Superfusion Apparatus Research Articles

A superfusion apparatus for ex vivo human eye irritation investigations

A superfusion apparatus (SA) was developed to maintain isolated human corneas ex vivo under conditions which mimic the natural eye environment in vivo, including controlled temperature, tear flow and intraocular pressure. The SA was designed, developed and tested for use in ophthalmic pre-clinical research and to test new pharmaceutical formulations. Corneas undergo an equilibration process in the new physiological environment for one day. The test was then initiated by the application of the test substance, incubation, and temporal assessment of corneal damage using various parameters. The effects of mild and severe irritant concentrations of NaOH (2% and 8%, respectively) on corneal opacity, swelling and epithelial integrity were studied, and the inflammatory status assessed using F4/80 and MPO as macrophages and neutrophils markers, respectively. The SA was then used to test new artificial tear formulations supplemented with silver ions as an active constituent, showing different degrees of inflammatory responses as indicated by the migration of MPO and F4/80 positive cells towards the epithelium. The human cornea superfusion apparatus was proposed as a model for acute eye irritation research.

Endocrine, behavioral and autonomic effects of neuropeptide AF

The actions of neuropeptide AF (NPAF), on the hypothalamic-pituitary-adrenal (HPA) axis, behavior and autonomic functions were investigated. NPAF (0.25, 0.5, 1, 2 nmol) was administered intracerebroventricularly to rats, the behavior of which was monitored by means of telemetry, open-field (OF) observations and elevated plus-maze (EPM) tests. The temperature and heart rate were recorded by telemetry, and the plasma ACTH and corticosterone levels were used as indices of the HPA activation. The dopamine release from striatal and amygdala slices after peptide treatment (100 nM and 1 μM) was measured with a superfusion apparatus. To establish the transmission of the HPA response, animals were pretreated with the corticotrophin-releasing hormone (CRH) receptor antagonist antalarmin or astressin 2B (0.5 nmol). In the OF test, the animals were pretreated with antalarmin or haloperidol (10 μg/kg), while in the EPM test they were pretreated with antalarmin or diazepam (1 mg/kg). NPAF stimulated ACTH and corticosterone release, which was inhibited by antalarmin. It activated exploratory locomotion (square crossings and rearings) and grooming in OF observations, and decreased the entries to and the time spent in the open arms during the EPM tests. The antagonists inhibited the locomotor responses, and also attenuated grooming and the EPM responses. NPAF also increased spontaneous locomotion, and tended to decrease the core temperature and the heart rate in telemetry, while it augmented the dopamine release from striatal and amygdala slices. These results demonstrate, that acute administration of exogenous NPAF stimulates the HPA axis and behavioral paradigms through CRH and dopamine release.

Endocrine and behavioral effects of neuromedin S

The present experiments focused on the effects of neuromedin S on hypothalamic–pituitary–adrenal (HPA) activation and behavior. The peptide (0.25–1 nmol) was administered intracerebroventricularly to rats, the behavior of which was monitored by means of telemetry, open field observations and an elevated plus-maze (EPM) test. Autonomic functions such as the temperature and the heart rate were recorded by telemetry. The action on the HPA axis was assessed via measurements of the plasma corticosterone and ACTH levels. To reveal the transmission of the endocrine responses, animals were pretreated with corticotrophin releasing hormone receptor (CRHR) antagonists (1 nmol). In the open field test, the animals were pretreated with either a CRHR 1 antagonist (antalarmin) or haloperidol (10 μg/kg), while in the EPM test they were pretreated with antalarmin or diazepam (1 mg/kg). The dopamine release from striatal and amygdala slices after peptide treatment was measured with a superfusion apparatus. Neuromedin S exerted dose-dependent effects on the HPA system, which were inhibited by antalarmin. It also activated grooming and decreased the entries to and time spent in the open arms during the EPM test. The grooming response was abolished by haloperidol and antalarmin pretreatment, while diazepam and antalarmin showed a tendency to attenuate the response evoked in the EPM test. In the superfusion studies, neuromedin S enhanced the dopamine release from the amygdala slices. These results demonstrate that neuromedin S stimulates the HPA axis through the CRHR 1 pathway and evokes stereotyped behavior and anxiety through mesolimbic dopamine and corticotrophin releasing hormone release.

Muscarinic receptor subtypes modulate the release of [ 3H]-noradrenaline in rat spinal cord slices

Spinal muscarinic receptors are involved in the mediation of antinociceptive effects. The modulation of noradrenaline (NA) release on muscarinic receptor subtypes in the rat spinal cord was investigated in in vitro perfusion experiments. After rat spinal cord slices were preincubated in [ 3H]NA, the slices were perfused with a superfusion apparatus. The slices were field stimulated during the 4th (S 1) and 11th (S 2) superfusion collection periods. Perfusion of drugs was initiated at the 8th collection period and was maintained until the 14th collection period. Fractional release was calculated as the percentage of the radioactivity present in the slices at the beginning of the stimulation period. Drugs were administered between S 1 and S 2. The following drugs were used: [ 3H]NA, neostigmine, pirenzepine (M 1 antagonist), AFDX116 (M 2 antagonist), atropine. Neostigmine significantly increased the release of [ 3H]NA in a concentration-dependent manner. Pirenzepine (1 μM) and atropine (0.3 μM) significantly reduced the release of [ 3H]NA, but AFDX116 (1 μM) did not significantly reduce release in the presence of neostigmine (1 μM). The results of this study indicate that neostigmine can enhance noradrenergic neurotransmission, and that acetylcholine can stimulate spinal cord NA release via M 1 muscarinic receptor subtypes.

Hydrogen ion concentration differentiates effects of methamphetamine and dopamine on transporter‐mediated efflux

Methamphetamine (METH) causes release of stored intracellular dopamine (DA). We explored the interactions of METH with the recombinant human vesicular monoamine (hVMAT2) and/or human DA transporters (hDAT) in transfected mammalian (HEK293) cells and compared the findings with those for DA. In 'static' release assays at 37 degrees C, less than 20% of pre-loaded [(3)H]DA was lost after 60 min, while nearly 80% of pre-loaded [(3)H]METH was lost at 37 degrees C under non-stimulated conditions. Results obtained by measuring substrate release using a superfusion apparatus revealed an even greater difference in substrate efflux. At pH 7.4, nearly all of the pre-loaded [(3)H]METH was lost after just 6 min, compared with the loss of 70-80% of pre-loaded [(3)H]DA (depending on cell type) after superfusion for 32 min. Increasing the extracellular pH from 7.4 to 8.6 had opposite effects on [(3)H]DA and [(3)H]METH retention. At pH 8.6, [(3)H]METH was retained more effectively by both hDAT and hDAT-hVMAT2 cells, compared with results obtained at extracellular pH 7.4. [(3)H]DA, however, was more effectively retained at pH 7.4 than at pH 8.6. These data suggest that DA and METH interact differently with the DAT and VMAT2, and require different H(+) concentrations to exert their effects.

Serotonin may alter the pattern of gonadotropin-induced progesterone release of human granulosa cells in superfusion system.

Serotonin plays a hormonal function in several nonneuronal peripheral tissues, such as the ovaries. Our aim was to investigate whether there is a modulatory action of serotonin on gonadotropin-induced steroid secretion of human granulosa cells. In granulosa cell culture, serotonin was administered alone or in combination with luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Also, granulosa cells were transferred into a dynamic superfusion apparatus and challenged by FSH and LH alone or along with serotonin. Estradiol and progesterone concentrations of samples were measured by radioimmunoassay. As expected, administration of FSH, LH, and serotonin alone resulted in a significant estradiol and progesterone release in cell culture, as well as a significant increase in progesterone release in dynamic superfusion system. In cell culture, co-administration of serotonin with gonadotropins had no additive effect on gonadotropin-induced secretion of progesterone, while it further augmented that of estradiol. In superfusion system, when gonadotropins were added along with serotonin, the increase in progesterone release was markedly less, while peaks of hormone response were remarkably prolonged compared to challenges by LH and FSH alone. The observed effects of serotonin on gonadotropin-induced steroid release of granulosa cells may reveal further details about the regulation of granulosa cell function.

Glucose-deprivation-induced [3H]D-aspartate release from isolated bovine and human retinae.

The glucose deprivation-induced release of [3H]D-aspartate was studied in bovine and human retinas in a superfusion apparatus. [3H]D-aspartate release was significantly increased upon omitting glucose in the superfusion buffer. This effect was dependent on external Ca2+ because L- and N-type Ca2+-channel blockers, such as diltiazem (1 microM), nitrendipine (1 microM), and omega-conotoxin (100 nM), significantly reduced the effect of glucose-deprivation induced release of [3H]D-aspartate. Furthermore, while glutamate receptor agonists (L-glutamate, N-methyl-D-aspartate, but not kainate) potentiated the effects of glucose deprivation, antagonists (MK-801, MCPG, ifenprodil, and L-AP3) at these receptors blocked the glucose deprivation-induced release process. Taken together, these studies have demonstrated that under conditions of glucose deprivation, as may happen during ischemic events in vivo, the retinal glutamatergic nerve endings and/or glial cells promote the efflux of [3H]D-aspartate into the extracellular environment. This process appears to be receptor-mediated and dependent on extracellular Ca2+ and is similar to previous reports pertaining to brain tissues.

The effect of catecholamines, acetylcholine and histamine on progesterone release by human granulosa cells in a granulosa cell superfusion system

There are experimental data demonstrating the presence and actions of various neurotransmitters in the ovary, thus supporting the view that they might play a role in intraovarian regulatory mechanisms,although their exact function in the regulation of ovarian hormone secretion is unclear. The objective of the present study was to investigate the direct action of catecholamines, acetylcholine and histamineon progesterone secretion of human granulosa cells in a superfused cell system. Human granulosa cells were isolated from preovulatory follicular fluid using a Percoll gradient centrifugation method. Approximately2 × 106 cells were mixed with Sephadex G-10 and were transferred into two chambers of the superfusion apparatus. The system was perfused with a culture medium and test materials were addedto the system at a dose of 100 pmol/ml. The progesterone concentration of samples was measured using an 125I radioimmunoassay. Administration of epinephrine (adrenaline), norepinephrine (noradrenaline),dopamine and histamine had no effect on progesterone release. However, acetylcholine produced a significant progesterone release, which could be blocked by atropine. The observed effect of acetylcholineon progesterone release of superfused human granulosa cells may reflect a physiological role of acetylcholine in the regulation of granulosa cell function during the menstrual cycle.

CLA reduces antigen-induced histamine and PGE2release from sensitized guinea pig tracheae

Conjugated linoleic acid (CLA) has been shown to enhance immune reactions such as lymphocyte blastogenesis and delayed-type hypersensitivity. We investigated the role of CLA in type I (immediate) hypersensitivity, using a guinea pig tracheal superfusion model for measuring antigen-induced airway smooth muscle contraction and inflammatory mediator release. Female Hartley guinea pigs were fed a diet supplemented with 0.25 g corn oil or linoleic acid/100 g of diet (control) or 0.25 g CLA/100 g of diet for at least 1 wk before and during active sensitization to ovalbumin antigen. Tracheae from sensitized guinea pigs were suspended in air-filled water-jacketed (37 degrees C) tissue chambers in a superfusion apparatus. Tracheae were superfused with buffer containing antigen, and tissue contraction was recorded. Superfusate was collected at 90-s intervals for evaluation of histamine and PGE(2) release. CLA did not affect antigen-induced tracheal contractions when expressed as gram contraction per gram tissue. CLA significantly reduced antigen-induced histamine and PGE(2) release. CLA appears to decrease release of some inflammatory mediators during type I hypersensitivity reactions.

Long-term role of nitric oxide in the enteric nervous system of the transplanted rat intestine.

We investigated the long-term changes of the nitric oxide (NO)-related neural component after syngeneic total small bowel transplantation in rats. In the present study, the NO-related neural component was examined using the electrophysiological and NADPH-diaphorase histochemical technique. The rats were divided into four groups: an untreated young adult control group, an untreated 2-year-old control group, a group killed 1 month after transplantation, and a group killed 2 years after transplantation. A superfusion apparatus was used to evaluate the response of jejunal strips to electrical transmural stimulation. In the presence of adrenergic and cholinergic blockade, the inhibitory effect of L-N(G)-nitro arginine (L-NNA; a nitric oxide synthesis inhibitor) on nonadrenergic, noncholinergic (NANC) relaxation was expressed as a L-NNA-sensitive component. The L-NNA-sensitive component accounted for 41.6 +/- 4.6% (mean +/- SE), 43.1 +/- 3.5%,54.6 +/- 4.1%, and 55.8 +/- 3.5% in the young control group, 2-year control group, 1-month transplant group, and 2-year transplant group, respectively, being significantly higher in the transplant groups (p < 0.05). The actual strength of the L-NNA-sensitive component was 0.24 +/- 0.03 (mean +/- SE), 0.26 +/- 0.02, 0.44 +/- 0.04, and 0.46 +/- 0.04 mg of tension per mg of wet weight, respectively, also being significantly higher in the transplant groups (p < 0.001). In addition, the percentage of NADPH-diaphorase-positive fibers was 24.1 +/- 1.1% (mean +/- SE), 25.5 +/- 1.4%, 31.0 +/- 1.6%, and 30.9 +/- 2.0%, respectively, being significantly higher in the transplant groups (p < 0.05). These results suggest that NO neurons in the intrinsic jejunal nervous system have an adaptive role in maintaining intestinal graft motility.

ATP is released from rabbit urinary bladder epithelial cells by hydrostatic pressure changes--a possible sensory mechanism?

1. The responses of rabbit urinary bladder to hydrostatic pressure changes and to electrical stimulation have been investigated using both the Ussing chamber and a superfusion apparatus. These experiments enabled us to monitor changes in both ionic transport across the tissue and cellular ATP release from it. 2. The urinary bladder of the rabbit maintains an electrical potential difference across its wall as a result largely of active sodium transport from the urinary (mucosal) to the serosal surface. 3. Small hydrostatic pressure differences produced by removal of bathing fluid from one side of the tissue caused reproducible changes in both potential difference and short-circuit current. The magnitude of these changes increases as the volume of fluid removed increases. 3. Amiloride on the mucosal (urinary), but not the serosal, surface of the membrane reduces the transepithelial potential difference and short-circuit current with an IC50 of 300 nM. Amiloride reduces the size of, but does not abolish, transepithelial potential changes caused by alterations in hydrostatic pressure. 4. Field electrical stimulation of strips of bladder tissue produces a reproducible release of ATP. Such release was demonstrated to occur largely from urothelial cells and is apparently non-vesicular as it increases in the absence of calcium and is not abolished by tetrodotoxin. 5. It is proposed that ATP is released from the urothelium as a sensory mediator for the degree of distension of the rabbit urinary bladder and other sensory modalities.

Human Anti-IgE Monoclonal Antibody Blocks Passive Sensitization of Human and Rhesus Monkey Bladder

IgE antibodies may play an important role in noninfectious urinary inflammation, including interstitial cystitis (IC). In this study, urinary bladder strips were passively sensitized for 20 hours with serum from a ragweed-sensitive patient in the absence or presence of an anti-human IgE monoclonal antibody (MaE11) at 0, 1-, or 5-fold IgE concentration. The urinary bladder strips then were suspended in a superfusion apparatus for measurement of contraction and histamine release in response to antigen E (AgE) challenge. Non-sensitized tissues did not react to AgE challenge, whereas AgE challenge of passively-sensitized tissues resulted in time-dependent bladder contraction and histamine release. MaE11 abolished AgE-induced contraction and histamine release in a concentration-dependent manner. The safety of MaE11 was confirmed by its failure to contract or release histamine from passively-sensitized bladder tissues. The results of this study suggest that MaE11 may have immunotherapeutic benefit for amelioration of IgE-mediated diseases of the urinary system.

Possible involvement of α 1-adrenoceptors in the modulation of [ 3H]noradrenaline release in rat brain cortical and hippocampal synaptosomes

Nerve terminals obtained from rat brain cortex and hippocampus, were labelled with 0.04 μM of [ 3H]noradrenaline ([ 3H]NA). Thereafter the basal release of [ 3H]NA was measured in a Brandel superfusion apparatus, in the presence of α 1-adrenoceptor agonists (phenylephrine or noradrenaline) or there α 1-adrenoceptor agonists along with prazosin, an α 1-adrenoceptor antagonists, In cortical synaptosomes both α 1-adrenoceptor agonists increased the basal release of [ 3H]NA in a concentration-dependent manner (EC 50=0.15 μM for phenylephrine and 12.6 μM for noradrenaline). Effects were reversed by 0.01 μM prazosin (EC 50 = 2.46 and 130.1 μM, respectively). In synaptosomes from rat brain hippocampus, phenylephrine (EC 50 = 1.28 μM) and noradrenaline (EC 50 = 33.7 μM) also increased the [ 3H]NA release and prazosin (0.01 μM) shifted the corresponding concentration-response curves to the right (EC 50 = 7.38 and 264.0 μM, respectively). Events produced by noradrenaline acting as α 1-adrenoceptor agonist did not show Ca 2+ dependence. These results suggest (1) the presence of functional α 1-adrenoceptors in nerve terminals from rat brain cortex and hippocampus, (2) that these receptors seem to play a role in the presynaptic modulation of [ 3H]NA release, and (3) that intraterminal Ca 2+ may be involved.

Nitric oxide-related neural components in the rat small intestine after transplantation.

The changes in nitric oxide (NO)-related neural components in the transplanted small intestine are unknown. In this study, the NO neural component was examined using electrophysiological and NADPH-diaphorase histochemical techniques in a rat small bowel transplantation model. Syngeneic total small bowel transplantation was performed in 26 male Lewis rats using microsurgical techniques. The rats were divided into four groups: an untreated control group and animals at 1 (G1), 2 (G2), and 4 (G4) weeks after transplantation. Jejunal strips were mounted in a superfusion apparatus for examination. In the presence of atropine and guanethidine, the effect of the NO synthesis inhibitor L-NG-nitro-arginine (L-NNA, 100 microM) relaxation mediated by the nonadrenergic, noncholinergic (NANC) neural system was assessed following electrical stimulation at 2Hz. The inhibitory effect of L-NNA on relaxation was taken as an indicator of NO production. The percentage of inhibition in the control group, and in G1, G2, and G4 was 43.30% +/- 6.08% (mean +/- SE), 42.10% +/- 6.69%, 43.62 +/- 10.00%, and 52.46% +/- 6.00%, respectively. Inhibition in G4 was significantly higher than in the other groups (P < 0.01). The percentage of NADPH-diaphorase-positive fibers in the control group, G1, G2, and G4 was 25.06% +/- 4.70% (mean +/- SE), 26.27% +/- 2.17%, 24.73% +/- 2.87%, and 30.76% +/- 3.19%, respectively. Again, G4 showed a significantly higher level than the other groups (P < 0.01). We conclude that increased NO production may play a significant role in maintaining the intrinsic nervous system of the small intestine after transplantation.

Human FcERI-IgG and humanized anti-IgE monoclonal antibody MaE11 block passive sensitization of human and rhesus monkey lung

IgE antibodies are thought to play an important role in the induction of allergic inflammation of the bronchi. In this study we assessed the capacity of two inhibitors, FcERI-IgG, an immunoadhesin made up of the a chain of the high-affinity IgE receptor joined to a truncated IgG heavy chain, and MaE11, a humanized murine anti-human IgE antibody, to prevent allergen sensitization. Lung parenchyma strips from rhesus monkeys and human beings were passively sensitized for 20 hours with serum from a ragweed-sensitive patient in the presence of 0, 1-, 5-, or 10-fold concentrations of the inhibitors relative to IgE. The parenchymal strips were then suspended in a superfusion apparatus for measurement of isometric tone and collection of superfusate for histamine analysis in response to challenge with antigen E (AgE). Nonsensitized tissues did not react to AgE challenge, whereas AgE challenge of passively sensitized tissues resulted in a time-dependent parenchymal contraction and histamine release. Both FcERI-IgG and MaE11 completely abolished the AgE-induced contraction and histamine release in a dose-dependent manner. In addition, passively sensitized lung tissues failed to respond to direct challenge with either FcERI-IgG or MaE11. The results of this study suggest that FcERI-IgG and MaE11 may have important immunotherapeutic benefit for the amelioration of IgE-mediated diseases.

Changes of intestinal motility after small bowel transplantation in the rat.

Small bowel transplantation disturbs the enteric neural network as a result of complete extrinsic denervation, but few data are available on the status of the intrinsic neurons in intestinal grafts. This study assessed intestinal motility and the intrinsic innervation of jejunal longitudinal muscle after transplantation in rats using electrical transmural stimulation. Syngeneic total small bowel transplantation was performed in male Lewis rats using microsurgical techniques. The rats were divided into four groups-i.e., a control group and groups examined at one (G1), two (G2), and four (G4) weeks after transplantation. Jejunal strips were mounted in a superfusion apparatus for examination. Functional innervation was assessed by comparing contraction and relaxation following stimulation at 10 Hz with or without various drugs. All grafts showed increased contractile motility when compared with the control group. The cholinergic excitatory components in the control, G1, G2, and G4 groups were 45%, 73%, 66%, and 24%, respectively, while nonadrenergic noncholinergic-excitatory components were 56%, 53%, 57%, and 78%. Thus, there was a marked change in excitatory innervation after transplantation. However, the tetrodotoxin-insensitive (myogenic) component showed no changes in active tone after transplantation. Adrenoceptor antagonists had a decreased effect on all grafts except for yohimbine (an alpha 2 blocker). The contractile motility of transplanted jejunum was increased compared with that of the control bowel. However, the dominant intrinsic component varied with the time after transplantation.

Inhibition in the human urinary bladder by gamma‐amino‐butyric acid

To investigate the effects of gamma-amino-butyric acid (GABA) on detrusor activity in man to determine whether it has any inhibitory effect on detrusor contraction. The inhibitory neurotransmitter GABA has been found in mammalian urinary bladders and the effects of GABA on detrusor activity in the rabbit bladder has previously been described [1]. Human detrusor muscle strips, obtained at cystectomy, were made to contract by electrical stimulation of their autonomic nerves or by the addition of carbachol in a superfusion apparatus. GABA and its analogues were added to the superfusion chamber and any changes in the responses were measured. The electrically evoked nerve-mediated contractions in human bladder muscle were exclusively cholinergic. GABA inhibited nerve-mediated contractions in human detrusor muscle-strips by the activation of the GABAB receptor, since baclofen (a GABAB receptor agonist) produced similar inhibition and muscimol (a GABAA receptor agonist) did not. There was no inhibition of carbachol-mediated contractions by GABA. This in vitro study shows that GABA has a peripherally mediated inhibitory effect on excitatory neurotransmission in human detrusor muscle. The site of action is on the post-ganglionic nerves and appears to be mediated via the GABAB receptor.

Screening of cattle urine samples for the presence of beta-agonists with a functional test: some preliminary results.

A new procedure based on the measurement of biological effects has been developed for the determination of residues of beta-agonists, such as clenbuterol, in urine. A multi-chamber superfusion apparatus containing isolated trachea strips from guinea pigs was used to detect smooth muscle relaxation induced via beta 2-adrenoceptor activation. The trachea tissue was pre-contracted with metacholine. Urine samples were extracted using a solid-phase column containing reversed-phase and anion-exchange materials. Extracts were introduced into the superfusion apparatus via flow injection. The intensity and response of relaxation are dependent on the type and concentration of the beta-agonist introduced. The sensitivity of the assay for clenbuterol in calf urine is about 1 microgram l-1. This methodology in the present form is especially suitable for survey screening analysis for several types of samples. An extensive validation of the procedure is performed to determine the range of analytes that can be detected, the possibilities of analysing urine samples obtained from mature cattle or other animal species and the influence of cross-reacting substances.

Opioid agonists modulate excitatory and inhibitory neurotransmission in human colon

AbstractThe effects of different opioid agonists on spontaneous mechanical activity and response to electrical transmural nerve stimulation of both longitudinal and circular muscle strips from the human colon were studied by using a superfusion apparatus to record isometric contractions. Exogenously added opioid agonists did not modify the spontaneous contractile activities of both types of strips. Nerve stimulation induced a triphasic response composed of a first contraction C1 followed by a relaxation C2 and an off‐contraction C3; this response was mediated by cholinergic excitatory nerves and non‐adrenergic, non‐cholinergic (NANC) excitatory and inhibitory nerves. The delta‐agonists methionine enkephalin, [D‐Pen2, D‐Pen5] enkephalin (DPDPE) and the kappa‐agonists dynorphin, trans‐3,4 dichloro‐N‐methyl‐N‐(2‐[1 pyrolidinyl]‐cyclohexyl) (U‐50488H) decreased the amplitudes of the contractions C1 and C3 of both strips in a dose‐dependent manner. The selective mu‐agonist D‐alaglymepheglyol (DAGO) decreased the contraction C1 of longitudinal and circular muscle at high dose (1 μM) and often reduced the relaxation C2 of both types of strips at low dose (0.05 μM), finally, naloxone, but only at higher concentrations (1 μM) decreased the C1 amplitude significantly in circular muscle. In conclusion, these data suggest that mu, delta and kappa opioid receptors are involved in the neuro‐regulation of smooth muscle of human colon and that opioid agonists modulate both excitatory and inhibitory neurotransmission through an action on cholinergic and non‐adrenergic, non‐cholinergic neurons.

Substance P excites neurons in the gustatory zone of the rat nucleus tractus solitarius

Whole-cell patch recordings of neurons in the rostal (gustatory) nucleus tractus solitarius (rNTS) were performed in a brain slice preparation from rat medulla. Neural responses to brief applications (10–45 s) of substance P (SP), via a constant superfusion apparatus, were recorded. SP transiently depolarized 80 of 117 (68%) rNTS neurons in a dose-dependent manner. Sub-micromolar concentrations of SP had potent excitatory effects, and the half maximal response occurred at 0.6 μM. The depolarizing effect of SP was accompanied by an increase in input resistance in 81% of the responsive neurons. The excitatory effects of SP persisted in low Ca 2+ (0.2 mM) and high Mg 2+ (12 mM) saline as well as in the presence of 2 μM TTX ( n = 5 for each), suggesting direct postsynaptic action on the recorded neurons. SP also hyperpolarized 4 neurons (4%) and had no effect on 33 neurons (28%). Each of the 4 neurons which were hyperpolarized by SP showed a decrease in input resistance. A more detailed assessment of the types of neurons in the rNTS which respond to SP was also conducted. Neurons were separated into 4 electrophysiological groups on the basis of their repetitive firing pattern induced by a hyperpolarizing and depolarizing current injection paradigm. Neurons belonging to each of the 4 electrophysiological groups responded to SP. Eighteen neurons, which were filled with 1% biocytin during recording, were categorized as ovoid, multipolar or fusiform based on their morphological characteristics. SP excited all 3 morphological types of neurons in similar proportion. These results suggest that SP is an excitatory neurotransmitter in the rNTS. The effects of SP are not restricted to a particular neuron type defined either biophysically or morphologically. The implications of these results on the possible role of SP in processing gustatory and somatosensory information within the rNTS are discussed.