Capsaicin induced release of multiple tachykinins (substance P, neurokinin A and eledoisin-like material) from guinea-pig spinal cord and ureter

The release of tachykinins from isolated slice preparations of the guinea-pig spinal cord and ureter was studied in vitro. Capsaicin (10 microM) caused release of substance P, neurokinin A and an eledoisin-like component from both the spinal cord and ureter. The release of tachykinins induced by capsaicin or potassium (60 mM) was calcium dependent. No detectable release of neurokinin B or neuropeptide K, an N-terminally extended form of neurokinin A, was induced by capsaicin. No detectable release of tachykinins could be demonstrated after exposure to agents which are known to activate C-fibre afferents, such as histamine, bradykinin, serotonin, prostaglandins E1, E2 or acetylcholine. Protein extravasation in the ureter, as determined by the Evans Blue extravasation technique was used as a functional correlate to the tachykinin release. Protein extravasation was induced in vivo by local intraluminal injections of capsaicin at several hundred-fold lower concentrations than those required to induce a detectable release of tachykinins in vitro. The difference may, however, partly depend on the experimental conditions and the detection limit of the tachykinin assay used. The protein extravasation response to capsaicin was absent after systemic capsaicin pretreatment, which causes a marked depletion of tachykinins in the ureter. In conclusion, capsaicin evokes release of several tachykinins from both central and peripheral endings of primary afferent neurons. The peptides released from sensory nerves in the periphery may induce effects such as protein extravasation and smooth muscle contraction.     

Neurotensin-like Peptides in the CNS of Lampreys: Chromatographic Characterization and Immunohistochemical Localization with Reference to Aminergic Markers

Neurotensin (NT)-like peptides in the CNS of the lamprey Lampetra fluviatilis were studied by radioimmunoassay (C-terminal specific NT antiserum), reverse-phase HPLC and immunohistochemistry. Multiple peaks of NT-immunoreactive (-ir) material were observed upon HPLC, of which a major peak eluted in the position of bovine NT. Immunofluorescence histochemistry showed that a monoclonal antibody recognizing the N-terminal (1 - 11) fragment of NT, as well as two polyclonal NT antisera labelled a large number of cell bodies in the periventricular area of hypothalamus, including the postinfundibular commissural nucleus and the ventral and dorsal hypothalamic nuclei. Additional groups of NT-ir cells were observed in the preoptic nucleus, the postoptic commissural nucleus, the mesencephalic tegmentum (L.fluviatilis), and in the spinal cord (L.fluviatilis and Ichtyomyzon unicuspis). Dense NT-ir fibre plexuses were present in the caudal hypothalamus, corpus striatum, ventral mesencephalon, and in the dorsal horn and lateral margin of the spinal cord. At the ultrastructural level the lateral spinal margin showed NT-ir terminal structures, which in most cases were not associated with synaptic specializations, although occasional synaptic contacts with unlabelled elements were found. The relation between NT-ir and monoamine-containing cells was examined with immunofluorescence double-staining, using antisera to tyrosine hydroxylase (TH), 5-hydroxytryptamine (5-HT), and histamine respectively. In the periventricular nuclei of hypothalamus numerous TH-, 5-HT-, as well as histamine-ir cells were located in close association with NT-ir cells, but none of the aminergic markers could be detected within NT-ir neurons. The chemical properties as well as the anatomical distribution of lamprey NT-like peptides show several similarities with those present in mammals, suggesting that NT-containing neuronal systems in the CNS developed early in vertebrate phylogeny.     

A subconvulsive dose of kainate selectively compromises astrocytic metabolism in the mouse brain in vivo

Despite the well-established use of kainate as a model for seizure activity and temporal lobe epilepsy, most studies have been performed at doses giving rise to general limbic seizures and have mainly focused on neuronal function. Little is known about the effect of lower doses of kainate on cerebral metabolism and particularly that associated with astrocytes. We investigated astrocytic and neuronal metabolism in the cerebral cortex of adult mice after treatment with saline (controls), a subconvulsive or a mildly convulsive dose of kainate. A combination of [1,2-¹³C]acetate and [1-¹³C]glucose was injected and subsequent nuclear magnetic resonance spectroscopy of cortical extracts was employed to distinctively map astrocytic and neuronal metabolism. The subconvulsive dose of kainate led to an instantaneous increase in the cortical lactate content, a subsequent reduction in the amount of [4,5-¹³C]glutamine and an increase in the calculated astrocytic TCA cycle activity. In contrast, the convulsive dose led to decrements in the cortical content and ¹³C labeling of glutamate, glutamine, GABA, and aspartate. Evidence is provided that astrocytic metabolism is affected by a subconvulsive dose of kainate, whereas a higher dose is required to affect neuronal metabolism. The cerebral glycogen content was dose-dependently reduced by kainate supporting a role for glycogen during seizure activity.     

Stress at three‐month immunization: Parents’ and infants’ salivary cortisol response in relation to the use of pacifier and oral glucose

The aims of the present study were to investigate (1) whether the salivary cortisol response could be dampened during a routine three‐month immunization if the infant received sweet‐tasting solution in combination with a pacifier and (2) stress experienced by parents during immunization of the infant. Ninety‐eight infants were included into one of four intervention groups: ‘glucose and pacifier’, ‘water and pacifier’, ‘glucose’, or ‘water’. Saliva was collected before and 30min after the immunization. Infants’ crying‐time and parents’ self‐reported stress (VAS) were measured before and after immunization. Infants in the ‘pacifier and glucose’ group had a significantly smaller change in salivary cortisol than infants in the other groups (F_3,72=3.1, p<0.05). In the ‘glucose and pacifier’ group the median salivary cortisol levels decreased 33% after the immunization. In the ‘water and pacifier’, ‘glucose’, and ‘water’ group median cortisol increased with 50%, 42%, and 8%, respectively. No significant differences in crying‐time were observed between the intervention groups. If the infant cried before the immunization, the crying‐time during the immunization was longer (p<0.01) and cortisol increased more (p<0.05). Median cortisol levels for parents decreased after the immunization (p<0.01). Median VAS increased 50% (p<0.0001) after immunization. First time parents rated higher stress on VAS before immunization (p<0.01). Parents’ change in cortisol and VAS were significantly related to infants’ crying time. In conclusion, the combination of oral glucose and pacifier dampen infants’ salivary cortisol in response to the three‐month immunization.     

Tachykinins Influence Interdigestive Rhythm and Contractile Strength of Human Small Intestine

The effect of the putative entericneurotransmitters neurokinin A and substance P wereinvestigated on human small intestinal motility. Eitherneurokinin A, at doses of 6-25 pmol/kg/min, or substanceP at doses of 1- 6 pmol/kg/min were administeredintravenously to healthy volunteers over 4 hr.Neurokinin A dose-dependently increased the fraction ofphase II of the migrating motor complex, contraction frequency, motility index, and amplitude ofcontractions. At the highest dose, neurokinin A induceda phase II-like pattern, disrupting the migratingmyoelectric complex. Substance P dose-dependentlyincreased phase II of the migrating motor complex. Thecontraction frequency increased slightly at the highestdose, but neither motility index nor contractionamplitude changed. It is concluded that neurokinin A and substance P stimulate small intestinalmotility in man, and it can be speculated that they playa role in the control of human small intestinalmotility.     

Substantial discrepancies in 17β‐oestradiol concentrations obtained with three different commercial direct radioimmunoassay kits in rat sera

The extensive use of oestrogen for contraception and amelioration of post‐menopausal symptoms has made it the subject of substantial recent research efforts, and ovariectomized (ovx) rats treated with exogenous ovarial hormones are important when investigating the effects and mechanisms of oestrogen actions. The crucial need to control and monitor plasma levels of 17β‐oestradiol calls for accurate, precise and robust assay methods. The performance of direct radioimmunoassays (RIAs) in measurement of 17β‐oestradiol has been reported previously for human samples, but to our knowledge not for rat samples. In the current study, 552 serum samples from ovx, native and hormone‐treated rats were used to compare the performance of three commercially manufactured direct RIAs from the companies DPC (Siemens Healthcare Diagnostics Inc., formerly Diagnostic Products Corporation), DSL (Diagnostic Systems Labs) and MPB (MP Biomedicals, formerly ICN Biomedicals). Substantial differences in results between the three assay methods were found when measuring serum 17β‐oestradiol concentrations. The following formulas describing the relation between the different methods were obtained using weighted Deming's orthogonal regression (based on pg/mL): DSL = 0.43*DPC+12.3, MPB = 2.1*DPC+84.7 and DSL = 4.8*MPB+22.2. Furthermore, a preceding diethyl ether extraction step of the serum appears to impair the performance of the RIAs in the present samples (based on pg/mL): DPC_ex = 0.39*DPC_unex+0.76, DSL_ex = 0.32*DSL_unex?1.7 and MPB_ex = 0.22*MPB_unex+1.4.     

Glial-neuronal interactions are impaired in the schizophrenia model of repeated MK801 exposure.

Schizophrenia-mimicking compounds such as phencyclidine (PCP) and MK801 are antagonists at the N-methyl-D-aspartate (NMDA) receptor and produce the whole spectrum of positive, negative, and cognitive symptoms. This is one of the most important pillars of the hypoglutamatergic hypothesis of schizophrenia. Since the synthesis of glutamate and GABA in neurons is closely connected to astrocyte metabolism, the study of astrocytic function is essential in this context. Dizocilpine-maleate (MK801) (0.5 mg/kg) was injected into rats every day for 6 days. The last dose was given together with [1-(13)C]glucose and [1,2-(13)C]acetate. Extracts from frontal, retrosplenial, and cingulate cortices (CRFC) and temporal lobes were examined by (13)C nuclear magnetic resonance spectroscopy, high pressure liquid chromatography, and light microscopy. In CRFC, significant increases in the levels of glutamate, glutathione, and taurine were seen, whereas amounts and turnover of noradrenaline, dopamine, and serotonin were unchanged. Glutamate and glutamine, derived from [1,2-(13)C]acetate and thus astrocytes, were significantly decreased in CRFC as compared to controls. Labeling from [1-(13)C]glucose and thus mostly neuronal metabolism was affected in the same brain region with decreased labeling of glutamate and GABA. The present model mimics the increased glutamate/glutamine activity found in drug-naive patients with first episode schizophrenia. Moreover, the decreased labeling indicates the transition to lower glutamatergic function seen in chronic schizophrenia patients. The disturbance in astrocytic function and the glutamine-glutamate-GABA cycle are of significant importance and might add to the malfunction of the cortico-striato-thalamo-cortical loop caused by NDMA receptor blockade.     

Modern anesthesia and peroperative monitoring methods reduce per- and postoperative mortality during transient occlusion of the middle cerebral artery in rats

Mortality and morbidity during and after occlusion of the middle cerebral artery in rats are important confounding factors which may be minimized by improved anesthesia and peroperative monitoring techniques. We describe state of the art techniques for inducing anesthesia, endotracheal intubation, ventilation and monitoring peroperatively in this context. Introducing the subtemporal approach of Tamura et al. in our laboratory 5 years ago, we experienced 25% peroperative and 24 h postoperative rat mortality when performing temporary clipping of the middle cerebral artery. This prompted us to abandon intraperitoneal anesthesia by chloral hydrate and ventilation by tracheotomy in favor of endotracheal intubation and isoflurane anesthesia (1% isoflurane in 30%:70% O(2)/N(2)O). These anesthetic techniques in combination with improved surgical skills have reduced our initial 25% peroperative- and 24 h postoperative mortality to 2.7% (1.8% peroperatively and 0.9% 24 h postoperatively). Furthermore, the following 14 days postoperative mortality rate was 1.8%. A total number of 203 rats have been operated with this method in different studies where a focal reperfusion stroke model combined with extended periods of observations were the cornerstone.