José Javier Miguel-Hidalgo

β-Amyloid(1–40)-induced neurodegeneration in the rat hippocampal neurons of the CA1 subfield

Abstract Small volumes of solutions injected into the hippocampus produce dramatic degeneration in dentate gyrus neurons, but not in neurons of the CA1 subfield. The aim of the present study was to ascertain whether solutions with different fragments of the β-amyloid protein (Aβ) could produce further degeneration in areas beyond the dentate gyrus. It was found that 5 days after injection of an aqueous solution containing the Aβ 1–40 fragment into the hippocampus, long stretches of the CA1 subfield were either deprived of neurons or most of the neurons were degenerating. By contrast, in animals with deposits containing Aβ 1–28, Aβ 1–42 or water, neuronal degeneration or depletion only occurred in a reduced area around the place where the implant needle penetrated the CA1 subfield. In animals injected with Aβ 1–40, many profiles in the CA1 subfield and dentate gyrus were undergoing apoptosis, as seen using preparations processed by routine histology or the TUNEL technique for detection of fragmented DNA. In addition, there was higher infiltration by ED1-positive, activated microglia-macrophagic cells in Aβ 1–42 deposits than in deposits of Aβ 1–40. The present results suggest that the intrahippocampal injection of toxic Aβ fragments produces neuronal degeneration in the rat CA1 subfield when using the appropriate protocol, and, thus, can provide an in vivo model to investigate the neurotoxic effects of Aβ and for the evaluation of drugs with potential anti-neurodegenerative activity.

Distribution of calbindinlike immunoreactive structures in the optic tectum of normal and eye-enucleated cyprinid fish

SummaryUsing the ABC immunohistochemical method, we investigated the distribution of calbindinlike immunoreactive structures in the optic tectum of normal fish, Tinca tinca, and from normal and unilaterally eye-enucleated fish, Cyprinus carpio. In nonoperated individuals of both species the optic tectum contained numerous immunoreactive neurons with strongly positive somata located in the stratum periventriculare and a thick immunolabeled dendritic shaft ascending radially toward the stratum fibrosum et griseum superficiale. The retinorecipient layers contained many fibrous immunoreactive structures. Some varicose fibers, isolated or in small bundles, were localized to the stratum album centrale, especially in the dorsal tectal half. Unilateral eye removal produced the disappearance of the immunoreactive fibrous structures located in the retinorecipient layers of the tectum contralateral to the enucleation. The present work shows that calbindinlike immunoreactive substances are localized in specific neural circuits of the fish optic tectum and suggests that the calbindin-like immunoreactive fibers in the retinorecipient strata are of retinal origin.

PLASTICITY OF CONGO RED STAINING DISPLAYED BY SUBPOPULATIONS OF NEURONS WITHIN THE RAT CENTRAL NERVOUS SYSTEM

Abstract We document the presence of subpopulations of neurons within the rat central nervous system that are labelled with a new Congo red staining technique. These neurons (CR neurons) show shrunken somata, and smaller and darker nuclei than Congo red-negative cells (non-CR cells). With the Bielschowsky and the cresyl violet Nissl staining methods, two comparable subpopulations of cells can be distinguished by the same morphometrical criteria as those used for CR and non-CR cells. CR neurons are located preferentially in some brain regions while in others they are virtually absent. Their distribution and proportion varied greatly from animal to animal and after particular treatments. Injections of water that damaged the hippocampal dentate gyrus, cortical lesions or eye enucleation decreased the number of CR-cells in the CA1 subfield, reflected in a shift from the CR-staining subclass to the non-CR subclass. Treatment with 200 mg/kg of CDP-choline also significantly reduced the number of CR cells observed in CA1. In the red nucleus, CR neurons showed a characteristic distribution of β-amyloid precursor protein (APP) immunoreactivity. The population of dendrites immunolabelled for microtubule-associated protein 2 was markedly decreased in the areas of the hippocampus with high numbers of CR cells. Therefore, it is proposed that neurons labelled with the present Congo red technique might be in a reversible degenerative state or represent a particular physiological state in some areas of the central nervous system.

Anapsos: New Therapeutic Strategies for Neurodegeneration and Brain Aging with Neuroimmunotrophic Factors

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by an heterogeneous etiopathogenesis on a genetic basis. The most prominent neuropathological findings in AD are senile plaques, neurofibrillary tangles (NFT), amyloid deposition in neural tissues and vessels, synaptic loss and subsequent neuronal death (Cacabelos et al., 1993, 1995; Hardy and Allsop, 1991; Terry et al., 1991). However, other neurochemical mechanisms may also account for cell death and neurodegeneration in AD, including neuroimmune dysfunction, free radical formation, neurotransmitter deficits and alterations in brain calcium homeostasis (Cacabelos et al., 1994, 1995; Richardson, 1993). So, new approaches to AD treatment are oriented to search for agents acting on etiopathogenic events and pleiotropic compounds displaying multifactorial effects.

Projections of tachykinin- and glutaminase-containing rat retinal ganglion cells.

Glutamate (Glu) and the tachykinin substance P (SP) have been proposed as neurotransmitters or neuromodulators of the retinal projection to the brain. In the present study, we demonstrate that tachykinin-like (TK) immunoreactivity (IR) accumulates in rat retinal axons following electrical lesions to the optic tract, indicating that SP is conveyed in the optic nerve to its central targets. In addition, we show that eye enucleation causes a dramatic decrease in TK-IR fibers in the pretectal olivary nucleus (PON), but not in other retinorecipient nuclei of the thalamus and the midbrain, and that Fluorogold injected into the pretectum is retrogradely transported to the somata of TK-IR retinal ganglion cells (RGCs), indicating an important projection of TK-IR RGCs to the PON. We also show that most rat RGCs are labeled with antibodies against phosphate-activated glutaminase, an enzyme considered to generate the transmitter pool of glutamate. Unlike TK-IR fibers, phosphate-activated glutaminase-IR structures disappear in most retinorecipient nuclei following eye enucleation. The present results give neuroanatomical support to the idea that glutamate is a neurotransmitter in the retinal projection and suggest an important role for TK-IR RGCs in the relay of visual information to the PON.

Ultrastructure and retinal innervation of deafferentation-induced enkephalin-immunoreactive elements in the superficial layers of the rat superior colliculus.

Leu-enkephalin-like immunoreactive (ENK-I) elements appearing in the superficial layers of the rat superior colliculus (SC) after eye-enucleation were examined by means of immunoelectronmicroscopy. ENK-I somata were of a single type and formed symmetric and asymmetric synapses with non-immunoreactive axon terminals. Some degenerating retinal terminals made synaptic contacts only with small ENK-I dendrites, suggesting that deafferentation-induced ENK-I neurons in the rat SC receive retinal input onto the distal portions of their dendrites.

In Vivo Neurotoxicity of β-Amyloid 1–40 in the Rat Hippocampus

The hippocampus is a privileged brain center for the study of neuronal plasticity and the responses of neurons to various types of injury. Specific degeneration of different neuronal populations occurs in the hippocampus after particular aggressions (Miguel-Hidalgo and Cacabelos, 1997). A classical example is the degeneration of CA1 neurons following a short period of ischemia (Pulsinelli, 1988). In this instance it is already known that excess extracellular glutamate caused by prolonged depolarization of hypoxic neurons contributes greatly to neuronal degeneration. In the dentate gyrus, granule cells are specifically sensitive to corticosteroid deprivation specially in rodents, although in this case the mechanisms of cell death are not yet well understood (Sloviter et al. 1993). Specific neuronal depletion is found also in the dentate gyrus as a consequence of small injections of fluids into the hippocampus (Vietje and Wells, 1989), but the mechanisms involved are unknown. Neuronal degeneration in the hippocampus, among other structures, is a characteristic of the Alzheimer’s disease (AD) that very likely contributes to the overhelming memory deficits observed in AD patients (Hyman et al., 1984; Hyman and Van Hoesen, 1989). Accordingly, models of degeneration involving hippocampal neurons are relevant to AD therapy whenever it is possible to show that particular pharmacological molecules are capable of protecting the neurons that degenerate in those models. With this consideration in mind, we have been studying the effects of injecting small volumes of β-amyloid peptide fragments dissolved with water or water alone into the rat hippocampus in order to assess the degenerative effects on hippocampal neurons at the morphological and behavioral levels.

Anapsos Improves Learning and Memory in Rats with βA(1–28) Deposits Into the Hippocampus

Anapsos is a vegetal extract obtained from dried rhizhomes of the fern Polypodium leucotomos growing in Central America. This extract is devoid of toxic effects in animals and humans at therapeutic doses and is commercially available in Spain. An antitumoral effect of anapsos was found in early studies (Horwath et al., 1967), and this effect was confirmed by other authors suggesting an interaction of anapsos with cytoplasm receptors (Vargas et al., 1981). These studies show that anapsos has anabolic effects in normal tissues in vivo oppossite to the catabolic action exerted by cytostatics. Furthermore, Anapsos exerts immunoregulatory effects in control subjects (Sempere et al., 1997; Vargas et al., 1983) and in patients with atopic dermatitis (Jimenez et al., 1987), psoriasis (Padilla et al., 1974) or vitiligo (Mohamed, 1989), and increases the allograft survival in rats and mice with skin transplants (Perez de las Casas et al., 1987; Tuominen et al., 1991). In healthy subjects anapsos increases the lymphoblast response to mitogens, serum immunoglobulin levels and the proportion of CD8+ cells in a dose-dependent manner (Vargas et al., 1983). Anapsos also stimulates cell proliferation, reduces LPS-stimulated IL-lβ levels and delays the peak response of IL-lβ to LPS+PHA in human PBMC cultures (Sempere et al., 1997). Recently, we have found that anapsos improves learning and reduces brain IL-lβ levels in normal rats; reverses learning impairment and brain IL-lβ overexpression in rats with lesions in the nucleus basalis of Meynert; improves motor functioning and influences the production of brain immune factors as histamine and IL-lβ in aged mice; and reduces behavioral deficits and neuronal degeneration induced by β-amyloid implants into the hippocampus, modulating IL-lβ and Superoxide dismutase (SOD) activity levels in the brain of βA-injected rats (Alvarez et al., 1992, 1995, 1996c, 1997; Fernandez-Novoa et al., 1997).