Nerve growth factor prevents apoptotic cell death in injured central cholinergic neurons.

Experimental lesions have been widely used to induce neuronal degeneration and to test the ability to trophic molecules to prevent lesion-induced alterations, but these studies have not demonstrated unequivocally that afflicted neurons die as a result of these manipulations. The documentation of neuronal death in the above-described models and the time when it occurs after injury are crucial for the interpretation of trophic effects. In the present study, we combined multiple approaches to investigate the nature of retrograde neuronal changes in cholinergic neurons of the medial septal nucleus (MSN) after complete, unilateral transection of the fimbria-fornix (F-F). Projections neurons of the MSN were prelabeled with the fluorescent tracer Fluoro-gold (FG) 1 week prior to lesion. By counting both FG-labeled and choline acetyltransferase (ChAT)-immunoreactive neurons in the MSN at multiple time points postaxotomy, we differentiated the phenotypic response to injury from the degenerative process and established a critical time between the third and fourth weeks postaxotomy, during which approximately 50% of fluorescent perikarya disappear. Working in the previous time window, we identified dying cells by electron microscopy (EM) and terminal transferase-mediated (TdT) deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL) and showed that MSN neurons die via apoptosis, beginning at 16 days postaxotomy. An additional group of animals was allowed to survive for 1 month (i.e., 10 days after cell death has been completed); during this period, animals were treated with intraventricular nerve growth factor (NGF). Quantitative analysis of surviving cholinergic perikarya showed that NGF prevented degeneration of the majority of neurons. In concert, the results of the present study establish that NGF does not merely protect the phenotype but also prevents cell death in lesioned central cholinergic neurons.

Comparison of new and traditional methods for detection of senile plaques in Alzheimer's disease.

The pathologic diagnosis of Alzheimer's disease (AD) rests upon the identification of senile plaques and neurofibrillary tangles (NFT) in brain tissue. Methods for staining these structures vary in their sensitivity. Six different silver stains and immunocytochemistry for the beta-amyloid (A beta) peptide were compared for sensitivity in staining of plaques and tangles. For staining of plaques, the silver stains fell into two groups: one group stained primarily large, distorted neurites in classic plaques, and the other stained primarily fine, filamentous elements in diffuse plaques and at the periphery of classic plaques. Our recently developed "quick silver" method demonstrated the highest number of plaques. Sensitivity of NFT staining also varied considerably. The quick silver and Yamamoto-Hirano methods were best for staining both plaques and NFT.

Phosphate/calcium alterations in the first stages of Alzheimer's disease: implications for etiology and pathogenesis.

The present study tested aspects of a novel etiological/pathogenetic hypothesis of Alzheimer's disease (AD), which proposes that alterations in endocrine regulation of peripheral calcium/phosphate (Ca/PO4) homeostasis (e.g., by glucocorticoids, vitamin D, etc.) induce and/or reflect altered calcium homeostasis and neurotoxicity in brain neurons. Two key predictions of this hypothesis were tested, namely that: (1) alterations in serum Ca and/or PO4 regulation should be present before or near the onset of AD and (2) these alterations should be found consistently in subjects with unconfounded AD. Previous studies have sought evidence of changes in Ca regulation primarily late in the disease, and usually in severely demented, thin and immobile inpatients in whom Ca/PO4 measures are likely to be confounded. In the present study, only mobile, relatively healthy, adequately nourished outpatients with probable AD were selected. In comparison to age-matched controls or demented subjects with even mild indications of vascular contributions, the AD subjects were characterized by lower serum PO4 and, to a lesser degree, lower serum Ca as well as a higher chloride/PO4 ratio. On serum chemistry tests from the first stages of AD (within 1 or 3 years after the onset of cognitive symptoms), these changes in serum PO4/Ca were as pronounced as they were later in the disease. Moderately low values of either PO4, Ca, or both (below -1.0 S.D. from the control group mean) identified 74% of all AD subjects and 100% of early onset AD subjects, in comparison to only 46% of mixed/vascular dementia subjects and 31% of normal age-matched controls. Thus, the results were consistent with the predictions and may have significant etiological/pathogenetic implications. If larger tests confirm these frequencies, serum Ca/PO4 indices may also prove useful in differential diagnosis.

Chronic stress-induced acceleration of electrophysiologic and morphometric biomarkers of hippocampal aging.

There is increasing evidence that experimental interventions that alter adrenal corticosteroid plasma concentrations can modulate aging changes in the rodent hippocampus. However, there still is very little evidence that elevation of endogenous corticosteroid levels within physiological ranges, such as occurs during chronic stress, can accelerate hippocampal aging-like changes. In addition, almost all prior intervention studies of corticosteroid effects on brain biomarkers of aging have utilized morphologic measures of aging, and it is not yet clear whether electrophysiologic biomarkers of hippocampal aging can also be accelerated by conditions that elevate corticosteroids. In the present studies, specific pathogen-free rats of three ages (4, 12, and 18 months at the start) were trained for 6 months (4 hr/d, 5 d/week) in a two-way shuttle escape task, using low intensity foot shock. This task induces "anxiety" stress, because animals receive little actual shock, but chronic training in the task has been shown to elevate plasma corticosteroids and to downregulate hippocampal corticosteroid receptors. At the end of 6 months, animals were allowed to recover for 3 weeks and were then assessed in acute, anesthetized preparations on a battery of hippocampal neurophysiological markers known to separate young from aged animals (frequency potentiation, synaptic excitability thresholds, EPSP amplitude). The brains were then fixed and sectioned for quantification of neuronal density in field CA1 (a highly consistent anatomic marker of hippocampal aging). The pattern of stress effects differed considerably across age groups. The two younger stress groups exhibited increased evidence of aging-like neurophysiologic change, but exhibited no indications of accelerated neuronal loss.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant human nerve growth factor prevents retrograde degeneration of axotomized basal forebrain cholinergic neurons in the rat.

Cholinergic neurons in the basal forebrain magnocellular complex (BFMC) respond to nerve growth factor (NGF) during development and in adult life, and it has been suggested that the administration of NGF might ameliorate some of the abnormalities that occur in neurological disorders associated with degeneration of this population of neurons. A prerequisite for the introduction of NGF in clinical trials is the availability of active recombinant human NGF (rhNGF). The present investigation was designed to test, in vivo, the efficacy of a preparation of rhNGF. Axons of cholinergic neurons of the BFMC in the rat were transected in the fimbria-fornix; this manipulation alters the phenotype and, eventually, causes retrograde degeneration of these neurons. Our investigation utilized two lesion paradigms (resection and partial transection of fibers in the fimbria-fornix), two different strains of rats, and two delivery systems. Following lesions, animals were allowed to survive for 2 weeks, during which time one group received intraventricular mouse NGF (mNGF), a second group received rhNGF, and a third group received vehicle alone. In animals receiving vehicle, there was a significant reduction in the number (resection: 70%; transection: 50%) and some reduction in size of choline acetyltransferase- or NGF receptor-immunoreactive cell bodies within the medial septal nucleus ipsilateral to the lesion. Treatment with either mNGF or rhNGF completely prevented these alterations in the number and size of cholinergic neurons. The rhNGF was shown to be equivalent in efficacy with mNGF. Thus, rhNGF is effective in preventing axotomy-induced degenerative changes in cholinergic neurons of the BFMC. Our results, taken together with the in vitro effects of rhNGF (42), indicate that an active rhNGF is now available for further in vivo studies in rodents and primates with experimentally induced or age-associated lesions of basal forebrain cholinergic neurons. These investigations provide essential information for the consideration of future utilization of rhNGF for treatment of human neurological disorders, including Alzheimer's disease.

Alterations in levels of mRNAs coding for neurofilament protein subunits during regeneration.

Animal models of neuronal injury can be used to explore mechanisms that regulate the expression of genes coding for cytoskeletal proteins and transmitter-related markers. In the present study, in situ hybridization was used to measure levels of messenger ribonucleic acid (mRNA) encoding each of the neurofilament subunits and beta-tubulin in spinal motor neurons at intervals (4 to 56 days) following a unilateral crush of the sciatic nerve. Levels of beta-tubulin mRNA increased (approximately twofold), peaked at 28 days postaxotomy, and returned to control values by 56 days postaxotomy. In contrast, levels of mRNA encoding neurofilament subunits were reduced and returned to control values at 56 days following the lesion. There were significant differences among relative levels of mRNAs coding for each subunit. Other studies have demonstrated that the ratio of pulse-labeled neurofilament subunits in motor axons remained unaltered during regeneration. Therefore, the ratios of neurofilament subunits in axons must be regulated at one of the steps that intervenes between the control of levels of mRNA and the anterograde axonal transport of assembled neurofilaments.

Aberrant phosphorylation of neurofilaments accompanies transmitter-related changes in rat septal neurons following transection of the fimbria-fornix.

Lesions of the fimbria-fornix (FF) have been reported to cause retrograde changes in neurons of the medial septal nucleus (MSN). To analyze the nature and time course of these events, we investigated changes in cytoskeletal elements (phosphorylated and non-phosphorylated neurofilament (NF) proteins) and transmitter-related enzymes (choline acetyltransferase (ChAT) in MSN neurons following FF transection. During the first week postlesion, ChAT immunoreactivity and size of many perikarya were reduced. Irregular, swollen cholinergic fibers appeared first at postlesion day 2 in caudal septum and soon spread rostrally, reaching rostral septum by day 7. A few perikarya developed abnormal accumulations of phosphorylated NFs. At postlesion days 7-10, many neurons did not stain for ChAT. Phosphorylated NFs were present in many perikarya. At this time, cell loss was apparent in Nissl-stained material. Cholinergic cell loss continued through postlesion weeks 6-8 but at a much slower rate than during the first week. Phosphorylated NF accumulations in MSN perikarya persisted until postlesion week 6, disappearing thereafter. Double-immunostaining procedures showed that MSN neurons expressed both ChAT and phosphorylated NF immunoreactivity at postlesion day 3; however, at days 7 and 14, cells that accumulated phosphorylated NFs did not stain for ChAT. The results of this study indicate that FF transection leads to perikaryal shrinkage with loss of ChAT immunoreactivity, perikaryal phosphorylation of NFs, cholinergic fiber abnormalities, and cell loss. Recent evidence suggests that reduction of transmitter markers and aberrant phosphorylation of NFs may be involved in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. Therefore, FF transection provides a useful animal model for further investigations of complex disorders of the central nervous system that involve degeneration of transmitter-specific pathways.

Synaptic vesicle redistribution during hippocampal frequency potentiation and depression in young and aged rats.

Technical complexities associated with studies of defined synapses in the mammalian CNS have made it difficult to determine whether CNS synaptic vesicles respond to repetitive monosynaptic activation in the same ways as do PNS vesicles. Moreover, even in the PNS, the specific ultrastructural correlates of short-term synaptic potentiation are not well characterized. In the present studies, therefore, the Schaffer collateral-commissural fiber projection to field CA1 of the hippocampus was used as a model brain system in which to assess quantitative relations between dynamic vesicle patterns and frequency potentiation and depression. Anesthetized rats were perfused with aldehyde fixative during stimulation trains and concomitant microelectrode recording in order to study temporal correlations between physiology and synaptic structure. In addition, young and aged animals were compared to determine whether there are ultrastructural correlates of a previously described age-related impairment of hippocampal frequency potentiation. After 1 min of 10 Hz synaptic stimulation (stim), frequency potentiation of the population spike was correlated with increases in local vesicles (LV; within 150 nm of the active zone) and in vesicles directly apposed (AV) to the active zone membrane of CA 1 synapses. In addition, the ratio LVD: DVD of local-to-distant vesicle (DV; beyond 150 nm) densities was increased under all stimulation conditions. After 10 min of 10 Hz stim, or 1 min of 40 Hz stim, some vesicle depletion (approximately 25%) was present, as was some spike depression or decline from peak potentiation. However, LV depletion was not well correlated with degree of spike depression. Mean dendritic spine area was also increased in association with potentiation (at 1 min of 10 Hz stim) in young rats. Membrane infoldings similar to the cisternae described in other systems were seen with repetitive stimulation, providing evidence of vesicle recycling in these CNS terminals. In comparison to young control animals, terminals in non-stimulated aged group control rats exhibited a higher LVD:DVD ratio and a reduced DVD. In addition, the stimulation-induced increase in AV at 1 min of 10 Hz stim was not present in aged rat synapses. However, no age-related differences in vesicle-density measures were found at any stimulation point.(ABSTRACT TRUNCATED AT 400 WORDS)

Redistribution of synaptic vesicles during long-term potentiation in the hippocampus.

Synaptic vesicles were quantified 20 min after the induction of long-term potentiation (LTP) in the Schaffer-commissural system of the hippocampus. With LTP, significant increases were found in vesicles attached to the active zone membrane, and in the percentage of vesicles adjacent to the active zone. In addition, as others have reported, overall vesicle density was decreased and spine area was increased. These results suggest that an increased probability of vesicle release may contribute to brain LTP.

The effects of high Mg2+-to-Ca2+ ratios on frequency potentiation in hippocampal slices of young and aged rats.

In some central systems, excitatory postsynaptic potential (EPSP) amplitude increases substantially during repetitive synaptic stimulation ("frequency potentiation"), as does the probability of spike generation. An apparently analogous phenomenon at the neuromuscular junction ("frequency facilitation") depends on residual Ca2+ in nerve terminals. However, the mechanisms of central frequency potentiation are not completely defined and it is therefore not clear whether the patterns of Ca2+-dependent synaptic plasticity are fully analogous in central and peripheral systems. In addition, an age-related deficit in hippocampal frequency potentiation has been previously described, and the degree of sensitivity of this deficit to Mg2+-to-Ca2+ balance could yield important insights into its nature. In these studies, we used the hippocampal slice preparation to examine the effects of varying Mg2+-to-Ca2+ ratios in the artificial cerebrospinal fluid (ACF) on frequency potentiation in aged and young rats. Extracellular and intracellular methods were used to assess the responses of hippocampal CA1 neurons during orthodromic stimulation of the monosynaptic Schaffer-commissural pathway. In experiment 1, frequency potentiation of the hippocampal population spike during 7-Hz stimulation was found to be significantly greater in an ACF with a high Mg2+-to-Ca2+ ratio (2.7) than in an ACF with a normal Mg2+-to-Ca2+ ratio (0.5), for both young and aged rat slices. Aged slices exhibited less frequency potentiation than young in both media. In experiment 2, the field EPSP and population spike were monitored concurrently, and the differences in Mg2+-to-Ca2+ ratio between the high Mg2+-to-Ca2+ ACF ratio (2.0) and normal Mg2+-to-Ca2+ ACF ratio (1.0) were reduced, to determine whether aged and young brains differed in sensitivity to smaller variations in Mg2+-to-Ca2+ balance. Under these conditions, the effects of high Mg2+-to-Ca2+ ratios on frequency potentiation (at 7 Hz) were found to be most pronounced in aged rat slices, particularly for potentiation of the spike. No effects were seen of age or Mg2+-to-Ca2+ ratios on presynaptic fiber volley amplitudes. Field EPSP (but not spike) amplitudes were reduced with aging, in an input-output (I/O) stimulation series at control frequency (0.2 Hz). However, the high Mg2+-to-Ca2+ ACF ratio of (2.0), which improved field EPSP frequency potentiation, did not decrease control field EPSP amplitudes in the I/O series. Therefore, the effects of high MG2+-to-Ca2+ ACF ratio on brain frequency potentiation seem to be mediated in part by mechanisms other than the classical reduction of release probability.(ABSTRACT TRUNCATED AT 400 WORDS)