBP), glutamic acid decarboxylase (GAD), and gamma-aminobutyric acid (GABA) in the somatosensory thalamus of the raccoon and to compare these features to those of other species, especially primates. Immunohistochemistry was used to study the location of these neurons in the ventroposterior nucleus (VP), ventroposterior inferior nucleus (VPI), posterior group of nuclei (Po), and reticular nucleus (Rt). A consistent differential pattern of PV-positive (PV+) and CaBP-positive (CaBP+) cells was found in the somatosensory thalamus. Many PV+ neurons were observed in VP and Rt, but very few were found in VPI or Po. In contrast, CaBP+ neurons were distributed throughout VP, VPI, and Po but were very sparse or absent in Rt. In the VP nucleus, PV+ cells were distributed in clusters separated by interclusteral regions with a sparse distribution of PV+ cell bodies. The distributions of PV+ and CaBP+ cells tended to be complementary to each other in VP; regions with a high density of PV+ neurons had a low density of CaBP+ cell bodies. Double-labeling experiments revealed very few neurons in which PV and CaBP immunoreactivities were colocalized. Cells immunoreactive for GAD or GABA were found in PV-dense clusters of VP; fewer GABAergic neurons were present in the CaBP-dense interclusteral regions of VP and in VPI and Po. GAD+ and GABA+ neurons were most prominently distributed in Rt. We conclude that the distributions of PV+ and CaBP+ cell bodies in the raccoon somatosensory thalamus are very similar to those in primates. The density of GABAergic neurons in the somatosensory thalamus of the raccoon is less than that in the cat and monkey, but the relative distribution of GABAergic neurons in the different somatosensory nuclei is very similar to that in the cat and monkey. These results are discussed in relation to findings in other species and are related to the functions of lemniscal and nonlemniscal somatosensory pathways.

Effects of cholinergic depletion on experience-dependent plasticity in the cortex of the rat.

Clinical and functional studies have strongly suggested that acetylcholine input from the nucleus basalis of Meynert is important for the cortex's adaptive response to experience. The purpose of this study was to investigate the effects of depletion of acetylcholine inputs from nucleus basalis of Meynert on experience-dependent plasticity in the cortex of young adult male rats. The posteromedial barrel subfield in the primary somatosensory cortex was studied. Experience-dependent plasticity was elicited using a whisker-pairing paradigm in which all whiskers except D2 and D3 were trimmed daily. Plasticity within barrel D2 of the posteromedial barrel subfield was measured using the electrophysiological extracellular recording technique. An index of plasticity was determined in two ways: as an increase in the magnitude of evoked activity to stimulation of whisker D2 and as a bias in the ratio of evoked activity for stimulation of paired whisker D3 and cut whisker D1 (D3/D1). Whiskers D2, D3 and D1 were stimulated (deflected) by a Chubbuck electromechanical stimulator. Cholinergic neurons in the nucleus basalis of Meynert were selectively lesioned with an immunotoxin, 192 IgG-saporin, injected into the left lateral ventricle. Lesions of cholinergic neurons in the nucleus basalis of Meynert were verified using choline acetyltransferase immunocytochemistry and radioenzymatic assay. Experience-dependent plasticity was significantly reduced in cholinergic-depleted animals. The magnitude of evoked activity to stimulation of whisker D2 increased by 16-100% in control animals compared with 0-20% in cholinergic-depleted animals. Similarly, compared to a 60-100% increase in the D3/D1 ratio of evoked activity for phosphate-buffered saline-injected control animals, cholinergic-depleted rats showed no significant increase in the D3/D1 ratio (0-15%) after undergoing the whisker-pairing paradigm. After whisker trimming, the D3/D1 response ratio in immunotoxin-treated animals was essentially the same as in control animals that had not been subjected to the whisker-pairing paradigm. This study showed that no significant plasticity response was observed in the absence of cholinergic input from the nucleus basalis of Meynert. The mechanisms of the action of acetylcholine in cortical plasticity are still not known, but we hypothesize that this type of plasticity is activity dependent and is significantly enhanced in the presence of acetylcholine.

Topography of cholinergic afferents from the nucleus basalis of Meynert to representational areas of sensorimotor cortices in the rat.

We investigated (1) the topography of projection neurons in the nucleus basalis of Meynert (NBM) with efferents to restricted regions of the primary somatosensory (SI), the second somatosensory (SII), and the primary motor (MI) cortices in the rat; (2) the percentage of these NBM projection neurons that were cholinergic; and (3) the collateralization, if any, of single NBM neurons to different subdivisions within SI, to homotopic areas of SI and SII, and to homotopic areas of SI and MI. Retrograde single- and double-labeling techniques were used to study NBM projections to electrophysiologically identified subdivisions of SI and to homotopic representational areas of SI and SII, and of SI and MI. Choline acetyltransferase immunocytochemistry was done to identify cholinergic NBM neurons. Of the retrogradely labeled NBM neurons that projected to selective subdivisions of SI, SII, and MI, 89%, 87%, and 88%, respectively, were cholinergic. We found a rostral-to-caudal progression of retrogradely labeled NBM neurons following a medial-to-lateral sequence of injections into subdivisions of SI. Overlapping groups of single-labeled NBM neurons were observed after injections of different tracers into adjacent subdivisions within SI or homotopic areas of SI and SII, and of SI and MI. We conclude that NBM innervation to SI, SII, and MI is mostly cholinergic in the rat, that each cortical area receives cholinergic afferents from neurons widely distributed within the NBM, and that each NBM neuron projects to a restricted cortical area without significant collateralization to adjacent subdivisions within SI or to homotopic areas of SI and SII, or SI and MI.