Hypothalamic sites responding to predator threats--the role of the dorsal premammillary nucleus in unconditioned and conditioned antipredatory defensive behavior

In this study we provide a comprehensive analysis of the hypothalamic activation pattern during exposure to a live predator or an environment previously associated with a predator. Our results support the view that hypothalamic processing of the actual and the contextual predatory threats share the same circuit, in which the dorsal premammillary nucleus (PMd) plays a pivotal role in amplifying this processing. To further understand the role of the PMd in the circuit organizing antipredatory defensive behaviors, we studied rats with cytotoxic PMd lesions during cat exposure and examined the pattern of behavioral responses as well as how PMd lesions affect the neuronal activation of the systems engaged in predator detection, in contextual memory formation and in defensive behavioral responses. Next, we investigated how pharmacological blockade of the PMd interferes with the conditioned behavioral responses to a context previously associated with a predator, and how this blockade affects the activation pattern of periaqueductal gray (PAG) sites likely to organize the conditioned behavioral responses to the predatory context. Behavioral observations indicate that the PMd interferes with both unconditioned and conditioned antipredatory defensive behavior. Moreover, we have shown that the PMd influences the activation of its major projecting targets, i.e. the ventral part of the anteromedial thalamic nucleus which is likely to influence mnemonic processing, and PAG sites involved in the expression of antipredatory unconditioned and conditioned behavioral responses. Of particular relevance, this work provides evidence to elucidate the basic organization of the neural circuits integrating unconditioned and contextual conditioned responses to predatory threats.     

酸敏感离子通道1α介导缺氧皮质神经元损伤及相关机制的探讨

目的探讨缺氧条件下阻断酸敏感离子通道1α(acid sensing ion channels 1α,ASIC1α)对大鼠皮质神经元存活及细胞内钙离子水平的影响,以及缺氧对皮质神经元ASIC1表达的影响。方法通过使用ASIC1α阻断剂、N甲基D门冬氨酸(NMDA)受体阻断剂、电压门控通道阻断剂,利用fura-3/AM荧光显像,乳酸脱氢酶(lactate dehydrogenase,LDH)释放实验等技术,比较观察对缺氧皮质神经元存活以及钙离子水平的影响,进一步利用Western印迹法检测ASIC1的表达变化。结果酸性环境中,缺氧皮质神经元形态明显改变,细胞内钙离子浓度升高;与加入NMDA受体阻断剂、电压门控通道阻断剂相比较,加入ASIC1α阻断剂明显抑制细胞内钙离子水平升高(P<0.05),对神经元起到保护作用。随缺氧时间的延长,神经元ASIC1蛋白表达增加。结论酸性环境中,缺氧皮质神经元的损伤与ASIC1α开放以及表达增加有关;阻断ASIC1α具有神经保护作用。     

rCBF differences between panic disorder patients and control subjects during anticipatory anxiety and rest.

BACKGROUND: Our goal was to identify brain structures involved in anticipatory anxiety in panic disorder (PD) patients compared to control subjects. METHODS: Seventeen PD patients and 21 healthy control subjects were studied with H(2)(15)O positron emission tomography scan, before and after a pentagastrin challenge. RESULTS: During anticipatory anxiety we found hypoactivity in the precentral gyrus, the inferior frontal gyrus, the right amygdala, and the anterior insula in PD patients compared to control subjects. Hyperactivity in patients compared to control subjects was observed in the parahippocampal gyrus, the superior temporal lobe, the hypothalamus, the anterior cingulate gyrus, and the midbrain. After the challenge, the patients showed decreases compared to the control subjects in the precentral gyrus, the inferior frontal gyrus, and the anterior insula. Regions of increased activity in the patients compared to the control subjects were the parahippocampal gyrus, the superior temporal lobe, the anterior cingulate gyrus, and the midbrain. CONCLUSIONS: The pattern of regional cerebral blood flow activations and deactivations we observed both before and after the pentagastrin challenge was the same, although different in intensity. During anticipatory anxiety more voxels were (de)activated than during rest after the challenge.     

Storm in a coffee cup: caffeine modifies brain activation to social signals of threat

Caffeine, an adenosine A₁ and A_2A receptor antagonist, is the most popular psychostimulant drug in the world, but it is also anxiogenic. The neural correlates of caffeine-induced anxiety are currently unknown. This study investigated the effects of caffeine on brain regions implicated in social threat processing and anxiety. Participants were 14 healthy male non/infrequent caffeine consumers. In a double-blind placebo-controlled crossover design, they underwent blood oxygenation level-dependent functional magnetic resonance imaging (fMRI) while performing an emotional face processing task 1 h after receiving caffeine (250 mg) or placebo in two fMRI sessions (counterbalanced, 1-week washout). They rated anxiety and mental alertness, and their blood pressure was measured, before and 2 h after treatment. Results showed that caffeine induced threat-related (angry/fearful faces > happy faces) midbrain-periaqueductal gray activation and abolished threat-related medial prefrontal cortex wall activation. Effects of caffeine on extent of threat-related amygdala activation correlated negatively with level of dietary caffeine intake. In concurrence with these changes in threat-related brain activation, caffeine increased self-rated anxiety and diastolic blood pressure. Caffeine did not affect primary visual cortex activation. These results are the first to demonstrate potential neural correlates of the anxiogenic effect of caffeine, and they implicate the amygdala as a key site for caffeine tolerance.     

The selective serotonin reuptake inhibitor citalopram increases fear after acute treatment but reduces fear with chronic treatment: a comparison with tianeptine.

BACKGROUND: Selective serotonin reuptake inhibitors (SSRIs) are efficacious in the treatment of a variety of fear or anxiety disorders. Although they inhibit the reuptake of serotonin within hours of administration, therapeutic improvement only occurs after several weeks. In this study, we used fear conditioning to begin to understand how acute and chronic SSRI treatment might differentially affect well-characterized fear circuits. METHODS: We evaluated the effects of acute and chronic treatment with the SSRI citalopram on the acquisition of auditory fear conditioning. To further understand the role of serotonin in modulating fear circuits, we compared these effects with those of acute and chronic administration of the antidepressant tianeptine, a purported serotonin reuptake enhancer. RESULTS: We found that acute administration of the SSRI citalopram enhanced acquisition, whereas chronic treatment reduced the acquisition of auditory fear conditioning. In comparison, treatment with tianeptine had no effect acutely but also reduced the acquisition of tone conditioning when administered chronically. CONCLUSIONS: Our findings with citalopram are consistent with the clinical effects of SSRI treatment seen in patients with anxiety disorders, in which anxiety is often increased during early stages of treatment and decreased after several weeks of treatment. The findings also indicate that auditory fear conditioning can be a useful tool in understanding differences in the effects of short-term and long-term antidepressant treatment with serotonergic medications.     

Genetic mapping of ASIC4 and contrasting phenotype to ASIC1a in modulating innate fear and anxiety

Although ASIC4 is a member of the acid‐sensing ion channel (ASIC) family, we have limited knowledge of its expression and physiological function in vivo. To trace the expression of this ion channel, we generated the ASIC4‐knockout/CreERT²‐knockin (Asic4^CreERT2) mouse line. After tamoxifen induction in the Asic4^CreERT2::CAG‐STOP^floxed‐Td‐tomato double transgenic mice, we mapped the expression of ASIC4 at the cellular level in the central nervous system (CNS). ASIC4 was expressed in many brain regions, including the olfactory bulb, cerebral cortex, striatum, hippocampus, amygdala, thalamus, hypothalamus, brain stem, cerebellum, spinal cord and pituitary gland. Colocalisation studies further revealed that ASIC4 was expressed mainly in three types of cells in the CNS: (i) calretinin (CR)‐positive and/or vasoactive intestine peptide (VIP)‐positive interneurons; (ii) neural/glial antigen 2 (NG2)‐positive glia, also known as oligodendrocyte precursor cells; and (iii) cerebellar granule cells. To probe the possible role of ASIC4, we hypothesised that ASIC4 could modulate the membrane expression of ASIC1a and thus ASIC1a signaling in vivo. We conducted behavioral phenotyping of Asic4^CreERT2 mice by screening many of the known behavioral phenotypes found in Asic1a knockouts and found ASIC4 not involved in shock‐evoked fear learning and memory, seizure termination or psychostimulant‐induced locomotion/rewarding effects. In contrast, ASIC4 might play an important role in modulating the innate fear response to predator odor and anxious state because ASIC4‐mutant mice showed increased freezing response to 2,4,5‐trimethylthiazoline and elevated anxiety‐like behavior in both the open‐field and elevated‐plus maze. ASIC4 may modulate fear and anxiety by counteracting ASIC1a activity in the brain.     

Health anxiety and fear of fear in panic disorder and agoraphobia vs. social phobia: a prospective longitudinal study

Background: This study is aimed to evaluate the role of two vulnerability factors, health anxiety and fear of fear, in the prediction of the onset of panic disorder/agoraphobia (PDA) relative to a comparison anxiety disorder. Methods: Young women, aged between 18 and 24 years, were investigated at baseline and, 17 months later, using the Anxiety Disorders Interview Schedule‐Lifetime and measures of health anxiety and fear of bodily sensations (subscale disease phobia of the Whiteley Index, and total score of the Body Sensations Questionnaire). First, 22 women with current PDA were compared to 81 women with current social phobia and 1,283 controls. Second, 24 women with an incidence of PDA were compared to 60 women with an incidence of social phobia and 1,036 controls. Results: Multiple logistic regression analyses adjusted for history of physical diseases, somatic symptoms, and other psychological disorders revealed that (a) fear of bodily sensations was elevated for women with PDA vs. controls as well as women with social phobia, and (b) health anxiety (and history of physical diseases) was elevated in women who developed PDA vs. controls and vs. women who developed social phobia. Conclusions: These results suggest that health anxiety, as well as history of physical diseases, may be specific vulnerability factors for the onset of PDA relative to social phobia. Whereas fear of bodily sensations was not found to be a risk factor for the onset of panic disorder/agoraphobia, it was a specific marker of existing PDA relative to social phobia. Depression and Anxiety, 2010. © 2010 Wiley‐Liss, Inc.     

Testosterone reduces unconscious fear but not consciously experienced anxiety: implications for the disorders of fear and anxiety.

BACKGROUND: The fear-reducing properties of testosterone have been firmly established in animals but not in humans. However, human data on the relation between testosterone, fear, and anxiety have predominantly involved questionnaires that index cortically executed conscious appraisal of anxious mood. Animal studies, on the other hand, indicate that the effects of testosterone on motivation and emotion are of subcortical origin and of unconscious nature. Presently, it was hypothesized that a single testosterone administration to humans would reduce unconscious fear but not consciously experienced anxiety. METHODS: In a placebo-controlled, double-blind crossover design, a single dose of testosterone (.5 mg) or placebo was administered to 16 healthy female volunteers. Afterward, a masked emotional Stroop task measured unconscious emotional responses to fearful faces, while multiple self-reports of mood indexed consciously experienced anxiety. RESULTS: As hypothesized, the habitual vigilant emotional response to the masked fearful face observed in the placebo condition was significantly reduced after testosterone was administered, while the self-reported measures of anxiety remained unaffected. CONCLUSIONS: These data provide the first direct evidence for fear-reducing properties of testosterone in humans. Furthermore, by dissociating specific aspects of fear and anxiety in humans, this outcome highlights that testosterone's effects on motivation and emotion concern the subcortical affective pathways of the brain.     

The effects of septal stimulation on spontaneous and tail-shock evoked neuronal activity in the brainstem of the rat

The effects of stimulation of the lateral septum (LS) and the medial septum (MS) with single pulses of current were assessed on the neural activity evoked by a noxious tail shock as well as on spontaneous activity in 72 cells located in the periventricular gray (PVG), periaqueductal gray (PAG), dorsal raphe (DR) and nucleus raphe magnus (NRM) of the anesthetized rat. Pronounced inhibitory effects of evoked activity, occurring with a 10–15 ms latency and lasting 20–100 ms, were found primarily in the DR and ventrolateral PAG. Inhibition was not confined to nociceptive cells, and could also be observed in some units unresponsive to noxious stimulation. Short-lived excitatory effects were encountered mostly in the PVG and dorsolateral PAG units. The results were interpreted as support for the notion of septal participation in pain inhibition, and the hypothesis was advanced that its mode of action may involve suppression of aspects of nociception at the supraspinal level.     

Extinction is not a sufficient condition to prevent fear memories from undergoing reconsolidation in the basolateral amygdala

Consolidated memories when reactivated may return to a state that requires protein synthesis in order to be restabilized (reconsolidation). It has been shown in a variety of systems that if reactivation induces significant extinction then extinction is the protein synthesis dependent memory state, rather than reconsolidation. Thus, extinction consolidation may prevent the memory from undergoing reconsolidation. We investigated whether such an interaction also exists between extinction and reconsolidation of fear memories within the amygdala, by using a within subjects experimental design. We found that inhibition of protein synthesis in the basolateral amygdala (BLA) impaired reconsolidation for both the briefly reactivated and extinguished fear memories suggesting that extinction is not a sufficient condition to prevent induction of reconsolidation in the amygdala. These findings demonstrate that extinction consolidation does not always interact with reconsolidation. Therefore, under these conditions, extinction is not a boundary condition on reconsolidation of fear memories in the basolateral amygdala.     

The role of the amygdala in the extinction of conditioned fear.

The amygdala has long been known to play a central role in the acquisition and expression of fear. More recently, convergent evidence has implicated the amygdala in the extinction of fear as well. In rodents, some of this evidence comes from the infusion of drugs directly into the amygdala and, in particular, into the basolateral complex of the amygdala, during or after extinction learning. In vivo electrophysiology has identified cellular correlates of extinction learning and memory in the lateral nucleus of that structure. Human imaging experiments also indicate that amygdaloid activity correlates with extinction training. In addition, some studies have directly identified changes in molecular constituents of the basolateral amygdala. Together these experiments strongly indicate that the basolateral amygdala plays a crucial role in extinction learning. Interpreted in the light of these findings, several recent in vitro electrophysiology studies in amygdala-containing brain slices are suggestive of potential synaptic and circuit bases of extinction learning.     

Effects of focal electrical stimulation and morphine microinjection in the periaqueductal gray of the rat mesencephalon on neuronal activity in the medullary reticular formation

Neurons in the medullary reticular formation (MRF; nucleus reticularis gigantocellularis and nucleus reticularis paragigantocellularis) were evaluated for their involvement in the analgesia produced by focal electrical stimulation and microinjection of morphine into the periaqueductal gray region (PAG) of the rat mesencephalon. Analgesia-producing PAG stimulation altered the spontaneous activity of 80% of the neurons in the MRF (both excitation and inhibition were observed) and inhibited the noxious-evoked excitation of 75% of MRF neurons. Microinjection of morphine into the PAG also increased (50%) and decreased (17%) the spontaneous activity of MRF units and inhibited the noxious-evoked excitation of 47% of MRF neurons. These effects were specific for analgesia produced by the PAG manipulations and were partially reversed by naloxone. The role of the MRF in PAG-induced analgesias and the degree of overlap in neuronal systems influenced by intracranial morphine and electrical stimulation is discussed.     

Modulation of fear-potentiated startle and vocalizations in juvenile rhesus monkeys by morphine, diazepam, and buspirone.

BACKGROUND: Modulation of the acoustic startle response by aversive sensory stimulation is a simple and objective indicator of emotionality in rodents and human beings that has been extremely valuable for the analysis of neural systems associated with fear and anxiety. We have described a paradigm for measuring fear-potentiated, whole-body acoustic startle in nonhuman primates and have developed a protocol for maintaining fear-potentiated startle over repeated sessions with minimal extinction to allow measurement of pharmacological effects on fear-potentiated startle by using within-subjects designs in relatively small groups of monkeys. METHODS: A novel, within-subjects testing protocol was used to examine the effects of three compounds in rhesus monkeys that have anxiolytic effects in rodents on fear-potentiated startle but that differ in their mechanism of action. Spontaneous vocalizations during testing also were recorded. Juvenile monkeys that were trained to associate a visual stimulus with a fear-inducing air blast to the face were tested after acute administration of different doses of buspirone diazepam, morphine, or vehicle. RESULTS: Monkeys rapidly developed a robust and persistent elevation of startle response in the presence of the CS during repeated testing sessions. Diazepam and morphine produced dose-related reductions of fear-potentiated startle. Buspirone did not significantly reduce fear-potentiated startle at the doses tested, although a trend was evident at the highest dose. All drugs reduced rates of coo vocalizations during startle testing. CONCLUSIONS: These fear-potentiated startle results suggest that rhesus monkeys have a pharmacological profile with respect to these compounds that is closer to humans than to rats. This demonstrates the value of examining the effects of drugs on fear-potentiated startle in nonhuman primates.     

Beneficial effect of mirtazapine on diabetes-induced hyperalgesia: involvement of TRPV1 and ASIC1 channels in the spinal cord and dorsal root ganglion

ABSTRACT

Objectives: Neuropathic pain reduces the life qualities of patients with Diabetes mellitus. Clinical guidelines recommend relief in diabetic neuropathic pain through the use of some antidepressants, anticonvulsants, opioids as well as capsaicin cream or lidocaine patches. However, since the majority of patients do not or partially respond to current treatments, there is a growing necessity for new drugs increasing the pain relief in patients with diabetes. Therefore, based on the therapeutic potential of antidepressants on neuropathic pain, we investigated the promising antihyperalgesic effect of mirtazapine (MRT) in painful diabetic neuropathy.

Methods: Experimental diabetes was induced in rats by single intraperitoneal injection of 55 mg/kg dose of streptozocin (STZ). After 4 weeks of injection of STZ, MRT was administrated for 14 days at 40 mg/kg dose. Randall–Selitto and Hargreaves tests were applied for paw-withdrawal threshold and paw-withdrawal latency measurement. TRPV1 and ASIC1 expressions measured by Western blot in dorsal root ganglion and spinal cord.

Results: Administration of MRT significantly improved both of the decreased paw-withdrawal threshold and shortened the paw-withdrawal latency of diabetic rats, respectively. Besides, increased levels of TRPV1 and ASIC1 channels in dorsal root ganglion and spinal cord of diabetic rats, evaluated by Western blot method, were decreased following the MRT treatment.

Discussion: These data show, for the first time, that MRT has beneficial effects against diabetes-induced hyperalgesia, and that suppressive effect of this drug on TRPV1 and ASIC1 levels, which are increased in diabetic rats, may be some of the pharmacological mechanisms underlying the exhibited antihyperalgesic effect of MRT.     

Individual differences in neural correlates of fear conditioning as a function of 5-HTTLPR and stressful life events

Fear learning is a crucial process in the pathogeneses of psychiatric disorders, which highlights the need to identify specific factors contributing to interindividual variation. We hypothesized variation in the serotonin transporter gene (5-HTTLPR) and stressful life events (SLEs) to be associated with neural correlates of fear conditioning in a sample of healthy male adults (n = 47). Subjects were exposed to a differential fear conditioning paradigm after being preselected regarding 5-HTTLPR genotype and SLEs. Individual differences in brain activity as measured by functional magnetic resonance imaging (fMRI), skin conductance responses and preference ratings were assessed. We report significant variation in neural correlates of fear conditioning as a function of 5-HTTLPR genotype. Specifically, the conditioned stimulus (CS⁺) elicited elevated activity within the fear-network (amygdala, insula, thalamus, occipital cortex) in subjects carrying two copies of the 5-HTTLPR S′ allele. Moreover, our results revealed preliminary evidence for a significant gene-by-environment interaction, such as homozygous carriers of the 5-HTTLPR S′ allele with a history of SLEs demonstrated elevated reactivity to the CS⁺ in the occipital cortex and the insula. Our findings contribute to the current debate on 5-HTTLPR x SLEs interaction by investigating crucial alterations on an intermediate phenotype level which may convey an elevated vulnerability for the development of psychopathology.