Label-free autofluorescence and hyperspectral imaging of cerebral amyloid-ß lesions in aged squirrel monkeys.

The observation of amyloid-ß (Aß) lesions using autofluorescence in transgenic mice and human Alzheimer disease patients has been reported frequently. However, no reports verify the autofluorescence of spontaneous Aß amyloidosis in animals, to our knowledge. We validated the autofluorescence of Aß lesions in spontaneous squirrel monkey cases under label-free conditions; lesions had intense blue-white autofluorescence in fluorescence microscopy using excitation light at 400-440 nm. Thioflavin S staining and immunohistochemistry of the same specimens revealed that this blue-white autofluorescence was derived from Aß lesions. Hyperspectral analysis of these lesions revealed a characteristic spectrum with bimodal peaks at 440 and 460 nm, as reported for Aß lesions in mice. Principal component analysis using hyperspectral data specifically separated the Aß lesions from other autofluorescent substances, such as lipofuscin. A non-labeled and mechanistic detection of Aß lesions by hyperspectral imaging could provide valuable insights for developing early diagnostic techniques.

Locomotor energetics in primates: gait mechanics and their relationship to the energetics of vertical and horizontal locomotion.

All primates regularly move within three-dimensional arboreal environments and must often climb, but little is known about the energetic costs of this critical activity. Limited previous work on the energetics of incline locomotion suggests that there may be differential selective pressures for large compared to small primates in choosing to exploit a complex arboreal environment. Necessary metabolic and gait data have never been collected to examine this possibility and biomechanical mechanisms that might explain size-based differences in the cost of arboreal movement. Energetics and kinematics were collected for five species of primate during climbing and horizontal locomotion. Subjects moved on a treadmill with a narrow vertical substrate and one with a narrow horizontal substrate at their maximum sustainable speed for 10­20 min while oxygen consumption was monitored. Data during climbing were compared to those during horizontal locomotion and across size. Results show that climbing energetic costs were similar to horizontal costs for small primates (<0.5 kg) but were nearly double for larger species. Spatio-temporal gait characteristics suggest that the relationship between the cost of locomotion and the rate of force production changes between the two locomotor modes. Thus, the main determinants of climbing costs are fundamentally different from those during horizontal locomotion. These new results combining spatiotemporal and energetic data confirm and expand on our previous argument (Hanna et al.: Science 320 (2008) 898) that similar costs of horizontal and vertical locomotion in small primates facilitated the successful occupation of a fine-branch arboreal milieu by the earliest primates.

Milk composition of captive tufted capuchins (Cebus apella).

Little is known about the milk composition of nonhuman primates, and it has never been examined in capuchin monkeys (genus Cebus). This article reports on the macronutrient milk composition (fat, crude protein (CP), lactose, dry matter (DM), and total gross energy (GE)) of captive housed tufted capuchins (Cebus apella) (n=8). C. apella milk averaged 5.22% fat, 2.40% CP, 6.94% lactose, 16.48% DM, and 0.89 kcal/g. Fat was the most variable macronutrient and was significantly higher in samples collected after 2 months of lactation. To explore the adaptive significance of C. apella milk composition, results were compared with data on milk composition from a closely related cebid, Saimiri boliviensis boliviensis, and another large-brained anthropoid, Homo sapiens. C. apella milk was only significantly different from Saimiri milk in CP and the proportion of energy from CP. Compared with human milk, C. apella milk was lower in lactose but higher in fat, CP, DM, GE, and the proportion of energy from CP. Results from this small dataset suggest that among anthropoid primates, the macronutrient composition of milk is influenced by phylogeny, may vary relative to infant growth rates, but may not be related in any direct way to relative brain size.

The Claustrum in the Squirrel Monkey.

The claustrum (CLA) is a subcortical structure that is reciprocally and topographically connected with the cerebral cortex. The complexity of the cerebral cortex varies dramatically across mammals, raising the question of whether there might also be differences in CLA organization, circuitry, and function. Species variations in the shape of the CLA are well documented. Studies in multiple species have identified subsets of neurochemically distinct interneurons; some data suggest species variations in the nature, distribution, and numbers of different neurochemically identified neuronal types. We have studied the CLA in a smooth-brained primate, the squirrel monkey, using Nissl-stained sections and immunohistochemistry. We found that the shape of the CLA is different from that in other primates. We found several different neurochemically defined populations of neurons equally distributed throughout the CLA. Immunoreactivity to GAD65/67 and GABAA receptors suggest that GABAergic interneurons provide widespread inhibitory input to CLA neurons. Immunoreactivity to glutamate transporters suggests widespread and overlapping excitatory input from cortical and possibly subcortical sources. Comparison of CLA organization in different species suggests that there may be major species differences both in the organization and in the functions of the CLA. Anat Rec, 303:1439-1454, 2020. © 2019 American Association for Anatomy.

Susceptibilities of nonhuman primates to chronic wasting disease.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy, or prion disease, that affects deer, elk, and moose. Human susceptibility to CWD remains unproven despite likely exposure to CWD-infected cervids. We used 2 nonhuman primate species, cynomolgus macaques and squirrel monkeys, as human models for CWD susceptibility. CWD was inoculated into these 2 species by intracerebral and oral routes. After intracerebral inoculation of squirrel monkeys, 7 of 8 CWD isolates induced a clinical wasting syndrome within 33-53 months. The monkeys' brains showed spongiform encephalopathy and protease-resistant prion protein (PrPres) diagnostic of prion disease. After oral exposure, 2 squirrel monkeys had PrPres in brain, spleen, and lymph nodes at 69 months postinfection. In contrast, cynomolgus macaques have not shown evidence of clinical disease as of 70 months postinfection. Thus, these 2 species differed in susceptibility to CWD. Because humans are evolutionarily closer to macaques than to squirrel monkeys, they may also be resistant to CWD.

Androgen resistance in squirrel monkeys (Saimiri spp.).

The goal of this study was to understand the basis for high androgen levels in squirrel monkeys (Saimiri spp.). Mass spectrometry was used to analyze serum testosterone, androstenedione, and dihydrotestosterone of male squirrel monkeys during the nonbreeding (n = 7) and breeding (n = 10) seasons. All hormone levels were elevated compared with those of humans, even during the nonbreeding season; the highest levels occurred during the breeding season. The ratio of testosterone to dihydrotestosterone in squirrel monkeys is high during the breeding season compared to man. Squirrel monkeys may have high testosterone to compensate for inefficient metabolism to dihydrotestosterone. We also investigated whether squirrel monkeys have high androgens to compensate for low-activity androgen receptors (AR). The response to dihydrotestosterone in squirrel monkey cells transfected with AR and AR-responsive reporter plasmids was 4-fold, compared with 28-fold in human cells. This result was not due to overexpression of cellular FKBP51, which causes glucocorticoid and progestin resistance in squirrel monkeys, because overexpression of FKBP51 had no effect on dihydrotestosterone-stimulated reporter activity in a human fibroblast cell line. To test whether the inherently low levels of FKBP52 in squirrel monkeys contribute to androgen insensitivity, squirrel monkey cells were transfected with an AR expression plasmid, an AR-responsive reporter plasmid, and a plasmid expressing FKBP52. Expression of FKBP52 decreased the EC50 or increased the maximal response to dihydrotestosterone. Therefore, the high androgen levels in squirrel monkeys likely compensate for their relatively low 5 alpha-reductase activity during the breeding season and AR insensitivity resulting from low cellular levels of FKBP52.

Glucocorticoid resistance in squirrel monkeys results from a combination of a transcriptionally incompetent glucocorticoid receptor and overexpression of the glucocorticoid receptor co-chaperone FKBP51.

Squirrel monkeys have high cortisol compared to Old World primates to compensate for glucocorticoid resistance. Glucocorticoid resistance in squirrel monkeys may result from mutations in the glucocorticoid receptor (GR) that render it less transcriptionally competent, or expression of the co-chaperone FKBP51 that reduces ligand binding. The goal of this study was to reconcile the contribution of each mechanism. Responsiveness of squirrel monkey GR in COS-7 cells was reduced compared to human GR, but induction of GR activity by maximum dexamethasone concentrations was similar. Also, expression of squirrel monkey FKBP51 reduced responsiveness of both squirrel monkey and human GR in T-REx-293 cells. The EC(50) for dexamethasone was 100-fold higher in cells expressing squirrel monkey GR and excess FKBP51 compared to cells expressing only human GR. Effects of FKBP51 expression and treatment with FK506 were also determined in squirrel monkey SQMK-FP cells that naturally express high levels of FKBP51. Overexpression of FKBP51 in SQMK-FP cells had little effect on GR responsiveness, but treatment with FK506 that blocks the effect of FKBP51 increased GR responsiveness. Thus, glucocorticoid resistance in squirrel monkey cells results from both expression of GRs that are less responsive and overexpression of FKBP51 that further reduces GR responsiveness.

Cortisol metabolism in the Bolivian squirrel monkey (Saimiri boliviensis boliviensis).

New World squirrel monkeys (Saimiri spp.) have high circulating cortisol levels but normal electrolytes and blood pressures. The goal of the present study was to gain insight into adaptive mechanisms used by Bolivian squirrel monkeys to minimize the effects of high cortisol on mineralocorticoid receptor (MR) activity and electrolyte and water balance. Aldosterone levels in serum from 10 squirrel monkeys were 17.7 +/- 3.4 ng/dl (normal range in humans, 4 to 31 ng/dl), suggesting that squirrel monkeys do not exhibit a compensatory increase in aldosterone. The squirrel monkey MR was cloned and expressed in COS-7 cells and found to have similar responsiveness to cortisol and aldosterone as human MR, suggesting that squirrel monkey MR is not inherently less responsive to cortisol. To determine whether altered metabolism of cortisol might contribute to MR protection in squirrel monkeys, serum and urinary cortisol and cortisone were measured, and a comprehensive urinary corticosteroid metabolite profile was performed in samples from anesthetized and awake squirrel monkeys. The levels of cortisone exceeded those of cortisol in serum and urine, suggesting increased peripheral 11beta-hydroxysteroid dehydrogenase 2 activity in squirrel monkeys. In addition, a significant fraction (approximately 20%) of total corticosteroids excreted in the urine of squirrel monkeys appeared as 6beta-hydroxycortisol, compared with that in man (1%). Therefore, changes in cortisol metabolism likely contribute to adaptive mechanisms used by Bolivian squirrel monkeys to minimize effects of high cortisol.

Characterization of the Sweet Taste Receptor Tas1r2 from an Old World Monkey Species Rhesus Monkey and Species-Dependent Activation of the Monomeric Receptor by an Intense Sweetener Perillartine.

Sweet state is a basic physiological sensation of humans and other mammals which is mediated by the broadly acting sweet taste receptor-the heterodimer of Tas1r2 (taste receptor type 1 member 2) and Tas1r3 (taste receptor type 1 member 3). Various sweeteners interact with either Tas1r2 or Tas1r3 and then activate the receptor. In this study, we cloned, expressed and functionally characterized the taste receptor Tas1r2 from a species of Old World monkeys, the rhesus monkey. Paired with the human TAS1R3, it was shown that the rhesus monkey Tas1r2 could respond to natural sugars, amino acids and their derivates. Furthermore, similar to human TAS1R2, rhesus monkey Tas1r2 could respond to artificial sweeteners and sweet-tasting proteins. However, the responses induced by rhesus monkey Tas1r2 could not be inhibited by the sweet inhibitor amiloride. Moreover, we found a species-dependent activation of the Tas1r2 monomeric receptors of human, rhesus monkey and squirrel monkey but not mouse by an intense sweetener perillartine. Molecular modeling and sequence analysis indicate that the receptor has the conserved domains and ligand-specific interactive residues, which have been identified in the characterized sweet taste receptors up to now. This is the first report of the functional characterization of sweet taste receptors from an Old World monkey species.

Structure-function analysis of squirrel monkey FK506-binding protein 51, a potent inhibitor of glucocorticoid receptor activity.

FK506-binding protein 51 (FKBP51) and FKBP52 are large molecular weight immunophilins that are part of the mature glucocorticoid receptor (GR) heterocomplex. These proteins possess peptidyl-prolyl isomerase (PPIase) and tetratricopeptide repeats (TPR) domains that are important for modulation of GR activity. A naturally occurring animal model of glucocorticoid resistance, the squirrel monkey, results from the relative overexpression of FKBP51 that renders the GR in a low-affinity state. In vitro studies demonstrated that the squirrel monkey form of FKBP51 is greater than 6-fold more potent than human FKBP51 in this respect. The goals of these studies were to determine the roles of the TPR and PPIase domains in the inhibitory activity of squirrel monkey FKBP51 and to gain insight into structural features of squirrel monkey FKBP51 responsible for potent inhibition of dexamethasone-stimulated GR activity. Mutations in the TPR of squirrel monkey FKBP51 that inhibit association with heat shock protein 90 blocked GR inhibitory activity. Mutations that abrogate the PPIase activity of squirrel monkey FKBP51 had no effect on GR inhibitory activity. Chimeras of squirrel monkey and human FKBP51 were tested to identify domains responsible for their different inhibitory potencies. Amino acid differences in domains FK1 and FK2 between squirrel monkey and human FKBP51 contribute equally to the enhanced inhibitory activity of squirrel monkey FKBP51. Furthermore, squirrel monkey FKBP51 in which either FK1 or FK2 was deleted lacked GR inhibitory activity. Thus, the potent inhibitory activity of squirrel monkey FKBP51 involves both FK domains and the heat shock protein 90-binding TPR domain.

Molecular phylogenetics and phylogeography of all the Saimiri taxa (Cebidae, Primates) inferred from mt COI and COII gene sequences.

Some previous genetic studies have been performed to resolve the molecular phylogenetics of the squirrel monkeys (Saimiri). However, these studies did not show consensus in how many taxa are within this genus and what the relationships among them are. For this reason, we sequenced 2,237 base pairs of the mt COI and COII genes in 218 Saimiri individuals. All, less 12 S. sciureus sciureus from French Guyana, were sampled in the wild. These samples represented all the living Saimiri taxa recognized. There were four main findings of this study. (1) Our analysis detected 17 different Saimiri groups: albigena, cassiquiarensis, five polyphyletic macrodon groups, three polyphyletic ustus groups, sciureus, collinsi, boliviensis, peruviensis, vanzolinii, oerstedii and citrinellus. Four different phylogenetic trees showed the Central American squirrel monkey (S. oerstedii) as the most differentiated taxon. In contrast, albigena was indicated to be the most recent taxon. (2) There was extensive hybridization and/or historical introgression among albigena, different macrodon groups, peruviensis, sciureus and collinsi. (3) Different tests showed that our maximum likelihood tree was consistent with two species of Saimiri: S. oerstedii and S. sciureus. If no cases of hybridization were detected implicating S. vanzolinii, this could be a third recognized species. (4) We also estimated that the first temporal splits within this genus occurred around 1.4-1.6 million years ago, which indicates that the temporal split events within Saimiri were correlated with Pleistocene climatic changes. If the biological species concept is applied because, in this case, it is operative due to observed hybridization in the wild, the number of species within this genus is probably more limited than recently proposed by other authors. The Pleistocene was the fundamental epoch when the mitochondrial Saimiri diversification process occurred.

Immunolocalization of growth, inhibitory, and proliferative factors involved in initial ovarian folliculogenesis from adult common squirrel monkey (Saimiri collinsi).

We performed an immunohistochemical (IHC) study to determine the follicular expression of growth differentiation factor 9 (GDF-9), anti-Müllerian hormone (AMH), Kit Ligand (KL), and c-Kit in squirrel monkey ovary. Ovarian tissue fragments from 4 squirrel monkeys were collected by laparotomy and processed for classical histology and IHC. Additionally, follicle development was assessed by Ki67 immunostaining to evaluate proliferative status of granulosa cells. A total of 4025 follicles were examined (1475 for classical histology and 2550 for immunohistochemistry). More than 80% of the evaluated follicles were morphologically normal. The GDF-9 protein was detectable in oocyte cytoplasm from primordial (100%), primary (99.1%), and secondary (100%) follicles. The AMH was not expressed in primordial follicles but just in few primary follicles (13.8%). On the other hand, it was highly expressed in granulosa cells from secondary follicles (67.9%). c-Kit, KL receptor, was found in the oolemma of primordial (100%), primary (100%), and secondary (100%) follicles. The KL expression was observed in oocytes and granulosa cells from primordial (94.9%), primary (91.6%) and secondary follicles (100%). Ki67 immunostaining was observed in granulosa cells from primary (5.7%) and secondary (54.8%) follicles but not in primordial follicles. In conclusion, we described the localization of GDF-9, KL, c-Kit, and Ki67 proteins and confirmed the presence of AMH protein in preantral follicles from squirrel monkey. Our results offer contribution for understanding of folliculogenesis in neotropical nonhuman primates. Moreover, these markers can be used to assess follicular viability and functionality after cryopreservation, transplantation, or in vitro culture of ovarian tissue.

Adaptation of the mineralocorticoid target tissues to the high circulating cortisol and progesterone plasma levels in the squirrel monkey.

Many New World primate species have elevated circulating free plasma cortisol concentrations, target tissue resistance to cortisol, and no evidence of sodium retention. A representative New World primate, the squirrel monkey (Saimiri sciureus), has plasma cortisol concentrations above those necessary to cause complete suppression of the renin-angiotensin-aldosterone axis in an Old World primate, the cynomolgus monkey (Macaca fascicularis). Despite this, the arterial blood pressure as well as the plasma sodium, potassium, and bicarbonate levels of the squirrel monkey are similar to those of the cynomolgus monkey, and its plasma aldosterone concentrations are approximately 2-fold higher. These findings suggest that cortisol has minimal sodium-retaining effects in this species. Renal cytosol aldosterone receptor concentrations are about 2- to 3-fold lower in the squirrel monkey than in the cynomolgus, whereas the receptor affinities for [3H]aldosterone are similar in the two monkeys. Higher concentrations of cortisol are needed to displace [3H]aldosterone from the mineralocorticoid receptor in the squirrel monkey than from the renal receptor in the cynomolgus [apparent equilibrium dissociation constant (Ki) = 7.8 X 10(-7) vs. 2.9 X 10(-8) M, respectively]. In addition, in contrast to man and presumably other Old World primates, plasma aldosterone concentrations in the female squirrel monkey do not increase during the reproductive cycle or pregnancy when progesterone concentrations are 10- to 20-fold higher than those of the male or the reproductively quiescent female. This suggests that progesterone is a poor aldosterone antagonist in this species. We conclude that a low concentration of mineralocorticoid receptors in New World Primates is compensated for by higher aldosterone levels, with a concomitant increase in receptor occupancy. The salt-retaining potency of cortisol is low, presumably because of a decrease in the affinity of the aldosterone receptor for glucocorticoids in New World primates.

Cloning and expression of the glucocorticoid receptor from the squirrel monkey (Saimiri boliviensis boliviensis), a glucocorticoid-resistant primate.

New World primates such as the squirrel monkey have elevated cortisol levels and glucocorticoid resistance. We have shown that the apparent binding affinity of the glucocorticoid receptor in squirrel monkey lymphocytes is 5-fold lower than that in human lymphocytes (apparent Kd, 20.9 +/- 1.8 and 4.3 +/- 0.2 nmol/L, respectively; n = 3), consistent with previous studies in mononuclear leukocytes isolated from the two species. As a first step in understanding the mechanism of decreased binding affinity in New World primates, we used reverse transcription-PCR to clone the glucocorticoid receptor from squirrel monkey liver and have compared the sequence to receptor sequences obtained from owl monkey liver, cotton-top tamarin B95-8 cells, and human lymphocytes. The squirrel monkey glucocorticoid receptor is approximately 97% identical in nucleotide and amino acid sequence to the human receptor. The ligand-binding domain (amino acids 528-777) of the squirrel monkey glucocorticoid receptor contains four amino acid differences (Ser551 to Thr, Ser616 to Ala, Ala618 to Ser, and Ile761 to Leu), all of which are present in owl monkey and cotton-top tamarin receptors. The DNA-binding domain (amino acids 421-486) is completely conserved among human, squirrel monkey, owl monkey, and cotton-top tamarin receptors. Twenty-two differences from the human sequence were found in the N-terminal region (amino acids 1-421) of the squirrel monkey receptor. None of the substitutions in the ligand-binding domain matched mutations known to influence binding affinity in other species. To determine whether the substitutions per se were responsible for decreased affinity, squirrel monkey and human glucocorticoid receptors were expressed in the TNT Coupled Reticulocyte Lysate System. Expressions of human and squirrel monkey glucocorticoid receptors and a squirrel monkey receptor in which Phe774 was mutated to Leu (F774L) were similar. When expressed in the TNT System, squirrel monkey and human glucocorticoid receptors had similar, high affinity binding for dexamethasone (apparent Kd, 5.9 +/- 1.2 and 4.3 +/- 0.5 nmol/L, respectively; n = 3), whereas the squirrel monkey F774L receptor had lower affinity binding (apparent Kd, 20.4 +/- 2.0 nmol/L; n = 3). Thus, substitutions within the ligand-binding domain of the squirrel monkey glucocorticoid receptor cannot account for the decreased binding affinity of these receptors in squirrel monkey cells. Rather, the binding affinity is probably influenced by the expression of cytosolic factors that affect glucocorticoid receptor function.

Quantitative light and electron microscopic analysis of cytochrome oxidase-rich zones in the striate cortex of the squirrel monkey.

Cytochrome oxidase activity was examined in the striate cortex (area 17) of squirrel monkeys at both the light and ultrastructural levels. Two prominent bands of reactivity were found in 4A and 4C with intermittent puffs of cytochrome oxidase reactivity in laminae 2 and 3. These puffs, spaced 0.5 mm apart, were in register with intermittent concentrations of activity in laminae 4B, 5, and 6. A thin band of reactivity was observed in lamina 1. The upper portion of 4C beta was less reactive than 4C alpha or the lower portion of 4C beta. Reactive neurons included stellate cells in all laminae and pyramidal cells in laminae 2 through 4B, 5, and 6. A row of large reactive pyramidal cells was observed in upper lamina 6. More reactive neurons were found in the puffs (laminae 2 and 3) than were observed in interpuff regions, and the reactive neurons were significantly larger than the nonreactive neurons. Reactive neurons contained two to three times as many reactive mitochondria as did the nonreactive neurons and often had indented nuclei. Based on the number of darkly or highly reactive, moderately reactive and lightly reactive mitochondria, puff regions were significantly different from nonpuff regions; there were approximately two times as many darkly reactive mitochondria in puff regions as compared to a similar nonpuff area. The majority of mitochondria (32% in puff; 44% in nonpuff) were found to reside in the dendritic profiles, which also contained the majority of highly reactive mitochondria. In a separate analysis, the total area of highly reactive mitochondria within puff regions was found to be twice the total area of highly reactive mitochondria in a comparable nonpuff region. An analysis of synapses showed that there were more asymmetrical synapses in both puff and nonpuff regions (55% and 54%, respectively) than symmetrical ones (45% in puff and 46% in nonpuff). There was an increase in mitochondrial reactivity in both asymmetrical and symmetrical synapses in the puff areas; however, the increased reactivity within asymmetrical terminals was significantly greater than that within symmetrical ones. Several somatodendritic synapses were observed and they were all of the symmetrical variety. Axospinous contacts were primarily of the asymmetrical type; however, symmetrical axospinous synapses were observed and were typically seen in association with an asymmetrical synapse. It was concluded that cytochrome oxidase activity is localized primarily within the dendritic profiles in both puff and nonpuff regions.(ABSTRACT TRUNCATED AT 400 WORDS)

Corticotropin-releasing factor immunoreactivity in monkey neocortex: an immunohistochemical analysis.

Corticotropin-releasing factor (CRF) has been implicated in the pathophysiology of certain human neuropsychiatric disorders that affect neocortical function. However, the anatomical organization of CRF-containing structures in the expanded and highly differentiated primate neocortex has not been previously described. In this study, the distribution of CRF-immunoreactive neurons and processes was characterized in the neocortex of New World squirrel monkeys (Saimiri sciureus). Substantial regional differences were present in the density, laminar distribution, and morphological appearance of CRF-immunoreactive neurons. The greatest density of labeled neurons was present in anterior cingulate cortex. A wide range of intermediate densities of CRF-immunoreactive neurons was evident in the association regions of the prefrontal, parietal, and temporal cortices. The lowest numbers of CRF-immunoreactive neurons were observed in the primary visual and primary motor cortices. For example, the density of labeled neurons was nearly five times greater in the anterior cingulate cortex than in the precentral cortex. CRF-immunoreactive neurons were also distributed in at least four different laminar patterns. For example, in the agranular anterior cingulate cortex, labeled cell bodies were distributed throughout layers II, III, and V. In other regions, such as the posterior cingulate cortex, labeled neurons were present in layers II, III, and IV. In contrast, labeled neurons were predominantly present in layers II and superficial III of the visual cortex, whereas in the inferior temporal cortex, they were present predominantly in layer IV. Regional and laminar differences were also present in the relative distributions of the two major morphological types (as defined by cell body shape) of CRF-immunoreactive neurons. Vertically oriented oval neurons, which frequently had a single dendritic process arising from each somal pole, were most frequently found in layer III. In contrast, the labeled neurons in layers II and IV tended to have a round- or triangular-shaped soma. In layer IV of some association cortices, these multipolar neurons were associated with a high density of rod-like structures composed of large immunoreactive varicosities clustered together in vertical arrays. These structures were frequently found to be located immediately below the soma of pyramidal neurons. Comparison of these findings with Golgi impregnation studies strongly suggests that CRF is present in the soma and axonal cartridges of a subset of chandelier neurons. The heterogeneous distribution and morphological diversity of CRF-containing neurons suggest that CRF may mediate distinct functions in different regions and layers of monkey neocortex.

Dopaminergic innervation of the basal ganglia in the squirrel monkey as revealed by tyrosine hydroxylase immunohistochemistry.

The organization of the dopaminergic mesostriatal fibers and their patterns of innervation of the basal ganglia in the squirrel monkey (Saimiri sciureus) were studied immunohistochemically with an antiserum raised against tyrosine hydroxylase (TH). Numerous fibers arose from midbrain TH-positive cell bodies of the substantia nigra pars compacta (group A9), the retrorubral area (group A8), and the lateral portion of the ventral tegmental area (group A10). These fibers accumulated dorsomedially to the rostral pole of the substantia nigra where they formed a massive bundle that coursed through the prerubral field and ascended along the laterodorsal aspect of the medial fore-brain bundle in the lateral hypothalamus. Some ventrally located fibers ran throughout the rostrocaudal extent of the lateral preopticohypothalamic area and could be followed up to the olfactory tubercle, whereas other fibers turned laterodorsally to invade the head of the caudate nucleus. At more dorsal levels in the lateral hypothalamus, many fiber fascicles detached themselves from the main bundle and swept laterally to reach the globus pallidus, the putamen, and the amygdala. Several TH-positive fibers coursed along the dorsal surface of the subthalamic nucleus, and some invaded the dorsomedial third of this structure. The remaining portion of the subthalamic nucleus contained relatively few TH-positive elements. In contrast, the globus pallidus received a dense dopaminergic innervation deriving mostly from two fascicles that coursed backward along the two major output pathways of the pallidum: the lenticular fasciculus caudodorsally and the ansa lenticularis rostroventrally. At the pallidal level, the labeled fibres merged within the medullary laminae and arborized profusely in the internal pallidal segment and less abundantly in the external pallidal segment. However, the caudoventral portion of the external pallidum displayed a dense field of TH-positive axonal varicosities. Other fibers ran through the dorsal two-thirds of the external pallidum en route to the putamen. The striatum contained a multitude of thin axonal varicosities among which a few long and varicosed fibers were scattered. These immunoreactive neuronal profiles were rather uniformly distributed along the rostrocaudal extent of the striatum but appeared slightly more numerous in the ventral striatum than in the dorsal striatum. The pattern of distribution of the TH-positive axonal varicosities in the dorsal striatum was markedly heterogeneous: it consisted of typical zones of poor TH immunoreactivity lying within a matrix of dense terminal labeling.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative light and electron microscopic analysis of cytochrome oxidase-rich zones in V II prestriate cortex of the squirrel monkey.

Area 18 of V II of the prestriate cortex of the squirrel monkey was examined at both the light and electron microscopic (EM) levels for cytochrome oxidase (C.O.) activity. At the 17/18 border, the intense C.O. staining of lamina 4 abruptly ended and a new pattern continued for approximately 6 mm into the adjacent prestriate cortex. Here, periodic puffs of high C.O. activity appeared in laminae (lam.) 2 and 3, with the highest activity in lower 3 (3B) extending slightly into upper 4. There was a hint of a columnar pattern in that lam. 4 and especially 5 below the puffs were slightly more reactive than adjacent areas. A thin band of activity could also be seen in upper 5 (5A) and another one between 5 and 6. Tangential sections revealed that the puffs were arranged in alternating wide and narrow rows that radiated orthogonally from the 17/18 border. The puffs in the wider rows tended to be larger (700-1,100 micrometers in diameter) than those in the narrow rows (400-890 micrometers in diameter). The center-to- center spacing between the puffs was approximately 1,100 micrometers. Both C.O.-reactive and nonreactive stellate and pyramidal cells were found between lam. 2 and 6. Quantitatively analysis of the supragranular layers indicated that the mean area of reactive neurons was significantly larger than that of nonreactive neurons in both the puffs and interpuff (nonpuff) regions. The relative density of reactive neurons was also significantly greater than that of nonreactive neurons, and was highest within the puffs. At the EM level, reactive neurons were medium to large pyramidal cells as well as medium-sized stellates with mild to severely indented nuclei and darker cytoplasm filled with reactive mitochondria. The majority of small stellates with scanty cytoplasm and few mitochondria were nonreactive. Extensive quantitative analysis of mitochondria number and level of reactivity in different neuronal profiles indicated that the number and area of darkly reactive mitochondria was significantly higher in the puffs than in the nonpuffs, and that the majority of them resided in dentritic profiles. Between a third to half of the mitochondria in axonal profiles were darkly reactive, the frequency being slightly higher in profiles with flattened vesicles making symmetrical synapses than those with round vesicles making asymmetrical synapses. Mitochondria in axonal trunks and myelinated axons contributed to only a small percentage of the total population. Glial cells, in general, were not very reactive.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterogeneity of chandelier neurons in monkey neocortex: corticotropin-releasing factor- and parvalbumin-immunoreactive populations.

Chandelier neurons are a unique subclass of cortical nonpyramidal neurons. The axons of these neurons terminate in distinctive vertically arrayed cartridges that synapse on the axon initial segment of pyramidal neurons. In this study, the rapid Golgi method and immunohistochemical techniques were used to characterize the morphology, regional distribution, laminar location, and biochemical content of chandelier neurons in the prefrontal and occipital cortices of three monkey species. As in our previous studies of visual areas V1 and V2 (Lund: Journal of Comparative Neurology 257:60-92, 1987; Lund et al.: Journal of Comparative Neurology 202:19-45, 1981, 276:1-29, 1988), Golgi impregnations of areas 46 and 9 of macaque prefrontal cortex show chandelier neurons to be present in layers 2 through superficial 5. The vertical arrays of terminal boutons (axon cartridges) typical of this neuron class are also present in layers 2-6 of the prefrontal cortex, but are not found in layer 1 or the subcortical white matter. In immunohistochemical studies, a calcium-binding protein, parvalbumin, and a neuropeptide, corticotropin-releasing factor (CRF), identify rod-like structures that are morphologically similar to the axon cartridges of chandelier neurons seen in the Golgi material. In addition, both parvalbumin- and CRF-immunoreactive cartridges are located below the somata of unlabeled pyramidal neurons and appear to outline the axon initial segment of these neurons. However, we find that parvalbumin and CRF are present in only subpopulations of chandelier axon cartridges. For example, in adult primary visual cortex, parvalbumin-labeled cartridges are present in very low numbers only in layers 2-3, whereas in prefrontal and occipital association cortices these cartridges are a very prominent component of layers 2-superficial 3 and are present in much lower density in the deeper cortical layers. In contrast to these findings in adult macaque monkeys, prefrontal and occipital association cortices of infant macaque monkeys contain a very high density of parvalbumin-labeled cartridges in layer 4 and relatively few in the superficial cortical layers. Furthermore, in adult squirrel monkey prefrontal cortex, CRF-labeled cartridges are predominately present in layer 4, but these CRF-immunoreactive structures have not been observed in the homologous regions of infant or adult macaque monkeys. These findings indicate that even for neurons of such distinctive morphology and presumably constant functional role as chandelier neurons, factors such as regional and laminar location, age, and primate species are associated with differences in the biochemical content of subpopulations of these neurons.

Distribution of corticotropin-releasing-factor-like immunoreactivity in brainstem of two monkey species (Saimiri sciureus and Macaca fascicularis): an immunohistochemical study.

Immunohistochemical methods were utilized to systematically map the distribution of corticotropin-releasing-factor-like immunoreactivity (CRF-LI) in the diencephalon, mesencephalon, and rhombencephalon of two monkey species (Saimiri sciureus and Macaca fascicularis). A primary antiserum directed against the human form of the peptide was utilized. Immunoreactive neuronal perikarya and processes were evident in numerous areas, and the distributions of these elements were similar for the two species. As previously reported for rats, monkeys, and human, intense immunoreactivity was evident in putative hypophyseal neurons in the parvicellular component of the paraventricular nucleus of the hypothalamus and in fibers extending from this area into the median eminence. The results for other brainstem regions, most of which have been previously examined for CRF-LI only in rats, indicate that many similarities exist between rats and monkeys in the distribution of this peptide in brainstem extrahypophyseal neuronal circuits, although substantial differences are also evident. For example, immunoreactive perikarya previously observed in other hypothalamic nuclei in rats were not evident in monkeys. Conversely, in monkeys, unlike rats, labeled perikarya were evident in several thalamic nuclei, especially in the intralaminar complex. Also, two large groups of immunoreactive neurons which have generally not been observed in rat studies were present in the mesencephalon and rhombencephalon. In the mesencephalon this consisted of a group of neurons just lateral to the mesencephalic tegmentum, extending throughout the rostral-caudal extent of the midbrain. In the rhombencephalon, labeled perikarya were observed throughout the inferior olive. Some of the differences between rats and monkeys in the locations of labeled perikarya may be due to differences in antiserum specificity and/or sensitivity, or they may result from the fact that colchicine pretreatment was not utilized in the present study. The distributions of immunoreactive fibers also exhibited similarities and differences between monkeys and rats. The most striking terminal fields observed in the present study which have not been previously described are a moderate-to-dense field within and adjacent to presumed dopamine-containing neurons in the substantia nigra pars compacta, a dense innervation of certain subdivisions of the interpeduncular nucleus, and a regionally and parasagittally organized distribution of fibers in the Purkinje cell and molecular layers of the cerebellar cortex.(ABSTRACT TRUNCATED AT 400 WORDS)