Hormonal and behavioral variation in pied tamarins housed in different management conditions.

Pied tamarins are an endangered Amazonian primate that has limited breeding success in zoos. Unfortunately, little is known about their reproductive biology and adrenocortical activity. Objectives were: (1) determine if fecal hormones could be utilized to monitor gonadal and adrenocortical activity; (2) characterize male and female gonadal and adrenocortical hormones; and (3) determine if there were differences between adrenocortical activity and behavior in a nonbreeding, on-exhibit (NB-ON) pair compared to a breeding, off-exhibit (B-OFF) pair. Fecal samples were collected from four (two males; two females) individuals. Hormones were analyzed for fecal progesterone (FPM), androgen (FAM), and glucocorticoid (FGM) metabolites by enzyme immunoassay. Behavioral observations were conducted for 6 months. Data were collected on instantaneous behavior, location, and all occurrences of intraspecific behaviors. Fecal progesterone metabolites were validated by pregnancy (mean ± SE, pregnant: 28.47 ± 1.60 µg/g; nonpregnant: 8.63 ± 0.89 µg/g). Fecal androgen metabolites were higher (T = 31,971, P < 0.05) in the B-OFF male (863.66 ± 46.30 µg/g) than the NB-ON male (838.63 ± 60.70 µg/g). Fecal glucocorticoid metabolites were validated by response to veterinary procedure with elevated values (7.31 ± 1.48 µg/g) seven times the baseline (0.37 ± 0.04 µg/g) at 24-hr postphysical. Females had higher baseline FGM than the males (P < 0.05). Baseline FGM were higher (P < 0.05) in the NB-ON female (0.93 ± 0.03 µg/g) compared to the B-OFF female (0.38 ± 0.02 µg/g). Similarly, the NB-ON male's FGM baseline (0.71 ± 0.03 µg/g) were higher (P < 0.05) than the B-OFF male (0.21 ± 0.01 µg/g). Behavioral data revealed stereotypical behaviors in the NB-ON pair but no stereotypical behaviors in the B-OFF pair. Fecal hormone monitoring and behavioral analysis may provide insight on the limited breeding success of pied tamarins in zoos.

Development of a field-friendly technique for fecal steroid extraction and storage using the African wild dog (Lycaon pictus).

Hormonal analysis provides information about wildlife populations, but is difficult to conduct in the field. Our goal was to develop a rapid and effective field method for fecal steroid analysis by comparing: (1) three extraction methods (laboratory (LAB), homogenize (HO) and handshake (HS)) and (2) two storage methods (solid-phase extraction (SPE) tubes vs. plastic tubes (PT)). Samples (n=23) from captive African wild dogs (Lycaon pictus) were thoroughly mixed, three aliquots of each were weighed ( approximately 0.5 g) and 5 ml of 90% ethanol was added. For LAB, samples were agitated (mixer setting 60; 30 min), centrifuged (1,500 rpm; 20 min) and poured into glass tubes. Or aliquots were HO (1 min) or HS (1 min) and poured through filter paper into glass tubes. Samples were split, analyzed for corticosterone (C) and testosterone (T) metabolites using enzyme immunoassays or stored in SPE or PT. Samples were stored (room temperature) for 30, 60 or 180 days, reconstituted in buffer and analyzed. Mean C and T recoveries of HO were greater (P=0.03) than HS compared with LAB, which was similar to HO (P>0.05). After 30 days <21% of C and T was recovered from SPE, but approximately 100% of each was recovered from HO-PT and HS-PT. Similarly, after 60 and 180 days, approximately 100% of C and T was recovered from HO-PT and HS-PT. Results demonstrated that, for C and T, HO was more comparable (P<0.001) to LAB than HS and PT storage was more efficient than SPE (P<0.001).

The loss of GluR2(3) immunoreactivity precedes neurofibrillary tangle formation in the entorhinal cortex and hippocampus of Alzheimer brains.

Double-immunolabeling techniques were employed to examine the distribution of GluR2(3) subunits and markers of early cytoskeletal changes (mab MC1) within the entorhinal cortex (EC) and hippocampus of cases with varying degrees of Alzheimer disease (AD) pathology (stages I-VI by Braak and Braak). In addition near-adjacent tissue sections were double-immunolabeled using antibodies against GluR2(3) and a marker of normal neuronal cytoskeleton (MAP2). In those cases classified as stages I-II, most layer II neurons of the EC and pyramidal neurons in the CA1/subiculum were double-labeled with GluR2(3) and MAP2. An occasional MC1-labeled cell was observed, yet in no instance were these neurons double-labeled with GluR2(3). In cases with moderate AD pathology (stages III-IV), layer II of the EC and CA1/subiculum were characterized by a substantial loss of GluR2(3)-labeled neurons, while many were still immunoreactive to MAP2. Notably, the loss of GluR2(3) immunolabeling was accompanied by an increasing number of MC1-positive neurons. In no instance were GluR2(3) and MC1 co-localized within the same neuron. In cases with severe AD pathology (stages V-VI), the EC and CA1/subiculum were almost completely devoid of GluR2(3)-positive neurons. MAP2-labeled neurons also were reduced in number. In contrast, both regions contained an abundance of MC1-positive cells. That GluR2(3) and MC1 are not observed in the same neuron, together with the observation that the number of GluR2(3)-labeled neurons decreases as the number of MC1-positive cells increases, suggest that a loss of GluR2(3) immunolabeling precedes the appearance of MC1 immunolabeling.

An immunohistochemical quantification of fibrous astrocytes in the aging human cerebral cortex.

In order to determine whether cortical fibrous astrocytes increase with age, we studied 25 patients ranging in age from 24 to 100 years with no clinical or pathological evidence of dementia or other cerebral disorder. Paraffin sections of mid-frontal cortex were obtained and stained with the avidin-biotin immunolabeling procedure for glial intermediate filament protein. The resulting immunolabeled fibrous astrocytes were then counted in the molecular and cellular (cortical laminae 2-6) layers. Populations of fibrous astrocytes in both layers varied widely among individuals, and in the molecular layer their numbers were not significantly correlated with advancing age. In the cellular layer, however, despite widely ranging cell counts among individuals within the same decades of life, there was a significant linear increase with age. Our data suggest that the increase occurs or accelerates significantly after age 70, but the case numbers preclude reaching such a conclusion with statistical confidence. However, when the patients are divided into those less than 70 and those older, fibrous astrocytes in the cellular layer are shown to be significantly increased in the latter group compared to the former.

Development of the basal forebrain cholinergic system: phenotype expression prior to target innervation.

We measured choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities in the rat to determine the time course of development, maturity, and senescence of ChAT activity. Tissue was obtained from Sprague-Dawley rats ranging in age from embryonic day 14 through 23 months. Seven regions were examined, including the magnocellular preoptic/substantia innominata region, frontal cortex, medial septal region, hippocampus, diagnoal band, and medial and lateral striatum. ChAT and AChE activities were first detected as early as E18 in the medial septum, diagonal band and magnocellular preoptic area, all regions of cholinergic cell bodies. Enzyme activity subsequently developed in terminal fields of these cholinergic perikarya (hippocampus and frontal cortex) as well as in the striatum. For all regions, enzyme activity rose during the first four postnatal weeks. This increase in enzyme activity was transient and, in most instances, decreases were observed between postnatal days 30 and 60. Most dramatic were the decreases in enzyme activity in the magnocellular preoptic/substantia innominata and diagonal band regions. Age-related declines also occurred in the frontal cortex, hippocampus, magnocellular preoptic/substantia innominata region, and the striatum. Cholinergic systems undergo dynamic changes especially during development and adulthood.

Morphologic alterations of cholinergic processes in the neocortex of aged rats.

In the present study we observed enlarged cholinergic processes in the neocortex of aged Fischer 344 rats. These swollen ChAT-positive profiles appeared either as a single axon enlargement or, in many instances, the bulbous processes coalesced to form grape-like clusters of immunoreactivity. The latter structures looked similar to the immunoreactive profiles observed in the cortex of patients with Alzheimer's disease and in the rat septum following fimbria-fornix transection. Together, these data provide evidence that morphologic changes occur within processes of cholinergic neurons in the aged rat. Moreover, the similarity in appearance between the axonal alterations in the aged rat and in patients with Alzheimer's disease suggests a common pathologic process.

Morphologic alterations of choline acetyltransferase-positive neurons in the basal forebrain of aged behaviorally characterized Fisher 344 rats.

We examined Fisher 344 female rats aged 6, 27, and 33 months old. Prior to sacrifice and morphometric analyses of forebrain cholinergic neurons all rats underwent behavioral characterization in a spatial learning task using the Morris water maze. Performance on the spatial task permitted subsequent grouping of the 27- and 33-month-old animals into impaired or nonimpaired groups. Importantly, the percentage of animals that displayed spatial impairments increased sharply with advancing age. Quantitative assessment of the size and density of choline acetyltransferase (ChAT)-positive neurons throughout the basal forebrain revealed a significant enlargement of forebrain cholinergic neurons within 27-month-old nonimpaired rats compared to 6-month-old rats and 27- and 33-month-old impaired animals. This increase in size was most noted in the medial septum and nucleus of the diagonal band. Significant decreases in the density of ChAT-positive neurons was observed only in the nucleus of the diagonal band of 27-month-old impaired rats compared to 6-month-old controls. Although the significance of enlarged forebrain cholinergic neurons is unclear, we discuss the possibility that within aged rodents neuronal swelling is an active event and represents an early manifestation of the aging process and may constitute a restorative and/or compensatory event in that these rats are relatively asymptomatic with respect to their behavioral deficits. In addition, we discuss in some detail various technical and life effect issues which may vary the outcome of investigations of aged rodents.

Ultrastructural characterization of choline acetyltransferase-containing neurons in the basal forebrain of rat: evidence for a cholinergic innervation of intracerebral blood vessels.

The ultrastructural morphology and vascular associations of cholinergic neurons in the horizontal limb of the nucleus of the diagonal band of Broca (nDBBhl) and amygdala of rat were determined by the immunocytochemical localization of choline acetyltransferase (ChAT), the acetylcholine biosynthetic enzyme. Within the nDBBhl peroxidase reaction product was distributed throughout the cytoplasm of selectively labeled neuronal perikarya and dendrites. Labeled perikarya were characterized by an oval cell body (7-10 microns X 17-26 microns in diameter) in which was located a large nucleus and often a prominent nucleolus. Dendrites were by far the most numerous immuno-labeled profiles in the nDBBhl. The labeled dendrites had a cross-sectional diameter of 0.4-4.6 microns and contained numerous mitochondria and microtubules. Approximately 10% of all immunolabeled dendrites received synaptic contacts from unlabeled presynaptic boutons. In contrast to the relatively large number of ChAT-labeled dendrites within the nDBBhl, ChAT-positive axons were less frequently observed and immunolabeled axon terminals were never detected. The labeled axons had an outside diameter of 0.4-1.4 micron and were myelinated. The absence or relative paucity of immunolabeled terminals in the nDBBhl indicates that most if not all of the cholinergic perikarya within this nucleus are efferent projection neurons. The nDBB is known to have widespread projections to many areas of the neocortex, hippocampus, and amygdala. In the present study we examined the amygdala and observed many ChAT-labeled axon boutons. The immunolabeled varicosities contained numerous agranular vesicles and although ChAT-positive terminals were in direct contact with unlabeled neuronal elements within the amygdala, few if any synaptic densities were detected in a single plane of section. With respect to the vasculature, immunolabeled perikarya and dendrites within the nDBBhl and axon terminals in the amygdala were often in direct apposition to blood vessels. In many instances the labeled profile was observed lying directly on the basal lamina of a capillary endothelial cell. In no instance, however, were membrane densities observed. The presence of cholinergic neuronal elements contacting the vessel wall provides morphologic evidence suggesting that the neurogenic control of cerebral vasculature is in part mediated via a cholinergic mechanism.

AMPA-selective glutamate receptor subtype immunoreactivity in the aged human hippocampal formation.

It has been hypothesized that, in Alzheimer's disease, glutamate-mediated excitotoxicity contributes to the degeneration of selected populations of neurons. In the present study, immunocytochemical techniques were used to determine the distribution and anatomical features of GluR1- and GluR2/3-immunolabeled cell bodies and processes within the hippocampal formation of normal (i.e., no pathology) elderly humans. The results of this study provide an essential baseline with which to compare the expression and distribution of glutamate receptor subunits within the brains of patients with Alzheimer's disease. With respect to GluR1 immunoreactivity, the molecular layer of the dentate gyrus displays the most intense immunolabeling of any hippocampal structure. Contributing to this intense labeling are apical dendrites that arise from neurons within the adjacent granule cell layer. Interestingly, GluR1-labeled neurons account for a relatively small percentage of the total number of neurons as revealed by Nissl staining in the granule cell layer. In contrast, GluR2/3-labeled neurons are densely distributed throughout the granule cell layer, yet they provide relatively few processes to the adjacent molecular layer compared to GluR1-positive processes. GluR1 labeling is also prominent within the CA fields of Ammon's horn, with CA2 > CA3 > CA1 > or = CA4. Most prominent within the CA fields are the labeled dendrites of pyramidal neurons. In many instances, apical dendrites can be traced into the adjacent stratum radiatum, where they impart a deep striated appearance to this region of the hippocampus. Robust GluR2/3 labeling is also observed within the pyramidal layer of Ammon's horn, with an order of staining intensity similar to that observed for GluR1. However, unlike GluR1 labeling, which is localized predominantly along dendrites, GluR2/3 labeling is observed primarily in association with cell bodies. Collectively, these data suggest that the molecular composition of the AMPA receptor complex may differ between the dendrite and soma of granule and pyramidal neurons within the hippocampal formation, so functionally we may predict that these two regions of the neuron would respond differently following glutamate receptor stimulation.

Cholinergic neurons within the rat hippocampus: response to fimbria-fornix transection.

The distribution and morphologic characteristics of choline acetyltransferase (ChAT)-containing neurons were studied throughout the rostrocaudal extent of the rat hippocampus and in a midline area just dorsal to the dorsal hippocampus. Peroxidase reaction product was observed with the aid of immunohistochemical methods and a high-titer polyclonal antibody against ChAT, the acetylcholine biosynthetic enzyme. ChAT-positive cells in the hippocampus were characterized by small, round or oval perikarya with two or more proximal processes. They were located within the caudal and temporal hippocampal formation, predominantly within the subiculum, in the stratum lacunosum moleculare, at the border of the stratum lacunosum moleculare and the stratum radiatum, and in the molecular layer of the dentate gyrus. The cells resembled in morphology the small, bipolar and multipolar neocortical ChAT-immunoreactive cells. In addition to the hippocampus, ChAT-positive neurons were observed caudally in a region just above the dorsal hippocampal commissure and rostrally in the columns of the fornix. These cells were large with an oval perikarya and darkly labeled compared to neurons in the hippocampus. They more closely resembled the ChAT-positive neurons in the midline raphe of the medial septal nucleus. Examination of the rat hippocampus 2 and 8 weeks following unilateral lesioning of the fimbria-fornix and supracallosal striae revealed a sparse innervation of ChAT-positive fibers in the hippocampus ipsilateral to the lesion. ChAT-labeled neurons in the hippocampus did not appear to sprout in response to the lesion. In contrast, ChAT-positive cells in the midline did appear to sprout into the medial dorsal subiculum and dorsal medial hippocampus. We conclude that these two populations of cells are distinct with respect to their response to hippocampal denervation and, furthermore, that this distinction may be attributed to a differential response to nerve growth factor.

Morphological response of axotomized septal neurons to nerve growth factor.

Septal efferent fibers from the neurons in the medial septal nucleus are destroyed by fimbria-fornix aspirative lesion. In the present study we used quantitative morphometric techniques to evaluate the response of these axotomized septal neurons to a constant infusion of nerve growth factor (NGF). By 2 weeks following the lesion, approximately 75% of the cholinergic neurons had degenerated in the untreated rats. The remaining cholinergic neurons showed few signs of the effect of the lesion when stained for a polyclonal antibody to ChAT and examined in 40-micron-thick sections. In 1-micron-thick sections the remaining ChAT-immunoreactive (IR) neurons also appeared no different from the intact ChAT neurons. However, non-ChAT-IR neurons had a shrunken nucleus, while all other morphometric parameters appeared normal. NGF infusion protected most of the ChAT-IR neurons from degenerating. The saved neurons had the same parameters as the undamaged ChAT-IR neurons when examined in either 40-micron- or 1-micron-thick sections. In addition, the shrunken appearance of the non-ChAT-IR neurons' nuclei was avoided by the NGF infusions. Enlarged ChAT-IR processes were evident in the dorsolateral quadrant of the septum following damage to the fimbria-fornix. NGF-infusions prevented the formation of these processes. Instead, in the treated animals the dorsal lateral quadrant contained a dense plexus of fine ChAT-IR varicosities. Taken together these results demonstrate that NGF not only can protect the cholinergic neurons from axotomy-induced degeneration but can also cause the saved neurons to maintain the same morphometric appearance as intact ChAT-IR neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Numerical aspects of pontine, lateral reticular, and inferior olivary projections to two paravermal cortical zones of the cat cerebellum.

Two different olivo-cortico-nuclear zones in the cat cerebellum have been compared quantitatively as regards the numbers of cells projecting to them from within several sources of mossy fibres (MFs), namely the basal pontine nuclei (BPN), nucleus reticularis tegmenti pontis (NRTP), and the ipsilateral lateral reticular nucleus (LRN). The zones chosen were the C3 zone in lobule V of the anterior lobe and the C1 zone in pars copularis of the paramedian lobule (PMLpc), localised by recording climbing fibre-mediated potentials evoked on their surface as a result of volleys set up in their spino-olivocerebellar paths. The zones were injected with fluorescent-labelled latex microspheres and cell bodies, retrogradely labelled in the MF source nuclei and in the contralateral inferior olive, were counted and mapped. Evidence was obtained that tracer efficiency was very high in both the MF projections and the olivo-cerebellar projection and that each olivocerebellar axon may provide only one climbing fibre to the upper part of a lobule V folium but an average of nearly two to the same part of a PML folium. When the numbers of labelled cells in each MF source nucleus were expressed as a percentage of the total number of labelled pontine cells, the biggest source for lobule V was the contralateral BPN, followed by LRN, contralateral NRTP, ipsilateral BPN, and ipsilateral NRTP. For PMLpc, the order was similar except that ipsilateral BPNs exceeded contralateral NRTPs, but the dominance of contralateral BPN as a source was much greater. Cell totals were converted into projection densities (i.e., numbers of cells labelled per square millimetre of cortical sheet involved in the injection site); densities for PMLpc were found to be almost three times greater than those for lobule V for contralateral BPN but the two densities were not significantly different for ipsilateral BPN. The three other MF sources projected at higher densities to lobule V than to PML. These findings indicate that two cortical zones, both of which receive climbing fibres from the rostral part of the dorsal accessory olive and project to nucleus interpositus anterior, nevertheless differ markedly in regard to both the relative and the absolute sizes of the projections they receive from several of their most important sources of MFs.

Evidence that transmitter-containing dystrophic neurites precede paired helical filament and Alz-50 formation within senile plaques in the amygdala of nondemented elderly and patients with Alzheimer's disease.

Immunocytochemical techniques were employed to examine the temporal ordering whereby amyloid beta-protein (A beta P) and neuronal elements collectively come together to form senile plaques in Alzheimer's disease (AD). Specifically, we addressed three questions: (1) whether A beta P deposition precedes or follows neuritic changes; (2) whether paired helical filament (PHF) formation is an early or late event in the genesis of the dystrophic neurites which participate in plaque formation; and (3) whether the density of senile plaques displays any relationship with the prevalence of PHF or Alz-50 containing neurons. To address these questions we studied the amygdala from a group of patients with AD, a group of nondemented age-matched individuals exhibiting a sufficient number of senile plaques to be classified by neuropathological criteria as AD, and a group of age-matched controls without AD pathology. Amyloid-bearing plaques were demonstrated by A beta P immunolabeling and thioflavine-S staining. Neuritic changes in the form of dystrophic neurites were observed with the aid of antibodies against PHF, Alz-50, as well as antibodies against several neuropeptides (i.e., substance P, somatostatin, and neurotensin) and the acetylcholine biosynthetic enzyme, choline acetyltransferase. By using a graded range of pathologic changes both within and across the patient population to provide us with a means of evaluating plaque deposition from its earliest to most advanced stages of development, we observed in patients and/or regions of the amygdala displaying a mild degree of pathologic change A beta P deposition in the absence of any neuritic changes. With increasing density of A beta P, however, we began to observe dystrophic neurites within plaques. In regions of relatively few plaques, the dystrophic neurites were immunolabeled only with antibodies against the various neurotransmitters and they lacked evidence of cytoskeletal pathology (i.e., Alz-50 or PHF). Only as the density of A beta P increased further within a region, were dystrophic neurites observed that exhibited Alz-50 or PHF. In no instance did we observe a relationship between the density of A beta P deposition and the density of Alz-50 or PHF-immunoreactive neurons. Collectively, our data suggest that the deposition of A beta P is an early pathologic event in senile plaque formation. Thereafter, swollen neurites can be seen in the vicinity of A beta P. This early neuritic response, which can first be visualized by immunolabeling for one or another transmitter substance, is followed by alterations in the cytoskeleton as recognized initially by antibodies to Alz-50 and subsequently by the presence of PHF.

Expression of choline acetyltransferase and nerve growth factor receptor within hypoglossal motoneurons following nerve injury.

In the present study we employed light microscopic immunocytochemical techniques in order to investigate the temporal response of choline acetyltransferase (ChAT) and nerve growth factor receptor (NGFr) within hypoglossal motoneurons following unilateral transection or crushing of the XII nerve or after intraneural injections of ricin into the nerve. In control rats (i.e., sham operated) virtually all the motoneurons of the XII nucleus displayed intense immunolabeling for ChAT and were devoid of NGFr immunoreactivity. As early as 3 days post-operative the intensity and the number of ChAT-labeled neurons were reduced on the axotomized side compared to the non-lesioned side. This decrease was maximal approximately two weeks post-operative when virtually no ChAT-labeled cells were present on the lesioned side. In contrast, no loss of hypoglossal neurons was found using Nissl stains. This absence of ChAT immunolabeling persisted for several days, yet by 30 days many of the motoneurons had begun to re-express the enzyme. In contrast to the decrease in ChAT immunoreactivity, transection of the XII nerve also resulted in the expression of NGFr immunoreactivity within the lesioned motoneurons. This response was detected as early as one day post-operatively and continued throughout all time points thus far examined including times after many of the motoneurons had begun to re-express ChAT. Crushing of the XII nerve effected the expression of ChAT and NGFr in a manner comparable to, yet less intense than, that observed following transection. Ricin injected directly into the XII nerve resulted in the loss of hypoglossal motoneurons as demonstrated both in immunohistochemical and Nissl-stained tissue preparations. The cell loss was readily apparent 3 days post-operatively, and ChAT immunoreactivity permanently disappeared. NGFr immunolabeling was seen only in scattered surviving neurons but not in ricin poisoned cells. The possible mechanisms underlying the differential expression of ChAT and NGFr are discussed.

The distribution of choline acetyltransferase in the rat amygdaloid complex and adjacent cortical areas, as determined by quantitative micro-assay and immunohistochemistry.

The distribution of choline acetyltransferase (ChAT) within the amygdaloid complex has been studied to evaluate what should primarily represent the terminal field of the cholinergic projection from the basal forebrain. Two currently available methods have been combined for the comparison: immunohistochemistry with a monoclonal antibody against ChAT, by a double peroxidase-antiperoxidase procedure, and quantitative histochemistry involving micro-assay of the ChAT activity of contiguous microdissected samples. Both methods indicate prominent ChAT activity in the basolateral amygdaloid nucleus (especially rostrally), the nucleus of the lateral olfactory tract (especially layer II), and the amygdalohippocampal area. Regions of lower ChAT activity were not accurately represented by the immunohistochemistry, but could be discriminated by the quantitative assays. Lowest activity was found in the medial nucleus of the amygdala. Most other regions had activities at least as high as average brain or neocortex. Gradients of enzyme activity were found within several regions, including the basolateral and lateral amygdaloid nuclei and the nearby posterior piriform cortex. In the piriform cortex, a region of particularly high ChAT activity was found at its medial edge near the nucleus of the lateral olfactory tract. The immunohistochemical method shows a few intensely reactive somata in layer III within this zone. Comparison of the results seen with immunohistochemistry and quantitative histochemistry suggests an advantage in using them together, since their respective strengths and weaknesses tend to complement each other.

Nerve growth factor receptor and choline acetyltransferase colocalization in neurons within the rat forebrain: response to fimbria-fornix transection.

Although it is well known that magnocellular cholinergic basal forebrain neurons are trophically responsive to nerve growth factor (NGF) and contain NGF receptors (NGFr), the exact distribution of forebrain NGFr-immunoreactive neurons and the degree to which cholinergic neurons are colocalized with them have remained in question. In this study we employed a very sensitive double-labelling method and examined in the same tissue section the distribution and cellular features of NGFr-positive and choline acetyltransferase (ChAT)-immunolabelled neurons within the rat basal forebrain. Throughout this region the majority of magnocellular basal forebrain neurons were immunoreactive for both NGFr and ChAT. However, a small percentage of neurons in the ventral portion of the vertical limb of the diagonal band of Broca were immunoreactive only for NGFr, whereas a larger population of magnocellular neurons in the substantia innominata exhibited only ChAT immunoreactivity. No NGFr-immunoreactive cells were found associated with ChAT-positive neurons in the striatum, neocortex, or hippocampus, and no single-labelled NGFr-immunoreactive neurons were found outside the basal forebrain area, except for a large number of positive-labelled cells along the ventricular walls of the third ventricle. In addition to its function in maintaining the normal integrity of the basal forebrain and cholinergic, peripheral sympathetic, and neural-crest-derived sensory neurons, NGF may also have a role in the growth of these neurons after damage to the nervous system. To examine this postulate the hippocampus was denervated of its septal input and examined 8 weeks later. Two populations of neurons were found to have undergone collateral sprouting--namely, the midline magnocellular cholinergic neurons of the dorsal hippocampus and the sympathetic noradrenergic neurons of the superior cervical ganglion. Both of these neuronal populations also stained strongly for NGFr. In contrast, the small intrinsic cholinergic neurons of the hippocampus exhibited neither sprouting response nor staining for NGFr. In view of these results, we suggest that the differing sprouting responses demonstrated by these three neuronal populations may be due to their responsiveness to NGF, as indicated by the presence or absence of NGF receptors.

Distribution of neurotensin immunoreactivity within the human amygdaloid complex: a comparison with acetylcholinesterase- and Nissl-stained tissue sections.

In a previous study, we reported marked depletion of neurotensin-immunoreactivity (NT-IR) within selected regions of the amygdala of patients with Alzheimer's disease. The significance of these observations was partly obscured largely because we lacked a thorough understanding of the innervation pattern of neurotensin in the normal human amygdala. Accordingly, in the present study, we used a polyclonal antibody against neurotensin to characterize the distribution and morphology of neurotensin-immunoreactive neuronal elements within the human amygdaloid complex. NT-IR occurred in a topographic manner that respected the cytoarchitectural boundaries of the amygdaloid subregions as defined by Nissl staining and acetylcholinesterase histochemistry. Most NT-IR in the amygdala was contained within beaded fibers and dot-like puncta. Within the subnuclei of the amygdala, immunoreactive neuritic elements were most dense within the central nucleus followed by the medial nucleus and intercalated nuclei. The anterior amygdaloid area, basal complex, paralaminar nucleus, cortical nucleus, cortical-amygdaloid transition area, and amygdalohippocampal area contained moderate densities of immunoreactivity. The accessory basal and lateral nuclei exhibited scant NT-IR. Immunoreactive neurons were found only within the anterior amygdaloid area and the central, medial, intercalated, and lateral capsular nuclei. The distribution of NT-immunoreactive processes and cell bodies within selected regions of the amygdala provides an anatomical substrate that may explain, in part, the neuromodulatory actions of neurotensin upon autonomic, endocrine, and memory systems.

Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase.

The neuroanatomical location and cytological features of cholinergic neurons in the rat brain were determined by the immunocytochemical localization of the biosynthetic enzyme, choline acetyltransferase (ChAT). Perikarya labeled with ChAT were detected in four major cell groups: (1) the striatum, (2) the magnocellular basal nucleus, (3) the pontine tegmentum, and (4) the cranial nerve motor nuclei. Labeled neurons in the striatum were observed scattered throughout the neostriatum (caudate, putamen) and associated areas (nucleus accumbens, olfactory tubercle). Larger ChAT-labeled neurons were seen in an extensive cell system which comprises the magnocellular basal nucleus. This more or less continuous set of neuronal clusters consists of labeled neurons in the nucleus of the diagonal band (horizontal and vertical limbs), the magnocellular preoptic nucleus, the substantia innominata, and the globus pallidus. Labeled neurons in the pontine tegmentum were seen as a group of large neurons in the caudal midbrain, dorsolateral to the most caudal part of the substantia nigra, and extended in a caudodorsal direction through the midbrain reticular formation into the area surrounding the superior cerebellar peduncle. The neurons in this latter group constitute the pedunculopontine tegmental nucleus (PPT). An additional cluster of cells was observed medially adjacent to the PPT, in the lateral part of the central gray matter at the rostral end of the fourth ventricle. This group corresponds to the laterodorsal tegmental nucleus. Large ChAT-labeled neurons were also observed in all somatic and visceral motor nerve nuclei. The correspondence of the distribution of ChAT-labeled neurons identified by our methods to earlier immunocytochemical and acetylcholinesterase histochemical studies and to connectional studies of these groups argues for the specificity of the ChAT antibody used.

Electron microscopic immunocytochemical localization of tyrosine hydroxylase in the area postrema of rat.

The ultrastructural morphology and specialized neuronal, vascular, and ventricular associations of tyrosine hydroxylase-labeled neurons are examined within the area postrema of rat brain. Specific antiserum to the purified enzyme is localized throughout the rostrocaudal and dorsoventral extent of the area postrema by means of the peroxidase-antiperoxidase technique. In all regions, peroxidase immunoreactivity for tyrosine hydroxylase is distributed throughout the cytoplasm of selectively labeled neuronal perikarya and processes. The perikarya contain a large nucleus, infolded nuclear membrane, numerous cytoplasmic organelles, and form axosomatic synapses with unlabeled terminals. The majority of the labeled processes are dendrites, which contain ribosomes, microtubules, mitochondria, and scattered vesicles. These dendrites are postsynaptic to unlabeled axon terminals and show membrane specializations with other labeled dendrites and perikarya. In contrast to dendrites, peroxidase-labeled profiles clearly distinguished as axons or axon terminals are sparse and never show membrane specializations with other neuronal or nonneuronal structures within the area postrema. Numerous large processes which could be either axons or dendrites are associated with blood vessels and the ventricular surface of the area postrema. With respect to blood vessels, processes are located either in direct apposition to the external glial membrane, or less frequently, within the perivascular space. The ventricular processes are either associated with blood vessels in the subpial space or distributed among the cilia and villi at the anterior margins of the area postrema. The neuronal and nonneuronal associations of the tyrosine hydroxylase-labeled processes are consistent with a receptor or chemosensor function for catecholamines in this circumventricular organ.

Distribution of dopamine-, noradrenaline-, and adrenaline-containing cell bodies in the rat medulla oblongata: demonstrated by the immunocytochemical localization of catecholamine biosynthetic enzymes.

The immunocytochemical localization of the biosynthetic enzymes--tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT)--was used to determine the cytological features and precise neuroanatomical location of catecholaminergic neurons in the medulla oblongata of rat. Perikarya labeled with TH were detected in two bilaterally symmetrical columns located in the ventrolateral and dorsomedial medulla. The distribution and the number of neuronal perikarya containing TH were the same as those containing DBH, except in the dorsal motor nucleus of the vagus at the level of the area postrema where the number of neurons immunocytochemically labeled for TH was considerably greater than those labeled for DBH. The detection of perikarya which show immunoreactivity for TH, used in the biosynthesis of dopamine, noradrenaline, and adrenaline, but not DBH, which converts dopamine to noradrenaline, suggests the existence of dopamine-synthesizing neurons in the medulla. Perikarya labeled with PNMT, used in the biosynthesis of adrenaline, were localized in more restricted regions corresponding to rostral subsets of the dorsal and ventral groups labeled for TH and DBH. Counts of neurons immunocytochemically labeled for TH or PNMT were obtained in order to determine the relative ratio of neurons which contain the enzymes necessary for the synthesis of dopamine, noradrenaline, or adrenaline at various levels of the medulla. At the most caudal levels no PNMT labeled neurons were detected. Further rostral, PNMT-labeled neurons were first detected in the ventrolateral medulla. At the level of the area postrema, the number of PNMT-labeled neurons in the ventrolateral medulla was approximately half of the number of cells showing immunoreactivity for TH. In contrast, few PNMT-labeled cells were detected in the dorsomedial medulla at the level of the area postrema compared to many neurons labeled for TH. At rostral medullary levels, in both the ventrolateral and the dorsomedial regions, the number of neurons labeled for TH and PNMT was essentially the same. Thus most, if not all, of the catecholaminergic neurons in the rostral medulla have PNMT, necessary for the synthesis of adrenaline.