J.N. Hingtgen

Postsynaptic action by four antidepressive drugs in an animal model of depression

To further test the new hypersensitive postsynaptic serotonin (5-HT) receptor theory of depression bases on or animal model, it was necessary to demonstrate that some of the currently used antidepressive drugs can block D,L-5-hydroxytryptophan (5-HTP) induced depression acting through postsynaptic rather than presynaptic mechanisms. Rats working for milk reinforcement and exhibiting behavioral depression following administration of 5-HTP (IP) were pretreated (1 hour before the 5-HTP injection) with fluoxetine (5 mg/kg IP) or methysergide (5 mg/kg IP) to establish a behavioral basis for distinguishing between pre- and postsynaptic events, respectively. Fluoxetine, a known specific uptake blocker of 5-HT, potentiated the depressive effect of 12.5 mg/kg 5-HTP by 200%. Methysergide, a postsynaptic blocker of 5-HT, almost completely (93%) abolished the depressive effect of 50 mg/kg 5-HTP. Since acute pretreatment with comparable clinical doses of the antidepressive drugs, mianserin, amitriptyline, imipramine, or iprindole, resulted in blockade of the 5-HTP induced depression by 70, 50, 40, and 20% respectively, these drugs can act as antagonists of 5-HT at the postsynaptic serotonin receptor. When these results are viewed in terms of recent data reported from CNS binding studies, the therapeutic effects of some antidepressants may be explained by their postsynaptic rather than presynaptic effects at central serotonergic receptors.

Pre- and postsynaptic serotonergic manipulations in an animal model of depression ☆

Rats working on a food-reinforced operant schedule and exhibiting behavioral depression following administration of D,L-5-hydroxytryptophan (5-HTP) were pretreated with one of three drugs: methysergide, fluoxetine, or amitriptyline. The former two drugs were used to establish a basis for distinguishing between pre- and postsynaptic events. We found that methysergide, a known postsynaptic blocker of serotonin, almost completely abolished the depressive effect of 5-HTP, whereas fluoxetine, a known specific uptake blocker of serotonin, potentiated the depressive effect of the 5-hydroxytryptamine (5-HT) precursor. Amitriptyline, one of the commonly prescribed antidepressive drugs, reduced the behavioral depression following 5-HTP by approximately 50%. These data indicate that amitriptyline can act as an antagonist of 5-HT at the postsynaptic receptor. The results of this study, as well as those recently reported from CNS membrane binding studies, suggest that the therapeutic effects of some antidepressive drugs may be explained by their postsynaptic rather than presynaptic properties at central serotonergic receptors. Thus, these studies support the hypothesis that some types of human depression may be primarily due to an excess of free 5-HT acting at postsynaptic receptors.

NEUROCHEMICAL CORRELATES OF BEHAVIOR

Abstract— Pigeons working on a multiple fixed ratio 50, fixed interval 10 schedule for food reinforcement were killed at 0, 50, 90, 150 and 240 min after an i.m. injection of 300mg/kg l‐tryptophan. The levels of tryptophan, 5‐hydroxytryptophan, 5‐hydroxytryptamine, 5‐hydroxyindole acetic acid, tyrosine, dopamine and norepinephrine were concurrently measured in crude nerve ending fractions (P2) isolated from the telencephalon, diencephalon plus mesencephalon and pons plus medulla‐oblongata of each pigeon. Increases in 5‐hydroxytryptamine levels in the nerve ending fraction from the telencephalon were correlated with the onset of the decreased response rates, whereas a return to baseline responding was correlated with a return to normal serotonin levels in this fraction. Changes in dopamine or norepinephrine were not related to the onset of or recovery from the decreased response rate. One group of pigeons were found which did not display any behavioral disruption even though each had received an injection of l‐tryptophan; the content of 5‐hydroxytryptophan, 5‐hydroxytryptamine and 5‐hydroxyindoleacetic acid in the nerve ending fraction isolated from the telencephalon of these birds did not differ from control values.

Serotonergic and cholinergic mechanisms during disruption of approach and avoidance behavior

Injections of D,L-5-hydroxytryptophan (D,L-5-HTP) into pigeons and rats working on approach schedules produce a period of behavioral depression that is temporally correlated to increased levels of total serotonin (5-?HT) in the telencephalon and diencephalon. Administration of alpha-methyl-meta-tyrosine (alpha-MMT) also results in depressed responding; however, the temporal correlation is with decreased levels of total 5-HT in brain. Our hypothesis to explain these two apparent opposite biochemical states which result in similar behavioral disruptions is that in both cases more 5-HT is released within certain key serotonergic synapses mediating this behavior. Evidence from subcellular studies supports this concept. tnot only are the levels of 5-HT significantly higher in preparations of nerve endings isolated from the telencephalon and diencephalon of pigeons given injections of D,L-5-HTP, but in vitro studies also show that low concentrations of L-5-HTP significantly increased the release of radioactive 5-HT from serotonergic nerve endings. On the other hand, L-5-HTP in much higher concentrations had no effect on the release of labeled dopamine or norephinephrine. A major metabolite of alpha-MMT, alpha-methyl meta tyramine, also caused a significant increase in the release of labeled 5-?HT from similar preparations of nerve endings. Whereas serotonin appears to be involved in the disruption of approach behavior, another series of studies have indicated that acetylcholine may play a role in excitation during avoidance behavior. Behavioral excitation observed following administration of tetrabenazine 18 hr after iproniazid pretreatment to rats working on shock-avoidance schedules was temporally correlated with lowered levels of acetylcholine in the telencephalon. Pretreatment with 0.8 mg/kg of atropine blocked excitation whereas one-eight of this dose increased the duration. Excitation in these rats was shortened by 50% following bilateral septal lesions, which lowered brain acetylcholine levels. Mechanisms to explain these neurochemical correlates of behavior are discussed.

In vivo determination of endogenous biogenic amines in rat brain using HPLC and push-pull cannula.

Abstract Combining the methods of push-pull cannulation with those of high performance liquid chromatography (HPLC), we have measured the content of a number of biogenic amines in the perfusate of freely moving rats. In an initial study, the lateral hypothalamus (LH) was chronically implanted with a push-pull cannula and was perfused with 0.9% NaCl. Fifteen minute samples were collected through the push-pull cannula (flow rate: 25 μl/min) and aliquots of 200 μl were injected into the HPLC without any extraction or prepurification procedure. Simultaneous determination of the levels of 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine (NE) and dopamine (DA) in the perfusate was accomplished by means of HPLC with electrochemical detection. The HPLC system utilized a C-18 reverse phase column coupled with a glassy carbon detector. Results indicate that this combination of push-pull perfusions and HPLC assay can provide a simple, rapid, and sensitive technique for the in vivo simultaneous determination of the compounds released in discrete brain areas. In preliminary studies in which these methods were used, 50 mg/kg D,L-5-HTP was injected subcutaneously (SC) into rats implanted with push-pull cannulae and working on a variable interval (VI 1) schedule of reinforcement. Increases in 5-HTP, 5-HT and 5-HIAA were measured during the period of behavioral depression following 5-HTP administration. This technique could provide a useful tool in the assessment of neurochemical changes in brain during ongoing steady-state behaviors or during the disruption of behavior following administration of drugs, precursors, or other perturbations.

Selective enhancement of shock avoidance responding elicited by GABA blockade in the posterior hypothalamus of rats

Recent studies have shown that blockade of gamma-aminobutyric acid (GABA) in the posterior hypothalamus in anesthetized rats elicits cardio-respiratory stimulation similar to that seen in emotional defense reactions and, in conscious rats, locomotor arousal suggesting a flight response. The present study was conducted in order to test the hypothesis that the behavioral effects elicited by GABA blockade in the posterior hypothalamus were the results of disinhibiting a mechanism whose activation selectively enhances reactivity to aversive stimuli. Male rats were trained on a Sidman shock avoidance schedule (RS20:SS10) as well as a food-reinforced approach schedule (VI 1). Under anesthesia, guide cannulae were stereotaxically implanted bilaterally in the posterior hypothalamus at sites where microinjection of the GABA antagonist, bicuculline methiodide (BMI) 25 ng, increased heart rate. After recovery, rats were tested in both the avoidance and VI 1 schedules after hypothalamic microinjection of saline, BMI 25 ng, and the GABA agonist, muscimol 25 ng. Microinjection of BMI significantly increased the avoidance responses but had no effect on the approach responses. Muscimol decreased both the avoidance and approach responses. When microinjected into the lateral hypothalamic area, BMI had no effect on the response rates in either schedule while muscimol decreased the approach responding only. Therefore, GABA blockade at the discrete area of the posterior hypothalamic nucleus appears to elicit a selective enhancement of avoidance responses. These results suggest that an endogenous GABAergic system in the posterior hypothalamus may tonically inhibit a constellation of autonomic, locomotor and motivational responses that are necessary for some types of defense reaction.

Postsynaptic serotonergic blockade following chronic antidepressive treatment with trazodone in an animal model of depression

Acute pretreatment with clinically equivalent doses of antidepressive drugs has been observed to block D,L-5-hydroxytryptophan (5-HTP) induced behavioral depression in rats working on a food-reinforced operant schedule. Data from studies designed to distinguish presynaptic from postsynaptic events, indicated that the antidepressants were acting in part as blockers of postsynaptic serotonergic receptors. Using the same 5-HTP model of depression, we studied both the chronic and acute effects of a recently introduced antidepressant, triazolopyridine compound. Rats working for milk reinforcement and exhibiting behavioral depression following administration of 50 mg/kg 5-HTP were pretreated (one hr before 5-HTP) with 1, 2, or 4 mg/kg trazodone with resulting blockade of 5-HTP induced depression of 35, 62 and 70% respectively. Chronic administration of trazodone (2 mg/kg trazodone/day) also resulted in a significant blockade of the 5-HTP effect (75%). Neither 2 mg/kg or 4 mg/kg trazodone was found to potentiate the shorter period of depression following 25 mg/kg 5-HTP. Chronic treatment with the antidepressant drugs, amitriptyline or mianserin also blocked 5-HTP depression. Thus, as in our earlier studies, these data suggest an important postsynaptic mechanism associated with chronic administration of trazodone, amitriptyline and mianserin which could be implicated in the therapeutic effectiveness of these drugs. The potency of trazodone in relation to other antidepressant drugs in our behavioral model of depression paralleled their potency in displacing radioligand binding to 5-HT receptors, and gives additional support for the new hypersensitive postsynaptic serotonin receptor theory of depression.

CHANGES IN ACETYLCHOLINE, NOREPINEPHRINE AND 5‐HYDROXYTRYPTAMINE CONCENTRATIONS IN SEVERAL DISCRETE BRAIN AREAS OF THE RAT DURING BEHAVIOURAL EXCITATION1,2

—Acetylcholine, 5‐hydroxytryptamine, and norepinephrine concentrations were measured in the telencephalon, diencephalon plus mesencephalon (midbrain), and pons‐medulla oblongata of rats exhibiting behavioral excitation while working on a Sidman avoidance schedule and injected with 50 mg/kg iproniazid 16 hr before being given 2 mg/kg tetrabenazine. Acetylcholine concentrations in all three brain areas decreased and returned to normal levels at different times. The time course of increased response rates correlated best with the acetylcholine levels in the telencephalon. Both the 5‐hydroxytryptamine and norepinephrine concentrations remained similar to the iproniazid control values during the period of behavioural excitation. However, the norepinephrine concentration in the midbrain showed a continuous decreasing trend toward naive control levels. These data suggest that changes in a cholinergic system in the telencephalon and a noradrenergic system in the midbrain operate in the maintenance of behavioural excitation.

Effects of 5-hydroxytryptophan on serotonin in nerve endings.

—Preparations of synaptosomes (P2) from the telencephalon and from the diencephalon plus optic lobes of the pigeon and from the telencephalon of the rat were used to study the effects of 5‐hydroxytryptophan (5‐HTP) on (a) the levels of serotonin (5‐HT) in nerve endings and (b) the release of 5‐HT from nerve endings. The levels of 5‐HT were significantly higher (3.21 × 0.35 nmol/g original tissue weight) in the P2 fraction isolated from the telencephalon of pigeons given intramuscular injections of 50mg/kg of d,l‐5‐HTP in comparison to control values (1.42 ± 0.07). A similar twofold increase was observed with the P2 fraction isolated from the diencephalon plus optic lobes. In addition, the levels of 5‐HTP and 5‐hydroxyindoleacetic acid also increased significantly in these P2 fractions isolated from pigeons given d,l‐5‐HTP injections in comparison to values obtained for pigeons given saline injections. In vitro studies using preparations of synaptosomes (from both pigeon and rat) labelled with [3H]5‐HT indicated that 0.10 mil l‐5‐HTP increased the release of [3H]5‐HT twofold over control values. A concentration as low as 0.001 mm l‐5‐HTP was tested on the P2 fraction from the telencephalon of the pigeon and was found to significantly increase the release of [3H]5‐HT over control values. This effect by l‐5‐HTP was blocked if a decarboxylase inhibitor was added to the medium. l‐5‐HTP at a concentration of 1.5 mm had no apparent effect on the release of [3H]norepinephrine or [3H]dopamine from synaptosomes prepared from the telencephalon of the rat or pigeon. The results are discussed in terms of the role of serotonin in producing certain types of behavioral depressions exhibited by pigeons and rats given injections of 5‐HTP.

Stress-induced changes of norepinephrine uptake sites in the locus coeruleus of C57BL/6J and DBA/2J mice: a quantitative autoradiographic study using [3H]-tomoxetine

Inbred C57BL/6J (C57) and DBA/2J (DBA) mice were subjected to open-field evaluation and Porsolt swim test after restraint stress. Norepinephrine (NE) uptake sites in the locus coeruleus (LC) of these inbred mice were studied by using [3H]-tomoxetine. Results showed that naive C57 mice were more active in the open field and possessed more NE uptake sites in the LC than naive DBA mice. Previous work has shown that restraint decreases open field activity in C57 mice, but not DBA mice, whereas the present study has demonstrated that, after restraint stress, C57 mice spent more time immobile than DBA mice did in the forced swim test. Furthermore, in these stressed animals, NE uptake sites in the LC were greatly increased with consistently more uptake sites in C57 mice. Collectively, results of this study and the literature suggest that enhanced NE function in the LC of C57 mice is associated with their susceptibility to stress-induced behavioral depression.