Amyloid-beta expression in retrosplenial cortex of triple transgenic mice: relationship to cholinergic axonal afferents from medial septum.

Triple transgenic (3xTg-AD) mice harboring the presenilin 1, amyloid precursor protein, and tau transgenes (Oddo et al., 2003b) display prominent levels of amyloid-beta (Abeta) immunoreactivity in forebrain regions. The Abeta immunoreactivity is first seen intracellularly in neurons and later as extracellular plaque deposits. The present study examined Abeta immunoreactivity that occurs in layer III of the granular division of retrosplenial cortex (RSg). This pattern of Abeta immunoreactivity in layer III of RSg develops relatively late, and is seen in animals older than 14 months. The appearance of the Abeta immunoreactivity is similar to an axonal terminal field and thus may offer a unique opportunity to study the relationship between afferent projections and the formation of Abeta deposits. Axonal tract tracing techniques demonstrated that the pattern of axon terminal labeling in layer III of RSg, following placement of DiI in medial septum, is remarkably similar to the pattern of cholinergic axons in RSg, as detected by acetylcholinesterase histochemical staining, choline acetyltransferase immunoreactivity, or p75 receptor immunoreactivity; this pattern also is strikingly similar to the band of Abeta immunoreactivity. In animals sustaining early damage to the medial septal nucleus (prior to the advent of Abeta immunoreactivity), the band of Abeta in layer III of RSg does not develop; the corresponding band of cholinergic markers also is eliminated. In older animals (after the appearance of the Abeta immunoreactivity) damage to cholinergic afferents by electrolytic lesions, immunotoxin lesions, or cutting the cingulate bundle, result in a rapid loss of the cholinergic markers and a slower reduction of Abeta immunoreactivity. These results suggest that the septal cholinergic axonal projections transport Abeta or amyloid precursor protein (APP) to layer III of RSg.

A role for neurotrophin-3 in targeting developing cholinergic axon projections to cerebral cortex.

This study examined the relationship between expression of neurotrophin-3 (NT-3) and the ingrowth of cholinergic axonal projections in cerebral cortex. Patterns of expression of NT-3 (defined by beta-galactosidase reporter expression in heterozygous offspring of transgenic NT-3(lacZneo/+) mice) revealed that limbic cortical regions (including frontal, cingulate, and insular cortex, as well as the dentate gyrus) express NT-3 and that these cortical regions receive early and relatively dense cholinergic axons (stained for acetylcholinesterase, AChE). Using the dentate gyrus as a model system, studies revealed that expression of the NT-3 reporter parallels, and precedes by approximately 2 days, the ingrowth of AChE positive cholinergic axons. Studies of forebrain organotypic slice cultures demonstrate that basal forebrain-derived cholinergic axons extend into cortical regions in a pattern that mimics the pattern of expression of the NT-3 reporter. Similarly, chimeric co-cultures, combining wild type septum with a slice of hippocampus from heterozygous NT-3(lacZneo/+) mice, demonstrate that cholinergic axons grow into regions of the dentate gyrus that express the NT-3 reporter. Hemisphere slice cultures made from NT-3 knockout mice reveal cholinergic axonal growth into cortex, but these axons do not form the regional pattern characteristic of slice cultures made from wild type or heterozygous NT-3(lacZneo/+) mice. Further, chimeric co-cultures made using slices of wild type septum combined with slices of hippocampus from NT-3 knockout mice demonstrate robust cholinergic axonal growth into the hippocampus, but the cholinergic axons do not form the characteristic preterminal pattern associated with the dentate gyrus. Slice cultures from limbic cortical tissue from the NT-3 null mice do not display exaggerated levels of cell death. In aggregate, these data support the hypothesis that expression of NT-3 by cortical neurons serves to attract basal forebrain cholinergic projections to their target cells in cerebral cortex.

Basal forebrain cholinergic cell attachment and neurite outgrowth on organotypic slice cultures of hippocampal formation.

Distributions of somata and neurites of cholinergic neurons were studied after seeding dissociated cells onto organotypic slice cultures. Slice cultures were made from hippocampal formation and adjacent cortical regions from rats or mice. Dissociated cell suspensions of basal forebrain tissue from rat or mouse fetuses were seeded onto the slice cultures. Combined cultures were maintained for 1-21 days in vitro. Cultures processed for acetylcholinesterase (AChE) histochemistry demonstrated non-random patterns of cholinergic cells and their neurites. Labeled cells appeared most frequently in the molecular layer of the dentate gyrus, and in the deeper layers of cortical regions adjacent to the hippocampus. Neurites extending from these labeled cells appeared to target the dentate molecular layer and the cortical subplate layer. By 4 days in vitro, AChE-positive basal forebrain cells display several short and thick neurites that appear to be dendrites, and one long process that appears to be an axon. By 5 days in vitro, dendrites are well developed; by 7 days the presumed axon has extended widely over the cortical target zone. These neurites are maintained through 3 weeks in culture. Distributions of cells varied with the age of the slice. AChE-labeled cells were not seen overlying hippocampal tissue when dissociated cells were seeded on slice cultures made from day 0 rats, but a few labeled cells were seen when seeded on slices from day 2 rats. Clear non-random patterns of labeled cells and neurite outgrowth were seen on slice cultures from day 5 or older pups. The non-random distribution seen with AChE-positive neurons was not seen using other techniques that labeled all cells (non-selective fluorescent labels) or all neurons; these techniques resulted in labeled cells scattered apparently homogenously across the slice culture.These studies demonstrate a non-random pattern of attachment or differentiation of basal forebrain cholinergic neurons when these cells are seeded onto cultured cortical slices; this pattern mimics the normal patterns of basal forebrain cholinergic projections to these cortical regions. These data suggest that the factors that normally guide basal forebrain-derived cholinergic axons to their target cells in vivo are present and detectable in this model system.

Evidence for target preferences by cholinergic axons originating from different subdivisions of the basal forebrain.

Possible target preferences of basal forebrain cholinergic neurons were studied in organotypic slice cultures. Cholinergic neurons in slices of medial septum or substantia innominata send axons into both hippocampus and neocortex when co-cultured together. However, septal cholinergic axons course through adjacent slices of neocortex to reach and branch densely in slices of hippocampus, but septal axons seldom grow beyond adjacent hippocampal tissue to reach neocortex. In contrast, cholinergic axons from substantia innominata commonly grow through hippocampus to reach neocortex, and also grow through neocortex to reach hippocampus, with similar branching densities in each target. The greater density of septal axonal branches in hippocampus than in neocortex suggests a preference of septal axons for the hippocampal target.

Arrival of afferents and the differentiation of target neurons: studies of developing cholinergic projections to the dentate gyrus.

This study examined the relationship between the development of cholinergic axons originating from the septum and a group of their target cells, the granule cells of the dentate gyrus of the rat. Acetylcholinesterase histochemistry was used to identify septal cholinergic afferents to the dentate gyrus; parallel studies used anterograde movement of a carbocyanine dye to label the septal projections. Septal cholinergic axons are present in the molecular layer of the internal blade of the dentate gyrus shortly after birth, but these axons do not reach the external blade until several days later. Results demonstrate that acetylcholinesterase positive septal axons grow into the external blade of the dentate gyrus only after the recently generated granule cells have coalesced to form a clearly defined layer. Results from studies using in situ hybridization techniques demonstrate that dentate gyrus granule cells express messenger RNAs for brain derived neurotrophic factor and for neurotrophic factor 3 shortly after formation of the granule cell layer. Ingrowth of septal cholinergic axons follows two days after the formation of the external blade of the dentate gyrus and the expression of neurotrophin messenger RNAs by the dentate granule cells. These data support the hypothesis that target cell development is a prerequisite for attracting the ingrowth of septal afferent axons.

In vivo induction of massive proliferation, directed migration, and differentiation of neural cells in the adult mammalian brain.

The development of an in vivo procedure for the induction of massive proliferation, directed migration, and neurodifferentiation (PMD) in the damaged adult central nervous system would hold promise for the treatment of human neurodegenerative disorders such as Parkinson's disease. We investigated the in vivo induction of PMD in the forebrain of the adult rat by using a combination of 6-hydroxydopamine lesion of the substantia nigra dopaminergic neurons and infusions of transforming growth factor alpha (TGFalpha) into forebrain structures. Only in animals with both lesion and infusion of TGFalpha was there a rapid proliferation of forebrain stem cells followed by a timed migration of a ridge of neuronal and glial progenitors directed toward the region of the TGFalpha infusion site. Subsequently, increasing numbers of differentiated neurons were observed in the striatum. In behavioral experiments, there was a significant reduction of apomorphine-induced rotations in animals receiving the TGFalpha infusions. These results show that the brain contains stem cells capable of PMD in response to an exogenously administered growth factor. This finding has significant implications with respect to the development of treatments for both acute neural trauma and neurodegenerative diseases.

Do subplate neurons comprise a transient population of cells in developing neocortex of rats?

Studies were undertaken to determine whether neurons of the subplate layer represent a transient or stable population of cells in developing neocortex of rat. The first set of studies sought to determine the fraction of subplate neurons that is lost during early postnatal development. The optical dissector method was used to analyze fluorescently stained material in animals the age of postnatal day 0 (P0) to P40. These results demonstrate a reduction of slightly less than half of the total number of subplate neurons from P0 to P40. Counts of labeled cells in littermates at varied ages after [(3)H]thymidine or BRDU treatment on gestational day 14 (G14 - birthdate of occipital subplate neurons) or G18 (birthdate of layers III-IV neurons) demonstrate loss of approximately 50% of neurons in the subplate layer between P0 and P40, somewhat greater than the loss of neurons from cortical layers III-IV. The second set of studies investigated whether subplate neurons display cellular atrophy during postnatal development. Analysis of subplate neurons injected intracellularly with Lucifer yellow in fixed slice preparations indicates no reduction in soma size, number of dendrites, or extent of dendritic fields of subplate neurons taken from animals age P0 to P60. The third set of studies investigated whether functional markers of subplate neurons are reduced during postnatal development. Analysis of tissue stained histochemically for cytochrome oxidase or acetylcholinesterase, or stained immunocytochemically for GABA, somatostatin, or neuropeptide Y, demonstrate a remarkable loss of expression of staining patterns from late gestational ages to P20. These data demonstrate that, although subplate neurons seem not to be a transient population of cells in the usual sense of being eliminated by cell death or structural atrophy, the loss of histochemical and immunocytochemical markers indicates that they may be a functionally transient population of cells.

Specificity of attachment and neurite outgrowth of dissociated basal forebrain cholinergic neurons seeded on to organotypic slice cultures of forebrain.

Development and differentiation of basal forebrain-derived cholinergic neurons were studied using a new technique that combines dissociated cell cultures with organotypic slice cultures. Slices of cerebral cortex or entire forebrain hemispheres were taken from early postnatal rat pups and maintained as organotypic cultures on membranes. Dissociated cell suspensions of basal forebrain tissue, taken from rat or mouse fetuses at gestational day 15-17, were seeded on to the slice cultures. Combined cultures were maintained for two to 14 days in vitro. Cultures processed for acetylcholinesterase histochemical staining demonstrated that stained neurons display regional variation in attachment to the slice, with most attachment occurring on cortex and with no detectable attachment on the caudate-putamen. Regional differences in attachment occur between cortical areas, with medial (cingulate) cortex showing much denser cell attachment than lateral (parietal) cortex, and across cortical layers, with layer I and deep layers showing more attachment than middle cortical layers. Similar patterns were observed on slices from rat brain irrespective of whether rat or mouse dissociated cells were used. Tyrosine hydroxylase-stained dissociated cells from ventral midbrain displayed a different pattern of attachment, with prominent attachment to the caudate putamen and less apparent specificity of regional and cortical laminar attachment. Little evidence of neurite outgrowth occurred during the first two days in vitro, but by four days, acetylcholinesterase-positive basal forebrain cells displayed several short and thick neurites that appeared to be dendrites, and one long process that appeared to be an axon. By seven days in vitro, dendrites are well developed and the presumed axon has extended branches over wide areas of cortex. These studies revealed several different types of cell-tissue interaction. The degree of cell growth and differentiation ranged from robust growth when dissociated cells were seeded on to slice cultures of normal target tissue, to apparently no attachment or growth when cells were seeded on to non-target tissue. This combined technique appears to be a useful method for studies of specificity of cell attachment and patterns of neurite outgrowth.

Cholinergic neurons from different subdivisions of the basal forebrain lack connectional specificity for cerebral cortical target sites in vitro.

Basal forebrain cholinergic neurons send their axons to cerebral cortex in a topographically organized projection. Experiments tested the hypothesis that this topographic organization results from target preferences of the cholinergic neurons. Slices containing either medial septum or substantia innominata were grown in co-culture with slices of lateral neocortex and hippocampus. Cholinergic neurons from septum and from substantia innominata projected axons into neocortex and hippocampus, without obvious differences in pattern or density. These data suggest that basal forebrain cholinergic neurons can innervate any portion of the cerebral mantle.

Cholinergic innervation of cerebral cortex in organotypic slice cultures: sustained basal forebrain and transient striatal cholinergic projections.

Slices of entire forebrain hemispheres were taken from early postnatal rat pups and maintained as organotypic slice cultures. Basal forebrain cholinergic neurons, identified by histochemical staining for acetylcholinesterase, develop axons that grow rapidly into cerebral cortex. Ingrowth occurs by two routes: some axons course laterally from the basal forebrain region to reach lateral neocortex; others course dorsally from the septum to reach medial cortex. By one to two weeks in vitro, acetylcholinesterase-positive axons have extended throughout most of the cortical territory. In addition to basal forebrain cholinergic axons, the normally local circuit cholinergic neurons of the striatum also send axons into cerebral cortex. These striatum-derived axons can be distinguished from basal forebrain axons by their distinct morphological characteristics and by their different response to excision of the striatum or basal forebrain. Further, acetylcholinesterase-positive axons in cortex that originate from striatum appear to retract or degenerate after about one week in culture, while those from basal forebrain remain present and apparently healthy beyond two weeks. These data document the basal forebrain cholinergic ingrowth into cerebral cortex using this whole hemisphere slice culture system and also demonstrate different degrees of maintenance of cortical afferents that are derived from different subcortical sources.

A surgical alternative in the management of chronic neurotrophic ulcerations of the foot.

Chronic neurotrophic ulcerations involving weightbearing portions of the foot present a unique management challenge. Under the appropriate clinical setting, muscle transposition can be a rewarding operation in managing these otherwise unresponsive pedal ulcerations. Presented herein is a case demonstrating the successful resolution of a recalcitrant ulceration. The authors recommend this approach, not as a replacement, but merely as an alternative to more traditional techniques.

Electrocardiographic changes during cesarean section: a cause for concern?

A Holter monitor was used to record ST segment changes during cesarean section in 170 consecutive healthy parturients starting 2 h before and ending 3 h after surgery. Lumbar epidural anesthesia (LEA, n = 120) or subarachnoid anesthesia (SA, n = 50) was used. Transthoracic 2-D echocardiograms were obtained in 30 patients from the LEA group. ST depression or elevation occurred 160 times in 44 patients from both groups. Ninety-eight percent of these changes occurred between induction of anesthesia and the end of surgery, with 78% of the episodes registering -1 mV. In the LEA group, the number of episodes tended to increase after delivery, but in the SA group, the frequency remained constant. ST segment depression was recorded in 38% and 14% of patients in the LEA and SA groups, respectively (P < 0.05, x2 analysis). No wall motion abnormality was noted in the echocardiogram during ST segment depression. Neither the 12-lead electrocardiogram nor plasma myocardial specific creatine kinase suggested myocardial damage. The operative events, alone or in combination, including hypertension, tachycardia, hypotension, bradycardia, air embolism (precordial Doppler) were neither specific nor sensitive as predictors of ST segment change (stepwise logistic regression). Tachycardia was associated with ST segment changes in 10% of time epochs (5 min) (P = 0.05, x2 analysis). Thus, ST segment changes during cesarean section are not caused by myocardial ischemia and are not of any clinical consequence.

Axillary artery disruption secondary to anterior dislocation of the shoulder.

We report a 13-year-old male who sustained a segmental injury to the third part of the left axillary artery following a subcoracoid shoulder dislocation while wrestling. The artery was repaired with an autogenous saphenous vein interposition graft. The patient's postoperative course was uneventful, and, in a 3-year followup, he has complete range of motion of the left shoulder without neurovascular compromise.