Julie G. Hensler

Serotonergic modulation of the limbic system.

The limbic system is composed of cortical as well as subcortical structures, which are intimately interconnected. The resulting macrostructure is responsible for the generation and expression of motivational and affective states. Especially high levels of serotonin are found in limbic forebrain structures. Serotonin projections to these structures, which arise from serotonergic cell body groups in the midbrain, form a dense plexus of axonal processes. In many areas of the limbic system, serotonergic neurotransmission can best be described as paracrine or volume transmission, and thus serotonin is believed to play a neuromodulatory role in the brain. Serotonergic projections to limbic structures, arising primarily from the dorsal and median raphe nuclei, compose two distinct serotonergic systems differing in their topographic organization, electrophysiological characteristics, morphology, as well as sensitivity to neurotoxins and perhaps psychoactive or therapeutic agents. These differences may be extremely important in understanding the role of these two serotonergic systems in normal brain function and in mental illness. Central serotonergic neurons or receptors are targets for a variety of therapeutic agents used in the treatment of disorders of the limbic system.

Regulation of 5-HT1A receptor function in brain following agonist or antidepressant administration

Adaptive changes in the serotonergic system are generally believed to underlie the therapeutic effectiveness of the azapirone anxiolytics and a variety of antidepressant drugs. The serotonin-1A (5-HT(1A)) receptor has been implicated in affective disorders. Thus, studies of the regulation of 5-HT(1A) receptor function may have important implications for our understanding the role of this receptor in the mechanism of action of these therapeutic agents. This review focuses on the regulation of central 5-HT(1A) receptor function following administration of 5-HT(1A) receptor agonists or antidepressant drugs expected to increase the synaptic concentration of the neurotransmitter 5-HT. The majority of evidence supports regional differences in the regulation of central 5-HT(1A) receptor function following repeated agonist or antidepressant administration, which may be due to differences in processes involved in desensitization of the receptor at the cellular level. Region-specific differences in the regulation of 5-HT(1A) receptor function may be based on compensatory changes distal to the receptor, such as regulatory changes at the level of effector (e.g. adenylyl cyclase or ion channel), or at the level of the G protein such as changes in G protein expression, or phosphorylation of the G protein. It may be that the increase in serotonin neurotransmission, due to somatodendritic autoreceptor desensitization following agonist or antidepressant treatment, to normo-sensitive 5-HT(1A) receptors in certain brain regions (e.g. hippocampus or cortex) and to sub-sensitive 5-HT(1A) receptors in other brain regions (e.g. amygdala or hypothalamus) underlies the therapeutic efficacy of these drugs.

Differential regulation of 5-HT1A receptor-G protein interactions in brain following chronic antidepressant administration

Changes in 5-HT1A receptor function or sensitivity following chronic antidepressant treatment may involve changes in receptor-G protein interaction. We have examined the effect of chronic administration of the SSRI fluoxetine or the tricyclic antidepressant amitriptyline on 5-HT1A receptor-stimulated [35S]GTPγS binding in serotonergic cell body areas, and cortical and limbic structures using quantitative autoradiography. Treatment of rats with fluoxetine, but not amitriptyline, resulted in an attenuation of 5-HT1A receptor-stimulated [35S]GTPγS binding in the dorsal and median raphe nuclei. The binding of the antagonist radioligand [3H]MPPF to 5-HT1A receptor sites was not altered, suggesting that the observed changes in 5-HT1A receptor-stimulated [35S]GTPγS binding were not due to changes in receptor number. Thus, the desensitization of somatodendritic 5-HT1A autoreceptors in the dorsal and median raphe following chronic SSRI treatment appears to be due to a reduced capacity of the 5-HT1A receptor to activate G protein. By contrast, no significant change in postsynaptic 5-HT1A receptor-stimulated [35S]GTPγS binding was observed in any of the forebrain areas examined following chronic antidepressant treatment. Thus, changes in postsynaptic 5-HT1A receptor-mediated responses reported to follow chronic SSRI or tricyclic antidepressant administration most likely occur distal to receptor-G protein interaction, perhaps at the level of effector, or involving changes in neuronal function at the system or circuit level.

Ethanol consumption and serotonin-1A (5-HT1A) receptor function in heterozygous BDNF (+/–) mice

Heterozygous brain‐derived neurotrophic factor (BDNF) (+/–) mice display abnormalities in central serotonergic neurotransmission, develop decrements in serotonergic innervation of the forebrain, and exhibit enhanced intermale aggressiveness. As disturbances of serotonin neurotransmission are implicated in alcohol abuse and aggression, we have examined in BDNF (+/–) mice alcohol drinking behavior, as well as central 5‐hydroxytryptamine (5‐HT)1A receptor function at the level of 5‐HT1A receptor–G protein interaction. BDNF (+/–) mice displayed increased ethanol intake in a two‐bottle choice procedure. There was no difference in the preference ratio for non‐alcoholic tastants (i.e. quinine or saccharin) between genotypes. In the brains of alcohol‐naive mice, we measured [35S]GTPγS binding stimulated by the 5‐HT1A receptor agonist (+/–)‐8‐hydroxy‐2‐dipropyl‐aminotetralin hydrobromide (8‐OH‐DPAT; 1 µm). In BDNF (+/–) versus wild‐type (WT) mice, 5‐HT1A receptor‐stimulated [35S]GTPγS binding was significantly attenuated in the median raphe nucleus. There was a decrease in (+/–)8‐OH‐DPAT‐stimulated [35S]GTPγS binding in the dorsal raphe, which did not reach statistical significance. In the hippocampus, 5‐HT1A receptor–stimulated [35S]GTPγS binding was significantly attenuated in BDNF (+/–) mice. 5‐HT1A receptor–stimulated [35S]GTPγS binding was attenuated in the anterior cingulate cortex and lateral septum, although these reductions did not reach statistical significance. 5‐HT1A receptor number was not different between genotypes in any area of brain examined, suggesting that 5‐HT1A receptor function, specifically the capacity of the 5‐HT1A receptor to activate G proteins, is attenuated in BDNF (+/–) mice.

Density and function of central serotonin (5-HT) transporters, 5-HT1A and 5-HT2A receptors, and effects of their targeting on BTBR T+tf/J mouse social behavior

J. Neurochem. (2011) 116, 291–303.

Effect of early rearing conditions on alcohol drinking and 5-HT1A receptor function in C57BL/6J mice

We have evaluated in C57BL/6J mice the effect of maternal separation and post-weaning social isolation on ethanol intake, and on serotonin1A (5-HT1A) receptor function at the level of receptor-G protein interaction in the hippocampus and dorsal raphe nucleus. From postnatal days 2-14, litters were separated from the mother for 15 min (Handled) or for 180 min (Maternal separation). After weaning, pups were housed in pairs or in social isolation. At 2 months of age, ethanol intake and preference in mice were assessed using the two-bottle choice paradigm. Maternal separation increased ethanol preference in female mice that were subsequently housed in isolation. By contrast, post-weaning isolation increased ethanol preference and consumption in male mice regardless of pre-weaning rearing conditions. The increased ethanol preference and intake were limited to a 5% (v/v) concentration of ethanol. Our data suggest that adolescent mice are susceptible to the effects of post-weaning social isolation as shown by increased ethanol preference and consumption. Using quantitative autoradiography, 5-HT1A receptor number and function were determined by the binding of [3H]WAY-100635, and by [35S]GTPgammaS binding stimulated by the 5-HT1A receptor agonist 8-OH-DPAT, respectively. The binding experiments were done at approximately 3 months after the end of the two-bottle choice test in an attempt to minimize direct effects of ethanol drinking on 5-HT1A receptor function and number. 5-HT1A receptor-stimulated [35S]GTPgammaS binding in the dorsal raphe nucleus was increased in animals reared after weaning in isolation vs. in pairs, regardless of gender or pre-weaning rearing conditions. Our data suggest that there are long-term neurochemical consequences of social isolation of adolescent mice, specifically increased 5-HT1A receptor function in the dorsal raphe nucleus.

Serotonin transporter function, but not expression, is dependent on brain-derived neurotrophic factor (BDNF): In vivo studies in BDNF-deficient mice

In the present study, we used high‐speed chronoamperometry to examine serotonin (5‐HT) transporter (5‐HTT) function in vivo in 2‐, 5‐, and 10‐month‐old brain‐derived neurotrophic factor (BDNF)+/− mice. The rate of clearance of exogenously applied 5‐HT was measured in CA3 region of hippocampus. In 2‐month‐old mice, the rate of 5‐HT clearance did not differ between BDNF+/+ and BDNF+/− mice. In BDNF+/+ mice, 5‐HT clearance rate (Tc) increased markedly with age. In contrast, Tc remained relatively static in BDNF+/− mice across 2‐, 5‐, and 10‐month age groups. At 5 months of age, female BDNF+/+ mice had a lower maximal velocity (Vmax) for 5‐HT clearance than male BDNF+/+ mice. There was a similar trend in 5‐month‐old BDNF+/− mice, but this did not reach statistical significance. There was an age‐dependent increase in KT value for 5‐HT clearance (i.e., decreased in vivo affinity of 5‐HTT), but no significant effect of genotype or gender. 5‐HTT density, as measured by [3H]cyanoimipramine binding, was not different between BDNF+/+ and BDNF+/− mice, although there was a significant increase in 5‐HTT binding with age. The selective 5‐HT reuptake inhibitor fluvoxamine (50 and 100 pmol) significantly decreased 5‐HT clearance in BDNF+/+ mice, but not in BDNF+/− mice. Our data suggest that the profoundly reduced ability of 5‐ and 10‐month‐old BDNF+/− mice to clear 5‐HT is not because of a decrease in the total number of 5‐HTTs, but may be due to functional deficits in the 5‐HTT, e.g., in the machinery/signaling required for insertion of 5‐HTTs into the plasma membrane and/or activation of the 5‐HTT once it is positioned to take up 5‐HT from extracellular fluid.

γ-Aminobutyric Acid (GABA)-A Function and Binding in the Paraventricular Nucleus of the Hypothalamus in Chronic Renal-Wrap Hypertension

The goal of this study was to determine whether &ggr;-aminobutyric acid (GABA)ergic transmission and GABA binding are altered in chronic renal-wrap hypertension. Three groups of hypertensive and sham-operated rats were prepared for separate protocols. Four weeks later, the animals were prepared with femoral artery catheters for the measurement of mean arterial pressure. In all groups, blood pressure was significantly higher in the renal-wrapped animals. In the first study, bilateral microinjection of the GABA-A antagonist, bicuculline (50 pmol/site), into the paraventricular nucleus of the hypothalamus (PVN) caused a greater increase in arterial pressure (21.9±1.4 versus 16.7±1.8 mm Hg, P <0.05) and heart rate (135±15 versus 98±12 bpm, P =0.064) in hypertensive rats. [3H]Flunitrazepam was used to measure binding to the GABA-A receptor. Magnocellular neurons and the adjacent medial parvicellular neurons had more intense binding compared with the remainder of the PVN. Bmax was greater for the higher density binding area; the Kd value was less in the high-density region. There were no differences in these parameters between normotensive and hypertensive animals. Competitive reverse transcription–polymerase chain reaction was used to measure the expression of mRNA for the &agr;1 subunit of the GABA-A receptor. No difference was observed in the mRNA between renal-wrapped and sham-operated rats. In summary, inhibition of GABA-A receptors in the PVN is augmented in the chronic phase of hypertension and is unrelated to a change in the expression of the number or affinity to the receptor. These findings suggest that the greater GABAergic activity is the result of an increase in GABA release in the PVN in chronic renal-wrap hypertension.

Regulation of 5-HT1A receptor-stimulated [35S]-GTPγS binding as measured by quantitative autoradiography following chronic agonist administration

Because changes 5‐HT1A receptor number do not occur following repeated agonist treatment, we hypothesized that the basis for 5‐HT1A receptor desensitization involves changes in receptor‐G protein coupling. We measured the effect of repeated agonist administration on 5‐HT1A receptor‐stimulated [35S]‐GTPγS binding in forebrain areas, (i.e. anterior cingulate cortex, lateral septum, hippocampus, entorhinal cortex), and serotonergic cell body areas, the dorsal and median raphe nuclei. Following treatment of rats with (±)8‐OH‐DPAT (1 mg kg−1, s.c.) for 7 or 14 days, 5‐HT1A receptor‐stimulated [35S]‐GTPγS binding was significantly attenuated in both the dorsal and median raphe nuclei. 5‐HT1A receptor‐stimulated [35S]‐GTPγS binding was significantly attenuated in the CA1 region of the hippocampus after 7, but not 14 days of 8‐OH‐DPAT administration. 5‐HT1A receptor‐stimulated [35S]‐GTPγS binding was not altered in other forebrain areas examined. The binding of [3H]‐MPPF to 5‐HT1A receptor sites was not altered in any brain region examined following repeated agonist administration, suggesting that the observed changes in (±)8‐OH‐DPAT‐stimulated [35S]‐GTPγS binding were not due to changes in 5‐HT1A receptor number. Our data indicate that in serotonergic cell body areas the regulation of presynaptic 5‐HT1A receptor function following repeated agonist administration occurs at the level of receptor‐G protein interaction. In forebrain areas, however, the regulation of postsynaptic 5‐HT1A receptor sensitivity appears not to be at the level of receptor‐G protein coupling.

Fluoxetine disrupts food intake and estrous cyclicity in Fischer female rats

Adult, regularly cycling female Fischer rats were injected daily with 10 mg/kg fluoxetine for 12-23 days. In the first experiment, body weight and vaginal smears were monitored daily. Fluoxetine treatment reduced body weight within the first 24 h of treatment. Fluoxetine treatment also elongated the estrous cycle, reduced blood levels of progesterone, and eliminated lordosis behavior. In the second experiment, body weight and food intake were examined and a pair-fed group was included to determine if fluoxetine-induced anorexia contributed to the disturbance of the estrous cycle. In pair-fed rats, effects similar to fluoxetine treatment were present. These results lead to the suggestion that fluoxetines anorectic properties could disrupt the females normal endocrine cyclicity and that this disruption could be relevant to the reduction in sexual behavior and motivation. However, when the duration of fluoxetine treatment was extended beyond 16 to 17 days, fluoxetine-treated female rats reinitiated vaginal cyclicity and showed evidence of normal sexual receptivity. In contrast, the estrous cycles of their pair-fed counterparts remained disrupted. Thus, restricted food intake appears to contribute to the disruption of the estrous cycle and elimination of sexual receptivity during fluoxetine treatment. However, compensatory changes in the serotonergic system that are associated with chronic fluoxetine administration may contribute to the gradual recovery of estrous cyclicity and sexual receptivity of the fluoxetine-treated animals.