New perspective on the pathophysiology of panic: merging serotonin and opioids in the periaqueductal gray

Panic disorder patients are vulnerable to recurrent panic attacks. Two neurochemical hypotheses have been proposed to explain this susceptibility. The first assumes that panic patients have deficient serotonergic inhibition of neurons localized in the dorsal periaqueductal gray matter of the midbrain that organize defensive reactions to cope with proximal threats and of sympathomotor control areas of the rostral ventrolateral medulla that generate most of the neurovegetative symptoms of the panic attack. The second suggests that endogenous opioids buffer normal subjects from the behavioral and physiological manifestations of the panic attack, and their deficit brings about heightened suffocation sensitivity and separation anxiety in panic patients, making them more vulnerable to panic attacks. Experimental results obtained in rats performing one-way escape in the elevated T-maze, an animal model of panic, indicate that the inhibitory action of serotonin on defense is connected with activation of endogenous opioids in the periaqueductal gray. This allows reconciliation of the serotonergic and opioidergic hypotheses of panic pathophysiology, the periaqueductal gray being the fulcrum of serotonin-opioid interaction.

The response of social anxiety disorder patients to threat scenarios differs from that of healthy controls

The objective of the present study was to evaluate the response of social anxiety disorder (SAD) patients to threat scenarios. First-choice responses to 12 scenarios describing conspecific threatening situations and mean scores of defensive direction and defensive intensity dimensions were compared between 87 SAD patients free of medication and 87 matched healthy controls (HC). A significant gender difference in the first-choice responses was identified for seven scenarios among HCs but only for two scenarios among SAD patients. A significantly higher proportion of SAD patients chose "freezing" in response to "Bush" and "Noise" scenarios, whereas the most frequent response by HCs to these scenarios was "check out". SAD males chose "run away" and "yell" more often than healthy men in response to the scenarios "Park" and "Elevator", respectively. There was a positive correlation between the severity of symptoms and both defensive direction and defensive intensity dimensions. Factorial analysis confirmed the gradient of defensive reactions derived from animal studies. SAD patients chose more urgent defensive responses to threat scenarios, seeming to perceive them as more dangerous than HCs and tending to move away from the source of threat. This is consistent with the hypothesis that the physiopathology of anxiety disorders involves brain structures responsible for defensive behaviors.

Effect of escitalopram on the processing of emotional faces

Serotonin has been implicated in the neurobiology of depressive and anxiety disorders, but little is known about its role in the modulation of basic emotional processing. The aim of this study was to determine the effect of the selective serotonin reuptake inhibitor, escitalopram, on the perception of facial emotional expressions. Twelve healthy male volunteers completed two experimental sessions each, in a randomized, balanced order, double-blind design. A single oral dose of escitalopram (10 mg) or placebo was administered 3 h before the task. Participants were presented to a task composed of six basic emotions (anger, disgust, fear, happiness, sadness, and surprise) that were morphed between neutral and each standard emotion in 10 percent steps. Escitalopram facilitated the recognition of sadness and inhibited the recognition of happiness in male, but not female faces. No drug effect on subjective measures was detected. These results confirm that serotonin modulates the recognition of emotional faces, and suggest that the gender of the face can have a role in this modulation. Further studies including female volunteers are needed.

Defensive responses to threat scenarios in Brazilians reproduce the pattern of Hawaiian Americans and non-human mammals

A former study with scenarios conducted in Hawaii has suggested that humans share with non-human mammals the same basic defensive strategies - risk assessment, freezing, defensive threat, defensive attack, and flight. The selection of the most adaptive strategy is strongly influenced by features of the threat stimulus - magnitude, escapability, distance, ambiguity, and availability of a hiding place. Aiming at verifying if these strategies would be consistent in a different culture, 12 defensive scenarios were translated into Portuguese and adapted to the Brazilian culture. The sample consisted of male and female undergraduate students divided into two groups: 76 students, who evaluated the five dimensions of each scenario and 248 medical students, who chose the most likely response for each scenario. In agreement with the findings from studies of non-human mammal species, the scenarios were able to elicit different defensive behavioral responses, depending on features of the threat. "Flight" was chosen as the most likely response in scenarios evaluated as an unambiguous and intense threat, but with an available route of escape, whereas "attack" was chosen in an unambiguous, intense and close dangerous situation without an escape route. Less urgent behaviors, such as "check out", were chosen in scenarios evaluated as less intense, more distant and more ambiguous. Moreover, the results from the Brazilian sample were similar to the results obtained in the original study with Hawaiian students. These data suggest that a basic repertoire of defensive strategies is conserved along the mammalian evolution because they share similar functional benefits in maintaining fitness.

Serotonergic modulation of face-emotion recognition: [review]

Facial expressions of basic emotions have been widely used to investigate the neural substrates of emotion processing, but little is known about the exact meaning of subjective changes provoked by perceiving facial expressions. Our assumption was that fearful faces would be related to the processing of potential threats, whereas angry faces would be related to the processing of proximal threats. Experimental studies have suggested that serotonin modulates the brain processes underlying defensive responses to environmental threats, facilitating risk assessment behavior elicited by potential threats and inhibiting fight or flight responses to proximal threats. In order to test these predictions about the relationship between fearful and angry faces and defensive behaviors, we carried out a review of the literature about the effects of pharmacological probes that affect 5-HT-mediated neurotransmission on the perception of emotional faces. The hypothesis that angry faces would be processed as a proximal threat and that, as a consequence, their recognition would be impaired by an increase in 5-HT function was not supported by the results reviewed. In contrast, most of the studies that evaluated the behavioral effects of serotonin challenges showed that increased 5-HT neurotransmission facilitates the recognition of fearful faces, whereas its decrease impairs the same performance. These results agree with the hypothesis that fearful faces are processed as potential threats and that 5-HT enhances this brain processing.

The size and prevalence of the cavum septum pellucidum are normal in subjects with panic disorder

Panic disorder is thought to involve dysfunction in the septohippocampal system, and the presence of a cavum septum pellucidum might indicate the aberrant development of this system. We compared the prevalence and size of cavum septum pellucidum in 21 patients with panic disorder and in 21 healthy controls by magnetic resonance imaging. The length of the cavum septum pellucidum was measured by counting the number of consecutive 1-mm coronal slices in which it appeared. A cavum septum pellucidum of >6 mm in length was rated as large. There was no significant difference in the proportion of patients (16 of 21 or 76.2 percent) and controls (18 of 21 or 85.7 percent) with a cavum septum pellucidum (P = 0.35, Fisher's exact test, one-tailed), and no members of either group had a large cavum septum pellucidum. The mean cavum septum pellucidum rating in the patient and control groups was 1.81 (SD = 1.50) and 2.09 (SD = 1.51), respectively. There were also no significant differences between groups when we analyzed cavum septum pellucidum ratings as a continuous variable (U = 196.5; P = 0.54). Across all subjects there was a trend towards a higher prevalence of cavum septum pellucidum in males (100 percent, 10 of 10) than females (75 percent, 24 of 32; P = 0.09, Fisher's exact test, one-tailed). Thus, we conclude that, while panic disorder may involve septo-hippocampal dysfunction, it is not associated with an increased prevalence or size of the cavum septum pellucidum.

Decreased left temporal lobe volume of panic patients measured by magnetic resonance imaging

Reported neuroimaging studies have shown functional and morphological changes of temporal lobe structures in panic patients, but only one used a volumetric method. The aim of the present study was to determine the volume of temporal lobe structures in patients with panic disorder, measured by magnetic resonance imaging. Eleven panic patients and eleven controls matched for age, sex, handedness, socioeconomic status and years of education participated in the study. The mean volume of the left temporal lobe of panic patients was 9 percent smaller than that of controls (t21 = 2.37, P = 0.028). In addition, there was a trend (P values between 0.05 and 0.10) to smaller volumes of the right temporal lobe (7 percent, t21 = 1.99, P = 0.06), right amygdala (8 percent, t21 = 1.83, P = 0.08), left amygdala (5 percent, t21 = 1.78, P = 0.09) and left hippocampus (9 percent, t21 = 1.93, P = 0.07) in panic patients compared to controls. There was a positive correlation between left hippocampal volume and duration of panic disorder (r = 0.67, P = 0.025), with recent cases showing more reduction than older cases. The present results show that panic patients have a decreased volume of the left temporal lobe and indicate the presence of volumetric abnormalities of temporal lobe structures

Pharmacology of human experimental anxiety

This review covers the effect of drugs affecting anxiety using four psychological procedures for inducing experimental anxiety applied to healthy volunteers and patients with anxiety disorders. The first is aversive conditioning of the skin conductance responses to tones. The second is simulated public speaking, which consists of speaking in front of a video camera, with anxiety being measured with psychometric scales. The third is the Stroop Color-Word test, in which words naming colors are painted in the same or in a different shade, the incongruence generating a cognitive conflict. The last test is a human version of a thoroughly studied animal model of anxiety, fear-potentiated startle, in which the eye-blink reflex to a loud noise is recorded. The evidence reviewed led to the conclusion that the aversive conditioning and potentiated startle tests are based on classical conditioning of anticipatory anxiety. Their sensitivity to benzodiazepine anxiolytics suggests that these models generate an emotional state related to generalized anxiety disorder. On the other hand, the increase in anxiety determined by simulated public speaking is resistant to benzodiazepines and sensitive to drugs affecting serotonergic neurotransmission. This pharmacological profile, together with epidemiological evidence indicating its widespread prevalence, suggests that the emotional state generated by public speaking represents a species-specific response that may be related to social phobia and panic disorder. Because of scant pharmacological data, the status of the Stroop Color-Word test remains uncertain. In spite of ethical and economic constraints, human experimental anxiety constitutes a valuable tool for the study of the pathophysiology of anxiety disorders

The brain decade in debate: II. Panic or anxiety? From animal models to a neurobiological basis

This article is a transcription of an electronic symposium sponsored by the Brazilian Society of Neuroscience and Behavior (SBNeC). Invited researchers from the European Union, North America and Brazil discussed two issues on anxiety, namely whether panic is a very intense anxiety or something else, and what aspects of clinical anxiety are reproduced by animal models. Concerning the first issue, most participants agreed that generalized anxiety and panic disorder are different on the basis of clinical manifestations, drug response and animal models. Also, underlying brain structures, neurotransmitter modulation and hormonal changes seem to involve important differences. It is also common knowledge that existing animal models generate different types of fear/anxiety. A challenge for future research is to establish a good correlation between animal models and nosological classification

Effects of tryptophan depletion on anxiety induced by simulated public speaking

Several lines of evidence point to the participation of serotonin (5HT) in anxiety. Its specific role, however, remains obscure. The objective of the present study was to evaluate the effect of reducing 5HT-neurotransmission through an acute tryptophan depletion on anxiety induced by a simulated public speaking (SPS) test. Two groups of 14-15 subjects were submitted to a 24-h diet with a low or normal content of tryptophan and received an amino acid mixture without (TRY-) or with (TRY+) tryptophan under double-blind conditions. Five hours later they were submitted to the SPS test. The state-trait anxiety inventory (STAI) and the visual analogue mood scale (VAMS) were used to measure subjective anxiety. Both scales showed that SPS induced a significant increase in anxiety. Although no overall difference between groups was found, there was a trend (P = 0.078) to an interaction of group x gender x phases of the SPS, and a separate analysis of each gender showed an increase in anxiety measured by the STAI in females of the TRY- group. The results for the female TRY- group also suggested a greater arousing effect of the SPS test. In conclusion, the tryptophan depletion procedure employed in the present study did not induce a significant general change in subjective anxiety, but tended to induce anxiety in females. This suggests a greater sensitivity of the 5HT system to the effects of the procedure in this gender.

Effect of electrolytic lesion of the median raphe nucleus on behavioral and physiological measures of stress

The effect of electrolytic lesion of the median raphe nucleus was measured on behavioral and physiological parameters related to stress 24 h after the lesion. In of the elevated plus-maze the lesion decreased the percentage of open arm entries and tended to shorten the time spent on the open arms indicating as increase in anxiety. In contrast, the lesion markedly increased the time spent in the bright (aversive) compartment of the light-dark box and decrease in attempts to cross from the dark toward the bright compartment, an anxiolyic effect. With the exception of plasma prolactin level, which was lowered by the lesion, the physiological measures used in the present study indicate that the lesioned animals are under stress. Thus, death rate and weight loss after the surgery were higher in lesioned than in control animals. In addition, lesioned animals showed higher plasma corticoster- one levels, a high incidence of gastric ulcers in the fundus and a depressed immune response to the mitogen concavaline A. These results highlight the importance of the median raphe nucleus in the regulation of stress and anxiety. They also show that behavioral and physiological measures of stress may be dissociated.

D-Fenfluramine selectively releases 5-HT from dorsal raphe terminals

The aim of this study was to investigate whether D-fenfluramine (FEN) releases 5-hydroxytryptamine (5-HT) selectively from dorsal raphe (DR) terminais. Male Wistar rats, 180-200 g, were implanted with microdialysis probes in the amygdala (Am; N = 5) and dorsal hippocampus (DH; N = 6) and 5-HT levels were measured by electrochemical detection. Under basal conditions, 5-HT levels were approximately 50 and 230 fmol per 30 min sample, in the Am and DH, respectively. FEN (1O mg/kg, ip) produced a 3-4-fold increase in 5HT relesse in the Am, but not in the DH. Since the Am is mainly innervated by DR fibers while the DH receives 5-HT input chiefly from the median raphe (MR), the present results support the view that FEN selectively releases 5-HT from DR terminals.

Behavioral effects of intra-amygdala injections of GABA and 5-HT acting drugs in the elevated plus-maze

The effect of drugs bilaterally injected into the basolateral/medial nuclei of the amygdala on the behavior of male Wistar rats (300-330 g) in the elevated plus-maze was measured. The benzodiazepine agonist midazolam (MDZ, 20 and 40 nmol, 0.2 µl; N = 8-14) significantly increased open-arm exploration (percent open-arm entries; control = 20.27 + or - 3.71; 40 nmol MDZ = 42.63 + or - 7.16), having thus an anxiolytic effect. On the contrary, the non-selective 5-HT2 antagonist ketanserin (KET, 1 and 10 nmol, 0.2 µl; N = 8-11) had an anxiogenic effect (percent open-arm entries: control = 35.61 + or - 6.41; 10 nmol KET = 18.65 + or - 3.89). The 5-HT1A full agonist 8-OH-DPAT (2, 4 and 8 nmol, 0.2 µl; N = 9.12) did not significantly achange rat behavior in the plus-maze. While the present anxiolytic effect of midazolam agrees with results reported by others using punished behavior, the effect of the serotonergic drugs does not. Therefore, the effect of 5-HT acting drugs injected into the amygdala may be determined by the type of experimental model of anxiety used

Regional distribution of Fos-like immunoreactivity in the rat brain after exposure to fear-inducing stimuli

Fos protein immunohistochemistry was used to identify the neural substrate of fear/anxiety. The structures activated by exposure of Long Evans male rats (280-300 g) to the elevated plus-maze, a widely used animal model of anxiety, were compared with those activated by chemical stimulation of two aversive areas of the brain, the dorsal periaqueductal gray matter and the medial hypothalamus. Three different patterns of activation were obtained: Pattern 1 resulted from microinjection of the excitatory amino acid kainate (60 pmol; N = 5) or of the GABA(A) receptor antagonist SR-95531 (16 pmol; N = 3) into the dorsal periaqueductal gray matter and consisted mainly of caudal structures; Pattern 2 was observed after kainate injection (60 pmol; N = 4) into the medial hypothalamus and had a predominantly prosencephalic distribution; Pattern 3 extended from rostral to caudal brain regions and was induced by microinjection of either SR-95531 (16 pmol; N = 1) or kainate (120 pmol; N = 3) into the medial hypothalamus, as well as by 15-min exposure to the plus-maze (N = 3). Control animals were either injected with saline into the MH (N = 3) or the PAG (N = 3) or were exposed for 15 s to the elevated plus maze (N = 3) and exhibited no significant labeling. These results further support the participation of periventricular structures in the regulation of fear and aversion.

D-fenfluramine reduces anxiety induced by simulated public speaking

To further explore the role of serotonin (5-HT) in anxiety, 28 healthy volunteers received in a double-blind study d-fenfluramine (30 mg, p.o.) or placebo, and were submitted to a simulated public speaking test (SPS), consisting of speaking in front of a video camera. The SPS induced significant increases in subjective anxiety evaluated by the visual analogue mood scale of Norris [MANCOVA, F(1.66,39.93) = 8.51, P < 0.001], as well as in systolic blood pressure [F(3,72) = 5.70, P = 0.001] and in heart rate [F(3,72) = 3.95, P = 0.012]. The drug decreased the anxiety factor [F(1,23) = 5.21, P = 0.032], without significantly affecting physical sedation, mental sedation or other feelings and attitudes. Also, the physiological measurements were not significantly changed by d-fenfluramine. Reported evidence shows that d-fenfluramine releases 5-HT from nerve endings and blocks 5-HT reuptake, indirectly stimulating postsynaptic 5-HT receptors. Therefore, the present results indicate that 5-HT inhibits the neural substrate of SPS-induced anxiety

Influence of stimulus preexposure and of context familiarity on conditioned freezing behavior

1. This paper reports an experiment examining the influence of context on latent inhibition using conditioned freezing behavior as an index. 2. Two groups of 8 Wistar rats (290-320 g) were placed in one chamber (Context 2) and either exposed 7 times or not to a sound stimulus (68 dB, 90 s). Two additional groups of 8 rats received the same stimulation in a different environment (Context 1). Next, each rat was required to form a sound-shock (0.2 mA, 1.5 s) association (20 trials) in Context 2. Freezing behavior was measured both during sound (CS) presentation and during an equal period of time immediately preceding the CS. 3. When the test environment was familiar, the conditioning of fear was greater in the non-preexposed than in the CS-preexposed group. Acquisition of conditioned freezing was intermediate when the test environment was unfamiliar, irrespective of CS preexposure. 4. These results further support the context specificity of latent inhibition. In addition, they suggest that novelty interferes with sound-shock associations

The elevated T maze, a new experimental model of anxiety and memory: effect of diazepam

In rats placed in a T maze consisting of an enclosed arm at right angles with two open arms elevated 50 cm above the ground, ip doses of 2 and 4 mg/kg diazepam (DZP) abolished the delay of withdrawal from the enclosed arm towards the open arms, measured by retesting in the presence of the drug soon after training, as well as by further retesting 72 h later, in the absence of the drug. Therefore, DZP had both anxiolytic and amnestic effects on this inhibitory avoidance task. In contrast, DZP did not affect the latency of withdrawal from one of the open arms towards the closed arm on the first day. Moreover, the latency of this escape response similarly decreased in all treatment groups in the retest performed 72 h later, indicating that memory of this task was resistant to DZP. These results support the view that the anxiolytic and amnestic effects of benzodiazepines are closely related, and suggest that this new elevated T maze model may be useful for simultaneous measurement of drug effects on anxiety and memory

Strain-dependent effects of morphine injected into the periaqueductal gray area of mice

Microinjection of morphine (0.31,1.25 and 5.0 *g) into the periaqueductal gray area (PAG) of C57BL/6 (C57) and DBA/2 (DBA) mice increased the pain threshold in the tail-flick test. The highest dose also caused a behavioral reaction in both strains characterized by periods of immobility laternating with explosive motor behavior. In the DBA strain, the analgesic effect was demonstrated with all doses of morphine,with in the C57 strain only the highest dose induced analgesia. DBA mice presented a decrease in activity with the lowest dose of morphine, whereas in the C57 strain, this effect was obtained only with the highest dose of morphine. These data corroborate at the PAG level the results of other studies which have shown that central and peripheral injections of morphine procedure analgesia and alter motor activity in C57 and DBA strains. They also confirm that these two strains of mice present genotype-dependent differences in sensitivity to opioids as determined after injections of morphine into the PAG

Anxiolytic effect of midazolam microinjected into the dorsal periaqueductal grey area of rats

In order to investigate the role of the dorsal periaqueductal grey (DPAG) area in the anxiolytic effect of benzodiazepines male Wistar rats (N=10), weighing 200-250 g at the time of surgery, were microinjected into this structure with midazolam (80 nmol) and submitted to the elevated plus-maze, an ethologically based model of anxiety. Midazolam significantly increased the percentage of open arm entries from 32.4 ñ 4.6 (control) to 49.5 ñ 3.0 and of time spent in the open arms from 21.0 ñ 4.5 (control) to 35.6 ñ4.8 without affecting the total number of entries into either open or enclosed arms. This effect typifies an anxiolytic effect in the test and was antagonized by the benzodiazepine receptor antagonist flumazenil (80 nmol) microinjection. Microinjection of flumazenil alone had no effect. These results provide additional evidence for the participation of the DPAG in the physiopathology of anxiety and suggest that it that it may be a site for the anxiolytic effect of systemically injected benzodiazepines