5-HT1A receptors of the prelimbic cortex mediate the hormonal impact on learned fear expression in high-anxious female rats.

Hormones highly influence female behaviors. However, research on this topic has not usually considered the variable hormonal status. The prelimbic cortex (PrL) is commonly engaged in fear learning. Connections from and to this region are known to be critical in regulating anxiety, in which serotonin (5-HT) plays a fundamental role, particularly through changes in 5-HT1A receptors functioning. Also, hormone fluctuations can greatly influence anxiety in humans and anxiety-related behavior in rodents, and this influence involves the functioning of 5-HT brain systems. The present investigation sought to determine whether fluctuations in ovarian hormones relative to the estrous cycle would influence the expression of learned fear in female rats previously selected as low- (LA) or high-anxious (HA). Furthermore, we investigate the role of the 5-HT system of the PrL, particularly the 5-HT1A receptors, as a possible modulator of estrous cycle influence on the expression of learned fear through intra-PrL microinjections of 5-HT itself or the full 5-HT1A agonist 8-OH-DPAT (8-hydroxy-2-(di-n-propylamine)tetralin). Behavioral changes were assessed using the fear-potentiated startle (FPS) procedure. The results showed that fear intensity is associated with hormonal decay, being more accentuated during the estrus phase. This increase in fear levels was found to be negatively correlated with the expression of potentiated startle. In rats prone to anxiety and tested during the proestrus and estrus phases, 5-HT mechanisms of the PrL seem to play a regulatory role in the expression of learned fear. These results were not replicated in the LA rats. Similar but less intense results were found regarding the early and late diestrus. Our data indicate that future studies on this subject need to take into account the dissociation between low- and high-responsive females to understand how hormones affect emotional behavior.

Antinociceptive action of cannabidiol on thermal sensitivity and post-operative pain in male and female rats.

This study investigated the antinociceptive potential of cannabidiol (CBD) in male and female Wistar rats. The assessment and analysis included tail withdrawal to thermal stimulation (tail flick test) and mechanical allodynia induced by plantar incision injury (von Frey test). CBD reduced acute thermal sensitivity in uninjured animals and post-operative mechanical allodynia in males and females. In the tail flick test, CBD 30 mg/kg i.p. was required to induce antinociception in males. During the proestrus phase, females did not show a statistically significant antinociceptive response to CBD treatment despite a noticeable trend. In contrast, in a separate group of rats tested during the late diestrus phase, antinociception varied with CBD dosage and time. In the post-operative pain model, CBD at 3 mg/kg decreased mechanical allodynia in males. Similarly, this dose reduced allodynia in females during proestrus. However, in females during late diestrus, the lower dose of CBD (0.3 mg/kg) reduced mechanical allodynia, although the latency to onset of the effect was slower (90 min). The effectiveness of a 10-fold lower dose of CBD during the late diestrus stage in females suggests that ovarian hormones can influence the action of CBD. While CBD has potential for alleviating pain in humans, personalized dosing regimens may need to be developed to treat pain in women.

D(1)-like receptors in the nucleus accumbens shell regulate the expression of contextual fear conditioning and activity of the anterior cingulate cortex in rats.

Although dopamine-related circuits are best known for their roles in appetitive motivation, consistent data have implicated this catecholamine in some forms of response to stressful situations. In fact, projection areas of the ventral tegmental area, such as the amygdala and hippocampus, are well established to be involved in the acquisition and expression of fear conditioning, while less is known about the role of the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) in these processes. In the present study, we initially investigated the involvement of the mPFC and NAc in the expression of conditioned fear, assessing freezing behaviour and Fos protein expression in the brains of rats exposed to a context, light or tone previously paired with footshocks. Contextual and cued stimuli were able to increase the time of the freezing response while only the contextual fear promoted a significant increase in Fos protein expression in the mPFC and caudal NAc. We then examined the effects of specific dopaminergic agonists and antagonists injected bilaterally into the posterior medioventral shell subregion of the NAc (NAcSh) on the expression of contextual fear. SKF38393, quinpirole and sulpiride induced no behavioural changes, but the D1-like receptor antagonist SCH23390 increased the freezing response of the rats and selectively reduced Fos protein expression in the anterior cingulate cortex and rostral NAcSh. These findings confirm the involvement of the NAcSh in the expression of contextual fear memories and indicate the selective role of NAcSh D1-like receptors and anterior cingulate cortex in this process.

Intraoperative dopamine release during globus pallidus internus stimulation in Parkinson's disease.

BACKGROUND: It is still unclear whether dopamine (DA) levels correlate with Parkinson's disease (PD) severity or play a role in the mechanisms of high-frequency stimulation (HFS). METHODS: We have used microdialysis to record pallidal DA in 5 patients with PD undergoing microelectrode-guided pallidotomy. RESULTS: We found that patients with more severe disease and, consequently, lower pallidal DA did poorly after pallidal lesions. In the operating room, 4 of 5 patients had a significant increase in DA levels during HFS (600%, on average). To test the hypothesis that DA was important for the effects of stimulation, we correlated the amelioration in rigidity observed in the operating room with pallidal DA release. Though rigidity was 56% better during stimulation, no correlation was found between such an improvement and DA release. CONCLUSIONS: These findings suggest that additional mechanisms not directly dependent on pallidal DA release may be involved in the clinical effects of HFS of the globus pallidus internus.

Low doses of fluoxetine for the treatment of emotional premenstrual syndrome: a randomized double-blind, placebo-controlled, pilot study.

INTRODUCTION: The neuroactive metabolite of progesterone, allopregnanolone (ALLO), has been implicated in premenstrual syndrome (PMS) physiopathology and preclinical studies suggested that low doses of fluoxetine increase the ALLO brain concentration. OBJECTIVES: To assess which low dose of fluoxetine (2 mg/d, 5 mg/d or 10 mg/d), administered exclusively during the luteal phase of menstrual cycle, has a potential effect for preventing or mitigating emotional PMS symptoms. METHODS: In this randomized, double-blind, placebo-controlled pilot study, we followed 40 women (mean age = 29.7 +/- 7.4 years) with emotional PMS, during two menstrual cycles: cycle 1, without pharmacological intervention; and cycle 2, with pharmacological intervention. Participants took capsules, on average, seven days preceding the likely date of menses. We assessed the severity of PMS symptoms in both cycles using the Daily Record of Severity of Problems scale (DRSP). RESULTS: There was an increase in the DRSP scores during the late luteal phase of cycle 1, confirming the diagnosis of emotional PMS. Low doses of fluoxetine (5 mg/d: 33.5%; 10 mg/d: 48.4%) reduced DRSP total score in the day before menses (day-1) at cycle 2 compared with day-1 at cycle 1. Fluoxetine 10 mg/d had the most consistent decline in emotional PMS symptoms; 70% of the participants reported a reduction greater than 40% in the DRSP score. CONCLUSIONS: Low doses of fluoxetine, which may have no or few effect on the serotonergic system, but may interfere in the progesterone metabolization, seem to have some potential to mitigate emotional PMS symptoms. While the 10 mg/d of fluoxetine had the best performance on reducing emotional PMS symptoms, the 5 mg/d dose also seems to have some effect on emotional PMS symptoms. Further larger studies will help establish the lowest effective dose of flouxetine for PMS treatment.

Conditioned fear is modulated by D2 receptor pathway connecting the ventral tegmental area and basolateral amygdala.

Excitation of the mesocorticolimbic pathway, originating from dopaminergic neurons in the ventral tegmental area (VTA), may be important for the development of exaggerated fear responding. Among the forebrain regions innervated by this pathway, the amygdala is an essential component of the neural circuitry of conditioned fear. The functional role of the dopaminergic pathway connecting the VTA to the basolateral amygdala (BLA) in fear and anxiety has received little attention. In vivo microdialysis was performed to measure dopamine levels in the BLA of Wistar rats that received the dopamine D(2) agonist quinpirole (1 µg/0.2 µl) into the VTA and were subjected to a fear conditioning test using a light as the conditioned stimulus (CS). The effects of intra-BLA injections of the D(1) antagonist SCH 23390 (1 and 2 µg/0.2 µl) and D(2) antagonist sulpiride (1 and 2 µg/0.2 µl) on fear-potentiated startle (FPS) to a light-CS were also assessed. Locomotor performance was evaluated by use of open-field and rotarod tests. Freezing and increased dopamine levels in the BLA in response to the CS were both inhibited by intra-VTA quinpirole. Whereas intra-BLA SCH 23390 did not affect FPS, intra-BLA sulpiride (2 µg) inhibited FPS. Sulpiride's ability to decrease FPS cannot be attributed to nonspecific effects because this drug did not affect motor performance. These findings indicate that the dopamine D(2) receptor pathway connecting the ventral tegmental area and the basolateral amygdala modulates fear and anxiety and may be a novel pharmacological target for the treatment of anxiety.

Activation of mineralocorticoid receptors facilitate the acquisition of fear memory extinction and impair the generalization of fear memory in diabetic animals.

RATIONALE: Studies point out a higher prevalence of posttraumatic stress disorder (PTSD) in individuals with diabetes mellitus. It is known that glucocorticoid (GR) and mineralocorticoid (MR) receptors are implicated in fear memory processes and PTSD. However, there is no preclinical studies addressing the involvement of these receptors on abnormal fear memories related to diabetic condition. OBJECTIVES: By inducing a contextual conditioned fear memory, we generate a suitable condition to investigate the extinction and the generalization of the fear memory in streptozotocin-induced diabetic (DBT) rats alongside the expression of the cytosolic and nuclear GR and MR in the hippocampus (HIP) and prefrontal cortex (PFC). Moreover, we investigated the involvement of the MR or GR on the acquisition of fear memory extinction and on the generalization of this fear memory. When appropriate, anxiety-related behavior was evaluated. METHODS: Male Wistar rats received one injection of steptozotocin (i.p.) to induce diabetes. After 4 weeks, the animals (DBTs and non-DBTs) were subjected to a conditioned contextual fear protocol. RESULTS: The expression of MR and GR in the HIP and PFC was similar among all the groups. The single injection of MR agonist was able to facilitate the acquisition of the impaired fear memory extinction in DBTs animals together with the impairment of its generalization. However, the GR antagonism impaired only the generalization of this fear memory which was blocked by the previous injection of the MR antagonist. All treatments were able to exert anxiolytic-like effects. CONCLUSIONS: The results indicate that MR activation in DBT animals disrupts the overconsolidation of aversive memory, without discarding the involvement of emotional behavior in these processes.

Dopamine modulates individual differences in avoidance behavior: A pharmacological, immunohistochemical, neurochemical and volumetric investigation.

Avoidance behavior is a hallmark in pathological anxiety disorders and results in impairment of daily activities. Individual differences in avoidance responses are critical in determining vulnerability or resistance to anxiety disorders. Dopaminergic activation is implicated in the processing of avoidance responses; however, the mechanisms underlying these responses are unknown. In this sense, we used a preclinical model of avoidance behavior to investigate the possibility of an intrinsic differential dopaminergic pattern between good and poor performers. The specific goal was to assess the participation of dopamine (DA) through pharmacological manipulation, and we further evaluated the effects of systemic injections of the dopaminergic receptor type 1 (D1 antagonist - SCH23390) and dopaminergic receptor type 2 (D2 antagonist - sulpiride) antagonists in the good performers. Additionally, we evaluated the effects of intra-amygdala microinjection of a D1 antagonist (SCH23390) and a D2 antagonist (sulpiride) in good performers as well as intra-amygdala microinjection of a D1 agonist (SKF38393) and D2 agonist (quinpirole) in poor performers. Furthermore, we quantified the contents of dopamine and metabolites (3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)) in the amygdala, evaluated the basal levels of tyrosine hydroxylase expression (catecholamine synthesis enzyme) and measured the volume of the substantia nigra, ventral tegmental area and locus coeruleus. Our results showed that it could be possible to convert animals from good to poor performers, and vice versa, by intra-amygdala (basolateral and central nucleus) injections of D1 receptor antagonists in good performers or D2 receptor agonists in poor performers. Additionally, the good performers had lower levels of DOPAC and HVA in the amygdala, an increase in the total volume of the amygdala (AMG), substantia nigra (SN), ventral tegmental area (VTA) and locus coeruleus (LC), and an increase in the number of tyrosine hydroxylase-positive cells in SN, VTA and LC, which positively correlates with the avoidance behavior. Taken together, our data show evidence for a dopaminergic signature of avoidance performers, emphasizing the role of distinct dopaminergic receptors in individual differences in avoidance behavior based on pharmacological, immunohistochemical, neurochemical and volumetric analyses. Our findings provide a better understanding of the role of the dopaminergic system in the execution of avoidance behavior.

Short-term, low-dose fluoxetine prevents oestrous cycle-linked increase in anxiety-like behaviour in female rats.

BACKGROUND AND AIMS: We sought a robust behavioural test that evoked increased anxiety-like behaviour during the late dioestrus phase of the oestrous cycle (similar to the premenstrual period in women) and tested whether this could be prevented by acute low-dose fluoxetine (FLX). METHODS: Female Wistar rats in different stages of their cycle were exposed to four different tests of anxiety-like behaviour. RESULTS: No oestrous cycle differences were detected in fear potentiated startle or conditioned freezing to an aversive context. In a light switch-off test where rats move from one compartment of a shuttle-box to the other to turn off an aversive light, females displayed enhanced responding in late dioestrus. During isolation restraint stress females in late dioestrus emitted three times more 22 kHz ultrasound vocalisations (USV) than at other cycle stages. Using the USV test, short-term administration of low-dose FLX (1.75 mg kg-1, i.p.) designed to blunt the sharp fall in brain allopregnanolone concentration during late dioestrus but without affecting 5-HT systems, prevented the increase in isolation stress-evoked USVs. CONCLUSIONS: The light switch-off and isolation restraint-induced USV tests evoke unconditioned adverse emotional responses that are ethologically relevant and sensitive to oestrous cycle stage. The USV test fulfils many criteria required of a model for premenstrual syndrome in women. Using the USV test, short-term administration of FLX to increase brain allopregnanolone concentration without affecting 5-HT systems prevented the increased USV responding in late dioestrus. Short-term low-dose FLX treatment may have potential to alleviate development of adverse premenstrual symptoms in women.

Intranasal dopamine attenuates fear responses induced by electric shock to the foot and by electrical stimulation of the dorsal periaqueductal gray matter.

PURPOSE: Intranasally applied dopamine (IN-DA), which likely reaches the brain via nasal-brain pathways and bypasses the blood-brain barrier, has been found to increase extracellular DA and bind to the DA2 transporter in the striatum. Recent studies suggest that DA plays a significant role in the processing of signaled and unconditioned aversive stimulation, including evidence that may attenuate responses to painful input. The purpose of this study was to examine the effects of IN-DA on fear-related behaviors induced by electric shock to the foot or by electrical stimulation of the dorsal periaqueductal gray matter (dPAG). METHODS: DA hydrochloride suspended in a viscous castor oil gel (1 or 2 mg/kg) was applied (IN-DA) in a volume of 5 µL into the nostrils of adult Wistar male rats in order to evaluate its effects on (a) freezing induced by electric shock to the foot and (b) thresholds of freezing and escape and duration of post-stimulation freezing induced by electrical stimulation of the dPAG. RESULTS: IN-DA attenuated freezing induced by electric shock to the foot in the three test trials, indicating that it reduced long-term fear responses. IN-DA also increased the threshold of dPAG stimulation-induced escape responses and reduced post-stimulation freezing. CONCLUSIONS: IN-DA, which has previously been shown to facilitate learning and to have antidepressive-like effects, attenuated unconditioned fear responses elicited by peripheral and intramesencephalic (dPAG) stimulation and reduced long-term conditioned fear responses.

Regulation of conditioned and unconditioned fear in rats by 5-HT1A receptors in the dorsal periaqueductal gray.

Studies on the involvement of 5-HT1-mediated mechanisms in the dorsal periaqueductal gray (dPAG) of animals with past stressful experiences have not been conducted so far. We investigated the role of 5-HT1 receptors in the dPAG of rats previously submitted to contextual fear conditioning. Defensive behaviors induced by activation of the dPAG were assessed by measuring the lowest electric current applied to this structure (threshold) able to produce freezing and escape responses during testing sessions of contextual fear conditioning, in which animals were placed in a context previously paired to footshocks. The 5-HT1A function of the dPAG was evaluated by local injections of 8-OH-DPAT (4 and 8 nmol/0.2 microL) and WAY-100635 (10 nmol/0.2 microL), selective agonist and antagonist of 5-HT1A receptors, respectively. In accordance with previous studies, 8-OH-DPAT increased aversive thresholds (antiaversive effects) but injections of WAY 100635 into the dPAG did not produce significant effects on the aversive thresholds in naive rats. However, the aversive thresholds of animals exhibiting contextual fear remained unchanged with both treatments. Moreover, 8-OH-DPAT and WAY 100635 did not change the dPAG post-stimulation freezing. The present results suggest that the stressful experience of being fear conditioned has an effect on the role of the 5-HT1A receptors in mediating unconditioned fear. Also, the reduction in the regulation of the defensive behaviors by 5-HT1A-mediated mechanisms in the dPAG of these animals may underlie the stress precipitated psychopathology associated with the neural substrates of aversion of the dPAG.

Fear extinction in an obsessive-compulsive disorder animal model: Influence of sex and estrous cycle.

Obsessive-compulsive disorder (OCD) is a neuropsychiatric condition that affects men and women equally, but with a sexually dimorphic pattern of development. Reproductive cycle events can influence symptom severity of OCD in females, indicating that ovarian hormones or their interaction with distinct neurotransmitter systems may play a role in OCD pathophysiology. Clinical studies and animal models have confirmed the importance of the serotonergic (5-HT) system in the neurobiology and treatment of OCD. Accordingly, the non-selective 5-HT2c agonist, meta-chlorophenylpiperazine (mCPP), exacerbates symptoms in untreated OCD patients. In rodents, it evokes repetitive behaviors that engage brain areas that are homologous with those found to be dysfunctional in OCD patients. These regions, including the medial prefrontal and anterior cingulate cortices, are also involved in fear inhibition, which is impaired in OCD. Here, we treated rats with mCPP (0.5 and 3.0 mg/kg) to evaluate its influence on self-grooming behavior and assess potential fear extinction retention deficits, taking into account sex differences and females' estrous cycle. We found that mCPP exacerbated grooming in male and female rats, irrespective of the estrous cycle phase. Fear extinction retention, however, was impaired only in females. Moreover, females undergoing fear extinction training during the metestrus/diestrus phases of the estrous cycle were more sensitive to the impairments induced by mCPP. Our results indicate that mCPP can induce OCD-like symptoms, exacerbating self-grooming and impairing fear extinction. It suggests that changes in 5-HT signaling through 5-HT2c receptors may have an important role in the OCD pathophysiology and that the influence of gonadal hormones in OCD should be further investigated.

5-HT2 receptor mechanisms of the dorsal periaqueductal gray in the conditioned and unconditioned fear in rats.

RATIONALE: It is well known that 5-HT(2) mechanisms modulate the defensive behavior produced by the stimulation of the dorsal periaqueductal gray (dPAG). However, in spite of the notion that past stressful experiences play a role in certain types of anxiety, only studies with the stimulation of the dPAG of rats without previous aversive experience have been conducted so far. OBJECTIVES: We investigated the mediation of 5-HT(2) receptors of the dPAG in rats previously submitted to contextual fear conditioning (CFC). Defensive behaviors induced by the activation of the dPAG were assessed by measuring the lowest intensity of electric current applied to this structure (threshold) able to produce freezing and escape responses during the testing sessions of CFC in which animals were placed in a context previously paired to footshocks. The 5-HT(2) function of the dPAG in this condition was evaluated by local injections of alpha-methyl-5-HT (20 nmol/0.2 mul) and ketanserin (5 and 10 nmol/0.2 mul), selective agonist and antagonist of 5-HT(2) receptors, respectively. RESULTS: In accordance with previous studies, alpha-methyl-5-HT increased the aversive thresholds (antiaversive effects) in naive rats, and injection of ketanserin into the dPAG did not produce significant effects. On the other hand, ketanserin decreased in a dose-dependent manner the freezing threshold (proaversive effect) determined by the dPAG electrical stimulation, whereas alpha-methyl-5-HT continued to show antiaversive effects in animals under CFC. CONCLUSIONS: The present results suggest that past stressful experience can produce changes in the synaptic function of 5-HT(2) receptors within the dPAG with important impact on the expression of defensive behaviors.

Substance P injected into the dorsal periaqueductal gray causes anxiogenic effects similar to the long-term isolation as assessed by ultrasound vocalizations measurements.

Housing conditions change the emotional state of the animals. Ultrasound vocalizations (USVs) termed as 22 kHz are the usual components of the defensive responses of rats exposed to threatening conditions such as isolation. The amount of emission of 22 kHz USVs depends on the intensity of the aversive stimuli. While short periods of isolation caused an anxiolytic-sensitive enhancement of the defensive responses, long-term isolation tended to reduce the defensive performance of the animals to aversive stimuli. The dorsal periaqueductal gray (dPAG) is an important vocal center and a crucial structure for the expression of defensive response. While it has been shown that Substance P (SP) at this midbrain level is involved in the modulation of the defensive response, its role in the emission of ultrasound vocalizations has not been evaluated. In this study we examined whether the resocialization and local injections of SP into the dPAG have an influence on the isolation-induced 22 kHz USVs recorded within the frequency range of 18-26 kHz. Rats isolated for 1 day showed a significant increase in the number and duration of USVs, which were reversed by resocialization. On the other hand, 2-week isolation reduced the number and duration of 22 kHz USVs, which could not be reversed by resocialization. SP injections into the dPAG (35 pmol/0.2 microL) caused a reduction in the 22 kHz USVs. Pretreatment with the NK-1 receptor antagonist spantide (100 pmol/0.2 microL) blocked these effects but exhibited no effect when given alone. These findings suggest that 1-day and 2-week isolation recruit distinct brain defensive systems. Also, in agreement with the notion that intense fear is associated with the neural substrates of fear of the dPAG, activation of NK-1 receptors of this midbrain structure reduces the 22 kHz USVs.

5-HT2- and D1-mechanisms of the basolateral nucleus of the amygdala enhance conditioned fear and impair unconditioned fear.

The inferior colliculus (IC) is involved in processing of auditory information, but also integrates acoustic information of aversive nature. In fact, chemical stimulation of the IC with semicarbazide (SMC) - an inhibitor of the GABA synthesizing enzyme glutamic acid decarboxylase - has been found to cause defensive behavior in an open-field test and functions as an unconditioned stimulus in the place conditioned aversion test (PCA). A question has arisen regarding whether the basolateral nucleus of the amygdala (BLA) is involved in the acquisition of the aversive information ascending from the IC and whether dopaminergic and serotoninergic mechanisms of the BLA regulate this process. Recent evidence has shown that inactivation of the BLA with muscimol inhibits the PCA and causes an increase in the aversiveness of the chemical stimulation of the IC. Based on this, we examined the effects of ketanserin and SCH-23390, antagonists of the 5HT(2) and D(1) receptors, respectively, on the conditioned and unconditioned fear elicited by IC stimulation with SMC. The results obtained confirm the crucial role of 5-HT(2)- and D(1)-mechanisms of the BLA on conditioned fear in that ketanserin and SCH-23390 injections into the BLA caused a reduction in the PCA. On the other hand, ketanserin and SCH-23390 injections into the BLA enhanced the aversiveness of the IC injections of SMC. These findings suggest that while 5-HT(2) and DA(1) mechanisms in the BLA appear to facilitate the conditioned fear they inhibit the unconditioned fear triggered by IC activation.

Brainstem areas activated by diazepam withdrawal as measured by Fos-protein immunoreactivity in rats.

In the 1970s, chronic treatment with benzodiazepines was supposed not to cause dependence. However, by the end of the decade several reports showed that the interruption of a prolonged treatment with diazepam leads to a withdrawal syndrome characterized, among other symptoms, by an exaggerated level of anxiety. In laboratory animals, signs that oscillate from irritability to extreme fear-like behaviors and convulsions have also been reported. In recent years many studies have attempted to disclose the neural substrates responsible for the benzodiazepines withdrawal. However, they have focused on telencephalic structures such as the prefrontal cortex, nucleus accumbens and amygdala. In this study, we examined the Fos immunoreactivity in brain structures known to be implicated in the neural substrates of aversion in rats under spontaneous diazepam-withdrawal. We found that the same group of structures that originally modulate the defensive responses evoked by fear stimuli, including the dorso-medial hypothalamus, the superior and inferior colliculus and the dorsal periaqueductal gray, were most labeled following diazepam withdrawal. It is suggested that an enhanced neural activation of neural substrates of fear in the midbrain tectum may underlie the aversive state elicited in diazepam-withdrawn rats.

Serotonergic mechanisms in the basolateral amygdala differentially regulate the conditioned and unconditioned fear organized in the periaqueductal gray.

The amygdala is an important filter for unconditioned and conditioned aversive information. The amygdala synthesizes the stimuli input from the environment and then signals the degree of threat that they represent to the dorsal periaqueductal gray (dPAG), which would be in charge of selecting, organizing and executing the appropriate defense reaction. In this study, we examined the influence of fluoxetine microinjections (1.75 and 3.5 nmol/0.2 microL) into the lateral (LaA) and basolateral (BLA) amygdaloid nuclei on the freezing and escape responses induced by electrical stimulation of the dPAG. Freezing behavior was also measured after the interruption of the electrical stimulation of the dPAG. On the following day, these rats were also submitted to a contextual fear paradigm to examine whether these microinjections would affect the conditioned freezing to contextual cues previously associated with foot shocks. Fluoxetine injections into both amygdaloid nuclei did not change the freezing and escape thresholds, but disrupted the dPAG-post-stimulation freezing. Moreover, the conditioned freezing was enhanced by fluoxetine. Whereas 5-HT mechanisms in the amygdala facilitate the acquisition of conditioned fear they inhibit the dPAG-post-stimulation freezing. However, the unconditioned fear triggered by activation of the dPAG is produced downstream of the amygdala. These findings have important implications for the understanding of the neurochemical substrates that underlie panic and generalized anxiety disorders.

The blockade of AMPA-kainate and NMDA receptors in the dorsal periaqueductal gray reduces the effects of diazepam withdrawal in rats.

It is well established that the most persistent sign of withdrawal from chronic benzodiazepine use in humans is anxiety. In contrast to other types of drugs of abuse, the emergence of this anxiety does not seem to be linked directly to alterations in the levels of dopamine in the mesolimbic system. Some studies have proposed that fear-like behaviors elicited by benzodiazepine withdrawal could be the result either of alterations in the sensitivity of GABAA receptors or in the neuronal hyperexcitability that results from neuroadaptative responses to chronic treatment, probably mediated by glutamate. The increased fear-like behaviors induced by benzodiazepine withdrawal are similar to the defense reaction displayed by animals exposed to dangerous situations or submitted to electrical or chemical stimulation of the dorsal periaqueductal gray (dPAG), a key structure of the brain aversive system. However, the involvement of the dPAG in drug abuse has been investigated only in the context of the physical effects of drug dependence. Thus, in this study we investigated the effects of injections into the dPAG of the glutamic acid diethyl ester (GDEE) and 2-amino-7-phosphonoheptanoate (AP-7) (AMPA-kainate and NMDA receptors antagonists, respectively) on fear-like behaviors promoted by benzodiazepine withdrawal in rats submitted to aversive events (foot-shocks) immediately before chronic diazepam administration in a conditioning place-preference paradigm, using a light-dark box. Our results showed that inhibition of the glutamatergic neurotransmission in the dPAG reduces the consequence of the diazepam withdrawal in rats, implicating the excitatory amino acids of the dPAG in the modulation of the aversive state induced by benzodiazepine drugs withdrawal.

Intranasal administration of dopamine attenuates unconditioned fear in that it reduces restraint-induced ultrasound vocalizations and escape from bright light.

BACKGROUND: Although substantial evidence suggests that dopamine (DA) enhances conditioned fear responses, few studies have examined the role of DA in unconditioned fear states. Whereas DA does not cross the blood-brain barrier, intranasally-applied dopamine reaches the brain directly via the nose-brain pathways in rodents, providing an alternative means of targeting DA receptors. Intranasal dopamine (IN-DA) has been demonstrated to bind to DA transporters and to increase extracellular DA in the striatum as well as having memory-promoting effects in rats. The purpose of this study was to examine the influence of IN-DA in three tests of fear/anxiety. METHODS: The three doses of DA hydrochloride (0.03, 0.3, or 1 mg/kg) were applied in a viscous castor oil gel in a volume of 5 µl to each of both nostrils of adult Wistar rats prior to testing of (a) escape from a bright light, using a two-chamber procedure, (b) restraint-induced 22 kHz ultrasound vocalizations (USVs), and (c) exploratory behavior in the elevated plus-maze (EPM). RESULTS: IN-DA dose-dependently reduced escape from bright light and the number of USV responses to restraint. It had no influence on the exploratory behavior in the EPM. CONCLUSIONS: IN-DA application reduced escape behavior in two tests of unconditioned fear (escape from bright light and USV response to immobilization). These findings may be interpreted in light of the known antidepressant action of IN-DA and DA reuptake blockers. The results also confirm the promise of the nasal route as an alternative means for targeting the brain's dopaminergic receptors with DA.

Pharmacological assessment of the freezing, antinociception, and exploratory behavior organized in the ventrolateral periaqueductal gray.

Opioid and serotonergic mechanisms of the ventrolateral periaqueductal gray (vlPAG) are recruited by conditioned freezing and antinociception. However, it is unclear whether freezing and antinociception induced by stimulation of the vlPAG are interrelated. To address this issue we looked at the effects of the opioid antagonist naltrexone, the 5-HT2 antagonist ketanserin, and the benzodiazepine agonist midazolam injected into the vlPAG on the freezing and antinociception induced by electrical stimulation of this region. This antinociception was evaluated by the tail-flick and formalin tests. To further characterize the involvement of the vlPAG in unconditioned fear, the effects of intra-vlPAG injections of midazolam on the exploratory behavior were also assessed in independent groups of rats submitted to the elevated plus-maze test (EPM). The data obtained showed that: (i) electrical stimulation of the vlPAG causes freezing blocked by midazolam but not by naltrexone and ketanserin; (ii) antinociception generated at the level of the vlPAG is inhibited by naltrexone, ketanserin, and midazolam; (iii) activation of benzodiazepine-mediated mechanisms in the vlPAG increased the exploratory behavior of rats in the closed arms but not the avoidance behavior of open arms of the EPM. Thus, freezing and antinociception generated in the vlPAG are dissociated pharmacologically. Whereas antinociception is a multimediated process sensitive to naltrexone, ketanserin, and midazolam, the freezing induced by vlPAG stimulation was reversed only by the benzodiazepine compound. As injections of midazolam into the vlPAG do not cause anxiolytic effects in the EPM, the aversive stimuli inherent of this test seem to bypass the vlPAG.