Cellular Activity Patterns Research Articles

DJ-1 and UCH-L1 gene activity patterns in the brains of controls, Parkinson and schizophrenia patients and in rodents

DJ-1 (PARK7) has been implicated in early onset and familial cases of Parkinson's disease (PD). We therefore mapped cellular activity patterns of the DJ-1 gene in human and rodent brain tissue with radioactive in-situ hybridization. In all three mammals mRNA expression was restricted mainly to neurons in all regions analyzed. White matter, such as crus cerebri and capsula interna appeared negative, suggesting that glial cells express DJ-1 at levels below the detection limit of our method. We compared DJ-1 mRNA expression to the neuronal marker UCH-L1, which has also been implicated in PD, and found lower levels for DJ-1 but very similar patterns of expression. Measurement of the signal intensity revealed that human frontal cortex of control cases expressed DJ-1 mRNA more abundantly than other regions such as substantia nigra in the midbrain. Comparing DJ-1 expression in dopamine neurons on hemi-sections from controls and patients we could not detect any difference between 14 controls, 8 idiopathic Parkinson and 5 schizophrenia cases. Of note, DJ-1 is expressed in several other tissues such as the liver, gastrointestinal tract, adrenal and pituitary gland and during embryonic development, while UCH-L1 has a strictly neuronal expression also outside the CNS. We conclude that DJ-1 and UCH-L1, like other genes linked to PD, are not expressed specifically in DA neurons, but instead generally in neurons. The abundant expression of DJ-1 in certain peripheral tissues and of UCH-L1 in peripheral neurons may also be of relevance for the spectrum of symptoms in different forms of PD.

Cellular activation patterns of the main olfactory bulb and accessory olfactory bulb following exposure to beddings soiled by same- or opposite-sex conspecifics in Mandarin voles (Microtus mandarinus)

Using induction of Fos as an index, we studied cellular activation patterns in the accessory olfactory bulb (AOB) and main olfactory bulb (MOB) of Mandarin voles ( Microtus mandarinus (Milne-Edwards, 1871)) following their exposure to beddings soiled by different sexes. Male and female Mandarin voles that were exposed to beddings soiled by the opposite sex produced significantly more Fos-immunoreactive (Fos-ir) neurons in the anterior portion of the AOB and significantly fewer Fos-ir neurons in the posterior portion of the AOB than voles exposed to beddings soiled by the same sex. Furthermore, male and female Mandarin voles exposed to bedding soiled by different sex produced different numbers of Fos-ir cells in MOB. Mandarin voles exposed to beddings soiled by the opposite sex produced significantly more Fos-ir neurons in MOB than voles exposed to beddings soiled by the same sex. Our results establish that Mandarin voles of each sex showed different cellular activation patterns in AOB and MOB following exposures to sex-specific beddings. We suggest that both AOB and MOB were involved in sexual activities induced by chemosensory signals.

Flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides

Lipopolysaccharides (LPS) from Gram-negative bacteria are strong stimulators of white river crayfish, Procambarus zonangulus, haemocytes in vitro. Following haemocyte treatment with LPS and with LPS from rough mutant R5 (LPS Rc) from Salmonella minnesota, flow cytometric analysis revealed a conspicuous and reproducible decrease in cell size as compared to control haemocytes. These LPS molecules also caused a reduction in haemocyte viability as assessed by flow cytometry with the fluorescent dyes calcein-AM and ethidium homodimer. The onset of cell size reduction was gradual and occurred prior to cell death. Haemocytes treated with LPS from S. minnesota without the Lipid A moiety (detoxified LPS) decreased in size without a reduction of viability. The action of LPS on crayfish haemocytes appeared to be related to the activation of the prophenoloxidase system because phenoloxidase (PO)-specific activity in the supernatants from control and detoxified LPS-treated cells was significantly lower than that from LPS and LPS-Rc treated cells ( P≤0.05). Furthermore, addition of trypsin inhibitor to the LPS treatments caused noticeable delays in cell size and viability changes. These patterns of cellular activation by LPS formulations indicated that crayfish haemocytes react differently to the polysaccharide and lipid A moieties of LPS, where lipid A is cytotoxic and the polysaccharide portion is stimulatory. These effects concur with the general pattern of mammalian cell activation by LPS, thereby indicating common innate immune recognition mechanisms to bacterial antigens between cells from mammals and invertebrates. These definitive molecular approaches used to verify and identify mechanisms of invertebrate haemocyte responses to LPS could be applied with other glycoconjugates, soluble mediators, or xenobiotic compounds.

Odor-evoked activity is spatially distributed in piriform cortex.

Much data on the olfactory bulb (OB) indicates that structural characteristics of odorant molecules are encoded as ordered, spatially consolidated sets of active cells. New results with "genetic tracing" (Zou et al. [2001] Nature 414:173-179) suggest that spatial order is also present in projections from the OB to the olfactory cortex. For the piriform cortex (PC), results with this technique indicate that afferents conveying input derived from single olfactory receptors (ORs) are distributed to well-defined patches in the anterior PC (APC) but that these patches are much larger than in the OB. We have used c-fos induction to examine how input patterning for single ORs is translated into patterns of odor-evoked cellular activity in the PC. The laminar distribution of labeled cells and dual-immunostaining for gamma-aminobutyric acid (GABA)ergic markers indicated that activity was detected largely in pyramidal cells. In odor-stimulated rats, labeled cells were present throughout the posterior PC (PPC) but were concentrated in prominent rostrocaudal bands in APC. Analysis of responses to different odorants and concentrations revealed that locations and shapes of bands conveyed no apparent information regarding odor quality, rather, they appeared to correspond to subregions of the APC distinguished by cytoarchitecture and connectivity. Small-scale variations in labeling density were observed within APC bands and throughout the PPC that could reflect the presence of a complex topographical order, but discrete patches at consistent locations as observed by genetic tracing were absent. This finding suggests that as a result of afferent overlap and intracortical processing, odor-quality information is represented by spatially distributed sets of cells. A distributed organization may be optimal for discriminating biologically relevant odorants that activate large numbers of ORs.

Bone turnover during distraction osteogenesis in an experimental sheep model.

Biochemical serum markers of osteoblastic activity and collagen turnover were measured in a sheep model of distraction osteogenesis. Significant increases of the bone formation marker osteocalcin were found during the first part of the consolidation phase and peaked at a time point equalling the distraction phase. Collagen turnover parameters pyridinoline and the specific type I collagen marker desoxypyridinoline consistently increased during the distraction and consolidation phases. While pyridinoline peaked at a time point similar to osteocalcin, desoxypyridinoline increased to a later stage in consolidation, indicating continuous turnover of bone-specific type I collagen. The results indicate a characteristic pattern of osteoblast cellular activation during distraction osteogenesis with possible consequences for the timing of treatment.

Protection from pulmonary fibrosis in the absence of CCR2 signaling.

Pulmonary fibrosis can be modeled in animals by intratracheal instillation of FITC, which results in acute lung injury, inflammation, and extracellular matrix deposition. We have previously shown that despite chronic inflammation, this model of pulmonary fibrosis is lymphocyte independent. The CC chemokine monocyte-chemoattractant protein-1 is induced following FITC deposition. Therefore, we have investigated the contribution of the main monocyte-chemoattractant protein-1 chemokine receptor, CCR2, to the fibrotic disease process. We demonstrate that CCR2(-/-) mice are protected from fibrosis in both the FITC and bleomycin pulmonary fibrosis models. The protection is specific for the absence of CCR2, as CCR5(-/-) mice are not protected. The protection is not explained by differences in acute lung injury, or the magnitude or composition of inflammatory cells. FITC-treated CCR2(-/-) mice display differential patterns of cellular activation as evidenced by the altered production of cytokines and growth factors following FITC inoculation compared with wild-type controls. CCR2(-/-) mice have increased levels of GM-CSF and reduced levels of TNF-alpha compared with FITC-treated CCR2(+/+) mice. Thus, CCR2 signaling promotes a profibrotic cytokine cascade following FITC administration.

Blood Cell Activation, Coagulation, and Inflammation in Men and Women With Coronary Artery Disease

We investigated in some detail the immunologic and procoagulant activation patterns in men and women with unstable (UA, n=26) versus stable (SA, n=40) coronary artery disease (CAD). Leukocyte activation and platelet–leukocyte complex formation were assessed by flow cytometry. Plasma markers of coagulation and inflammation were analyzed. Unstable patients displayed higher levels of platelet–leukocyte complexes ( P<.001), of leukocyte CD11b ( P<.01), and of T cell HLA-DR ( P<.05) than healthy controls did. Female UA patients presented the highest degree of complexes. SA patients only differed significantly from controls with respect to HLA-DR ( P=.02). UA patients had higher levels of C-reactive protein ( P<.01), IL-6 ( P<.001), IL-10 ( P<.01), and soluble fibrin ( P<.001) than did stable ones. Regarding P-selectin levels, 25% of SA and 50% of UA patients were above normal range. Again, UA women presented the highest marker amounts. As for soluble tissue factor, women had higher levels than men regardless of the severity of disease ( P<.001 in SA). We conclude that in unstable coronary syndromes, there is an activation of both coagulation and inflammation that coincides with an increased activation of platelets and leukocytes. Cellular interactions may contribute to the systemic responses observed. Women have different patterns of cellular activation than men, indicating differences in pathogenetic mechanisms.

Agonistic Induction of a Covalent Dimer in a Mutant of Natriuretic Peptide Receptor-A Documents a Juxtamembrane Interaction That Accompanies Receptor Activation

The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular domain with a ligand binding site, a transmembrane-spanning domain, a kinase homology domain, and a guanylyl cyclase domain. In response to agonists (atrial natriuretic peptide (ANP) and brain natriuretic peptide), the kinase homology domain-mediated guanylate cyclase repression is removed, which allows the production of cyclic GMP. Previous work from our laboratory strongly indicated that agonists are exerting their effects through the induction of a juxtamembrane dimeric contact. However, a direct demonstration of this mechanism remains to be provided. As a tool, we are now using the properties of a new mutation, D435C. It introduces a cysteine at a position in NPR-A corresponding to a supplementary cysteine found in NPR-C6, another receptor of this family (a disulfide-linked dimer). Although this D435C mutation only leads to trace levels of NPR-A disulfide-linked dimer at basal state, covalent dimerization can be induced by a treatment with rat ANP or with other agonists. The NPR-A(D435C) mutant has not been subjected to significant structural alterations, since it shares with the wild type receptor a similar dose-response pattern of cellular guanylyl cyclase activation. However, a persistent activation accompanies NPR-A(D435C) dimer formation after the removal of the inducer agonist. On the other hand, a construction where the intracellular domain of NPR-A(D435C) has been truncated (DeltaKC(D435C)) displays a spontaneous and complete covalent dimerization. In addition, the elimination of the intracellular domain in wild type DeltaKC and DeltaKC(D435C) is associated with an increase of agonist binding affinity, this effect being more pronounced with the weak agonist pBNP. Also, a D435C secreted extracellular domain remains unlinked even after incubation with rat ANP. In summary, these results demonstrate, in a dynamic fashion, the agonistic induction of a dimeric contact in the juxtamembrane domain of NPR-A. In addition, this process seems to require membrane attachment of the receptor. Finally, the intracellular domain represses this contact at the basal state, showing its potent influence on the outer juxtamembrane domain.

Effects of selective sinoaortic denervations on phenylephrine-induced activational responses in the nucleus of the solitary tract

Intravenous administration of phenylephrine provokes a pattern of cellular activation in the nucleus of the solitary tract that resembles the central distributions of primary baroreceptor afferents supplied by the carotid sinus and aortic depressor nerves. Transganglionic transport and denervation methods were used in an experimental setting to test the dependence of phenylephrine-induced Fos immunoreactivity on the integrity of buffer nerve afferents, and to identify the subregions of the nucleus of the solitary tract supplied by each. Cholera toxin B–horseradish peroxidase injections into either or both nerves revealed terminal labeling concentrated in, but not restricted to, the dorsal commissural part of the nucleus of the solitary tract at the level of the apex of calamus scriptorius, and extending into the dorsal subnucleus at the level of the area postrema. Preferential ramifications of carotid sinus and aortic depressor nerve afferents at the levels of the commissural part of the nucleus and the area postrema, respectively, were reflected in the extent to which labeled fibers comingled with neurons exhibiting phenylephrine-induced Fos in dual labeling experiments. Complete sinoaortic denervation reduced by 90% the number of neurons exhibiting drug-induced Fos expression. Selective carotid and aortic sinus denervations effected partial reductions manifest preferentially in the caudal and rostral foci of the distribution, respectively. Reduced activational responses at the level of the area postrema of aortic sinus-denervated rats were accompanied by a reduction in cellular nicotinamide adenine dinucleotide phosphate-diaphorase activity in this region. Animals killed 30 days after complete sinoaortic denervation displayed no evidence of recovery of phenylephrine-induced Fos, while the strength and distribution of the response in rats that received selective carotid sinus denervation were indistinguishable from those seen in controls. These findings (i) support the dependence of phenylephrine-induced Fos expression on the integrity of carotid sinus and aortic depressor nerve afferents, (ii) provide anatomical and functional evidence that the two buffer nerves distribute differentially within the nucleus of the solitary tract, and (iii) implicate central reorganization as a likely basis for functional recovery of baroreflex mechanisms following partial sinoaortic denervation.

Cochleotopic fos immunoreactivity in cochlea and cochlear nuclei evoked by bipolar cochlear electrical stimulation

Fos-like immunoreactivity evoked by basal, second or apical turn bipolar intracochlear electrical stimulation was evaluated in the spiral ganglion and cochlear nuclei. At stimulation levels of six times the electrically evoked auditory brain stem response thresholds, immunoreactive neurons were observed at appropriate discrete cochleotopic regions relative to stimulation site. The number of neurons increased with stimulus level and closely correlated to wave I amplitude. At 10 times thresholds, some spread in fos-like immunoreactivity to adjacent cochlear turns was found. However, fos-like immunoreactivity at this high level of stimulation still clearly showed a differential distribution in density of expression. These results indicated that the restricted topographic distribution of activity evoked by high levels of electrical stimulation is initiated at first order primary neurons of the system. For the profoundly deaf with cochlear implants, this indicates that place (channel) information can be maintained in the spiral ganglion and central nervous system even at very high levels of electrical stimulation. Together with our previous studies, these results indicate that fos provides a marker which can be used for evaluation of extent and pattern of cellular activation throughout the central auditory pathways, including activation of auditory nerve cells.

Chronic food restriction increases Fos-like immunoreactivity (FLI) induced in rat forebrain by intraventricular amphetamine

Chronic food restriction enhances behavioral responsiveness to amphetamine and other abused drugs. Because this effect is evident when drugs are administered intracerebroventricularly (i.c.v.) as well as systemically, it would seem to reflect increased sensitivity of a neural substrate rather than a change in drug disposition. In the present study, c-Fos immunohistochemistry was used to evaluate whether the magnitude and pattern of cellular activation induced by i.c.v. amphetamine is altered by a regimen of food restriction previously shown to potentiate amphetamine reward. In the absence of amphetamine challenge, there was generally no difference in brain Fos-like immunoreactivity (FLI) between ad libitum fed and food-restricted rats. In response to amphetamine (50 μg), both groups displayed increased FLI in caudate–putamen, nucleus accumbens, bed nucleus of the stria terminalis, ventral pallidum, central nucleus of the amygdala, and cingulate cortex. With the exception of cingulate cortex and caudal caudate–putamen, a significantly greater response was observed in brain regions of food-restricted rats. These results indicate that food restriction augments a cellular immediate early gene (IEG) response to acute amphetamine in brain regions known to mediate rewarding and other behavioral effects of psychostimulants. The difference between these results and those produced by sensitizing regimens of psychostimulant exposure are discussed, as are possible endocrine factors that could be involved in the modulatory effect of food restriction on cellular and behavioral responses to amphetamine.

Functional Dichotomy within the Vomeronasal System: Distinct Zones of Neuronal Activity in the Accessory Olfactory Bulb Correlate with Sex-Specific Behaviors

Chemosensory neurons in the vomeronasal organ (VNO) detect pheromones that elicit social and reproductive behaviors in most terrestrial vertebrates. Vomeronasal receptor neurons are chemoarchitecturally divided into two populations based on their position in the VNO, the type of G-protein subunit expressed, the family of putative pheromone receptor expressed, and termination site of their axons in the accessory olfactory bulb (AOB). To investigate the functional implications of these two segregated VNO-AOB pathways, we stimulated mice with pheromonal cues associated with different behavioral contexts and examined cellular activation patterns in the AOB. Exposure of ICR male mice to BALB/c males resulted in aggressive behavior, accompanied by a VNO-dependent increase in c-fos immunoreactivity in a cluster of cells located almost exclusively in the caudal AOB in both strains. This caudal cluster of activated cells did not appear to require the overt display of aggressive behavior because it was present in both the dominant and submissive males and could be evoked when the stimulus animal was anesthetized. In contrast, exposure of an ICR male to an ICR female in diestrus resulted in activation of cells located predominantly in the rostral AOB. Our findings indicate that male-to-male interactions involving interstrain recognition activate a separate population of vomeronasal receptor neurons than chemosensory cues detected in a sexual context. The results suggest that the dichotomy in the peripheral vomeronasal system serves to separate pheromones based on the behaviors they drive. As such, the results provide a bioassay for identifying pheromone molecules.

Treatment of flexor tendon injuries: Surgeons' perspective

Medical researchers continue to explore the flexor tendon's response to injury and repair. In recent years, hand surgery and therapy publications have focused on the biomechanics of suture techniques and the benefits of early postoperative motion on surgically repaired flexor tendons. Laboratory and clinical studies have shown that stronger suture techniques can withstand the strain of immediate active motion without a significant risk of tendon rupture or gap formation. Newly proposed therapy techniques and anatomic studies defining the effects of wrist and digital position on tendon excursion share the goals of achieving early motion and reducing restrictive adhesions. Clinical studies have evaluated the various imaging modalities used to diagnose postoperative adhesions. Other clinical surveys have detailed the use of pedicled autograft and allograft tendons in staged reconstruction. Histologic and immunologic researchers have concentrated on cellular activation patterns following tendon injury and the effects of pharmacologic agents, such as hyaluronan and aprotinin, on tendon healing and adhesion formation.

Systemic inflammatory responses in acute coronary syndrome: increased activity observed in polymorphonuclear leukocytes but not T lymphocytes

Background: Local inflammation within the coronary arteries is involved in the pathogenesis of acute coronary syndrome. However, the contribution of a systemic inflammatory response to the pathogenesis of this syndrome has not been well characterized. Accordingly, we investigated systemic inflammatory responses in patients with acute coronary syndrome. Methods: A total of 83 patients with ischemic heart disease (15 with stable exertional angina and 68 with acute coronary syndrome) were studied. The luminol-dependent chemiluminescence (CL) response of polymorphonuclear leukocytes (PMNs), which reflects their ability to generate oxygen species, was used as a marker for PMN activation. Soluble interleukin-2 receptor (sIL-2R) levels were measured to assess T-lymphocyte activation. Results: CL counts of whole blood from patients with acute coronary syndrome were twice those of patients with stable angina (2.38±0.22 vs 1.10±0.17×10 6 counts, P<0.05). A comparison of CL counts between patients with unstable angina and those with acute myocardial infarction revealed no significant differences. T-lymphocyte activity, measured by serum sIL-2R, was significantly lower in patients with acute coronary syndrome than those with stable angina (214.3±11.5 vs. 358.3±115.7 U/ml, P<0.05). Conclusions: This investigation shows that there is a systemic increase in PMN activity and a decrease in T-lymphocyte activity in patients with acute coronary syndrome. This contrasts with the pattern of cellular activation seen at sites of local inflammation within atherosclerotic plaques, suggesting that two independent inflammatory processes (local and systemic) may be involved in the pathogenesis of this syndrome.

Evidence for an Intramedullary Prostaglandin-Dependent Mechanism in the Activation of Stress-Related Neuroendocrine Circuitry by Intravenous Interleukin-1

We have provided evidence that the stimulatory effects of intravenous interleukin-1 (IL-1) on neurosecretory neurons in the paraventricular nucleus (PVH) that express corticotropin-releasing factor (CRF) depend specifically on the integrity of catecholaminergic projections originating in caudal medulla. Here we report on experiments designed to test alternative means by which circulating IL-1 might access medullary aminergic neurons, including mechanisms involving sensory components of the vagus, the area postrema, or perivascular cells bearing IL-1 receptors. Neither abdominal vagotomy nor area postrema lesions reliably altered Fos expression induced in the medulla or PVH in response to a moderately suprathreshold dose of IL-1beta. Cytokine-stimulated increases in CRF mRNA in the PVH were also unaffected by either ablation. By contrast, systemic administration of the cyclooxygenase inhibitor indomethacin resulted in parallel dose-related attenuations of IL-1 effects in hypothalamus and medulla. Microinjections of prostaglandin E2 (PGE2; >/=10 ng) in rostral ventrolateral medulla, the principal seat of IL-1-sensitive neurons that project to the PVH, provoked discrete patterns of cellular activation in hypothalamus and medulla that mimicked those seen in response to intravenous IL-1. We interpret these findings as supporting the hypothesis that paracrine effects of PGE2 released from perivascular cells in the medulla as a consequence of IL-1 stimulation and, acting through prostanoid receptors on or near local aminergic neurons that project to the PVH, contribute to the stimulatory effects of increased circulating IL-1 on neurons constituting the central limb of the hypothalamo-pituitary-adrenal axis.

Role of T cells in the mediation of Heymann nephritis. ii. Identification of Th1 and cytotoxic cells in glomeruli

The role of immunoglobulin (Ig) and complement as mediators of Heymann nephritis (HN) has been questioned by recent studies showing that HN can be induced in a C6-deficient rat that cannot assemble the membrane attack complex of complement. Also, the severity of HN can be reduced by therapy directed at CD8+ T cells, which has no effect on antibody (Ab) production or immune deposits. To identify whether T cells may contribute to the glomerular injury of active HN in Lewis rats, the mononuclear infiltrate and cytokine mRNA in glomeruli and kidney interstitium were examined. Groups of Lewis rats immunized with Fx1A in CFA developed HN, and were compared to controls that received CFA only. Proteinuria, the marker of glomerular filtration barrier dysfunction, was absent at four weeks but present at eight weeks in HN. Serum anti-Fx1A Ab and glomerular Ig were present in HN at both time points. Immunoperoxidase staining with monoclonal Abs identified, at eight weeks, a glomerular infiltrate of CD4+ and CD8+ T cells, and macrophages, but not NK cells. Semiquantitative RT-PCR of isolated glomeruli at eight weeks demonstrated expression of cytokine mRNA for Th1 CD4+ cells (IFN-gamma and TNF-beta/LT, but not IL-2), cytotoxic CD8+ T cells (granzyme A and perforin), and macrophages (TNF-alpha and IL-10), but not Th2 CD4+ cells (no increase in IL-4, IL-5 and IL-6). At eight weeks, the cellular infiltrate and pattern of cellular activation in glomeruli was different to that in renal cortex. In the cortical infiltrate CD8+ cells were a lesser component, and NK cells were increased, as were CD4+ cells and macrophages. RT-PCR identified increased cytokine mRNA for macrophages, Th1 and Th2 cells, but not cytotoxic effector T cells. At four weeks, T cells including CD4+ and CD8+ cells were identified in the isolated glomeruli of rats with HN, but there was no increase in cytokine mRNA expression. There was no infiltrate or increase in cytokine mRNA detected in renal cortex at four weeks. Anti-Fx1A Ab's and glomerular deposition of Ig develop many weeks before the onset of proteinuria, when there is only a small cellular infiltrate present. The progressive development of infiltrates of activated T cells, principally Th1 and cytotoxic effector cells, and macrophages, within glomeruli is coincident with the development of proteinuria. These findings raise the possibility that these cells contribute to the mediation of the glomerular injury and proteinuria of HN.

Patterns of cellular activation after tendon injury

Mechanisms which lead to disabling adhesions following flexor tendon surgery of the hand were investigated in a rabbit model which was used to assess the relative response of the cells of the synovial sheath, epitenon and the endotenon to injury. A transverse laceration, cutting through 50% of the tendon, was made just outside the synovial sheath on the flexor aspect of the flexor digitorum profundus tendon. The synovial sheath was preserved intact. Using monoclonal antibodies for localizing specific inflammatory markers, we were able to follow the response and activity of the synovial sheath, epitenon and endotenon with respect to these markers at various times after surgery. Our findings suggest that the synovial sheath and the epitenon are relatively more reactive in the early period after injury, as judged by a range of inflammatory indices with the notable exception of the expression of the potent neovascularizing agent, basic fibroblast growth factor (bFGF).

State-dependent cellular activity patterns of the cat paraventricular hypothalamus measured by reflectance imaging

Activity within the cat paraventricular hypothalamus (PVH) during sleep and waking states was measured by quantifying intrinsic tissue reflectivity. A fiber optic probe consisting of a 1.0 mm coherent image conduit, surrounded by plastic fibers which conducted 660 nm source light, was attached to a charge-coupled device camera, and positioned over the PVH in five cats, Electrodes for assessing state variables, including electroencephalographic activity, eye movement, and somatic muscle tone were also placed. After surgical recovery, reflected light intensity was measured continuously at 2.5 Hz during spontaneously varying sleep/waking states. Sequential state transitions from active waking to quiet waking, quiet sleep and active sleep were accompanied by progressively increased levels of PVH activity, Overall activity was highest during active sleep, and decreased markedly upon awakening. Moment-to-moment activity oscillated in the 0–0.1 Fiz range. especially during active sleep and active waking; this oscillation diminished during quiet sleep, Distinct sub-regions of enhanced or diminished activity emerged within the imaged area in a state-dependent manner, We conclude that PVH activity changes with behavioral state in a regionally specific manner, and that overall activity increases during quiet sleep, and is even more enhanced in active sleep, PVH activation could be expected to stimulate pituitary release of adrenocorticotropic hormone (ACTH) and affect input to autonomic regulatory sites. Since ACTH and corticotropin releasing factor elicit arousal. and since the PVH projects to other brain areas which modulate state, we speculate that the PVH plays a role in shaping characteristics of sleep /waking states.

State-dependent cellular activity patterns of the cat paraventricular hypothalamus measured by reflectance imaging

Activity within the cat paraventricular hypothalamus (PVH) during sleep and waking states was measured by quantifying intrinsic tissue reflectivity. A fiber optic probe consisting of a 1.0 mm coherent image conduit, surrounded by plastic fibers which conducted 660 nm source light, was attached to a charge-coupled device camera, and positioned over the PVH in five cats, Electrodes for assessing state variables, including electroencephalographic activity, eye movement, and somatic muscle tone were also placed. After surgical recovery, reflected light intensity was measured continuously at 2.5 Hz during spontaneously varying sleep/waking states. Sequential state transitions from active waking to quiet waking, quiet sleep and active sleep were accompanied by progressively increased levels of PVH activity, Overall activity was highest during active sleep, and decreased markedly upon awakening. Moment-to-moment activity oscillated in the 0–0.1 Fiz range. especially during active sleep and active waking; this oscillation diminished during quiet sleep, Distinct sub-regions of enhanced or diminished activity emerged within the imaged area in a state-dependent manner, We conclude that PVH activity changes with behavioral state in a regionally specific manner, and that overall activity increases during quiet sleep, and is even more enhanced in active sleep, PVH activation could be expected to stimulate pituitary release of adrenocorticotropic hormone (ACTH) and affect input to autonomic regulatory sites. Since ACTH and corticotropin releasing factor elicit arousal. and since the PVH projects to other brain areas which modulate state, we speculate that the PVH plays a role in shaping characteristics of sleep /waking states.

Distribution of A-kinase anchoring proteins in parietal cells

Recent investigations have suggested that subcellular compartmentalization of second messenger responsive enzyme systems may be responsible for specific patterns of cellular activation. The type II cAMP-dependent kinase (A-kinase) is localized to particular subcellular domains through the binding of the regulatory subunit (R II) dimer to A-kinase anchoring proteins (AKAPs). Using a [ 32P]R II overlay assay, we have investigated the presence of AKAPs throughout the gastrointestinal tract, with specific emphasis focused on the gastric parietal cell. All gastrointestinal tissues contained at least one detectable AKAP (60 kDa), with five AKAPs (50–140 kDa) in fundic and antral mucosa. Isolated gastric glands contained four AKAPs. Two AKAPs (50 and 78 kDa) were detected in purified parietal cells, with the 78 kDa AKAP (AKAP78) specific to parietal cell enriched populations. R II-binding to all of these AKAPs was abolished by preincubation of [ 32P]R II with a synthetic peptide representing the R II-binding region of the AKAP, HT-31. AKAP78 was distributed throughout all membrane fractions of subfractionated parietal cells, with the largest amount of R II-binding detected in the light membrane fraction. Identification of A-kinase regulatory subunits by photoaffinity labeling with 8-azido-[ 32P]cAMP demonstrated that R II segregated into the same parietal cell subfractions as AKAP78. A majority (∼ 60%) of AKAP78 was detected in the Triton X-100-insoluble fraction, suggesting that this protein resides in a cytoskeletal domain. AKAP78 may be involved in localizing the type II A-kinase to specific intracellular locations in the parietal cell.