Anxiolytic effect of estradiol in the median raphe nucleus mediated by 5-HT1A receptors.

Estrogen deficiency has been associated with stress, anxiety and depression. Estrogen receptors have been identified in the median raphe nucleus (MRN). This structure is the main source of serotonergic projections to the hippocampus, a forebrain area implicated in the regulation of defensive responses and in the resistance to chronic stress. There is evidence showing that estrogen modulates 5-HT1A receptor functions. In the MRN, somatodendritic 5-HT1A receptors control the activity of serotonergic neurones by negative feedback. The present study evaluated the effect of intra-MRN injection of estradiol benzoate (EB) (600 or 1200ng/0.2microl) on the performance of ovariectomised rats submitted to the elevated plus-maze test of anxiety and to the open-field test. Additionally, the same effect was evaluated with a previous intra-MRN injection of WAY 100635(100ng/0.2microl), an antagonist of 5-HT1A receptors. The results showed that both doses of EB increased the percentage of entries and the percentage of time spent into the open arms, suggestive of an anxiolytic effect. The highest dose of the drug also increased the number of entries into the enclosed arm and locomotion in the open field, indicating a stimulatory motor effect. WAY 100635 antagonised the effect of estradiol in the elevated plus-maze and in the open-field. The results show that estrogen receptors of the MRN are implicated in the regulation of anxiety-related behaviour. The results also support claims that the effect of estrogen involves a change in 5-HT1A receptor function.

5-HT mechanisms of median raphe nucleus in the conditioned freezing caused by light/foot-shock association.

We have shown that 5-HT mechanisms of the median raphe nucleus (MRN) are involved in contextual fear-conditioning processes as electrolytic or neurotoxic lesions with N-methyl-D-aspartate (NMDA) or injections of 8-hydroxy-2-(di-n-propilamino)-tetralin (8-OH-DPAT) into this structure inhibit freezing behavior in a contextual fear paradigm. In this work, we extend these studies by analyzing the behavioral responses in a classical fear-conditioning paradigm (light or tone/foot-shock association) in rats with either neurochemical lesion with NMDA or injected with 8-OH-DPAT into the MRN. The animals received NMDA or 8-OH-DPAT or saline microinjections into the MRN and were submitted to conditioning trials in an experimental chamber, where they received 10 foot-shocks (0.6 mA, 1 s, variable interval between 10 and 50 s) paired with tone or light (CS). On the next day, they were tested in a different experimental chamber, with or without CS presentation, where the duration of freezing and the number of rearing episodes were recorded. Light or tone alone caused a significant amount of freezing. NMDA lesions or 8-OH-DPAT injections into the MRN clearly inhibited freezing behavior in rats conditioned to light/foot-shock association, but not in the conditioning sessions with tones. Besides the proposed role in contextual fear conditioning, these results clearly show that MRN is involved in the fear conditioning with light as conditioned stimuli. Distinct neural substrates seem to subserve conditioning fear with acoustic stimuli.

Distinct contributions of median raphe nucleus to contextual fear conditioning and fear-potentiated startle.

Ascending 5-HT projections from the median raphe nucleus (MRN), probably to the hippocampus, are implicated in the acquisition of contextual fear (background stimuli), as assessed by freezing behavior. Foreground cues like light, used as a conditioned stimulus (CS) in classical fear conditioning, also cause freezing through thalamic transmission to the amygdala. As the MRN projects to the hippocampus and amygdala, the role of this raphe nucleus in fear conditioning to explicit cues remains to be explained. Here we analyzed the behavior of rats with MRN electrolytic lesions in a contextual conditioning situation and in a fear-potentiated startle procedure. The animals received MRN electrolytic lesions either before or on the day after two consecutive training sessions in which they were submitted to 10 conditioning trials, each in an experimental chamber (same context) where they received foot-shocks (0.6 mA, 1 sec) paired to a 4-sec light CS. Seven to ten days later, the animals were submitted to testing sessions for assessing conditioned fear when they were placed for five shocks, and the duration of contextual freezing was recorded. The animals were then submitted to a fear-potentiated startle in response to a 4-sec light-CS, followed by white noise (100 dB, 50 ms). Control rats (sham) tested in the same context showed more freezing than did rats with pre- or post-training MRN lesions. Startle was clearly potentiated in the presence of light-CS in the sham-lesioned animals. Whereas pre-training lesions reduced both freezing and fear-potentiated startle, the post-training lesions reduced only freezing to context, without changing the fear-potentiated startle. In a second experiment, neurotoxic lesions of the MRN with local injections of N-methyl-D-aspartate or the activation of 5-HT1A somatodendritic auto-receptors of the MRN by microinjections of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) before the training sessions also reduced the amount of freezing and the fear-potentiated startle. Freezing is a prominent response of contextual fear conditioning, but does not seem to be crucial for the enhancement of the startle reflex by explicit aversive cues. As fear-potentiated startle may be produced in post-training lesioned rats that are unable to freeze to fear contextual stimuli, dissociable systems seem to be recruited in each condition. Thus, contextual fear and fear-potentiated startle are conveyed by distinct 5-HT-mediated circuits of the MRN.

Activation of somatodendritic 5-HT(1A) autoreceptors in the median raphe nucleus disrupts the contextual conditioning in rats.

We have shown that the median raphe nucleus (MRN) is involved in the control of contextual fear conditioning. Also, electrolytic lesion in the MRN causes signs of behavioral disinhibition, with an increase in locomotor activity measured in an open field. In this work, we have extended this study by analyzing the behavioral and autonomic responses in a contextual conditioning paradigm in rats with either neurochemical lesions from N-methyl-D-aspartate (NMDA), or microinjected with 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) in the MRN. The animals received NMDA, 8-OH-DPAT or saline microinjections into the MRN before the conditioning experiments, during which they were subjected to ten conditioning trials in an experimental chamber (same context) where they received foot-shocks (0.7 mA, 1 s with 20 s intervals). The next day, the animals were tested again either in the same or in a different experimental chamber, where the duration of freezing, number of rearings, bouts of micturition and number of fecal boli were recorded. Control rats placed in the same chamber showed more freezing than rats exposed to a different context. This freezing behavior was clearly inhibited in rats with NMDA lesions or with 8-OH-DPAT microinjections into the MRN. These MRN manipulations also produced a reduction in micturition. Such effects were context-specific since 8-OH-DPAT in MRN did not affect fear conditioned responses to tone previously associated with foot-shocks. In the open-field test, rats with neurotoxic lesions or with 8-OH-DPAT microinjections into the MRN had higher horizontal locomotor activity than control rats, without the number of rearings changing. As rats injected with 8-OH-DPAT into the MRN may freeze to a tone in a conditioned test paradigm and also show increased activity in an open field test the functional role of MRN on locomotor activity and contextual fear conditioning are clearly dissociated. Altogether, these results clearly show that such 5-HT mechanisms of the MRN serve as a neural substrate for the storage process of the context fear.

Antinociception elicited by aversive stimulation of the inferior colliculus.

We have shown that the inferior colliculus is involved in the integration of defensive reactions. Electrical and chemical stimulation of this structure elicits fear and escape behavior, expressed respectively by immobility (freezing) and wild running, followed by jumps. In this study, we analyzed whether the defensive behavior integrated at this level of the midbrain tectum is also followed by antinociception and its chemical mediation. In addition, we further addressed whether or not the aversive states and the stress-induced analgesia share the same neural substrates in the inferior colliculus. To this end, animals chronically implanted with a chemitrode, an electrode glued to a guide cannula, in the inferior colliculus were injected with naltrexone, methysergide, ketanserin, and midazolam. The animals were submitted to gradual increases in the electrical stimulation of the inferior colliculus, which allowed the measurement of the thresholds for aversive responses--vigilance, freezing, and escape. Following the induction of the aversive behavioral responses the animals were submitted to the tail-flick test. The results obtained show that midazolam was the only treatment that changed the aversive thresholds. On the other hand, while naltrexone and midazolam did not affect the fear-induced analgesia, it was inhibited by microinjections of the serotonergic blockers, methysergide and ketanserin. These results emphasize previous data demonstrating the nonopioid nature of the unconditioned analgesia to brain-aversive stimulation. Because methysergide is a nonspecific antagonist of 5-HT receptors, and ketanserin acts with a high degree of specificity at 5-HT2/5-HT1C receptors, the present results suggest that activation of 5-HT2/5-HT1C receptors may be implicated in the antinociception induced by stimulation of the inferior colliculus. Moreover, the present data also indicate that aversive reactions and analgesia from inferior colliculus stimulation can be pharmacologically dissociated.

Regulation of contextual conditioning by the median raphe nucleus.

The median raphe nucleus (MRN) has been suggested as the origin of a behavioral inhibition system that projects to the septum and hippocampus. Electrical stimulation of this mesencephalic area causes behavioral and autonomic manifestations characteristic of fear such as, freezing, defecation and micturition. In this study we extend these observations by analyzing the behavioral and autonomic responses of rats with lesions in the MRN submitted to a contextual conditioning paradigm. The animals underwent electrolytic or sham lesions of the median raphe nucleus. One day (acute) or 7 days (chronic) later they were tested in an experimental chamber where they received 10 foot-shocks (0.7 mA, 1 s with 20-s interval). The next day, sham and MRN-lesioned animals were tested again either in the same or in a different experimental chamber. During this, the duration of freezing, rearings, bouts of micturition and number of fecal boli were recorded. Sham-operated rats placed in the same chamber showed more freezing than rats exposed to a different context. This freezing behavior was clearly suppressed in rats with acute or chronic lesions in the MRN. MRN lesions also reduced the bouts of micturition and number of fecal boli. These rats showed a reduced number of rearings than sham-lesioned rats. This effect is probably the result of the displacement effect provoked by freezing since no significant differences in the number of rearings could be observed between these animals and the NMR-lesioned rats tested in an open field. This lesion produced higher horizontal locomotor activity in this test than the controls (sham-lesioned rats). These results point to the importance of the median raphe nucleus in the processing of fear conditioning with freezing being the most salient feature of it. Behavioral inhibition is also under control of MRN but its neural substrate seems to be dissociated from that of contextual fear.