Plasma Aß42/40 Ratio Detects Early Stages of Alzheimer's Disease and Correlates with CSF and Neuroimaging Biomarkers in the AB255 Study.

BACKGROUND: Easily accessible biomarkers are needed for the early identification of individuals at risk of developing Alzheimer's disease (AD) in large population screening strategies. OBJECTIVES: This study evaluated the potential of plasma ß-amyloid (Aß) biomarkers in identifying early stages of AD and predicting cognitive decline over the following two years. DESIGN: Total plasma Aß42/40 ratio (TP42/40) was determined in 83 cognitively normal individuals (CN) and 145 subjects with amnestic mild cognitive impairment (a-MCI) stratified by an FDG-PET AD-risk pattern. RESULTS: Significant lower TP42/40 ratio was found in a-MCI patients compared to CN. Moreover, a-MCIs with a high-risk FDG-PET pattern for AD showed even lower plasma ratio levels. Low TP42/40 at baseline increased the risk of progression to dementia by 70%. Furthermore, TP42/40 was inversely associated with neocortical amyloid deposition (measured with PiB-PET) and was concordant with the AD biomarker profile in cerebrospinal fluid (CSF). CONCLUSIONS: TP42/40 demonstrated value in the identification of individuals suffering a-MCI, in the prediction of progression to dementia, and in the detection of underlying AD pathology revealed by FDG-PET, Amyloid-PET and CSF biomarkers, being, thus, consistently associated with all the well-established indicators of AD.

Correlations between plasma and PET beta-amyloid levels in individuals with subjective cognitive decline: the Fundació ACE Healthy Brain Initiative (FACEHBI).

BACKGROUND: Peripheral biomarkers that identify individuals at risk of developing Alzheimer's disease (AD) or predicting high amyloid beta (Aß) brain burden would be highly valuable. To facilitate clinical trials of disease-modifying therapies, plasma concentrations of Aß species are good candidates for peripheral AD biomarkers, but studies to date have generated conflicting results. METHODS: The Fundació ACE Healthy Brain Initiative (FACEHBI) study uses a convenience sample of 200 individuals diagnosed with subjective cognitive decline (SCD) at the Fundació ACE (Barcelona, Spain) who underwent amyloid florbetaben(18F) (FBB) positron emission tomography (PET) brain imaging. Baseline plasma samples from FACEHBI subjects (aged 65.9 ± 7.2 years) were analyzed using the ABtest (Araclon Biotech). This test directly determines the free plasma (FP) and total plasma (TP) levels of Aß40 and Aß42 peptides. The association between Aß40 and Aß42 plasma levels and FBB-PET global standardized uptake value ratio (SUVR) was determined using correlations and linear regression-based methods. The effect of the APOE genotype on plasma Aß levels and FBB-PET was also assessed. Finally, various models including different combinations of demographics, genetics, and Aß plasma levels were constructed using logistic regression and area under the receiver operating characteristic curve (AUROC) analyses to evaluate their ability for discriminating which subjects presented brain amyloidosis. RESULTS: FBB-PET global SUVR correlated weakly but significantly with Aß42/40 plasma ratios. For TP42/40, this observation persisted after controlling for age and APOE ε4 allele carrier status (R2 = 0.193, p = 1.01E-09). The ROC curve demonstrated that plasma Aß measurements are not superior to APOE and age in combination in predicting brain amyloidosis. It is noteworthy that using a simple preselection tool (the TP42/40 ratio with an empirical cut-off value of 0.08) optimizes the sensitivity and reduces the number of individuals subjected to Aß FBB-PET scanners to 52.8%. No significant dependency was observed between APOE genotype and plasma Aß measurements (p value for interaction = 0.105). CONCLUSION: Brain and plasma Aß levels are partially correlated in individuals diagnosed with SCD. Aß plasma measurements, particularly the TP42/40 ratio, could generate a new recruitment strategy independent of the APOE genotype that would improve identification of SCD subjects with brain amyloidosis and reduce the rate of screening failures in preclinical AD studies. Independent replication of these findings is warranted.

FACEHBI: A Prospective Study of Risk Factors, Biomarkers and Cognition in a Cohort of Individuals with Subjective Cognitive Decline. Study Rationale and Research Protocols.

BACKGROUND: Long-term longitudinal studies with multimodal biomarkers are needed to delve into the knowledge of preclinical AD. Subjective cognitive decline has been proposed as a risk factor for the development of cognitive impairment. Thus, including individuals with SCD in observational studies may be a cost-effective strategy to increase the prevalence of preclinical AD in the sample. OBJECTIVES: To describe the rationale, research protocols and baseline characteristics of participants in the Fundació ACE Healthy Brain Initiative (FACEHBI). DESIGN: FACEHBI is a clinical trial (EudraCT: 2014-000798-38) embedded within a long-term observational study of individuals with SCD. SETTING: Participants have been recruited at the memory clinic of Fundació ACE (Barcelona) from two different sources: patients referred by a general practitioner and individuals from an Open House Initiative. PARTICIPANTS: 200 individuals diagnosed with SCD with a strictly normal performance in a comprehensive neuropsychological battery. MEASUREMENTS: Individuals will undergo an extensive neuropsychological protocol, risk factor assessment and a set of multimodal biomarkers including florbetaben PET, structural and functional MRI, diffusion tensor imaging, determination of amyloid species in plasma and neurophthalmologic assessment with optical coherence tomography. RESULTS: Two hundred individuals have been recruited in 15 months. Mean age was 65.9 years; mean MMSE was 29.2 with a mean of 14.8 years of education. CONCLUSIONS: FACEHBI is a long-term study of cognition, biomarkers and lifestyle that has been designed upon an innovative symptom-based approach using SCD as target population. It will shed light on the pathophysiology of preclinical AD and the role of SCD as a risk marker for the development of cognitive impairment.

Beyond the Controversy on Aß Blood-Based Biomarkers.

Central biomarkers of Alzheimer's disease (AD) have been proven to have diagnostic and prognostic capacity. However, both amyloid positron emission tomography and cerebrospinal fluid collection studies present problems that limit their widespread acceptability in global clinical trials. Thus, development of other measures as potential surrogates of amyloid positivity should be pursued. Results from numerous experimental studies strongly suggest that the association between Aß plasma levels, particularly the Aß42/Aß40 ratio, and AD diagnosis goes beyond what could be attributable to pure chance, although this association is still controversial. The aim of this review is to consider selected works that may help to improve the design of blood based biomarkers studies by controlling a number of confounding sources related to the clinical gold standard, the time-course of central and peripheral biomarkers, and the metabolism of Aß in blood that may be blurring the presumptive association between Aß blood levels and AD. Based on these data and to get pass the controversy, we tentatively postulate that at early stages of preclinical AD, blood Aß levels and central Aß biomarkers would follow parallel but temporally displaced trajectories. This association would become eventually lost as the disease progresses and the clearance mechanisms in the blood brain barrier are increasingly impaired.

Changes in the brain and plasma Aß peptide levels with age and its relationship with cognitive impairment in the APPswe/PS1dE9 mouse model of Alzheimer's disease.

Double transgenic mice expressing mutant amyloid precursor protein (APPswe) and mutant presenilin 1 (PS1dE9) are a model of Alzheimer-type amyloidosis and are widely used in experimental studies. In the present work, the relationships between brain and plasma amyloid-ß peptide (Aß) levels and cognitive impairments were examined in male APPswe/PS1dE9 double transgenic mice at different ages. When compared with non-transgenic littermates, APPswe/PS1dE9 mice exhibited significant learning deficits from the age of 6months (M6), which were aggravated at later stages of life (M8 and M12). Sporadic brain amyloid plaques were observed in mice as early as M3 and progressively increased in number and size up to M12. A similar increase was observed in brain insoluble Aß levels as assessed by enzyme-linked immunosorbent assay (ELISA). In particular, the levels of brain insoluble Aß peptides rose steeply from M4 to M6. Interestingly, this pronounced amyloid deposition was accompanied by a temporary fall in the concentration of brain soluble and membrane-bound Aß peptides at M6 that rose again at M8 and M12. The plasma levels of Aß40 and Aß42 decreased with advancing age up to M8, when they stabilized at M12. This decrease in plasma Aß levels coincided with the observed increase in insoluble brain Aß levels. These results could be useful for developing plasma Aß levels as possible biomarkers of the cerebral amyloidosis and provide advances in the knowledge of the Aß peptide biochemical changes that occur in the brain of Alzheimer's disease patients.

Effect of age and severity of cognitive dysfunction on two simple tasks in pet dogs.

Dogs exhibit age-dependent losses in learning and memory as well as a progressive accumulation of neuropathology that parallels that observed in normal human aging and early Alzheimer's disease. These deficits have been extensively studied using a number of standard cognitive tasks in the laboratory; however, appropriate tools for their assessment in veterinary clinics are still lacking. The aim of this study was to evaluate the effect of age and the severity of cognitive dysfunction syndrome (CDS) on two simple tests conducted in a clinical setting. A food searching (FS) task and a problem-solving (PS) task were administered to young (1-4 years, n=9), middle-aged (5-8 years, n=10), cognitively unimpaired aged (≥9 years, n=31), and cognitively impaired aged (≥ 9 years, n=37) dogs. Cognitive status was classified using an owner-based questionnaire, and in the impaired group, dogs were categorized as having either mild or severe CDS. During the FS task, younger dogs (<9 years) were able to locate the food more quickly and with more success than the aged groups (≥9 years). Dogs with severe CDS exhibited poorer performance than those with mild CDS or their healthy counterparts. In the PS task, younger dogs performed better than the aged dogs in obtaining food, but there were no differences related to CDS severity. The FS task might help to better characterize cognitively affected dogs in the clinical setting than the PS task. These and similar tasks require further investigations in the field.

Effect of age and severity of cognitive dysfunction on spontaneous activity in pet dogs - part 1: locomotor and exploratory behaviour.

Age-related cognitive dysfunction syndrome (CDS) has been reported in dogs and it is considered a natural model for Alzheimer's disease in humans. Changes in spontaneous activity (including locomotor and exploratory behaviour) and social responsiveness have been related to the age and cognitive status of kennel-reared Beagle dogs. The aim of this study was to assess the influence of age and severity of CDS on locomotor and exploratory behaviour of privately owned dogs. This is the first part of a two-part report on spontaneous activity in pet dogs. An open-field (OF) test and a curiosity test were administered at baseline and 6 months later to young (1-4 years, n=9), middle-aged (5-8 years, n=9), cognitively unimpaired aged (≥ 9 years, n=31), and cognitively impaired aged ( ≥ 9 years, n=36) animals. Classification of cognitive status was carried out using an owner-based observational questionnaire, and in the cognitively impaired group, the dogs were categorised as having either mild or severe cognitive impairment. Dogs were recorded during sessions in the testing room and the video-recordings were subsequently analysed. The severity of CDS (but not age) influenced locomotion and exploratory behaviour so that the more severe the impairment, the higher the locomotor activity and frequency of corner-directed (aimless) behaviours, and the lower the frequency of door-aimed activities. Curiosity directed toward novel stimuli exhibited an age-dependent decline although severely affected animals displayed more sniffing episodes directed towards the objects. OF activity did not change after 6 months. Testing aged pet dogs for spontaneous behaviour might help to better characterise cognitively affected individuals.

Effect of age and severity of cognitive dysfunction on spontaneous activity in pet dogs - part 2: social responsiveness.

Changes in social interactions with owners and other dogs are frequently observed in dogs with cognitive dysfunction syndrome (CDS). The aim of this work was to assess the effect of age and severity of CDS on social responsiveness. This is the second part of a 2-part report on spontaneous activity in pet dogs. A human interaction test and a mirror test were administered at baseline and 6 months later to assess social responses to humans and conspecifics, respectively, to four groups of privately-owned dogs: young (n=9), middle-aged (n=9), cognitively unimpaired aged (n=31), and cognitively impaired aged (n=36). The severity of cognitive impairment was considered in the last group and dogs were categorised as having either mild or severe CDS. The influence of the person and the mirror on locomotion and exploratory behaviour was also studied. Dogs were recorded in a testing room and the video recordings were subsequently analysed. Young dogs displayed more interactions involving physical contact with a person. Young and middle-aged dogs showed more vocalisations in response to social isolation. In contrast, aged animals spent more time in front of the mirror. Changes in social responsiveness associated with severe CDS included decreased response to social isolation and human interaction and increased time in front of the mirror, suggesting a deficit in habituation. Testing of spontaneous activity might help to characterise CDS in aged dogs, a condition increasingly diagnosed in veterinary clinics and a potentially useful natural model of Alzheimer's disease in humans.

Cloning, sequencing and expression in the dog of the main amyloid precursor protein isoforms and some of the enzymes related with their processing.

Alzheimer's disease (AD) is characterized by neuronal loss and the presence of both neurofibrillary tangles and senile plaques in the brain. These plaques arise from the deposition of beta-amyloid (Aß) peptides (38-43 amino acids), which are generated from enzymatic cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases. In the present work, we cloned the principal APP isoforms as well as some enzymes that have been implicated in their amyloidogenic and non-amyloidogenic processing in dogs. Additionally, the main proteases implicated in the degradation of Aß were also studied. We also investigated the level of expression of these APP isoforms and enzymes in different brain regions and in peripheral tissues. Our data demonstrate that these canine proteins are highly homologous to their human counterparts. In addition, the expression pattern of these proteins in dogs is consistent with previous data reported in human beings. Thus, dogs may be a natural model to study the biology of AD and could also serve as an animal model for Aß-targeted drugs against this devastating disease.

The neonatal treatment of rats with monosodium glutamate induces morphological changes in the subfornical organ.

The parenteral administration of monosodium glutamate (MSG) to neonatal rats induces specific lesions in the central nervous system that lead to a well characterized neuroendocrinological dysfunction. Additionally, it has been shown that MSG-treated rats present a blunted blood pressure response to the injection of nitric oxide synthase inhibitors. Recently, a similar cardiovascular alteration has been reported after the electrolytic lesion of the anteroventral region of the third ventricle affecting the connections of the subfornical organ (SFO). We hypothesized that the treatment of neonatal rats with MSG could affect the nitrergic cells of the SFO. In the present work, we have looked for alterations in the NADPH-diaphorase activity (a commonly used marker for nitrergic neurons) in the SFO of MSG-treated rats of either sex and at two different ages. Our results shown that the treatment of neonatal rats with MSG induced a substantial reduction in the volume of the SFO and in the number of its nitrergic cells with regard to control animals. These findings suggest that the SFO could be implicated in some of the cardiovascular alterations observed in MSG-treated rats.

Distribution of ACTH immunoreactivity in the diencephalon and the brainstem of the dog.

The present work describes for the first time the anatomical distribution of adrenocorticotropic hormone (ACTH) in the diencephalon and the brainstem of the dog by means of the indirect immunoperoxidase technique. The distribution found in this species agrees well with the pattern found in other mammals and particularly confirms much of the findings reported in the cat. An exception to that concordance is the presence of ACTH perikarya in the nucleus of the solitary tract of the dog, a population that has been described in the rat but not in the cat, and in the ventral mesencephalon. This last population spread across the ventral tegmental area from the raphe to the cerebral peduncle and appeared to be a specific feature of the canine brain. On the other hand, we can not see ACTH fibers in the substantia nigra of the dog which could be a characteristic of the domestic carnivores, opposite to rodents, since these fibers appeared to be also lacking in the cat. Nevertheless, the widespread distribution of ACTH fibers in the brain of the dog included many other nuclei containing monoaminergic neurons which supported a possible role for ACTH in the regulation of these neurotransmitter systems.

Distribution of alpha-neoendorphin immunoreactivity in the diencephalon and the brainstem of the dog.

Alpha-neoendorphin (alpha-NE) is an opiate decapeptide derived from the prodynorphin protein. Its anatomical distribution in the brain of mammals other than the rat, particularly in carnivores, is less well known than for other opiate peptides. In the present work, we have charted the distribution of alpha-NE immunoreactive fibers and perikarya in the diencephalon and the brainstem of the dog. The highest densities of labeled fibers were found in the substantia nigra and in patches within the nucleus of the solitary tract. Moderate densities appeared in the arcuate nucleus (Ar), median eminence, entopeduncular nucleus, ventral tegmental area, retrorubral area, periaqueductal central gray, interpeduncular nucleus and lateral parabrachial nucleus. Groups of numerous labeled perikarya were localized in the magnocellular hypothalamic nuclei, Ar and in the central superior and incertus nuclei in the metencephalon. Moreover, less densely packed fibers and cells appeared widely distributed throughout many nuclei in the region studied. These results are discussed with regard to the pattern described in other species. In addition, the present results were compared with the distribution of met-enkephalin immunoreactivity in the diencephalon and the brainstem of the dog that we have recently described. Although the distributions of these two peptides overlap in many areas, the existence of numerous differences suggest that they form separate opiate systems in the dog.

Distribution of somatostatin-28 (1-12) immunoreactivity in the diencephalon and the brainstem of the dog.

The term somatostatin refers to a family of peptides, mainly somatostatin-14, somatostatin-28 and somatostatin-28 (1-12), which are the cleavage products of a single 116 amino acid-long preprosomatostain molecule. The production of antibodies to these peptides allows their localization in a number of neuronal populations throughout the entire neuroaxis in many mammals. The dog has been pointed out as an extremely useful animal model for studying age-related cognitive dysfunction and other neuronal changes associated with aging in which somatostatin appears to be involved. However, only very scanty information is available with regard to the distribution of somatostatin in the brain of the dog. In the present work we have determined the pattern of the distribution of somatostatin-28 (1-12) immunoreactivity in the diencephalon and the brainstem of the dog. High to moderate densities of labeled perikarya were found in the anterior periventricular and arcuate hypothalamic nuclei, the reticular thalamic nucleus, in delimited parts of the nucleus of the brachium inferior colliculus, the retrorubral area, the dorsal raphe nucleus, the myelencephalic reticular formation and the dorsal motor nucleus of the vagus. Less dense population of somatostatin cells were localized in other diencephalic and brainstem nuclei. The distribution of labeled fibers was even broader as in addition to those above mentioned there were a number of areas that appeared devoid of labeled perikarya. Many of the findings were similar to those reported in earlier works while others underlined the existence of inconsistencies in the distribution pattern of this peptide in the brain of mammals.

Distribution of met-enkephalin immunoreactivity in the diencephalon and the brainstem of the dog.

The endogenous opioid system, in particular the enkephalins, has been implicated in a vast array of neurological functions. The dog could be a suitable model for the study of complex interactions between behavioral state and regulatory physiology in which the opioid system appeared to be implicated. Moreover, opiate derivatives are currently used in veterinary clinic and sometimes pharmacologically tested in the dog. However, there are no anatomical data regarding the organization of the opioid system in this species. The present work represents the first attempt to map the distribution of Met(5)-enkephalin-like-immunoreactive (Met-enk-li) cell bodies and fibers in the diencephalon and the brainstem of the dog. In the diencephalon, labeled cells were present in all the mid-line and intralaminar thalamic nuclei; the lateral posterior, pulvinar and suprageniculate nuclei; the ventral nucleus of the lateral geniculate body and the medial geniculate body. Additionally, Met-enk-li cells were seen in every hypothalamic nucleus except in the supraoptic. Variable densities of labeled fibers were also seen in all these nuclei except in the medial geniculate body and in most areas of the lateral posterior and pulvinar nuclei. In the mesencephalon, positive cells were found in the periaqueductal gray, the Edinger-Westphal and interpeduncular nuclei, delimited areas of the superior and inferior colliculi and the ventral tegmental area. In the rhombencephalon, labeled cells were seen in the majority of the nuclei in the latero-dorsal pontine tegmentum, the nuclei of the lateral lemniscus, the trapezoid, vestibular medial, vestibular inferior and cochlear nuclei, the prepositus hypoglossal, the nucleus of the solitary tract and the dorsal motor nucleus of the vagus, the infratrigeminal nucleus and the caudal part of the spinal trigeminal nucleus and in the rhombencephalic reticular formation. The distribution of fibers included additionally the substantia nigra, all the trigeminal nerve nuclei, the facial nucleus and a restricted portion of the inferior olive. These results are discussed with regard to previous reports on the distribution of Met-enk in other species.

An immunohistochemical marker for Wallerian degeneration of fibers in the central and peripheral nervous system.

This work was prompted by the accidental observation that a newly developed, affinity purified polyclonal antibody against the C-terminus of the neuropeptide tyrosine (NPY) Y1-receptor protein decorates degenerating fibers in the central nervous system (CNS). This staining did not appear in control animals in which the antibody marked perikarya and dendrites at previously described locations [X. Zhang, L. Bao, Z.-Q. Xu, J. Kopp, U. Arvidsson, R. Elde, T. Hökfelt, Localization of neuropeptide Y Y1-receptors in the rat nervous system with special reference to somatic receptors on small dorsal root ganglion neurons, Proc. Natl. Acad. Sci. USA 91 (1994) 11738-11742]. Three models of experimental lesions were studied: sciatic nerve transection, spinal cord transection and parietal cortex thermocoagulation. In each model, animals were divided in groups (n=2) and processed for indirect immunofluorescence at different time intervals up to 28 days post-lesion (PL) (see below). All three experimental lesions produced a very intense immunolabeling of fibers in the projection pathways of the lesioned structures, strongly reminding of Wallerian degeneration (WD). In the sciatic nerve, the staining first appeared on day 1 PL, was strongly increased on day 3 PL, then declined after 7 days and had almost completely disappeared after 14 days. In the CNS, the staining appeared later and was first observed on day 3 PL and remained for a longer period, thus showing different time courses in the brain and spinal cord as compared to the sciatic nerve. The labeling was completely abolished, both in the CNS and in the sciatic nerve, by pre-incubation of the Y1-R antibody with the immunogenic peptide at a dilution of 10-6 M. The appearance of the staining and its time course strongly suggest that the process was related to degenerating axons. Although the protein actually detected remains to be determined, it is suggested that the staining ability of this antibody could be used as a positive marker of axonal degeneration following experimental or naturally occurring lesions of the nervous system.

Distribution of a glucocorticoid-induced orphan receptor (JP05) mRNA in the central nervous system of the mouse.

JP05 (originally referred to as glucocorticoid-induced receptor gene or cDNA clone 4.2) designates a gene originally isolated from murine thymoma WEHI-7TG cells after being treated with glucocorticoids and forskolin. This gene is also induced by dexamethasone (a potent glucocorticoid receptor agonist) in isolated normal murine thymocytes. The predicted amino acid sequence was found to share significant similarity to the family of G-protein-coupled receptors, in particular to the tachykinin receptors NK-1, NK-2 and NK-3, with which it has an overall identity of 32%, 31% and 33%, respectively. The results of the present in situ hybridization analysis reveal that JP05 mRNA containing cells are extensively distributed throughout the rostrocaudal extension of the brain and spinal cord. However, the vast majority of the areas with high to moderate levels of JP05 mRNA were localized in the forebrain, primarily within limbic system structures, the dorsal and ventral striatum and in some hypothalamic nuclei. These results are discussed in relation to the central nervous system distribution of glucocorticoid receptor-containing cells and to the tachykinin system.

The projections to the medulla of neurons innervating the carotid sinus in the dog.

The localization and brain stem projections of neurons innervating the carotid sinus of the dog were studied by horseradish peroxidase histochemistry following microinjection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) under the adventitia of the carotid sinus. Within the brain stem, labeled afferent fibers and presumptive terminals were found bilaterally in the caudal nucleus tractus solitarius (nTS), the area postrema (AP), and the lateral tegmental field (LTF), reaching the area of the nucleus ambiguus (nA). Sparse labeling was also seen in the ipsilateral spinal trigeminal nucleus (SpV) and lateral cuneatus nucleus (LCn). These findings suggest the existence of multiple pathways by which peripheral baroreceptor inputs may influence central cardiovascular-related neurons. In addition to classically defined relay in the nTS, carotid sinus afferents may also interact more directly with these neurons in other brain stem regions.

The localization of neurons innervating the carotid sinus in the dog.

The localization of neurons innervating the carotid sinus of the dog was studied by horseradish peroxidase histochemistry following microinjection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) under the adventitia of the carotid sinus. Labeled cell bodies were found in the petrosal (198 +/- 108; mean +/- S.D.) and superior cervical ganglia (SCG) (890 +/- 354 mean +/- S.D.) supporting the existence of both a sensory afferent and a sympathetic efferent innervation of the carotid sinus. Labeled neurons in the petrosal ganglion were round pseudounipolar neurons of variable size. Labeled neurons in the SCG were multipolar and appeared distributed over the whole ganglion, but with a higher density toward its caudal half. No labeled perikarya appeared either in the brainstem or in the nodose or jugular ganglia, suggesting that in the dog a vagal pathway for carotid sinus baroreceptor afferents does not exist.

Coexistence of serotonin and cholecystokinin in paraneurons of the foetal sheep lung.

The coexistence of serotonin and cholecystokinin was studied in foetal sheep lungs at pseudoglandular stage of development by light microscopic immunohistochemistry. The coexistence was examined by staining consecutive sections with the different antibodies. Serotonin and cholecystokinin immunoreactivity was found within consecutive sections of most bronchopulmonary neuroepithelial bodies and in consecutive sections of the same intrapulmonary autonomic ganglia.

Localization of serotonin, cholecystokinin and somatostatin immunoreactivity in the lower respiratory tract of embryonic, foetal and postnatal sheep.

The lower respiratory tract of the sheep was studied by light-microscopical immunocytochemistry for serotonin, cholecystokinin, somatostatin, bombesin and calcitonin during different periods of lung development; embryonic, foetal and postnatal. At embryonic period only intraepithelial serotonin-containing cells as solitary neuroendocrine cells (NEC) and neuroepithelial bodies (NEB) were found. At foetal stages, immunoreactive cells to serotonin, cholecystokinin and somatostatin were observed in airway epithelium, as solitary NEC and NEBs, and in autonomic intrapulmonary ganglia as single or clusters of small intensely-fluorescent (SIF) cells. In postnatal sheep, serotonin- and cholecystokinin-containing cells were found within airway mucosa as solitary NECs and NEBs. No immunoreactive cells were observed with antiserum to bombesin and calcitonin. Quantitative studies showed that serotonin was the predominant substance, and that solitary neuroendocrine cells were more numerous in distal conducting airways and at foetal stages.