Neuropathology in cats experimentally infected with feline immunodeficiency virus: a morphological, immunocytochemical and morphometric study

Head flexion and extension movements near the natural head position (NHP) were analysed for the location of the mean instantaneous centre of rotation (ICR). Forty-six healthy young adults (30 women and 16 men) with sound dentitions, free from cranio-cervical disorders, performed habitual movements that were automatically detected and measured by an infrared three-dimensional motion analyser. ICR and curvature radius were calculated for each movement and subject. In both extension and flexion, ICR position changed during the motion. The movement was symmetrical in all subjects. No gender or flexion/extension differences were found for both ICR position and relevant curvature radius. On average, ICR relative to NHP soft-tissue nasion was located at about 150% of the soft-tissue nasion-right tragus distance, with an angle of about 220 degrees relative to the true horizontal. Results suggest that head flexion or extension is always performed with a combination of rotation (atlanto-occipital joint) and translation (cervical spine) even in the first degrees of motion. Moreover, NHP at rest seems to be some degree more flexed and anterior than head position during movements. These relative positions and their muscular determinants could also influence mandibular posture at rest and during functional movements.     

Colocalization of calretinin and calbindin-D28k with oxytocin and vasopressin in rat supraoptic nucleus neurons: a quantitative study

Leptin is a peptide hormone that appears critical in regulating Fat metabolism. Recently, circulating leptin levels were reported higher in patients with alcoholic cirrhosis. In health, hepatic stellate cells store retinoids, but following liver injury they transdifferentiate into myofibroblast-like cells with loss of the retinoid stores. Leptin expression was demonstrated by detection of leptin mRNA by RT-PCR analysis and by immunohistochemistry viewed with confocal microscopy in transdifferentiated stellate cells after 14 days, or more, of culture. Leptin expression was not found in freshly isolated quiescent stellate cells. Leptin expression was not demonstrated in freshly isolated or cultured Kupffer cells. Treatment of activated stellate cells with either 1 microM retionic acid or 10 microM retinol acetate resulted in the inhibition of leptin mRNA expression. The observation that activated stellate cells in culture can express leptin has implications for understanding adipocyte biology in liver disease and treatment of malnutrition in cirrhotics.     

kappa-opioid regulation of neuronal activity in the rat supraoptic nucleus in vivo

We investigated the influence of endogenous kappa-opioids on the activity of supraoptic neurons in vivo. Administration of the kappa-antagonist nor-binaltorphimine (200 micrograms/kg, i.v.), increased the activity of phasic (vasopressin), but not continuously active (oxytocin), supraoptic neurons by increasing burst duration (by 69 +/- 24%) and decreasing the interburst interval (by 19 +/- 11%). Similarly, retrodialysis of nor-binaltorphimine onto the supraoptic nucleus increased the burst duration (119 +/- 57% increase) of vasopressin cells but did not alter the firing rate of oxytocin cells (4 +/- 8% decrease). Thus, an endogenous kappa-agonist modulates vasopressin cell activity by an action within the supraoptic nucleus. To eliminate kappa-agonist actions within the supraoptic nucleus, we infused the kappa-agonist U50,488H (2.5 micrograms/hr at 0.5 micrograms/hr) into one supraoptic nucleus over 5 d to locally downregulate kappa-receptor function. Such infusions reduced the spontaneous activity of vasopressin but not oxytocin cells and reduced the proportion of cells displaying spontaneous phasic activity from 26% in vehicle-infused nuclei to 3% in U50, 488H-infused nuclei; this treatment also prevented acute inhibition of both vasopressin and oxytocin cells by U50,488H (1000 micrograms/kg, i.v.), confirming functional kappa-receptor downregulation. In U50, 488H-infused supraoptic nuclei, vasopressin cell firing rate was increased by nor-binaltorphimine (100 and 200 micrograms/kg, i.v.) but not to beyond that found in vehicle-treated nuclei, indicating that these cells were not U50,488H-dependent. Thus, normally functioning kappa-opioid mechanisms on vasopressin cells are essential for the expression of phasic firing.     

Cytodifferentiation of the supraoptic nucleus correlated with vasopressin synthesis in the rat

This study demonstrates that neurons in the supraoptic nucleus attain many of the prerequisites for functional activity prior to birth. Immunoassayable vasopressin (VP) was detected in the hypothalamo-neurohypophyseal system (HNS) of the rat as early as 17 days post-coitus (dpc). Vasopressin concentrations increased 3--6-fold daily from an average of 21 pg/animal on 17 dpc to 5984 pg/animal at 21 dpc. The daily increases were highly significant (P less than 0.001). Between 21 dpc and the morning of the day of birth on 22 dpc, a further significant increase (P less than 0.05) occurred to a mean level of 9672 pg VP/animal. Birth usually occurred on the afternoon of the 22nd day. Parturition did not seem to deplete VP stores in the HNS. Differentiation of the magnocellular neurons in the supraoptic nucleus closely paralleled the appearance and increases in VP. It was first possible to dintinguish a supraopic nucleus in the 17 dpc rat and to identify dense core granules in the developing neurons of the nucleus. Cytodifferentiation of the magnocellular neurons was essentially complete by 21 dpc. Synaptic contacts could not be found on the soma and dendrites of the supraoptic neurons until 21 dpc and were extremely rare throughout the period examined.     

Autoinhibition of supraoptic nucleus vasopressin neurons in vivo: a combined retrodialysis/electrophysiological study in rats

To examine the role of endogenous vasopressin on the electrical activity of vasopressin neurons within the supraoptic nucleus of the rat brain in vivo, we have developed a novel technical approach for administering neuroactive drugs directly into the extracellular environment of the neuronal dendrites. A microdialysis probe was used for controlled local drug administration into the dendritic area of the nucleus during extracellular recording of single neurons in vivo. Vasopressin or selective V1 receptor antagonists were administered for between 10 and 30 min via a U-shaped microdialysis probe placed flat on the surface of the supraoptic nucleus after transpharyngeal exposure of the nucleus in urethane-anaesthetized rats. Microdialysis administration (retrodialysis) of vasopressin inhibited vasopressin neurons by reducing their firing rate, sometimes to total inactivity. Retrodialysis of V1-receptor antagonists partially reversed the effect of vasopressin, and a subsequent vasopressin administration was not effective in reducing the activity of these neurons, suggesting a receptor-mediated action of endogenous vasopressin. In addition, the duration of the periods of activity and the mean frequency during the active phase were increased in vasopressin neurons after retrodialysis of V1-receptor antagonist, indicating a physiological role of endogenous vasopressin. Neither vasopressin nor the antagonists altered the activity of continuously firing oxytocin neurons. Thus, vasopressin released within the supraoptic nucleus may act via V1 receptors located specifically on vasopressin neurons to regulate their phasic activity by an auto-inhibitory action. Since vasopressin release from the dendrites of vasopressin neurons is increased and prolonged after various forms of stimulation, it is proposed that this mechanism will act to limit excitation of vasopressin neurons, and hence secretion from the neurohypophysis. In addition, combined in vivo retrodialysis/ single cell recording allows controlled introduction of neuroactive substances into the extracellular fluid in the immediate vicinity of recorded neurons. This is shown to provide a novel approach to study neurotransmitter actions on supraoptic neurons in vivo.     

Circannual morphometric investigations of the rat suprachiasmatic nucleus after pinealectomy, ganglionectomy and thyroidectomy

With the advent of combinatorial chemistry a new paradigm is evolving in the field of drug discovery. The approach is based on an integration of chemistry, high-throughput screening and automation engineering. The chemistry arm is usually based on solid-phase synthesis technology as the preferred approach to library construction. One of the most powerful of the solid-phase methods is encoded split synthesis, in which the reaction history experience by each polymeric bead is unambiguously recorded. This split-and-pool approach, employing chemically robust tags, was used to construct a 85,000-membered dihydrobenzopyran library.     

Whole-cell recordings of the supraoptic nucleus neurons from rat hypothalamic slices in vitro

Two patients presented with very different signs of central anticholinergic syndrome following general anaesthesia for which they had received premedication with hyoscine. Both responded dramatically to 1 mg of intravenous (i.v.) physostigmine, which produced a rapid return to a normal level of consciousness. The aetiology of central anticholinergic syndrome is multi-factorial, but the diagnosis should be considered in all patients who demonstrate abnormal post-anaesthetic awakening. It is recommended that 1 mg of intravenous physostigmine is a safe and effective treatment for central anticholinergic syndrome, and that a supply of this important drug must be kept readily available in the recovery area of the operating theatre department.     

Kindled seizures increase metabotropic glutamate receptor expression and function in the rat supraoptic nucleus

The spread of experimentally kindled seizures in rats results in sustained increases in plasma vasopressin (VP) and VP mRNA in the supraoptic nucleus (SON). These increases provide an excellent example of the pathological plasticity that can develop in normal cells exposed to recurrent seizure activity. To test whether this plasticity might be due in part to changes in metabotropic glutamate receptors (mGluRs), we examined mGluR mRNA expression in the SON 1 month after stage 5 amygdala kindling. Three mGluR subtypes were detected by in situ hybridization in the SON in the following relative levels: mGluR3 > mGluR1 > mGluR7. Both mGluR1 and mGluR3 mRNAs were significantly increased in the SON (+28-61%) and cortex (+27-42%) after kindling. Immunoreactivity for mGluR1 but not mGluR2/3 was significantly increased in vivo in the SON. Receptor protein expression and intracellular calcium accumulation in response to the mGluR agonist, 1S,3R ACPD, were evaluated after in vitro "kindling" of neuroendocrine cells by Mg2+ deprivation. Increased immunoreactivity for mGluR1 and mGluR2/3 was seen in all cultures 3 days after a brief exposure to Mg2+-free medium. 1S,3R 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) induced rapid peak responses and gradual accumulations of intracellular Ca2+ in neurons. Both responses were increased in the "kindled" cells. Increases in the expression of functional mGluR1 and perhaps mGluR3 receptors may contribute to the development of long-lasting plastic changes associated with seizure activity.     

Glutamate microstimulation of local inhibitory circuits in the supraoptic nucleus from rat hypothalamus slices

The hypothesis of a local inhibitory input to the hypothalamic supraoptic nucleus was tested with combined glutamate microstimulation and whole cell patch-clamp recordings in slices from rat hypothalamus. Synaptic activity in supraoptic magnocellular neuroendocrine cells (MNCs) was monitored and glutamate microdrops were applied in the perinuclear region of the supraoptic nucleus to evoke firing of action potentials in putative presynaptic inhibitory cells. The effect of glutamate microdrops applied in the perinuclear region was tested on 57 supraoptic MNCs. In control conditions, spontaneous excitatory (EPSCs) and inhibitory (IPSCs) postsynaptic currents were observed at resting membrane potential in all MNCs tested. Glutamate microstimulation evoked an abrupt increase in the frequency and size of spontaneous IPSCs in eight MNCs. Forty-nine MNCs did not show any change in the inhibitory synaptic input. Microapplication of glutamate in the periphery of the supraoptic nucleus did not modify the amplitude or the frequency of spontaneous EPSCs in any of the 57 MNCs tested. In the group of eight MNCs that responded to glutamate microstimulation by an increase in inhibitory input, two types of responses were observed. Four MNCs showed an increase in both size and frequency of spontaneous IPSCs through the entire range of amplitude. In the other four MNCs, local glutamate stimulation produced a dramatic increase in the size of IPSCs and a lesser increase in the frequency of the smaller IPSCs. The potential effect of the glutamate-evoked increase in inhibitory input on the firing activity of MNCs was tested in current-clamp conditions. Intracellular current injection was applied to evoke firing of action potentials in six MNCs that had responded to local glutamate microstimulation by an increase in inhibitory input. Glutamate microdrop applications inhibited the evoked action potential firing in all six cells. These results suggest 1) that local inhibitory interneurons are present in the periphery of the supraoptic nucleus, 2) that they contain functional glutamate receptors, 3) that they form inhibitory synapses with supraoptic MNCs, and 4) that activation of these interneurons inhibits firing in MNCs. These results support the hypothesis that local inhibitory interneurons play a important role in the firing activity of supraoptic MNCs.     

Differential colocalization of estrogen receptor beta (ERbeta) with oxytocin and vasopressin in the paraventricular and supraoptic nuclei of the female rat brain: an immunocytochemical study

Evidence exists for the localization of the newly identified estrogen receptor beta (ERbeta) within the rat paraventricular nucleus (PVN) and supraoptic nucleus (SON), regions which lack ERalpha. Presently, we investigate whether ERbeta-like-immunoreactivity (-ir) is found within cells of several major neuropeptide systems of these regions. Young adult Sprague-Dawley rats were ovariectomized (OVX), and 1 week later half of the animals received estradiol-17beta (E). Dual-label immunocytochemistry was performed on adjacent sections by using an ERbeta antibody, followed by an antibody to either oxytocin (OT), arginine-vasopressin (AVP), or corticotropin releasing hormone. Nuclear ERbeta-ir was identified within SON and retrochiasmatic SON, and in specific PVN subnuclei: medial parvicellular part, ventral and dorsal zones, dorsal and lateral parvicellular parts, and in the posterior magnocellular part, medial and lateral zones. However, the ERbeta-ir within magnocellular areas was noticeably less intense. OT-/ERbeta-ir colocalization was confirmed in neurons of the parvicellular subnuclei, in both OVX and OVX+E brains ( approximately 50% of OT and 25% of ERbeta-labeled cells between bregma -1.78 and -2.00). In contrast, few PVN parvicellular neurons contained both AVP- and ERbeta-ir. As well, very little overlap was observed in the distribution of cells containing corticotropin releasing hormone- or ERbeta-ir. In the SON, most nuclear ERbeta-ir colocalized with AVP-ir, whereas few OT-/ERbeta-ir dual-labeled cells were observed. These findings suggest that estrogen can directly modulate specific OT and AVP systems through an ERbeta-mediated mechanism, in a tissue-specific manner.     

Structure and plasticity of newly formed adult synapses: a morphometric study in the rat hippocampus

Increasing evidence suggests that synaptic structure represents a plastic feature of the neuron, although the plastic nature of newly formed and existing adult synapses has not yet been fully characterized. Following ipsilateral entorhinal cortical lesions, the rat dentate gyrus offers an excellent model for studying synaptogenesis and plasticity in the adult central nervous system. Unilateral entorhinal lesions were performed in young adult male rats. Synaptic counts and structural features were quantified at 3, 6, 10, 15, and 30 days post-lesion. The lesions resulted in an 88% synaptic loss in the denervated dentate middle molecular layer, which was followed by a period of rapid synaptogenesis. Synaptic element size decreased during the period of maximal synaptogenesis, which was associated with a peak in the presence of non-vesicular and perforated synapses. Following this period, synapses showed a gradual increase in the size of their pre- and postsynaptic elements. These data support the suggestion that newly formed adult synapses have smaller synaptic components than existing adult synapses (resembling synapses seen during development), and increase in size over time with usage. The results are discussed in terms of synaptic structural development and plasticity in the adult central nervous system.     

GM1 ganglioside potentiates trimethyltin-induced expression of interleukin-1 beta and the nerve growth factor in reactive astrocytes in the rat hippocampus: an immunocytochemical study

An approach to the evaluation and comparison of reversed-phase high-performance liquid chromatography stationary phases with particular emphasis on data analysis and presentation is described. Assessment is based on the peak efficiency, asymmetry (USP tailing factor) and relative retention properties shown by 24 basic compounds having a wide range of structural and physico-chemical properties. A novel approach to data normalisation and presentation is described. This overcomes the problems associated with the quality of the column packing process, as well as differences in stationary phase selectivity which in conjunction with extra column band broadening effects can make comparisons meaningless.     

Distribution of striatin, a newly identified calmodulin-binding protein in the rat brain: an in situ hybridization and immunocytochemical study

The worm-like chain model has often been employed to describe the average conformation of long, intrinsically straight polymer molecules, including DNA. The present study extends the applicability of the worm-like chain model to polymers containing bends or sections of different flexibility. Several cases have been explicitly considered: (i) polymers with a single bend; (ii) polymers with multiple coplanar bends; (iii) polymers with two non-coplanar bends; and (iv) polymers comprised of sections with different persistence lengths. Expressions describing the average conformation of such polymers in terms of the mean-square end-to-end distance have been derived for each case. For cases (i) and (iv), expressions for the projection of the end-to-end vector onto the initial orientation of the chain are presented. The expressions derived here have been used to investigate DNA molecules with sequence-induced bending (A-tracts). Mean-square end-to-end distance values determined from a large number of A-tract containing DNA molecules visualized by scanning force microscopy resulted in an average bend angle of 13.5 degrees per A-tract. A similar study was performed to characterize the flexibility of double-strandedDNA molecules containing a single-stranded region. Analysis of their mean-square end-to-end distance yielded a persistence length of 1.3 nm for single-stranded DNA.     

Ultrastructural immunocytochemical localization of neurotensin and the dopamine D2 receptor in the rat nucleus accumbens

The neuroleptic-like effects of neurotensin (NT) are thought to be due to interactions with dopamine (DA) acting primarily at D2 receptors within the nucleus accumbens septi (Acb). Using electron microscopic dual labeling immunocytochemistry, we sought to demonstrate cellular substrates for functional interactions involving NT and DA D2 receptors in the adult rat Acb. Peroxidase reaction product representing D2 receptor-like immunoreactivity (D2-LI) was seen along membranes of Golgi lamellae and multivesicular bodies of perikarya containing immunogold labeling representing NT-LI. Dually labeled somata usually contained highly indented nuclei, a characteristic of aspiny neurons. Dendrites also occasionally colocalized the two immunomarkers. Other somata, dendrites, and all axon terminals were singly labeled with either NT-LI or D2-LI. In distinct sets of terminals, NT-LI was commonly associated with large, dense-cored vesicles, whereas D2-LI was found along the plasmalemma and over nearby small clear vesicles. Each type of terminal comprised approximately 20% of synaptic input to NT-immunoreactive dendrites. Similar proportions of terminals containing NT-LI or D2-LI contacted unlabeled (approximately 55%) or NT-labeled (approximately 35%) dendrites and, occasionally, were observed converging onto common dendrites. Terminals containing NT-LI or D2-LI also were often closely apposed. These findings provide the first ultrastructural evidence that: (1) NT and D2 receptors are colocalized in aspiny neurons and dendrites, (2) NT may produce a direct postsynaptic effect on neurons receiving input from terminals which are presynaptically modulated by DA via D2 receptors, and (3) NT and DA acting at D2 receptors may interact through presynaptic modulation of common axon terminals.     

Neuronal loss and plasticity in the supraoptic nucleus in Parkinson's disease

Disturbed circadian control of renal water excretion and blood pressure adaptation in Parkinson's disease (PD) suggest impaired hypothalamic magnocellular neurosecretion. To test the hypothesis that this may relate to specific hypothalamic pathology in PD, we studied morphometrically the neuronal population of the supraoptic nucleus (SON) in PD patients and controls. Neuronal loss in the SON of PD patients was associated with increased somatic, nuclear, and nucleolar size of remaining neurons, suggesting compensatory response of these cells. We conclude that SON pathology is a feature of PD and may account for specific signs of neurohumoral dysfunction.     

The endolymphatic duct and sac of the rat: a histological, ultrastructural, and immunocytochemical investigation

A study of the ultrastructure, vascularization, and innervation of the endolymphatic duct and sac of the rat has been performed by means of light- and electron-microscopic and immunocytochemical methods. Two different types of epithelial cells have been identified: the ribosome-rich cell and the mitochondria-rich cell. These two cell types make up the epithelium of the complete endolymphatic duct and sac, although differences in their quantitative distribution exist. The morphology of the ribosome-rich cells varies between the different parts of the endolymphatic duct and sac; the morphology of the mitochondria-rich cells remains constant. According to the epithelial composition, vascularization, and structural organization of the lamina propria, both duct and sac are subdivided into three different parts. A graphic reconstruction of the vascular network supplying the endolymphatic duct and sac shows that the vascular pattern varies among the different parts. In addition, the capillaries of the duct are of the continuous types, whereas those supplying the sac are of the fenestrated type. Nerve fibers do not occur within the epithelium of the endolymphatic duct and sac. A few nerve fibers regularly occur in the subepithelial compartment close to the blood vessels; these fibers have been demonstrated in whole-mount preparations by the application of the neuronal marker protein gene product 9.5. Single beaded fibers immunoreactive to substance P and calcitonin-gene related peptide are observed within the same compartment. Dopamine-beta-hydroxylase-immunoreactive axons are restricted to the walls of arterioles. Morphological differences between the different portions of the endolymphatic duct and sac are discussed with regard to possible roles in fluid absorption and immunocompetence.