Gerald L. Kramer

Prevention of learned helplessness: In vivo correlation with cortical serotonin

Learned helplessness (LH) is prevented by pretreatment with acute benzodiazepines (BDZs), subchronic tricyclic antidepressants (TCAs), or escapable stress (ES). We have investigated the role of serotonin (5-HT) in LH prevention by these three prevention paradigms, using microdialysis to measure in vivo 5-HT release in frontal cortex (FC) after LH testing. Rats receiving pretreatment before inescapable stress with any of the three methods of prevention--BDZs, TCAs, or ES--showed escape behavior in the shuttle-box test for LH comparable to naive unstressed controls. K(+)-stimulated 5-HT release in all three groups receiving pretreatment was also similar to naive unstressed controls. Rats receiving saline before inescapable stress showed significantly more LH behavior in the shuttle-box task and had significantly lower 5-HT release as well. This suggests that LH correlates with a significant decrease in intracellular releasable 5-HT in FC, and that three different techniques for LH prevention, acute BDZs, subchronic TCAs, and ES all similarly prevent this 5-HT depletion.

Dominant Role of Paraoxonases in Inactivation of the Pseudomonas aeruginosa Quorum-Sensing Signal N-(3-Oxododecanoyl)-L-Homoserine Lactone

ABSTRACT The pathogenic bacterium Pseudomonas aeruginosa causes serious infections in immunocompromised patients. N-(3-Oxododecanoyl)-l-homoserine lactone (3OC12-HSL) is a key component of P. aeruginosas quorum-sensing system and regulates the expression of many virulence factors. 3OC12-HSL was previously shown to be hydrolytically inactivated by the paraoxonase (PON) family of calcium-dependent esterases, consisting of PON1, PON2, and PON3. Here we determined the specific activities of purified human PONs for 3OC12-HSL hydrolysis, including the common PON1 polymorphic forms, and found they were in the following order: PON2 ≫ PON1192R > PON1192Q > PON3. PON2 exhibited a high specific activity of 7.6 ± 0.4 μmols/min/mg at 10 μM 3OC12-HSL, making it the best PON2 substrate identified to date. By use of class-specific inhibitors, approximately 85 and 95% of the 3OC12-HSL lactonase activity were attributable to PON1 in mouse and human sera, respectively. In mouse liver homogenates, the activity was metal dependent, with magnesium- and manganese-dependent lactonase activities comprising 10 to 15% of the calcium-dependent activity. In mouse lung homogenates, all of the activity was calcium dependent. The calcium-dependent activities were irreversibly inhibited by extended EDTA treatment, implicating PONs as the major enzymes inactivating 3OC12-HSL. In human HepG2 and EA.hy 926 cell lysates, the 3OC12-HSL lactonase activity closely paralleled the PON2 protein levels after PON2 knockdown by small interfering RNA treatment of the cells. These findings suggest that PONs, particularly PON2, could be an important mechanism by which 3OC12-HSL is inactivated in mammals.

Low plasma GABA is a trait-like marker for bipolar illness.

Plasma gamma-aminobutyric acid (pGABA) is an index of brain GABA activity and a peripheral marker of mood disorder. Previous research has indicated that pGABA is abnormally low in approximately 40% of patients symptomatic with primary unipolar depression. We have now measured pGABA in a series of patients with bipolar disorder. Blood samples for GABA determinations were collected soon after admission to hospital or clinic while patients were symptomatic. In both manic and depressed phase bipolar patients, mean levels of pGABA were significantly lower than in healthy control subjects. The distribution of pGABA in bipolar patients, whether manic or depressed, was similar to that in symptomatic unipolar depression, with 30% to 40% having pGABA levels lower than the control range. These data indicate that low pGABA is not specific to the depressed state, as it is also found in the manic phase of bipolar disorder. Low pGABA may represent a shared biologic correlate between bipolar and unipolar illness.

Low plasma γ-aminobutyric acid levels in male patients with depression ☆

Abstract Plasma levels of γ-aminobutyric acid (GABA) were significantly lower in males with primary unipolar major depressive disorder than in healthy controls. Although the difference in means between control and symptomatic depressed patient groups was small, the distribution of plasma GABA in the depressed patients was markedly different from controls. Forty percent of depressed patients had plasma GABA levels below those of controls. Plasma GABA levels correlated positively with duration of illness, and negatively with age at onset of the mood disorder and the total Endogenomorphic Symptom Score on the Hamilton Rating Scale. Plasma GABA levels may be a biochemical marker of vulnerability to depression, as opposed to a consequence of the illness. A low GABA condition in depression fits and complements the prevailing biogenic amine hypotheses of depression.

Plasma GABA levels correlate with aggressiveness in relatives of patients with unipolar depressive disorder

Plasma gamma-aminobutyric acid (GABA) levels are decreased in some patients with depression, mania and alcoholism. Medications which increase plasma GABA improve symptoms of mood disorders and can decrease aggression. We examined the relationship between plasma GABA and aggressiveness on the Buss-Durkee Hostility Inventory in 77 psychiatrically healthy adults. In subjects selected for having a first-degree relative with primary unipolar depressive disorder (FH+, n=33), plasma GABA was negatively correlated with aggressiveness (beta=-0.338, P=0.036), as was age (beta=-0.483, P=0.005). A relationship between plasma GABA levels and aggressiveness was not observed in subjects with no such family history (FH-, n=44). Moreover, FH+ subjects had significantly lower plasma GABA concentrations than FH- subjects. These data suggest that low GABA levels may correlate with some aspects of aggressiveness and may be genetically regulated.

In vivo serotonin release and learned helplessness

Learned helplessness, a behavioral depression caused by exposure to inescapable stress, is considered to be an animal model of human depressive disorder. Like human depression, learned helplessness has been associated with a defect in serotonergic function, but the nature of this relationship is not entirely clear. We have used in vivo microdialysis brain perfusion to measure serotonin (5-hydroxytryptamine, 5HT) in extracellular space of medial frontal cortex in conscious, freely moving rats. Basal 5HT levels in rats perfused before exposure to tail-shock stress did not themselves correlate with subsequent learned helplessness behavior. However, 5HT release after stress showed a significant increase with helpless behavior. These data support the hypothesis that a cortical serotonergic excess is causally related to the development of learned helplessness.

Previous stress increases in vivo biogenic amine response to swim stress

In vivo microdialysis was used to determine biogenic amines in medial prefrontal cortex of rats exposed to eight minutes of swim stress on two consecutive days. On the first day of stress, norepinephrine (NE) efflux increased by 183% over baseline after stress, while dopamine (DA) and serotonin (5-HT) remained stable throughout. On the second day of stress, a robust increase was observed in all 3 neurotransmitters measured, with (NE), (DA), and (5-HT) increasing by 310%, 441% and 496% respectively, and remaining elevated for an hour or more after stress. This suggests that the first exposure to swim stress, while not causing dramatic changes in biogenic amine release, may sensitize biogenic amines in medial prefrontal cortex to subsequent swim stress. Our results also serve as preliminary data concerning the neurochemical changes which might underlie the forced swimming model of “behavioral despair”.

Learned helplessness increases 5-hydroxytryptamine1B receptor mRNA levels in the rat dorsal raphe nucleus

Learned helplessness is a behavioral condition induced by exposure to inescapable stress that models aspects of stress-related disorders including depression and posttraumatic stress disorder, and has been associated with diminished serotonin release in the rat frontal cortex. Our hypothesis was that presynaptic 5-hydroxytryptamine1B (5-HT1B) receptors, which inhibit the synthesis and release of serotonin in nerve terminals, may be increased in learned helplessness. Postsynaptic 5-HT1B mRNA hybridization levels in the hippocampus or frontal cortex were unchanged following induction of learned helplessness; however, presynaptic 5-HT1B mRNA hybridization signal in the dorsal raphe nucleus of helpless rats was 25% higher than control values. There was no change in dorsal raphe serotonin transporter mRNA level. The detection of increased 5-HT1B mRNA levels in the dorsal raphe nucleus suggests an increased capacity to synthesize presynaptic 5-HT1B receptors and could account for diminished serotonin neurotransmission in learned helplessness.

Decrease in stress-induced c-Fos-like immunoreactivity in the lateral septal nucleus of learned helpless rats.

Inescapable stress can induce learned helplessness in many species of animals. Learned helplessness is a phenomenon which has some behavioral and neurotransmitter analogies with human clinical depression. Stress can also induce the expression of immediate early genes, including c-fos in many areas of the central nervous system. We examined stress-induced c-Fos-like immunoreactivity (FLI) using the learned helplessness paradigm. Naive rats showed significantly higher FLI than the tested groups in all the amygdaloid regions and in the hypothalamic paraventricular nucleus. However, in the lateral septal nucleus, helpless animals showed significantly reduced FLI in response to stress, compared to the other groups. These, and other previous data, highlight the importance of the septal area in mediating behavioral responses to inescapable stress.

Serotonin and learned helplessness: a regional study of 5-HT1A, 5-HT2A receptors and the serotonin transport site in rat brain

Serotonin (5-HT) plays a central role in the neurochemistry of the learned helplessness animal model of depression. Using quantitative autoradiography, we measured the density of 5-HT1A and 5-HT2A receptors and of 5-HT transport sites in medial prefrontal cortex, dorsal hippocampus, septum, hypothalamus, and amygdala in learned helpless rats, and in rats that were nonhelpless after inescapable stress, as well as in shuttlebox-tested and nonhandled controls. We found no changes in 5-HT1A receptor density among the groups in any region studied. In dorsal hippocampus, 5-HT2A receptor density was decreased in nonhelpless rats, while in amygdala 5-HT2A receptor density was decreased in both groups of stressed rats, whether helpless or nonhelpless. In the hypothalamus 5-HT2A receptor density, was decreased in helpless rats as compared to controls. In medial prefrontal cortex, the serotonin transport sites showed decreased density in helpless rats as compared to controls but not to nonhelpless rats. These findings further highlight the complexity of regional 5-HT effects in the learned helplessness animal model.