Modification of AgNOR staining to reveal the nucleolus in thick sections specified for stereological and pathological assessments of brain tissue.

BACKGROUND: Various stains have been devised to reveal degenerative or reactive cell phenotypes, or the disintegrative and/or neuropathic lesions associated with Alzheimer's, Parkinson's, and Pick's diseases, Down's syndrome, or chemical toxicity. Utilization of silver staining has allowed researchers to elucidate neural pathways promoting a greater understanding of the functional connections between brain regions. All of these methods employing silver can be characterized as 'directed staining technologies'. NEW METHODS: The argyrophilic proteins (AgNOR) staining protocol was modified to stain nucleoli in thick sections prepared for stereological evaluation of brain tissue. Nucleoli appeared as black dots against a pale amber background. Tissue sections were counterstained with Toluidine Blue, or reduced-strength Tyrosine Hydroxylase immunohistochemistry to facilitate visualization of basic cellular morphology and regional nucleus identification. Here, we present a modified method for nucleolar staining in free-floating thick sections of brain embedded in a gelatin matrix. The modifications in our procedure include incubation in HCl to denature ('unravel') the DNA, a bleaching step to reduce non-specific background silver staining, and counterstaining with Toluidine Blue or reduced-strength tyrosine hydroxylase immunohistochemistry. COMPARISON WITH OLD METHODS: Prior to the development of immunohistochemistry, silver staining was used primarily to identify pathological profiles and trace axon pathways; however, in many cases, a combination of silver staining and immunohistochemistry are required to fully visualize pathomorphology. The mechanism of these stains requires the binding of silver ions to cellular components and the subsequent reduction of the ions to metallic silver. Dilutions of TH primary antibody were evaluated to maximize identification of neurons and the nucleolus amongst the soma and processes present in the thick section. The use of stereology as a tool to estimate cell number has become increasingly prevalent in neuroscience experiments. As requirements for the preparation of experimental tissue have been refined, researchers have begun to use thicker sections, between 40 to 80 microns, to increase the number of optical planes available for analysis. These thick sections require modified staining protocols to assure complete penetration of stains throughout the tissue section. CONCLUSIONS: This method is particularly useful in nucleolar identification for Stereology, and automated counting methods. Use of the nucleolus avoids some of the problems associated with use of the nucleus. The nucleolus is smaller than the nucleus and is less susceptible to transection during sectioning. It has a higher density than the nucleus and is easier to visualize. It is generally darker staining than the immunohistochemical reaction product that provides the identification marker for the cells to be counted. Examples of the method in several brain sections of the rat are shown, though the method has been also proven in other mammalian models.

Regional variations in the physiology of the rat caudate-putamen. 1. In vivo subregional effects of amphetamine on extracellular dopamine concentration.

The caudate putamen is a neurochemically and functionally heterogeneous nucleus. Understanding the correlation between these regional variations in neurochemistry and function could greatly aid in the treatment of neurological disorders associated with this area of the brain. Since dopaminergic dysfunction has been implicated in some of these disorders, regional variations in the neurochemistry of this transmitter system are of particular interest. The dopaminergic response to 2.5 mg/Kg D-amphetamine was examined by in vivo voltammetry in 7 dorsal and 7 ventral regions of the caudate-putamen in the urethane anaesthetized rat. Extracellular dopamine concentration increased in all the areas examined. However, the effect was regionally heterogeneous-areas separated by as little as 1 mm showing significantly different responses in terms of both the absolute change and the rate of change in dopamine concentration. A significant general trend was also evident. Amphetamine produced an increasing effect in extracellular dopamine concentrations in the dorsal and lateral areas of the nucleus. It was concluded that the regionally heterogeneous effects of amphetamine on extracellular dopamine could be attributed to regional variations in the density of dopamine transport sites within the caudate-putamen. Since this transport site is the site of entry of a number of neurotoxins this finding may contribute to our understanding of the functional loss associated with disorders such as Parkinson's disease.

Regional variations in the physiology of the rat caudate-putamen. 2. Effects of amphetamine and amphetamine induced dopamine release on basal and cortical stimulation evoked multiple unit activity.

Dopaminergic terminals within the caudate-putamen are located in an ideal position to modulate the corticostriatal system. Since this is the major afferent system of the striatum, dopamine has very powerful effects on striatal electrophysiological activity. The striatum is a regionally specialized multifunctional nucleus. It is therefore important to determine if dopamine has the same modulatory effects within different areas of the nucleus. The effects of 2.5 mg/Kg D-amphetamine (IP) on cortical stimulation evoked and basal multiple unit activity (MUA) was measured in 7 dorsal and 7 ventral striatal areas of the urethane anaesthetized rat. In general, amphetamine caused an increase in the basal activity and a decrease in the cortical stimulation evoked activity. However, there were both qualitative and quantitative regionally dependent differences in these responses. The effect on basal MUA was more pronounced in the dorsal and caudal areas whereas the effect on cortical stimulation evoked MUA was more pronounced in the ventral areas. The electrophysiological effects of amphetamine within the striatum were correlated with its regionally dependent effects on extracellular dopamine. This produced a measure of the effects of striatal dopamine on regional electrophysiological activity. This information was also used to determine the mathematical relationship between dopamine concentration change and the change in MUA. These data indicate that the excitatory effects of amphetamine-induced dopamine release on the non-stimulated MUA progressively increase along the rostro-caudal axis of the nucleus. In addition, the effects were more pronounced in the ventromedial as compared to the ventrolateral areas. These effects correlated best with the rate of change in dopamine concentration. In the dorsal striatum amphetamine-induced increases in dopamine had a regionally homogeneous inhibitory effect on the stimulated MUA. In the ventral striatum however, it had a progressively stronger effect along the rostro-caudal axis. These effects correlated best with the absolute change in dopamine concentration.

Three-dimensional electrophysiological topography of the rat corticostriatal system.

Projections from the cerebral cortex are the major afferents of the caudoputamen and probably determine the functions subserved by each region of the nucleus. The corticostriatal system has been mapped using cytological techniques which give little information on the physiological importance of projections from individual cortical areas. The objective of this study was to characterize the three-dimensional topography of the corticostriatal system in the rat and to determine the physiological significance of these projections using electrophysiological techniques. Eight functionally distinct areas of the cerebral cortex (prefrontal, primary motor, rostral and caudal primary somatosensory, hindlimb, auditory, occipital and primary visual) were stimulated while recording the multiple unit activity in seven dorsal and seven ventral areas of the caudoputamen. Each stimulation site produced a distinctive pattern of activation within the caudoputamen. There was also a large site-dependent variation in electrophysiological activation produced by each stimulation. The motor and somatosensory areas produced the most powerful overall activation. In addition, a number of trends were obvious. There was a rostrocaudal topographical relationship between the site of stimulation and the area of the caudoputamen activated. Furthermore, more caudally and medially placed stimulation sites produced greater dorsal activation of the caudoputamen relative to ventral.

Meta-analysis of functional outcome in Parkinson patients treated with unilateral pallidotomy.

Parkinson's disease (PD) profoundly affects activities of daily living (ADL) and quality of human life. Although unilateral pallidotomy has become a common surgical treatment for persons with advanced PD, functional outcome data from previous reports have failed to uniformly support this procedure. In the present investigation, results from 12 studies meeting specific inclusion criteria were subjected to meta-analysis. Only reports featuring unilateral pallidotomy as the exclusive surgery, a sample size of at least five patients, explicit assessment of ADL, and sufficient quantitative data were subjected to analysis. Type of research design was not a factor in the selection process. The results of our analysis suggest that unilateral pallidotomy successfully enhances functional outcome in patients with clinically advanced PD.