Ana G. Gutiérrez-García

The lowest effective dose of fluoxetine in the forced swim test significantly affects the firing rate of lateral septal nucleus neurones in the rat

The administration of a relatively high dose of antidepressant drugs produces an increased neuronal firing rate of the lateral septal nucleus (LSN) in the rat and a decreased immobility in rats forced to swim. However, it is unknown whether a minimally effective low-dose 21-day treatment with the selective serotonin reuptake inhibitor, fluoxetine, while reducing immobility in the forced swim test, also increases the neuronal firing rate of the LSN in Wistar rats. The total time of immobility decreased with a daily injection of 0.5, 1.0 or 2.0 mg/kg of fluoxetine (p< 0.001), and the lowest dose increasing the latency to the first immobility period (p < 0.0001) was 1.0 mg/kg. Therefore, the action of the 21-day fluoxetine treatment (1.0 mg/kg) on the firing rate of LSN neurones was tested in another group of rats. A total amount of 78 single-unit extracellular recordings was taken from the LSN of eight control rats (n = 40) and eight fluoxetine treated rats (n = 38). The LSN firing rate in the fluoxetine group was double (18.3 ± 2.5 spikes per 10 s, p< 0.05) that in the control group (7.0 ± 0.9 spikes per 10 s), and the first order interval of firing proved to be significantly lower in the fluoxetine group compared to the control group (384.3 ± 22.3 and 639.7 ± 27.5 ms, respectively; p < 0.05). In conclusion, the increased neuronal firing rate of the LSN in the animals treated with a low dose of fluoxetine may be associated with an increased motivation to escape from the stressful situation that the forced swim represents.

Allopregnanolone reduces immobility in the forced swimming test and increases the firing rate of lateral septal neurons through actions on the GABAA receptor in the rat

Since allopregnanolone reduces the total time of immobility in rats submitted to the forced swimming test, we decided to explore whether this neuroactive steroid shares other antidepressant-like actions, such as increasing the neuronal firing rate in the lateral septal nucleus (LSN). In order to discard the influence of the oestrous cycle on immobility and on the firing rate of LSN neurons, all Wistar rats used in the study underwent ovariectomy before treatments. A group of rats received different doses of allopregnanolone (0.5, 1.0, 2.0 and 3.0mg/kg, i.p.) 1 hour before being forced to swim in order to identify the minimum effective dose diminishing immobility. None of the tested doses of allopregnanolone produced significant changes in motor activity in the open-field test. The minimum dose of allopregnanolone producing a significant reduction in the total time of immobility (p<0.05) against the vehicle was 1.0mg/kg, while 2.0mg/kg and above also increased the latency to the first period of immobility (p<0.05). The minimum effective dose of allopregnanolone reducing immobility in the forced swimming test (1.0mg/kg) significantly (p <0.05) produced a higher (twofold) neuronal firing rate in LSN neurons, but did not produce any change in septofimbrial nucleus neurons, which fired at a rate similar to that of vehicle-treated rats. The pretreatment with the non-competitive GABAA receptor antagonist, picrotoxin (1.0mg/kg), blocked the aforementioned actions of allopregnanolone on both immobility and LSN firing rate. In conclusion, allopregnanolone produces an antidepressant-like effect in the forced swimming test, associated with an increase in the LSN neuronal firing rate, seemingly mediated by the GABAA receptor.

Anxiolytic-like effects of human amniotic fluid and its fatty acids in Wistar rats.

Odors from amniotic fluid produce signs of calmness in mammals suggesting some anxiolytic-like properties. Experimental models, such as the defensive burying, elevated plus maze, and open field tests offer well-controlled approaches to the study of putative anxiolytic substances using rats. Using gas chromatography–mass spectrometry, we first identified eight fatty acids (lauric, myristic, palmitic, palmitoleic, stearic, oleic, elaidic, and linoleic acids) as consistently present in human amniotic fluid. We then used the defensive burying and elevated plus maze tests to compare the action of diazepam (2 mg/kg), fresh amniotic fluid, and a mixture of its fatty acids with two vehicles (i.e. propylene glycol and centrifuged amniotic fluid with a low fatty acid content). No significant differences in estradiol or progesterone content were found between fresh amniotic fluid and centrifuged amniotic fluid using the microparticle enzyme immunoassay. Compared with the vehicle, diazepam, fresh amniotic fluid, and the fatty acid mixture increased burying latency, reduced cumulative burying, and increased the time spent in the open arms of the elevated plus maze in both sexes without altering general locomotor activity. We conclude that the fatty acids contained in human amniotic fluid exert anxiolytic-like effects, with minimal or no participation of female gonadal steroids.

Stressors can affect immobility time and response to imipramine in the rat forced swim test.

We subjected Wistar rats to the forced swim test (FST) to compare the effects of two doses of imipramine in physically stressed rats (P: unavoidable electric footshocks), emotionally stressed rats (E: odors), or non-stressed rats (C). Stress or control sessions lasted 35 days. Drug treatments began on day 21 and continued for the next 14 days. E rats were placed for 10 min, once per day for 35 days, in a small non-movement-restricting cage impregnated with urine collected from a P rat. E and P rats exhibited opposite changes in locomotion. After 21 days of stress sessions, P rats displayed the longest immobility times in the FST, followed by E rats. In the P group, on day 7 of treatment (day 28 of the study), imipramine (2.5 mg/kg) reduced immobility time to baseline values. In the E group, immobility time decreased only after 14 days of treatment with the low imipramine dose. The high dose of imipramine (5.0 mg/kg) reduced immobility time at day 7 of treatment in all groups. In conclusion, physical and emotional stress similarly increased immobility time in the FST, but emotional stress appears to be more resistant to imipramine treatment.

Amniotic fluid elicits appetitive responses in human newborns: Fatty acids and appetitive responses

In humans, maternal cues guide newborns to the maternal breast, and transitional cues may be present in maternal-fetal fluids. The aim of the present study was to determine the consistent presence of sensorial cues in three maternal-fetal fluids--amniotic fluid, colostrum, and milk--and test the ability of these cues to produce appetitive responses in newborns. In the analytical study, gas chromatography-mass spectrometry (GC-MS) detected eight fatty acids consistently present in the amniotic fluid, colostrum, and milk from 12 healthy volunteers, but we do not find a mammalian pheromone, identified in another mammalian species (rabbits), in another 30 volunteers. In the behavioral study, we explored the ability of amniotic fluid or its fatty acids to produce appetitive responses in 19 human newborns <24u2009hr after birth. Exposure to swabs impregnated with amniotic fluid or an artificial fatty acid mixture produced a longer duration of facial reactions that suggested appetitive (sucking) movements compared with respective vehicles (i.e., propylene glycol or centrifuged amniotic fluid with a low fatty acid content verified by GC-MS). We conclude that the fatty acids contained in amniotic fluid may constitute a transitional sensorial cue that guides newborns to the maternal breast.

Testosterone reduces cumulative burying in female Wistar rats with minimal participation of estradiol

Testosterone exerts anxiolytic effects, but the participation of its aromatase metabolic product estradiol is controversial. Therefore, we used the defensive burying paradigm in female Wistar rats to explore testosterones (1.0 mg/rat, s.c.) interactions with picrotoxin (a noncompetitive gamma-aminobutyric acid-A receptor [GABA(A)] antagonist; 1.0 mg/kg, i.p.), formestane (an aromatase inhibitor; 3.0 mg/rat, s.c.), and tamoxifen (an estrogen receptor-beta antagonist; 1.0 mg/kg, s.c.). Serum levels of testosterone, estradiol, and progesterone were determined in the same rats. Burying latency and locomotion did not significantly change. Systemic testosterone administration enhanced serum testosterone and estradiol levels and reduced defensive burying. This reduction in total burying was blocked by pretreatment with picrotoxin and tamoxifen, but not formestane. We conclude that testosterone produced anxiolytic-like effects in female rats that were mediated by actions at the GABA(A) receptor, with participation of the estradiol receptor-beta, rather than estradiol aromatization.

2-Heptanone Increases the Firing Rate of the Basal Amygdala: Role of Anterior Olfactory Epithelial Organs

Wistar rats subjected to physical stress release a urine alarm pheromone (2-heptanone) that produces signs of anxiety and despair in receptor rats not subjected to physical stress. However, unknown are the effects of 2-heptanone on the firing rate of the basal amygdala, a structure that participates in the expression of fear, and the participation of anterior olfactory epithelial organs, namely the septal organ and vomeronasal organ (SO-VNO). We explored the effects of 2-heptanone applied near the nostrils on single-unit extracellular recordings from the basal amygdala in a sham-operated group and rats that underwent removal of the SO-VNO. The firing rate of basal amygdala neurons in the SO-VNO removal group was significantly higher than in the sham-operated group. In both groups, recordings were classified according to the responses to 2-heptanone (i.e., increased firing rate, decreased firing rate, and no response). SO-VNO removal was associated with an increased firing rate in the three types of neurons. A similar number of neurons increased their firing rate during and after 2-heptanone stimulation in both groups, but such an increase in firing rate was longer in the group of rats subjected to SO-VNO removal. The results indicate that the SO-VNO is not essential for the effect of 2-heptanone on the firing rate of basal amygdala neurons. SO-VNO ablation did not block but rather accentuated the response of amygdala neurons to 2-heptanone.

Anxiety: An Adaptive Emotion

Anxiety as an adaptive response is a natural emotion that occurs in response to danger and prepares an organism to cope with the environment, playing a critical role in its survival. Among the components of anxiety, the expression of fear may inform other members of the group about the presence of imminent danger (i.e., an alarm cue). The environment is per‐ ceived by a filtering process that involves sensorial receptors. While coping with a stressful situation, an individual may simultaneously emit vocalizations, perform movements to es‐ cape, freeze, and deliver to the environment chemicals called alarm pheromones. These cues are recognized by the receptor-individual by specific sensory systems located in the legs and antennae in insects and olfactory sensorial systems in other organisms. In mammals, the sensorial information is integrated by anatomical and functional pathways, with the partici‐ pation of structures related to emotional memory, namely deep temporal lobe structures. Some stimuli are perceived as relevant when they contain relevant meaning according to previous experience and learning. The participation of ventral striatum and prefrontal cor‐ tex connections then leads to the selection of an adequate strategy for survival. The percep‐ tion of these cues by other individuals in the group establishes intraspecies communication and causes striking behavioral responses in the receptor subject, namely anxiety, but the consequence is likely different. While the emitting subject may be in an emergency situation that is perhaps devoid of a solution, the receptor subject may have the chance to cope with the dangerous situation by employing efficacious strategies, depending on previous experi‐ ence. The aim of this chapter is to review the participation of such anatomical pathways, their neurotransmission systems, and the resulting behavioral patterns.

Progesterone modifies the responsivity of the amygdala-mPFC connection in male but not female Wistar rats

Amygdala-medial prefrontal cortex (mPFC) connections partially regulate fear, anxiety, and the acquisition of conditioned fear. Progesterone exerts some effects on anxiety and fear. Currently unknown, however, are the actions of progesterone on the responsivity of amygdala-mPFC connections and possible sex differences. We performed single-unit extracellular recordings from the prelimbic (PL) and infralimbic (IL) cortices of the mPFC during stimulation of the basal amygdala (BA) in anesthetized male and diestrus female rats. Basal amygdala stimulation produced an initial excitatory paucisynaptic response that was similar between sexes and unaffected by progesterone. A long-lasting inhibitory response followed the initial brief excitatory response, which was more pronounced in the PL region in males. The unit activity ratio analysis indicated that progesterone negated the sex difference in the PL region response to BA stimulation. The results suggest that progesterone decreases the responsivity to amygdala stimulation, particularly in males compared with diestrus females, which may be related to sex differences in the strategies to cope with threatening situations.

Amniotic Fluid or Its Fatty Acids Produce Actions Similar to Diazepam on Lateral Septal Neurons Firing Rate

Human amniotic fluid (AF) contains eight fatty acids (FATs), and both produce anxiolytic-like effects in adult rats and appetitive responses in human newborns. The medial amygdala and lateral septal nucleus function are related to social behavior, but the action of AF or its FATs in this circuit is known. We obtained 267 single-unit extracellular recordings in Wistar rats treated with vehicle (1u2009mL, s.c.; n = 12), human AF (1u2009mL, s.c.; n = 12), a FAT mixture (1u2009mL, s.c.; n = 13), diazepam (1u2009mg/kg, i.p.; n = 11), and fluoxetine (1u2009mg/kg, p.o.; n = 12). Compared with the vehicle group, the spontaneous septal firing rate in the AF, FAT mixture, and diazepam groups was the lowest and in the fluoxetine group the highest. Cumulative peristimulus histograms indicated that the significant change in septal firing occurred only in the AF and FAT mixture groups and exclusively in those neurons that increased their firing rate during amygdala stimulation. We conclude that human AF and its FATs produce actions comparable to anxiolytic drugs and are able to modify the responsivity of a circuit involved in social behavior, suggesting facilitation of social recognition processes by maternal-fetal fluids.