Postsynaptic 5-HT1A receptors mediate 5-hydroxytryptamine release in the amygdala through a feedback to the caudal linear raphe.

Using brain microdialysis, it was demonstrated that the release of 5-hydroxytryptamine (5-HT) in the central nucleus of the amygdala is under inhibitory control of somatodendritic and postsynaptic 5-HT1A receptors. Systemic administration of flesinoxan, a selective 5-HT1A receptor agonist, significantly reduced the extracellular levels of 5-HT in the central nucleus of the amygdala. This effect could be completely antagonized by the 5-HT1A receptor antagonist N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)-N-(2-pyridyl)cyclohexane carboxamine trihydrochloride (WAY 100635). Local administration of these compounds by reversed microdialysis into the raphe nuclei revealed that extracellular 5-HT levels in the central nucleus of the amygdala can be regulated through 5-HT1A receptors in the caudal linear raphe nucleus, but not in the dorsal and median raphe nuclei. Interestingly, administration of flesinoxan into the central nucleus of the amygdala also decreased dialysate 5-HT levels both locally and in the caudal linear raphe nucleus. The former effect could be blocked by pretreatment with WAY 100635 when applied into the central nucleus of the amygdala, but not when applied into the caudal linear raphe nucleus. These data provide circumstantial evidence for the existence of a 5-HT1A receptor mediated feedback loop from the central nucleus of the amygdala to the caudal linear raphe nucleus.

The effects of a 5-HT1A receptor agonist and antagonist on the 5-hydroxytryptamine release in the central nucleus of the amygdala: a microdialysis study with flesinoxan and WAY 100635.

The modulation of extracellular 5-hydroxytryptamine (5-HT) in the central nucleus of the amygdala (CeA) by 5-HT1A receptors was studied by intracerebral microdialysis in awake and freely moving rats. Local administration of 1 microM tetrodotoxin (TTX), 60 mM K+ and perfusion with Ca(2+)-free Ringer containing EGTA confirmed that the major part of dialysate 5-HT levels from the CeA is of neuronal origin. Administration of 300 nM of RU 24969, a 5-HT1B receptor agonist, through the probe into the CeA decreased dialysate 5-HT levels to 67.2% of the baseline value. Systemic administration of the 5-HT1A receptor agonists 8-OH-DPAT and flesinoxan dose-dependently decreased 5-HT levels in the CeA. The effect of 0.3 mg/kg of flesinoxan could be completely antagonized by systemic administration of 0.05 mg/kg WAY 100635, a 5-HT1A receptor antagonist. WAY 100635 alone had only minimal effects at this dose. These data show that a major part of the extracellular 5-HT in the CeA stems from 5-HT neurons and that the amount of 5-HT released into this brain region can be modulated by 5-HT1A receptors.

Flesinoxan dose-dependently reduces extracellular 5-hydroxytryptamine (5-HT) in rat median raphe and dorsal hippocampus through activation of 5-HT1A receptors.

The effects of systemic administration of the serotonin (5-hydroxytryptamine) 5-HT1A receptor agonists flesinoxan and 8-hydroxy-2-(di-n-propylamino)tetralin on extracellular 5-HT were measured using microdialysis probes in both median raphe nucleus and dorsal hippocampus. Both 5-HT1A agonists dose-dependently decreased dialysate 5-HT levels from both brain regions. The effects of flesinoxan in the median raphe (0.3 mg/kg) and dorsal hippocampus (1.0 mg/kg) could be blocked by the 5-HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)cyclohexane carboxamide trihydrochloride (WAY 100,635) at a dose of 0.05 mg/kg s.c. The antagonist itself had no effect at this dosage. Local perfusion of flesinoxan for 30 min through the dialysis probe into the median raphe region at concentrations of 20, 100, and 1,000 nM resulted in a significant decrease in dialysate 5-HT content from both regions. The effect of 100 nM flesinoxan could be blocked by coperfusion of 1,000 nM WAY 100,635. The data indicate that flesinoxan is a potent 5-HT1A receptor agonist and also support the notion that somatodendritic 5-HT1A autoreceptors regulate both terminal and somatodendritic 5-HT release.

Effects of single and repeated oral administration of fluvoxamine on extracellular serotonin in the median raphe nucleus and dorsal hippocampus of the rat.

The delay in clinical effects of selective serotonin reuptake inhibitors (SSRIs) suggest the existence of adaptive phenomena, such as receptor sensitivity changes. To examine the effects of repeated administration of SSRIs on serotonin neurotransmission, we investigated the effects of acute and chronic administration of the SSRI fluvoxamine on the extracellular levels of 5-HT in the median raphe nucleus and dorsal hippocampus of conscious rats by means of brain microdialysis. A single oral dose of fluvoxamine (30 mg/kg) augmented extracellular 5-HT in the median raphe and dorsal hippocampus to 270 and 191% of baseline level, respectively. Administration of fluvoxamine (30 mg/kg) or vehicle for 14 days did not affect 5-HT baseline levels. Moreover, the increase in extracellular 5-HT in the median raphe nucleus and dorsal hippocampus after an oral dose of fluvoxamine (30 mg/kg) in rats chronically treated with fluvoxamine was not different from rats treated with vehicle. Using RU 24969 as a probe for the sensitivity of the 5-HT1B autoreceptors in the dorsal hippocampus, no change in receptor sensitivity could be observed. These results demonstrate that repeated oral treatment with fluvoxamine does not affect extracellular 5-HT in the median raphe and dorsal hippocampus, suggesting that presynaptic functional changes of 5-HT in the brain areas tested are not implicated in the observed delayed onset of action of this SSRI in humans.

Chronic treatment with fluvoxamine by osmotic minipumps fails to induce persistent functional changes in central 5-HT1A and 5-HT1B receptors, as measured by in vivo microdialysis in dorsal hippocampus of conscious rats.

This study investigated the alterations of the 5-HT1A and 5-HT1B autoreceptor function following chronic treatment with fluvoxamine using osmotic minipumps. The 5-HT1A and 5-HT1B autoreceptor function were studied using microdialysis in the dorsal hippocampus. The effect of the 5-HT1A receptor agonist 8-OH-DPAT (0.3 mg/kg, SC) and the 5-HT1B receptor agonist RU-24969 (100 nM through the dialysis probe for 30 min) on 5-HT release was compared with rats chronically treated with saline. 8-OH-DPAT decreased 5-HT release to 55% and 60% of baseline, while RU-24969 decreased 5-HT release to 66% and 70% of baseline value in the saline and fluvoxamine group, respectively. In both cases, differences between the saline and fluvoxamine groups were not statistically significant. Plasma levels of fluvoxamine after 21 days of treatment ranged from 3 to 5 ng/ml. Fluvoxamine concentration in rat brain during treatment was estimated between 100 and 200 nM, which approximates to the IC50 value of fluvoxamine on the 5-HT transporter in synaptosomes and is 50 times higher than the Kd value for the 5-HT reuptake site. In conclusion, no evidence was found for changes in 5-HT1A,B receptor function using 8-OH-DPAT and RU-24969 as probes after continuous treatment with fluvoxamine by means of osmotic minipumps.

Extracellular 5-hydroxytryptamine in median raphe nucleus of the conscious rat is decreased by nanomolar concentrations of 8-hydroxy-2-(di-n-propylamino) tetralin and is sensitive to tetrodotoxin.

Extracellular 5-hydroxytryptamine (5-HT) in the median raphe and dorsal hippocampus was measured using in vivo microdialysis. Administration of 60 mM K+ through the probe into the median raphe region significantly increased 5-HT output from the median raphe and the right dorsal hippocampus. Local infusion of 10 microM tetrodotoxin into the median raphe region substantially decreased 5-HT in the median raphe and left and right dorsal hippocampus. Systemic administration (0.3 mg/kg s.c.) of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) decreased the 5-HT levels in the dialysates from both the median raphe region and dorsal hippocampus. Administration of 30 nM 8-OH-DPAT through the dialysis probe into the median raphe region decreased 5-HT output from the median raphe and dorsal hippocampus significantly, whereas at concentrations from 60 nM to 10 microM, no significant effects were found in either region. With 100 microM 8-OH-DPAT, a significant increase was seen in the median raphe region, but not in dorsal hippocampus. Similar findings were obtained following microinjections of different doses of the compound into the median raphe region. The results of this study indicate that the somatodendritic release of 5-HT is impulse flow-dependent. Moreover, the decrease of 5-HT in the median raphe region by low nanomolar concentrations of 8-OH-DPAT supports the notion that somatodendritic 5-HT release is subject to a local negative feedback mechanism through 5-HT1A autoreceptors.

5-Hydroxytryptamine release in dorsal hippocampus of freely moving rats: modulation by pindolol.

1. The aim of the present study was to investigate the origin and modulation of 5-hydroxytryptamine release in dorsal hippocampus of freely moving rats using brain microdialysis. 2. Basal release of 5-hydroxytryptamine and 5-hydroxyindole-acetic acid was 3.0 +/- 0.2 pg/15 min and 519 +/- 18 pg/15 min, respectively. Stimulation by 60 mM K+ increased 5-hydroxytryptamine release by 66%. Inclusion of 10 microM tetrodotoxin in the perfusion medium reduced 5-hydroxytryptamine release to approximately 5% of basal levels. 3. In addition, release could be modulated by the 5-hydroxytryptamine receptor agonist 8-hydroxy-2-(di-N-propylamino)-tetralin (50 % decrease) and this effect was completely blocked by the 5-hydroxytryptamine-1 receptor antagonist pindolol. 4. These data are in concordance with results from the ventral hippocampus and indicate that release of 5-hydroxytryptamine is predominantly of neuronal origin. In contrast with other studies, the authors found the non-selective 5-hydroxytryptamine-1 antagonist, pindolol, to increase 5-hydroxytryptamine release by 65% following systemic administration. This may hint at a different autoreceptor control in serotonergic fibers ascending from the median raphe as compared to the dorsal raphe nucleus.

Behavioral biochemical and neuroendocrine concomitants of lactate-induced panic anxiety.

In a single-blind study using sodium lactate infusions to provoke panic attacks, 11 of 15 patients with panic disorder panicked with lactate. None of the 15 control subjects panicked during lactate administration. Before receiving lactate, higher preinfusion anxiety levels were present in the patient group as compared to controls. Preinfusion Acute Panic Inventory (API) scores were significantly higher in patients who panicked compared to nonpanicking patients. In addition, patients who panicked during lactate infusion showed a higher mean plasma MHPG level at baseline. During lactate infusion, however, no increase in plasma MHPG was seen in patients who panicked, nor in nonpanickers and controls. Several other biochemical and hormonal variables were measured. No single biochemical or neuroendocrine variable was found to correlate with lactate-induced panic attacks. It is argued that the baseline arousal level of patients with panic disorder may be increased, which renders these patients more vulnerable to panic attacks.

Healing of experimental colonic anastomoses: effect of antineoplastic agents.

The effect of a combination of antineoplastic agents (bleomycin (2 mg/kg/d), 5-fluorouracil (10 mg/kg/d) and cisdiamminedichloroplatinum (0.35 mg/kg/d)), given intravenously, on the healing of colonic anastomoses was investigated in rats. Ninety-six male Wistar rats were divided into four groups. A control group of rats underwent surgery only. The remaining rats were operated either 2 days before, 2 days after or during the 5-day chemotherapy course. In all groups rats were killed after 3, 7 and 21 days. Wound healing of the anastomosis was assessed by measuring the bursting pressures and hydroxyproline contents. Operating in the middle of the 5-day chemotherapy course had the greatest effect on anastomotic healing. This was most apparent on the seventh day postoperatively. When surgery was performed on the second day after the chemotherapy course there was an early delay in wound healing as measured by hydroxyproline content. Seven days postoperatively concentrations were in the same range as those in the control group. Chemotherapy commencing 2 days after surgery did not affect anastomotic healing as compared to the control group. Bursting pressure and hydroxyproline content at the end of the experiment were more or less comparable in all groups.

The effect of antineoplastic agents on the healing of small intestinal anastomoses in the rat.

The influence of an intravenous 5-day combined chemotherapy with bleomycin (2 mg/kg/d), 5-fluorouracil (10 mg/kg/d) and cis-diamminedichloroplatinum (0.35 mg/kg/d) on the healing of ileal anastomoses was investigated in rats. Ninety-six male Wistar rats were used, divided into four groups. The rats in the control group had surgery without administration of cytostatic agents. The other rats were operated either 2 days after, 2 days before, or during the 5-day chemotherapy course. In each group, rats were killed after 3, 7, and 21 days. Anastomotic healing was assessed by measurement of bursting pressures and hydroxyproline levels. Intestinal healing appeared to be impaired most if the operation was performed in the middle of the antineoplastic chemotherapy course. The effects were most pronounced on the seventh postoperative day. Surgery on the second day after the chemotherapy course led to a slight and early delay in wound healing as measured by the hydroxyproline content. Seven days postoperatively, concentrations had returned to preoperative values. Surgery 2 days before chemotherapy induced only minor differences with respect to the control group. In all groups, bursting pressure and hydroxyproline content at 21 days were similar. Thus, antineoplastic agents retard but do not prevent healing of intestinal anastomoses. The effects are most pronounced when surgery is performed during chemotherapy. If possible, surgery should be performed prior to chemotherapy. Increasing the time interval between surgery and chemotherapy may decrease the delay in intestinal woundhealing.

A spectrophotometric microassay for sulfated glycosaminoglycans using a laser densitometer.

The absorption spectrum of the dye 1,9-dimethylmethylene blue shifts if complexed with sulfated glycosaminoglycans. The present method uses the decrease in A633 rather than the increase in A535, described in a recent method, to measure the sulfated glycosaminoglycan content of biological samples. A conventional spectrophotometer was used to estimate the levels of sulfated glycosaminoglycan in papain extracts from intestinal wall tissue, by measuring both the A535 and the A633 and comparing them with a chondroitin sulfate standard: a highly significant correlation (r = 0.974, n = 17) was obtained. Also, interference by substances like RNA, DNA, and hyaluronic acid was similar for both methods. These results allowed us to employ a laser densitometer with a helium/neon laser emitting at 633 nm to improve the sensitivity and the capacity of the assay. The combination of a small reaction volume and a high-intensity light source allows the detection of less than 0.1 microgram chondroitin sulfate, a 40-fold improvement in sensitivity as compared with the original method. A very significant correlation (r = 0.885, n = 17) existed between results obtained with the macroassay, using a spectrophotometer, and those found by employing the microassay, using the laser densitometer. The use of microtiter plates and the screening potential of the densitometer yields an assay which is fast, very sensitive, and suitable for processing large numbers of samples.

Solubility of tissue hydroxyproline in experimental intestinal anastomoses.

Salt and acid solubility of collagen are thought to reflect its degree of crosslinking. In order to examine postoperative changes in crosslinking of intestinal collagen, which are of importance to the stability of the intestinal wall, we have investigated the solubility of hydroxyproline in rabbit ileum and colon, both in unwounded intestine and after construction of an anastomosis. Solubility in salt and dilute acid was increased by a sonication procedure. This way, 9% of total hydroxyproline in the unwounded colonic wall was salt soluble and 65% acid soluble. A similar distribution was observed in ileum. Three days after operation the salt-soluble fraction was significantly elevated in samples from the anastomotic area. In colon, this increase also persisted 7 days postoperatively. Comparison of anastomotic samples collected 3 and 7 days after surgery shows a significant decrease in the acid-soluble and a significant increase in the solid fraction. This phenomenon occurs both in ileum and colon. These results indicate that during the first days after operation the integrity of the intestinal wall is weakened not only by a loss of collagen, but also by a changed solubility of the remaining collagen.

Transbilayer distribution of phospholipid fatty acyl chains in photoreceptor membrane.

The transverse distribution of the fatty acyl chains of the major phospholipids over the two faces of the photoreceptor membranes has been determined in bovine rod outer segment (stacked disk) preparations. For this purpose, the fatty acid composition of the phospholipids has been analyzed before and after treatment with trinitrobenzenesulfonic acid and phospholipase D. The latter agents are used under conditions in which they have been demonstrated to attack only the outer (cytoplasmic) face of the membrane. After treatment with trinitrobenzenesulfonic acid or phospholipase D, the fatty acid composition of the unreacted phospholipids is the same as that before treatment, regardless of the extent of modification or hydrolysis attained. The fatty acid composition of phosphatidic acid, resulting from phospholipase D action, also remains unchanged during progressive hydrolysis. These results indicate that the fatty acyl chains of the major phospholipids have the same composition on either side of the disk membrane. Together with our previously published evidence for the distribution of the major phospholipids in rod outer segment disk membranes, this means that both the phospholipids and their fatty acyl chains have a remarkably symmetrical distribution over the two membrane faces. On the basis of literature data it is concluded that this approximate symmetry reflects the high mobility of the entire phospholipid pool of disk membranes, thus including appreciable transbilayer movements of the phospholipids.

The isolation of stable cattle rod outer segments with an intact plasma membrane.

A procedure is described to purify and stabilize cattle rod outer segments with an intact plasma membrane. Three criteria are applied to assess the integrity of the latter. Upon photolysis in these rod outer segments: (1) exogenous ATP cannot phosphorylate rhodopsin located in the disk membrane. (2) Endogenous cofactors (NADPH, NADPH-regenerating system) are still available in the rod cytosol and consequently retinol is the final photoproduct of photolysis of rhodopsin. (3) The rod cytosol can maintain a pH different from that of the medium, since the later stages of rhodopsin photolysis are independent of the medium pH. The stability and homogeneity of the preparation appear to be much better than those of freshly isolated frog rod outer segments, which have been used most frequently so far for experiments on the physiology of rod outer segments. In addition, these cattle rod outer segments remain intact during various manipulations and therefore considerably extend the experimental possibilities when intact rod outer segments are required.