The effect of the novel antidepressant tianeptine on the concentration of 5-hydroxytryptamine in rat hippocampal dialysates in vivo.

Tianeptine is a novel tricyclic antidepressant which, in marked contrast to other antidepressants, increases the uptake of 5-HT in brain tissue and blood platelet preparations ex vivo. In the present study it was shown, by microdialysis, that tianeptine (10 mg/kg i.p.), whether given as a single dose or as daily doses for 14 days, attenuated the K(+)-evoked rise of extracellular 5-HT in the hippocampus in vivo. Tianeptine (10 mg/kg i.p.), given acutely, did not reduce basal levels of 5-HT in hippocampal dialysates in the presence of the 5-HT reuptake inhibitor, citalopram. These results suggest mutually opposing effects of tianeptine and citalopram on neuronal uptake of 5-HT. Their significance in relation to the role of 5-HT in the action of antidepressants is discussed.

Detection of interleukin-1 and interleukin-6 in adult rat brain, following mechanical injury, by in vivo microdialysis: evidence of a role for microglia in cytokine production.

In vivo levels of interleukin-1 (IL-1) and IL-6, present in the interstitial spaces of brain, have been repeatedly monitored up to 7 days after insertion of a microdialysis probe, designed to induce mechanical trauma to the brain. IL-1 is barely detectable immediately after implantation but over a 24-48 h period a 15-fold increase is seen. In contrast IL-6 levels at day 0 are high, increasing slightly (10%) by day 1 but decreasing to 40% by day 2. The temporal pattern of IL-6 recovery in the cerebrospinal fluid was similar to that in the dialysate but the levels were significantly lower and may reflect diffusion from the site of the probe lesion. Cellular sources of these cytokines include macrophages and neutrophils, which have infiltrated the lesion and microglia resident in the brain, which can be identified at the lesion site within 24 h of probe implantation. The astrocytic response to injury, evidenced by increased glial fibrillary acidic protein staining occurs much later, by day 7, and is unlikely to be responsible for IL-1 and IL-6 production found at 24-48 h. Since upon isolation and stimulation of microglia in vitro with lipopolysaccharide IL-1 and IL-6 can be measured in the supernatant, it would appear that they have the capacity to produce cytokines in vivo. Localised synthesis of cytokines at sites of brain injury by microglia would further stimulate microglia in an autocrine manner and also propagate the astrocytic reaction.

Evidence for postsynaptic mediation of the hypothermic effect of 5-HT1A receptor activation.

1. The 5-HT1A ligand BMY 7378 (8-[2[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]8-azaspirol [4,5]-decane-7,9-dione dihydrochloride, 0.032-2 mg kg-1, s.c.) caused hyperphagia, a response to the activation of presynaptic 5-HT1A receptors. 2. BMY 7378 (8 mg kg-1, s.c.) and the 5-HT1A agonist (8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), 0.10 and 0.25 mg kg-1 s.c.) also caused hypothermia. This was inhibited by (-)-pindolol (1-mg kg-1, i.p.) and not prevented by pretreatments with p-chlorophenylalanine which grossly depleted 5-hydroxytryptamine (5-HT) from terminal regions. The hypothermic effects are explicable by activation of postsynaptic 5-HT1A receptors. Infusion of BMY 7378 (8-64 micrograms) into the dorsal raphe was without convincing hypothermic effect. 3. BMY 7378 (8 mg kg-1, s.c.) inhibited another effect of activation of postsynaptic 5-HT1A receptors, i.e., the induction of components of the 5-HT syndrome by 8-OH-DPAT (0.5, 1.0 mg kg-1, s.c.) which suggests that BMY 7378 has antagonistic as well as agonistic effects at these sites. 4. Partial agonist properties of BMY 7378 at postsynaptic sites were also indicated by doses for hypothermia being much greater than those for hyperphagia i.e., ED50 (hypothermia) greater than 2 mg kg-1, ED50 (hyperphagia) = 0.010 mg kg-1. This contrasts with the similar ED50 values for both the hypothermic (ED50 = 0.08-0.10 mg kg-1) and hyperphagic (ED50 = 0.06-0.10 mg kg-1) effects of 8-OH-DPAT.5. The evidence obtained for mediation of the hypothermic response to 5-HTIA agonists by postsynaptic sites is relevant to the interpretation of the effects on it of antidepressant treatments and depressive illness.

An in vivo dialysis and behavioural study of the release of 5-HT by p-chloroamphetamine in reserpine-treated rats.

1. Reserpine (2.5 mg kg-1 i.p.) decreased rat brain 5-hydroxytryptamine (5-HT) by 86% 24 h later but most components of the 5-HT-dependent behavioural syndrome induced by p-chloroamphetamine (PCA, 5 mg kg-1 i.p.) or 5-methoxy-N,N-dimethyltryptamine (5-MeODMT, 5 mg kg-1 i.p.) over 1 h after administration were unaffected. However, Straub tail was increased after giving PCA or 5-MeODMT and head weaving was decreased after giving 5-MeODMT. 2. Frontal cortex extracellular 5-HT concentrations of vehicle pretreated rats before injection of PCA, as calculated from dialysate 5-HT concentrations, were about 1/1000th of corresponding brain values. Extracellular 5-hydroxyindoleacetic acid (5-HIAA) and brain values were comparable with each other. Dialysate 5-HT increased after PCA with peak values at 20-40 min. 3. Reserpine pretreatment reduced dialysate 5-HT concentration before PCA was given but the net increase (AUC) over the 1 h after PCA did not differ significantly from that seen in animals pretreated with vehicle. Dialysate 5-HIAA values slowly decreased after PCA injection in both reserpine and vehicle pretreated groups. 4. The results suggest that PCA causes the 5-HT syndrome by releasing 5-HT from the neuronal cytoplasm but that physiological release of 5-HT occurs from vesicular stores.

Effect of p-chlorophenylalanine on release of 5-hydroxytryptamine from the rat frontal cortex in vivo.

1. Rats were given p-chlorophenylalanine (PCPA, 150 mg kg-1, i.p.) to inhibit partially 5-hydroxytryptamine (5-HT) synthesis so that its concentration in the frontal cortex fell by about half. The effects of this treatment on frontal cortex dialysate 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentrations were determined before and after stimulation by increasing K+ concentration in the perfusion fluid by 100 mM for 20 min. Rates of 5-HT synthesis as indicated by the effects of 3-hydroxybenzylhydrazine (NSD 1015, 150 mg kg-1, i.p.) on frontal cortex tissue and dialysate 5-hydroxytryptophan (5-HTP) and dialysate 5-HIAA were also measured in rats that had not been stimulated with K+. 2. Dialysate 5-HT and 5-HIAA concentrations of both vehicle- and PCPA-treated rats fell into major (group 1) and minor (group 2) populations statistically distinguishable from each other by the high 5-HT and low 5-HIAA values of the latter group. 3. In group 1 animals, PCPA decreased both the dialysate 5-HT concentration and its rise following stimulation by K+ in proportion with the decrease of 5-HT in frontal cortex tissue. 5-HIAA fell more markedly than 5-HT and in similar proportion in both tissue and dialysate. The fall of dialysate 5-HIAA on stimulation by K+ was also attenuated to the same degree. The elevated 5-HT/5-HIAA ratios after PCPA treatment imply increased conservation of the depleted 5-HT stores. 4. PCPA decreased the above 5-HIAA values and the effects of NSD 1015 on tissue 5-HTP or dialysate 5-HIAA concentrations in similar proportion. However, PCPA had little effect on corresponding dialysate 5-HTP values. 5. The results are discussed with respect to relationships between synthesis, storage and release of 5-HT. They indicate that (under the conditions of the present study) the availability of 5-HT to receptors is directly proportional to total vesicular stores under both basal conditions and during neuronal firing.

Measurements of tacrine and monoamines in brain by in vivo microdialysis argue against release of monoamines by tacrine at therapeutic doses.

1. The concentration of tacrine (tetrahydroaminoacridine or THA) in plasma, regions of brain and cerebral extracellular fluid has been studied in the rat at various times following injection of a dose of 5 mg kg-1, i.p. 2. The peak plasma THA concentration was 2.46 nmol ml-1, and occurred 30 min post injection and clearance was first order (t1/2 = 90 min). The concentration in the brain peaked between 30-60 min, and was around 30 times plasma concentration (striatum peak concentration = 65 +/- 3 nmol g-1). Extracellular cerebral concentration measured by in vivo microdialysis was similar to plasma concentration with the peak occurring 100 min post-injection. 3. No evidence was obtained by in vivo dialysis for THA inducing dopamine release from striatum or 5-hydroxytryptamine (5-HT) release from the frontal cortex. Enhanced release of dopamine did occur after (+)-amphetamine (5 mg kg-1, i.p.) injection, while KCl (100 mM) in the probe released both dopamine and 5-HT. 4. Since the minimum plasma THA concentration achieved in this study was at least twice that found in the plasma of patients given THA for the treatment of dementia, these results suggest that monoamine release in the brain does not occur during therapy.

Effect of portacaval anastomosis and chronic underfeeding on the hypothalamic-pituitary-gonadal axis in the rat.

Portacaval anastomosis (PCA) in the rat may be a useful experimental model for examining endocrine changes that occur during cirrhosis of the liver. A marked reduction in diet intake and body weight occurs in rats after establishing the shunt and studies were undertaken to determine the relationship of these effects to the testicular atrophy that also follows PCA. Control, sham-operated animals, experiencing a reduction in food intake similar to that of the animals with a PCA, showed reduced plasma levels of LH and testosterone but also exhibited a marked testicular response to LH. This was consistent with increased sensitivity of the hypothalamic-pituitary axis to the negative feedback of gonadal steroids in chronically underfed animals. Male rats with a PCA exhibited similarly reduced levels of LH and testosterone, but showed poor secretory responses of the pituitary gland to LH releasing hormone (LH-RH) and of the testis to LH. Testicular atrophy and cessation of spermatogenesis occurred in the animals with a PCA. These results suggested that the effects of PCA on the pituitary-gonadal axis cannot simply be explained as a consequence of the restricted intake of diet. This was confirmed by the responses to castration. In both fed and underfed sham-operated rats, castration resulted in a rapid and sustained increase in plasma LH and both groups showed a marked LH secretory response to LH-RH. In contrast, in animals with a PCA castration had little effect on plasma LH and the pituitary response to LH-RH was still poor. The effects of PCA cannot be simply explained by impeded metabolism of gonadal steroids causing increased negative feedback on the hypothalamic-pituitary axis.

Brain edema following an experimental missile wound to the brain.

Brain edema occurs after brain injury and may be life threatening. The occurrence and type of brain edema, its magnitude, and time course have never been ascertained following a missile wound to the brain. We therefore measured the development of brain edema in a series of cats in which a right cerebral hemisphere wound was made with a 2.0 mm, 31.7 mg steel sphere with a velocity of 240-300 m/s (0.9-1.4 J). The entire brain was surveyed for postwounding brain edema by determining the wet weight minus dry weight. Brain edema was seen to develop only in the white matter about the missile wound track in the injured cerebral hemisphere. There, brain water rose from 66 to 73%; white matter sodium also increased significantly from approximately 150 mEq/kg dry weight to 254 mEq/kg dry weight. Potassium levels remained essentially unchanged. This vasogenic edema was relatively mild, peaked at 24-48 h after wounding, and resolved spontaneously within a week without specific treatment. We infer that brain edema following an uncomplicated cerebral missile wound in the human brain is also mild, self-limited, and may resolve spontaneously without special treatment.

Effects of tianeptine on stress-induced behavioural deficits and 5-HT dependent behaviour.

The novel tricyclic antidepressant drug tianeptine had an antidepressant-like effect on a rat model of depression based on the deficit in open field activity observed on the day after 2 h restraint. Thus, when tianeptine (10 mg/kg IP) was given 2 h after the end of the restraint to either untreated rats or to animals previously given 10 mg/kg of the drug per day for 13 days, then the deficit was opposed. Tianeptine, given acutely but not chronically, moderately enhanced the 5-HT1C receptor-dependent hypolocomotor effect of m-chlorophenylpiperazine (mCPP) but did not alter other 5-HT1 receptor subtype-dependent behaviour. Acute but not chronic tianeptine also decreased 5-HT2 receptor-dependent body shakes induced by 5-hydroxytryptophan. Shakes induced by the 5-HT2 agonist 1-(2,5-dimethoxy-4-iodophenyl)-2 aminopropane (DOI) were unaffected. The results are discussed in relation to the possible mechanism of antidepressant action of tianeptine.

Association between learning and cortical catecholamines in non-drug-treated rats.

Seventeen male Sprague-Dawley rats were trained to eight to nine correct responses on a delayed spatial alternation test performed on alternate days in a T-maze. Locomotor activity in an observation box was scored on 2 consecutive days. The animals were killed 2 weeks after the end of behavioural testing and dopamine (DA), noradrenaline (NA), 5-hydroxytryptamine (5HT), the DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and the 5HT metabolite 5-hydroxyindoleacetic acid (5HIAA) determined in cortex, hippocampus, striatum and hypothalamus. Cortical concentrations of both DA and NA correlated negatively and significantly with the number of errors made in learning the alternation task, though the latter correlation was less striking and became negligible after the correlation between DA and NA was partialled out. Concentrations of DA and NA in the other regions did not correlate significantly with errors. None of the other neurochemical variables correlated significantly with either errors or locomotor activity, except for hypothalamic HVA concentration which showed a marginally significant correlation with locomotor activity. The above results, together with effects of brain lesions reported by other authors, strongly indicate that cortical catecholamines facilitate learning in the normal non-drug-treated rat.

Monitoring dopamine metabolism in the brain of the freely moving rat.

Determination of DOPAC and HVA in cisternal CSF taken repeatedly from freely moving rats provides a useful means of monitoring central DA metabolism. A large proportion of both metabolites occurs in cisternal CSF as conjugates from which they are liberated by acid hydrolysis. The method enables DA turnover values to be determined for individual rats. Drug experiments indicate that these values reflect brain DA metabolism and that most of this occurs in extrastriatal DA neurons. Concurrent determination of 5HT turnover on the same CSF samples revealed a significant positive correlation between the turnovers of the two transmitters together with considerable inter-individual differences. The turnover method was particularly convenient when investigating daily variations of turnover. Repeated CSF withdrawal also appears to be useful in the analysis of stress-provoked changes of the metabolism of DA and other transmitters. For example, it was used to show that central DA metabolism becomes highly responsive to tyrosine availability if rats are subjected to immobilization stress. The method can also be used to compare the time dependencies of both metabolic and behavioral responses to the stress in the same animal. Preliminary results suggest that the increase of 5HT metabolism during immobilization (rather than that of DA) may oppose the suppression of open field activity that occurs 24 hr later.

Monitoring 5HT metabolism in the brain of the freely moving rat.

The techniques of repeated CSF withdrawal and intracerebral dialysis are described. They were used to monitor 5HT metabolism in the brains of freely moving rats. Evidence indicates that results obtained proportionately reflect changes of 5HT metabolism in whole brain (CSF method) and in the striatum (intracerebral dialysis). The potential value of the methods is illustrated by two behavioral studies. The first suggested that 5HT turnover values (determined two weeks prior to behavioral testing by the CSF method) were predictive for neck + body biting in a social interaction test. The second study showed a consistent pattern of change of hypothalamic dialysate 5HIAA concentration associated with feeding in rats on a restricted food intake schedule.

Combined intracerebral microdialysis and electrophysiological recording: methodology and applications.

A microdialysis probe is described that can simultaneously monitor indices of electrical activity, ionic homeostasis and changes in the composition of the extracellular fluid at the same brain site in anaesthetised laboratory animals. The probe is no larger than its conventional counterpart and avoids tissue injury problems due to implantation of separate recording electrodes. Examples are given of its application to the study of changes following probe implantation, cerebral ischaemia and local high K(+)-induced depolarisation.

A study of the kinetic behaviour of glucose based on simultaneous estimates of influx and phosphorylation in brain regions of rats in different physiological states.

Results from the application of a procedure that allows simultaneous estimation of the rates of glucose transport from blood and of glucose phosphorylation in discrete regions of the brain are given for groups of rats displaying chemically induced motor disturbances. The procedure is based on sequential injections of [14C]- and [3H]-2-deoxyglucose followed a few minutes later by focused microwave irradiation to the head. Control conscious rats were used and rats displaying either whole body tremors, hind-limb rigidity or choreoathetotic movements induced by synthetic pyrethroid compounds. For individual rats estimates of 5 parameters were obtained in up to 16 brain regions. In addition brain tissue and plasma glucose concentrations were determined. The 5 parameters were: (1) rate of total glucose influx from plasma; (2) rate of glucose phosphorylation (equivalent to net influx); (3) rate of glucose efflux; (4) half-life of free glucose in brain; and (5) PS-product expressed as the ratio of the rate of glucose influx to plasma glucose concentration. For each parameter significant differences between regions were found in all groups of animals including conscious controls. The mean values for the somatosensory cortex of control rats were parameter: (1) 2.02 mumol/g/min; (2) 1.11 mumol/g/min; (3) 0.91 mumol/g/min; (4) 0.94 min; and (5) 0.196 ml/g/min. A high correlation was observed between the rate of total glucose influx and the rate of glucose phosphorylation for all brain regions in all groups of rats. This finding is discussed in terms of a synchronized regulatory mechanism on the glucose transport carrier of capillary endothelial cells and on the functional hexokinase Vmax activity within brain cells.

CSF tryptophan and transmitter amine turnover may predict social behaviour in the normal rat.

Central 5-hydroxytryptamine (5-HT) and dopamine (DA) turnovers and tryptophan concentrations were estimated in individual male rats using repeated CSF withdrawal. On subsequent pairing, the major biters of each pair (neck + body bites) were predicted by their higher concentrations of the 5-HT precursor tryptophan but not by 5-HT turnover. However, bites/pair correlated highly significantly with the lower 5-HT and DA turnover values in each pair. The investigation illustrates a new and flexible approach to the neurochemistry of social behaviour.

Brain metabolism and specific transport at the blood-brain barrier after portocaval anastomosis in the rat.

The rapid metabolism of [1-14C]butyrate and [2-14C]pyruvate in the brain was studied after intracarotid injection in control rats and rats given an end-to-side portocaval anastomosis (PCA). At 10 or 50 sec after injection there was a 40-50% lowering of the total amount of radioactivity in the brain of operated rats for both compounds. The percentage distribution of label in various metabolic fractions, e.g. amino acids, was unaltered by PCA. The results from the metabolic studies focused on an impaired transport of short chain monocarboxylic acids between blood and brain in rats with a PCA. The brain uptake index (BUI) of acetate, butyrate, pyruvate and glucose was determined by rapid intracarotid injection of the 14C test compound plus 3H2O as a reference marker. At 3 weeks after PCA, transport of all 3 monocarboxylic acids into brain was reduced by 40-50%. The reduction in [2-14C]pyruvate uptake was absent at one week, but thereafter, up to 50 weeks after operation, was consistently present. Eadie-Hofstee analysis of influx at varying substrate concentrations indicated a reduction in Vmax values of pyruvate and butyrate uptake without changes in Km. The calculated influx rate of glucose in operated rats was reduced in direct proportion to the lowering of plasma glucose. Portocaval anastomosis in the rat induces selective changes on substrates that are transported across the blood-brain barrier via a facilitated transport process.

Effect of alpha-methyl fluorodopa on dopamine metabolites: importance of conjugation and egress.

The effects of D,L-alpha-monofluoromethyldopa (MFMD), an inhibitor of aromatic L-amino acid decarboxylase, on the metabolism of dopamine (DA) and 5-hydroxytryptamine was investigated using rat brain and cerebrospinal fluid (CSF). Vehicle or MFMD (100 mg/kg) was given p.o. and 16 h later probenecid (200 mg/kg i.p.). CSF was sampled and the rats killed immediately or after 1 h. Vehicle treated rats showed regional differences of percentage conjugation of DA metabolites: 3,4-dihydroxyphenylacetic acid (DOPAC), striatum 10%, rest of brain 45%, CSF 67%; homovanillic acid (HVA), striatum 20%, rest of brain 35%, CSF 53%. These differences and the proportionately greater increases of conjugates than of free acids after probenecid vitiate regional comparisons of DA metabolism if conjugates are not included. MFMD alone decreased neither 5HIAA (except in the striatum) nor the free DA metabolites but decreased both conjugates in CSF and conjugated DOPAC in rest of brain. The inhibitory effects of MFMD on 5HT and DA synthesis were most evident when measured by the accumulation of 5HIAA or total (DOPAC + HVA) after giving probenecid. MFMD may also inhibit amine metabolite egress and the conjugation of DOPAC and HVA.

Hippocampal 5-HT synthesis and release in vivo is decreased by infusion of 8-OHDPAT into the nucleus raphe dorsalis.

Infusion of the serotonin-1A (5-HT1A) agonist, 8-hydroxy-2-di(n-propylamino)tetralin (8-OHDPAT) (0.5, 1.0 and 2.0 micrograms) into the dorsal raphe, decreased extracellular 5-HT as measured by dialysis in the ventral hippocampus and also decreased 5-HT synthesis in both the hippocampus and the rest of the brain as measured by 5-hydroxytryptophan (5-HTP) accumulation following decarboxylase inhibition by NSD 1015.

Changes in rat brain extracellular glutamate concentration during seizures induced by systemic picrotoxin or focal bicuculline injection: an in vivo dialysis study with on-line enzymatic detection.

An on-line enzymatic assay of dialysis fluid has been used to monitor the extracellular glutamate concentration in the rat hippocampus. Perfusion with artificial cerebrospinal fluid containing a glutamate uptake inhibitor (either dihydrokainate or 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid) produced a marked stable increase in glutamate concentration; 10 min perfusion with 100 mM K+ produced a transient increase. Sustained epileptiform EEG discharges were induced in the hippocampus by focal injection of bicuculline into the piriform cortex or by systemic injection of picrotoxin. Extracellular glutamate did not change significantly during seizure activity, either in the absence or in the presence of glutamate uptake inhibitors. It is concluded that seizure activity is not necessarily accompanied by an overall increase in extracellular glutamate concentration.

Experimental missile wound to the brain.

Among civilians in the United States, 33,000 gunshot wound deaths occur each year; probably half of these involve the head. In combat, head wounds account for approximately half of the immediate mortality when death can be attributed to a single wound. No significant reduction in the neurosurgical mortality associated with these wounds has occurred between World War II and the Vietnam conflict, and very little research into missile wounds of the brain has been undertaken. An experimental model has been developed in the anesthetized cat whereby a ballistic injury to the brain may be painlessly reproduced in order that the pathophysiological effects of brain wounding may be studied and better treatments may be designed to lower the mortality and morbidity rates associated with gunshot wounds. Prominent among physiological effects observed in this model was respiratory arrest even though the missile did not injure the brain stem directly. The incidence of prolonged respiratory arrest increased with increasing missile energy, but arrest was often reversible provided respiratory support was given. It is possible that humans who receive a brain wound die from missile-induced apnea instead of brain damage per se. The mortality rate in humans with brain wounding might be reduced by prompt respiratory support. Brain wounding was associated with persistently increased intracranial pressure and reduced cerebral perfusion pressure not entirely attributable to intracranial bleeding. The magnitude of these derangements appeared to be missile energy-dependent and approached dangerous levels in higher-energy wounds. All wounded cats exhibited postwounding increases in blood glucose concentrations consistent with a generalized stress reaction. A transient rise in hematocrit also occurred immediately after wounding. Both of these phenomena could prove deleterious to optimal brain function after injury.