William A. Falls

Fear-potentiated startle : a neural and pharmacological analysis

The fear-potentiated startle paradigm has proven to be a useful system with which to analyze neural systems involved in fear and anxiety. This test measures conditioned fear by an increase in the amplitude of a simple reflex (the acoustic startle reflex) in the presence of a cue previously paired with a shock. Fear-potentiated startle is sensitive to a variety of drugs such as diazepam, morphine, and buspirone that reduce anxiety in people and can be measured reliably in humans when the eyeblink component of startle is elicited at a time when they are anticipating a shock. Electrical stimulation techniques suggest that a visual conditioned stimulus ultimately alters acoustic startle at a specific point along the acoustic startle pathway. The lateral, basolateral and central amygdaloid nuclei and the caudal branch of the ventral amygdalofugal pathway projecting to the brainstem are necessary for potentiated startle to occur. The central nucleus of the amygdala projects directly to one of the brainstem nuclei critical for startle and electrical stimulation of this nucleus increases startle amplitude. Chemical or electrolytic lesions of either the central nucleus or the lateral and basolateral nuclei of the amygdala block the expression of fear-potentiated startle. The perirhinal cortex, which projects directly to the lateral and basolateral amygdaloid nuclei, plays a critical role in the expression of fear-potentiated startle using either visual or auditory conditioned stimuli. These latter amygdaloid nuclei may actually be the site of plasticity for fear conditioning, because local infusion of the NMDA antagonist AP5 into these nuclei blocks the acquisition of fear-potentiated startle. On the other hand, the expression of fear-potentiated startle is blocked by local infusion of the non-NMDA ionotropic antagonist CNQX or the G-protein inactivating toxin, pertussis toxin, but not by AP5. Finally, we have begun to investigate brain systems that might be involved in the inhibition of fear. Local infusion of AP5 into the amygdala was found to block the acquisition of experimental extinction, a prototypical method for reducing fear. We have also established a reliable procedure for producing both external and conditioned inhibition of fear-potentiated startle and hope to eventually understand the neural systems involved in these phenomena.

Extinction of fear-potentiated startle: blockade by infusion of an NMDA antagonist into the amygdala

Data derived from in vitro preparations indicate that NMDA receptors play a critical role in synaptic plasticity in the CNS. More recently, in vivo pharmacological manipulations have suggested that an NMDA- dependent process may be involved in specific forms of behavioral plasticity. All of the work thus far has focused on the possible role of NMDA receptors in the acquisition of responses. However, there are many examples in the behavioral literature of learning-induced changes that involve the reduction or elimination of a previously acquired response. Experimental extinction is a primary example of the elimination of a learned response. Experimental extinction is well described in the behavioral literature, but has not received the same attention in the neurobiological literature. As a result, the neural mechanisms that underlie this important form of learning are not at all understood. In the present experiments, the fear-potentiated startle paradigm was employed to begin to investigate neural mechanisms of extinction. The results show that infusion of the NMDA antagonist D,L-2- amino-5-phosphonovaleric acid (AP5) into the amygdala, a limbic structure known to be important for fear conditioning, dose-dependently blocked extinction of conditioned fear. Control experiments showed that the blockade of extinction was neither the result of the permanent disruption of amygdaloid function nor the result of decreased sensitivity of the animals to the conditioned stimulus. Infusion of AP5 into the interpositus nucleus of the cerebellum, a control site, did not block extinction. Finally, intra-amygdala infusion of a selected dose of the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione did not block extinction of conditioned fear. These results, together with a previous report from our laboratory (Miserendino et al., 1990), demonstrate the importance of the amygdala in the elaboration of conditioned fear and suggest that an NMDA-dependent process might underlie the extinction of conditioned fear.

Infusion of the non-NMDA receptor antagonist CNQX into the amygdala blocks the expression of fear-potentiated startle

The involvement of non-N-methyl-D-aspartate receptors in the amygdala in the expression of conditioned fear was examined using the fear-potentiated startle paradigm. Rats implanted with bilateral cannulae in the basolateral amygdaloid nuclei received 10 pairings of either a visual or auditory conditioned stimulus with footshock on each of 2 days. The next day, they were tested by eliciting the acoustic startle reflex in the presence or absence of the conditioned stimulus and divided into groups with equivalent levels of potentiation. One or two days later, rats were tested again following intra-amygdala infusion of vehicle or 0.025, 0.25, or 2.5 micrograms of 6-cyano-7-nitroquinoxaline-2,3-dione. The drug dose-dependently blocked the expression of potentiated startle in both sensory modalities, indicating that activation of non-NMDA receptors in the amygdala is necessary for the expression of conditioned fear.

Chronic stress increases pituitary adenylate cyclase-activating peptide (PACAP) and brain-derived neurotrophic factor (BDNF) mRNA expression in the bed nucleus of the stria terminalis (BNST): Roles for PACAP in anxiety-like behavior

Exposure to chronic stress has been argued to produce maladaptive anxiety-like behavioral states, and many of the brain regions associated with stressor responding also mediate anxiety-like behavior. Pituitary adenylate cyclase activating polypeptide (PACAP) and its specific G protein-coupled PAC(1) receptor have been associated with many of these stress- and anxiety-associated brain regions, and signaling via this peptidergic system may facilitate the neuroplasticity associated with pathological affective states. Here we investigated whether chronic stress increased transcript expression for PACAP, PAC(1) receptor, brain-derived neurotrophic factor (BDNF), and tyrosine receptor kinase B (TrkB) in several nuclei. In rats exposed to a 7 days chronic variate stress paradigm, chronic stress enhanced baseline startle responding induced by handling and exposure to bright lights. Following chronic stress, quantitative transcript assessments of brain regions demonstrated dramatic increases in PACAP and PAC(1) receptor, BDNF, and TrkB receptor mRNA expression selectively in the dorsal aspect of the anterolateral bed nucleus of the stria terminalis (dBNST). Related vasoactive intestinal peptide (VIP) and VPAC receptor, and other stress peptide transcript levels were not altered compared to controls. Moreover, acute PACAP38 infusion into the dBNST resulted in a robust dose-dependent anxiogenic response on baseline startle responding that persisted for 7 days. PACAP/PAC(1) receptor signaling has established trophic functions and its coordinate effects with chronic stress-induced dBNST BDNF and TrkB transcript expression may underlie the maladaptive BNST remodeling and plasticity associated with anxiety-like behavior.

The effects of intra-amygdaloid infusions of a D2 dopamine receptor antagonist on Pavlovian fear conditioning.

The present study examined the effects of bilateral intra-amygdaloid infusions of the D2 receptor antagonist, eticlopride, on the acquisition and expression of Pavlovian fear conditioning as measured by freezing to acoustic and background contextual stimuli in the rat. Infusions of eticlopride before acquisition or before both acquisition and retention testing significantly attenuated conditioned freezing to tone presentations during the retention test 24 hr later. No effects, however, were observed on freezing that emerged during acquisition. Furthermore, these effects were not attributable to state-dependent learning effects or alterations in baseline activity or shock reactivity. In conclusion, these results suggest that amygdaloid dopamine transmission at D2 receptors contributes to the formation and/or consolidation of fear memories.

Neural Systems Involved in Fear Inhibition: Extinction and Conditioned Inhibition

“I can’t get the memories out of my mind! The images come flooding back in vivid detail, triggered by the most inconsequential things, like a door slamming or the smell of stir-fried pork. Last night, I went to bed, was having a good sleep for a change. Then in the early morning a storm-front passed through and there was a bolt of crackling thunder. I awoke instantly, frozen in fear. I am right back in Viet Nam, in the middle of the monsoon season at my guard post. I am sure I’ll get hit in the next volley and convinced I will die. My hands are freezing, yet sweat pours from my entire body. I feel each hair on the back of my neck standing on end. I can’t catch my breath and my heart is pounding. I smell a damp sulfur smell. Suddenly I see what’s left of my buddy Troy, his head on a bamboo platter, sent back to our camp by the Viet Cong. Propaganda messages are stuffed between his clenched teeth. The next bolt of lightning and clap of thunder makes me jump so much that I fall to the floor..... ” (Paraphrased from a war veteran’s conversations with Dr. R. L. Gelman, Dept. of Psychiatry, Yale University School of Medicine).

Voluntary exercise in C57 mice is anxiolytic across several measures of anxiety

Voluntary wheel running in rodents is associated with a number of adaptive behavioral and physiological effects including improved learning, reduction in stress-associated behaviors, neurogenesis, angiogenesis, increases in neurotrophic factors, and changes in several signaling molecules. Exercise has also been reported to reduce anxiety-like behaviors. However, other studies have failed to find an anxiolytic effect of exercise. The inconsistencies in the literature may contribute to the scarcity of data examining the physiological correlates of the anxiolytic effect of exercise. Here we show that 2 weeks of voluntary exercise in male C57 mice is associated with reduced anxiety as measured with acoustic startle, stress-induced hyperthermia, social interaction, light-enhanced startle, and some, but not all, measures in the open field. A great deal is known about the neural circuits underlying anxiety. Given the consistency of the anxiolytic effect of voluntary exercise across several measures, it is now possible to begin a systematic analysis of the physiological basis of the anxiolytic effect of exercise.

Deletion in Catna2, encoding alpha N-catenin, causes cerebellar and hippocampal lamination defects and impaired startle modulation.

Mice homozygous for the cerebellar deficient folia (cdf) mutation are ataxic and have cerebellar hypoplasia and abnormal lobulation of the cerebellum. In the cerebella of cdf/cdf homozygous mice, approximately 40% of Purkinje cells are located ectopically in the white matter and inner granule-cell layer. Many hippocampal pyramidal cells are scattered in the plexiform layers, and those that are correctly positioned are less densely packed than are cells in wild-type mice. We show that fear conditioning and prepulse inhibition of the startle response are also disrupted in cdf/cdf mice. We identify a deletion on chromosome 6 that removes approximately 150 kb in the cdf critical region. The deletion includes part of Catna2, encoding αN-catenin, a protein that links the classical cadherins to the neuronal cytoskeleton. Expression of a Catna2 transgene in cdf/cdf mice restored normal cerebellar and hippocampal morphology, prepulse inhibition and fear conditioning. The findings suggest that catenin–cadherin cell-adhesion complexes are important in cerebellar and hippocampal lamination and in the control of startle modulation.

Roles for pituitary adenylate cyclase-activating peptide (PACAP) expression and signaling in the bed nucleus of the stria terminalis (BNST) in mediating the behavioral consequences of chronic stress.

Anxiety disorders are frequently long-lasting and debilitating for more than 40 million American adults. Although stressor exposure plays an important role in the etiology of some anxiety disorders, the mechanisms by which exposure to stressful stimuli alters central circuits that mediate anxiety-like emotional behavior are still unknown. Substantial evidence has implicated regions of the central extended amygdala, including the bed nucleus of the stria terminalis (BNST) and the central nucleus of the amygdala as critical structures mediating fear- and anxiety-like behavior in both humans and animals. These areas organize coordinated fear- and anxiety-like behavioral responses as well as peripheral stress responding to threats via direct and indirect projections to the paraventricular nucleus of the hypothalamus and brainstem regions (Walker et al. Eur J Pharmacol 463:199–216, 2003, Prog Neuropsychopharmacol Biol Psychiatry 33(8):1291–1308, 2009; Ulrich-Lai and Herman Nat Rev Neurosci 10:397–409, 2009). In particular, the BNST has been argued to mediate these central and peripheral responses when the perceived threat is of long duration (Waddell et al. Behav Neurosci 120:324–336, 2006) and/or when the anxiety-like response is sustained (Walker and Davis Brain Struct Funct 213:29–42, 2008); hence, the BNST may mediate pathological anxiety-like states that result from exposure to chronic stress. Indeed, chronic stress paradigms result in enhanced BNST neuroplasticity that has been associated with pathological anxiety-like states (Vyas et al. Brain Res 965:290–294, 2003; Pego et al. Eur J Neurosci 27:1503–1516, 2008). Here we review evidence that suggests that pituitary adenylate cyclase-activating polypeptide (PACAP) and corticotropin-releasing hormone (CRH) work together to modulate BNST function and increase anxiety-like behavior. Moreover, we have shown that BNST PACAP as well as its cognate PAC1 receptor is substantially upregulated following chronic stress, particularly in the BNST oval nucleus where PACAP-containing neurons closely interact with CRH-containing neurons (Kozicz et al. Brain Res 767:109–119, 1997; Hammack et al. Psychoneuroendocrinology 34:833–843, 2009). We describe how interactions between PACAP and CRH in the BNST may mediate stress-associated behaviors, including anorexia and anxiety-like behavior. These studies have the potential to define specific mechanisms underlying anxiety disorders, and may provide important therapeutic strategies for stress and anxiety management.

Lesions of the central nucleus of the amygdala block conditioned excitation, but not conditioned inhibition of fear as measured with the fear-potentiated startle effect.

Lesions of the amygdala block the expression of fear-potentiated startle following either moderate or extensive light + shock training. The present experiment assessed whether lesions of the amygdala would also block the expression of conditioned inhibition of fear. Rats were given conditioned inhibition training in which a light was paired with shock and a noise and light compound was presented in the absence of shock. Then half of the rats were given bilateral electrolytic lesions of the amygdala and the remaining rats were sham operated. Lesions of the amygdala blocked the expression of fear-potentiated startle to the light. To assess whether conditioned inhibition was disrupted, rats were retrained with light + shock pairings with no further conditioned inhibition training. Amygdala lesioned rats reacquired fear-potentiated startle to the light (Kim & Davis, 1993). Importantly, the noise conditioned inhibitor retained its ability to inhibit fear-potentiated startle to the retrained light. These results indicate that areas of the amygdala critical for initial performance of fear-potentiated startle are not critical for the expression of conditioned inhibition.