Neuroprotocols最新文献

Changes in the synaptic input to aging neurons can best be evaluated using electron microscopy (EM). Immunocytochemistry is used to identify neuronal populations and to distinguish the chemical identity of their synaptic input, using a double-label protocol (e.g., diaminobenzidine for the sites of the first antigen/antibody complexes and tetramethylbenzidine for the second). In preparing tissue for EM examination, neurons are sectioned in the plane of the nucleus at 70 nm and sections collected on slot hole grids. Photographic montages of neurons are made at a minimum of 3 depths of section, with about 1 μm intervening. The original micrographs are taken at 10,000× and printed at 25,000×. Morphometric analyses are performed using the Bloquant program (R&M Biometrics) and an IBM computer. The perikaryal membrane is outlined on an X-Y digitizing pad, and regions along which there is synaptic modification are measured. These synaptic regions are expressed as a percentage of the perikaryal membrane measured. Data are tested using a non-parametric statistic (Mann-Whitney U, P < 0.05). In some cases, the entire neuronal soma is serially sectioned in order to determine whether synapses are randomly distributed over the neuronal surface.

We describe the methods and rationale for using rats with a partial unilateral lesion of the nigrostriatal system as an animal model for studying neural plasticity in both young and aged brains. The rats are lesioned with 6-hydroxydopamine injected into the substantia nigra or the medial forebrain bundle. Amphetamine- and apomorphine-induced rotational behaviors are tested 3 and 4 weeks following the lesion. Based on the rotational responses to amphetamine and apomorphine administration, animals can be classified into one of three groups: unaffected, partially lesioned, or severely lesioned. Animals classified as displaying unaffected rotational behavior are those that do not respond to either amphetamine or apomorphine stimulation. Partially lesioned animals rotate ipsilateral to the lesioned side upon amphetamine injection, but do not display a significant number of rotations in response to apomorphine administration. In contrast, severely lesioned rats rotate after both amphetamine and apomorphine injections. Cell counts reveal that the mean number of dopamine neurons in the ventral mesencephalon of partially lesioned animals is reduced to 40% of that of the intact side. Also in partially lesioned animals, dopamine concentrations on the lesion side are even more severely depleted, averaging about 20% of levels in the contralateral intact striatum. Striatal dopamine concentrations correlate well with the number of surviving dopamine neurons in the ventral mesencephalon (r² = 0.66, P < 0.05). Amphetamine-induced rotation rates also show a moderate correlation with both striatal dopamine concentrations and mesencephalic dopamine neuron cell counts. Therefore, rotational behavior induced by amphetamine and apomorphine stimulation can be used to identify partially lesioned rats following unilateral 6-hydroxydopamine lesions. It is also possible to estimate the extent of nigrostriatal system damage from the rate of amphetamine-induced rotation.

A variety of methodologies, including fetal transplantation, electrophysiology, and molecular biological techniques, are now available to the neuroscientist interested in determining how the central nervous system and neuroendocrine systems change with age. The aim of our studies has been to examine the morphology, biochemistry, and physiology of neurons that are important to the modulation of gonadotropin hormone-releasing hormone (GnRH). This review focuses on neuroanatomical analyses. The nature of morphological changes in the CNS that result in the loss of normal reproductive cyclicity with aging have been unknown until now. Our studies have attempted to examine cytoarchitectural relationships between peptidergic and GnRH neurons in the rostral forebrain and the hypothalamus in order to determine the mechanism for the loss of reproductive capacity in the aging female C57BL/6J mouse. We have employed light and electron microscopic, morphometric, and immunocytochemical techniques. Our studies have shown that only specific subregions, and specific neurons within these regions, are susceptible to age-related changes. Removal of the ovary over the long term has failed to protect against any observed neuroanatomical alteration. Our neuroanatomical data clearly suggest that functional changes in neuroendocrine systems occur both with and without concomitant death of neurons.

One of the difficulties in the field of the neurobiology of aging is that many of the studies are necessarily correlative in nature. Investigators interested in the neurotransmitter or neuropeptide control of a given brain function often describe the situation in young animals and compare these observations with findings for groups of aged animals. However, ascribing causation with such studies is difficult at best. The use of antisense oligodeoxynucleotides described in this article allows the investigator to target one or more neuropeptides expressed in a confined area of the brain for selective abiation. The assessment of cause and effect on the physiological endpoint of Interest then becomes more tractable. Having previously identified age-related changes in neuropeptide dynamics that may underlie certain aspects of endocrine aging, we used this method to determine whether specific antisense oligodeoxynucleotide treatment of young animals mimicked the effect of age on the endocrine system. The theoretical background and practical aspects of the method are presented in sufficient detail to allow investigators not familiar with the technique to design appropriate oligodeoxynucleotides and use them in their research.

This article considers the problems encountered in the study of basic electrophysiologlcal properties of aged animal brain cells and describes several methods that are useful for such studies. Specific methods for rat brain slice preparations are reviewed, with an emphasis on factors relevant to aging animals. Alternative approaches (acutely dissociated cells) are also considered. Methods for investigating pharmacologically isolated and defined calcium potentials and calcium currents in aged rat brain neurons also are described. These may play an important role in the brain aging process.

For a given neuron, the development of its axonal and dendritic arborizations depends on many external factors, such as matrix molecules, growth factors, depolarization, electric fields, and adhesion molecules. In this paper, we summarize and comment on several protocols that can be used to modulate axonal or dendritic elongation and/or modify the shape of the neurites. A first series of protocols is based on the modulation of neuron-substratum adhesion by the addition of extra cellular matrix molecules. Indeed, axons initiate and elongate under low adhesion conditions, whereas dendrites grow only on highly adhesive substrata. A second series of protocols involves the use of drugs affecting the organization of the cytoskeleton. They suggest that the different behaviors of the axonal and dendritic compartments, in particular under low adhesion conditions, are due partly to the organization of the microtubule and actin networks. Third, we describe a protocol based on the internalization of Antennapedia homeodomain that translocates through the cell membrane and is conveyed to neuronal nuclei. Using this technique, we demonstrated that homeoproteins are involved in the morphological differentiation of postmitotic neurons and, in the case of the motoneurons, in axonal elongation. Furthermore, fusion polypeptides up to 109 amino acids and encompassing the 60-amino-acid translocating homeodomain are also transported through the membrane, thus offering a way to introduce exogenous biologically active peptides into live neurons.