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GSK3β plays an essential role in promoting cell death and is emerging as a potential target for neurological diseases. Understanding the mechanisms that control neuronal GSK3β is critical. A ubiquitous mechanism to repress GSK3β involves Akt-mediated phosphorylation of Ser9. Here we show that phosphorylation of GSK3β on Ser389 mediated by p38 MAPK specifically inactivates nuclear GSK3β in the cortex and hippocampus. Using GSK3β Ser389 to Ala mutant mice, we show that failure to inactivate nuclear GSK3β by Ser389 phosphorylation causes neuronal cell death in subregions of the hippocampus and cortex. Although this focal neuronal death does not impact anxiety/depression-like behavior or hippocampal-dependent spatial learning, it leads to an amplified and prolonged fear response. This phenotype is consistent with some aspects of post-traumatic stress disorder (PTSD). Our studies indicate that inactivation of nuclear GSK3β by Ser389 phosphorylation plays a key role in fear response, revealing new potential therapeutic approaches to target PTSD.

The bed nucleus of the stria terminalis (BNST) is a brain region important for regulating anxiety-related behavior in both humans and rodents. Here we used a chemogenetic strategy to investigate how engagement of G protein-coupled receptor (GPCR) signaling cascades in genetically defined GABAergic BNST neurons modulates anxiety-related behavior and downstream circuit function. We saw that stimulation of vesicular γ-aminobutyric acid (GABA) transporter (VGAT)-expressing BNST neurons using hM3Dq, but neither hM4Di nor rM3Ds designer receptors exclusively activated by a designer drug (DREADD), promotes anxiety-like behavior. Further, we identified that activation of hM3Dq receptors in BNST VGAT neurons can induce a long-term depression-like state of glutamatergic synaptic transmission, indicating DREADD-induced changes in synaptic plasticity. Further, we used DREADD-assisted metabolic mapping to profile brain-wide network activity following activation of Gq-mediated signaling in BNST VGAT neurons and saw increased activity within ventral midbrain structures, including the ventral tegmental area and hindbrain structures such as the locus coeruleus and parabrachial nucleus. These results highlight that Gq-mediated signaling in BNST VGAT neurons can drive downstream network activity that correlates with anxiety-like behavior and points to the importance of identifying endogenous GPCRs within genetically defined cell populations. We next used a microfluidics approach to profile the receptorome of single BNST VGAT neurons. This approach yielded multiple Gq-coupled receptors that are associated with anxiety-like behavior and several potential novel candidates for regulation of anxiety-like behavior. From this, we identified that stimulation of the Gq-coupled receptor 5-HT2CR in the BNST is sufficient to elevate anxiety-like behavior in an acoustic startle task. Together, these results provide a novel profile of receptors within genetically defined BNST VGAT neurons that may serve as therapeutic targets for regulating anxiety states and provide a blueprint for examining how G-protein-mediated signaling in a genetically defined cell type can be used to assess behavior and brain-wide circuit function.

Recent reports demonstrate that DNA damage is induced, and rapidly repaired, in circuits activated by experience. Moreover, stress hormones are known to slow DNA repair, suggesting that prolonged stress may result in persistent DNA damage. Prolonged stress is known to negatively impact physical and mental health; however, DNA damage as a factor in stress pathology has only begun to be explored. Histone H2A-X phosphorylated at serine 139 (γH2AX) is a marker of DNA double-strand breaks (DSB), a type of damage that may lead to cell death if unrepaired. We hypothesized that a 14-day period of variable stress exposure sufficient to alter anxiety-like behavior in male C57BL/6J mice would produce an increase in γH2AX levels in the bed nucleus of the stria terminalis (BNST), a region implicated in anxiety and stress regulation. We observed that 14 days of variable stress, but not a single stress exposure, was associated with increased levels of γH2AX 24 h after termination of the stress paradigm. Further investigation found that phosphorylation levels of a pair of kinases associated with the DNA damage response, glycogen synthase kinase 3 β (GSK3β) and p38 mitogen-activated protein kinase (MAPK) were also elevated following variable stress. Our results suggest that unrepaired DNA DSBs and/or repetitive attempted repair may represent an important component of the allostatic load that stress places on the brain.

Chronic or repeated stressor exposure can induce a number of maladaptive behavioral and physiological consequences and among limbic structures, the bed nucleus of the stria terminalis (BNST) has been implicated in the integration and interpretation of stress responses. Previous work has demonstrated that chronic variate stress (CVS) exposure in rodents increases BNST pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) and PAC1 receptor (Adcyap1r1) transcript expression, and that acute BNST PACAP injections can stimulate anxiety-like behavior. Here we show that chronic stress increases PACAP expression selectively in the oval nucleus of the dorsolateral BNST in patterns distinct from those for corticotropin releasing hormone (CRH). Among receptor subtypes, BNST PACAP signaling through PAC1 receptors not only heightened anxiety responses as measured by different behavioral parameters but also induced anorexic-like behavior to mimic the consequences of stress. Conversely, chronic inhibition of BNST PACAP signaling by continuous infusion with the PAC1 receptor antagonist PACAP(6-38) during the week of CVS attenuated these stress-induced behavioral responses and changes in weight gain. BNST PACAP signaling stimulated the hypothalamic-pituitary-adrenal (HPA) axis and heightened corticosterone release; further, BNST PACAP(6-38) administration blocked corticosterone release in a sensitized stress model. In aggregate with recent associations of PACAP/PAC1 receptor dysregulation with altered stress responses including post-traumatic stress disorder, these data suggest that BNST PACAP/PAC1 receptor signaling mechanisms may coordinate the behavioral and endocrine consequences of stress.

Single-nucleotide polymorphisms (SNPs) in the genes for pituitary adenylyl cyclase-activating peptide (PACAP) and the PAC1 receptor have been associated with stress-related psychiatric disorders. Although, from recent work, we have argued that stress-induced PACAP expression in the bed nucleus of the stria terminalis (BNST) may mediate stress-related psychopathology, it is unclear whether stress-induced increases in BNST PACAP expression require acute or repeated stressor exposure and whether increased BNST PACAP expression is related to stress-induced increases in circulating glucocorticoids. In the current work, we have used real-time quantitative polymerase chain reaction (qPCR) to assess transcript expression in brain punches from rats after stressor exposure paradigms or corticosterone injection. BNST PACAP and PAC1 receptor transcript expression was increased only after 7 days of repeated stressor exposure; no changes in transcript levels were observed 2 or 24 hours after a single-restraint session. Moreover, repeated corticosterone treatment for 7 days was not sufficient to reliably increase BNST PACAP transcript levels, suggesting that stress-induced elevations in corticosterone may not be the primary drivers of BNST PACAP expression. These results may help clarify the mechanisms and temporal processes that underlie BNST PACAP induction for intervention in stress-related anxiety disorders.

PurposeThis study assessed indices of autonomic arousal and vocal tension during challenge in preschool-age children who do stutter (CWS) and do not stutter (CWNS).MethodParticipants were preschool-age CWS (n = 10) and gender- and age-matched CWNS (n = 10) who performed in two speaking conditions: (1) ‘low’ challenge – naming age-appropriate pictures in a familiar room with the same examiner who administered his/her speech-language prescreening test (2) ‘high’ challenge – recalling the pictures named in condition (1) in a different room with an unfamiliar examiner while wearing acoustic startle electrodes on his/her face. Immediately following the ‘high’ challenge speaking task, the participants’ acoustic startle eyeblink response (ASEB) was measured. Dependent variables were ASEB in the ‘high’ challenge condition and the acoustic measure of mean fundamental frequency (Fo) in both challenge conditions.ResultsFindings indicated no significant between-group (CWS vs. CWNS) differences in Fo or ASEB r...

Exercise promotes stress resistance and is associated with reduced anxiety and reduced depression in both humans and in animal models. Despite the fact that dysfunction within the hypothalamic pituitary adrenal (HPA) axis is strongly linked to both anxiety and depressive disorders, the evidence is mixed as to how exercise alters the function of the HPA axis. Here we demonstrate that 4 weeks of voluntary wheel running was anxiolytic in C57BL/6J mice and resulted in a shorter time to peak corticosterone (CORT) and a more rapid decay of CORT following restraint stress. Wheel running was also associated with increased adrenal size and elevated CORT following systemic administration of adrenocorticotropic hormone. Finally, the HPA-axis response to peripheral or intracerebroventricular administration of dexamethasone did not suggest that wheel running increases HPA-axis negative feedback through GR-mediated mechanisms. Together these findings suggest that exercise may promote stress resilience in part by insuring a more rapid and shortened HPA response to a stressor thus affecting overall exposure to the potentially negative effects of more sustained HPA-axis activation.

Chronic or repeated exposure to stressful stimuli can result in several maladaptive consequences, including increased anxiety-like behaviors and altered peptide expression in anxiety-related brain structures. Among these structures, the bed nucleus of the stria terminalis (BNST) has been implicated in emotional behaviors as well as regulation of hypothalamic–pituitary–adrenal (HPA) axis activity. In male rodents, chronic variate stress (CVS) has been shown to increase BNST pituitary adenylate cyclase activating polypeptide (PACAP) and its cognate PAC1 receptor transcript, and BNST PACAP signaling may mediate the maladaptive changes associated with chronic stress. Here, we examined whether CVS would sensitize the behavioral and/or endocrine response to a subthreshold BNST PACAP infusion. Male and cycling female rats were exposed to a 7 day CVS paradigm previously shown to upregulate BNST PAC1 receptor transcripts; control rats were not stressed. Twenty-four hours following the last stressor, rats were bilaterally infused into the BNST with a normally subthreshold dose of PACAP. We found an increase in startle amplitude and plasma corticosterone levels 30 min following intra-BNST PACAP infusion in male rats that had been previously exposed to CVS. CVS did not enhance the startle response in cycling females. Equimolar infusion of the VPAC1/2 receptor ligand vasoactive intestinal polypeptide (VIP) had no effect on plasma corticosterone levels even in previously stressed male rats. These results suggest that repeated exposure to stressors may differentially alter the neural circuits underlying the responses to intra-BNST PACAP, and may result in different anxiety-like responses in males and females.

Recent reports demonstrate that the beneficial effects of voluntary exercise may be sensitive to stress prior to and during the wheel access period. Here, a variate stress procedure is used with socially isolated mice for 7 days prior to the introduction of running wheels to assess the impact of prior and concurrent stress on the anxiolytic effect of exercise. Following stress exposure, functioning or nonfunctioning running wheels were introduced into stressed and unstressed group-housed control cages. Following 3 weeks of wheel access, the anxiolytic effect of exercise was assessed using acoustic startle, stress-induced hyperthermia, and a challenge with the anxiogenic drug metachlorophenylpiperazine (mCPP). Variate stress was demonstrated to interfere with normal weight gain. Further, exercise was not anxiolytic in stressed mice. Consistent with previous reports unstressed exercising mice demonstrated reduced acoustic startle, attenuated stress induced hyperthermia, and a blunted increase in startle following mCPP administration when compared with unstressed sedentary controls. Stressed exercising mice were indistinguishable from stressed sedentary and unstressed sedentary controls on each anxiety measure. Although running distance varied between individual mice, the distance run did not predict the level of anxiety on any measure. These findings suggest that prior and ongoing stress delays or prevents the anxiolytic effect of exercise without affecting exercise itself.

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.

Exercise is associated with improved cognitive function in humans as well as improved learning across a range of tasks in rodents. Although these studies provide a strong link between exercise and learning, to date studies have largely focused on tasks that principally involve the hippocampus. However, exercise has been shown to produce alterations in other brain areas suggesting that the cognitive enhancing effects of exercise may be more general. Therefore we set out to examine the effects of voluntary exercise on cued Pavlovian fear conditioning, a form of learning that is critically dependent on the amygdala. In Experiment 1 we showed that mice given 2 weeks of access to a running wheel prior to tone and foot shock fear conditioning showed enhanced conditioned fear as measured by fear-potentiated startle. This effect was not the result of altered shock reactivity nor was it to due to reduced baseline startle amplitude in exercising mice. In subsequent experiments we sought to examine whether the enhanced cued conditioned fear was the result of an improvement in learning, consolidation or retrieval of conditioned fear. In separate groups of mice, two weeks of access to a running wheel was begun either prior to fear conditioning, immediately after fear conditioning (consolidation period) or 2 weeks after fear conditioning. Compared to sedentary mice, mice that exercised either prior to fear conditioning, or immediately after fear conditioning, showed enhanced cued conditioned fear. Fear conditioning was not enhanced in mice that began exercising 2 weeks after fear conditioning. Taken together these results suggest that voluntary exercise improves the learning and consolidation of cued conditioned fear but does not improve the retrieval or performance of conditioned fear. Because a great deal is known about the neural circuit for cued conditioned fear, it is now possible to examine the cellular, molecular and pharmacological changes associated with exercise in this well-understood neural circuit.

Metabolic mapping of downstream network activity following CNO-induced activation of hM3Dq in BNST VGAT neurons

The objective of this research was to measure the effects of increased stocking density of stalls and headlocks on the short-term behavioural responses of lactating Holstein cows. Multiparous ( n =92) and primiparous ( n =44) cows were assigned to four pens of 34 cows each and stocking densities (100, 113, 131, or 142%) were imposed in a 4×4 Latin square design with 7-d periods. Stocking density was adjusted by altering access to free stalls and headlocks. Activity of the cows was recorded continuously with digital video cameras for the last 2d at each stocking density and time spent as well as mean percentage of cows engaged in lying, standing, and feeding at each 10min scan sample was determined. Mean percentage of cows feeding over a 24-h period was approximately 20.5% and cows spent approximately 5h/d feeding, but the percentage of cows feeding during the first 60min following return from the milking parlour declined ( P P P

The degradation of the extracellular matrix is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Matrix components of the basement membrane play critical roles in the development and maintenance of the neuromuscular junction (NMJ), yet almost nothing is known about the regulation of MMP and TIMP expression in either the pre- or postsynaptic compartments. Here, we demonstrate that TIMP-2 is expressed by both spinal motor neurons and skeletal muscle. To determine whether motor function is altered in the absence of TIMP-2, motor behavior was assessed using a battery of tests (e.g., RotaRod, balance beam, hindlimb extension, grip strength, loaded grid, and gait analysis). TIMP-2−/− mice fall off the RotaRod significantly faster than wild-type littermates. In addition, hindlimb extension is reduced and gait is both splayed and lengthened in TIMP-2−/− mice. Motor dysfunction is more pronounced during early postnatal development. A preliminary analysis revealed NMJ alterations in TIMP-2−/− mice. Juvenile TIMP-2−/− mice have increased nerve branching and acetylcholine receptor expression. Adult TIMP-2−/− endplates are enlarged and more complex. This suggests a role for TIMP-2 in NMJ sculpting during development. In contrast to the increased NMJ nerve branching, cerebellar Purkinje cells have decreased neurite outgrowth. Thus, the TIMP-2−/− motor phenotype is likely due to both peripheral and central defects. The tissue specificity of the nerve branching phenotype suggests the involvement of different MMPs and/or extracellular matrix molecules underlying the TIMP-2−/− motor phenotype. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006

While a number of studies have examined the acquisition and expression of conditioned fear in inbred mice, very few have examined extinction of conditioned fear in inbred mice and few attempts have been made to compare extinction learning between inbred strains. Because inbred strains differ in a number of physiological and biochemical variables, differences in extinction learning may provide insight into the genetic influence of extinction learning. The purpose of this study was to examine extinction and renewal of conditioned fear in two common inbred strains of mice. C57BL/6J and DBA/2J mice were conditioned with pairings of either a tone or light and foot shock in a single session. On the following 4 days, mice were given extinction training, consisting of tone or light alone trials (Experiment 1A). C57 mice exhibited robust spontaneous recovery between sessions, but did extinguish both within and between sessions. DBA mice extinguished more quickly relative to C57 mice, and this extinction was stable between sessions (i.e., DBA mice did not exhibit spontaneous recovery). The rapid loss of fear in DBA relative to C57 mice was extinction-dependent and not merely due to poor long-term memory (Experiment 1B). Renewal testing (Experiment 2) replicated the strain difference in extinction and also showed that DBA mice have a deficit in the context specificity of extinction. C57 mice, but not DBA mice showed renewal of extinguished fear when tested in a context different from the one in which extinction training took place. These data suggest that the nature of extinction learning is influenced by characteristics of the inbred mouse strain.

The inferior colliculus (IC) is the major source of auditory information involved in processing the behavioral significance of acoustic stimuli. In the current study, we assessed whether the IC is a critical source of information which mediates the expression of fear and the inhibition of fear conditioned to an auditory stimulus. Fear and the inhibition of fear were tested by measuring fear-potentiated startle. In Experiment 1, we demonstrated that rats which received electrolytic lesions of the IC failed to show fear-potentiated startle in the presence of a noise previously conditioned to elicit fear. In Experiment 2, we demonstrated that rats with similarly placed lesions of the IC failed to inhibit fear-potentiated startle in the presence of a noise previously conditioned to inhibit fear to a light. Thus, in both Experiments 1 and 2, lesions of the IC disrupted the behavioral significance of the noise stimulus. Together with previous findings, these results are consistent with the view that the IC is a common source of diverging auditory information used to mediate the fear eliciting and safety signal properties conditioned to auditory stimuli.

Previous studies have suggested that the hippocampus may play an important role in some forms of inhibitory learning. The goal of the present study was to assess whether the hippocampus is also important for inhibition of fear acquired after serial feature-negative discrimination training. Rats were given aspiration lesions of the hippocampus either before or after training in which a target light was paired with shock when presented alone, but not paired with shock when presented in serial compound with a noise feature (light+/noise-->light-). Conditioned fear to the target stimulus and feature-target compound were measured with fear-potentiated startle. Pre-training lesion of the hippocampus did not disrupt feature-negative discrimination performance relative to sham-operated and cortical lesioned controls. In contrast, hippocampal lesions performed after training severely disrupted performance. Specifically, rats with hippocampal lesions failed to inhibit fear when the noise feature was presented in compound with the target. However, these rats could reacquire the feature-negative discrimination. These observations suggest that the hippocampus may normally be involved in retention or retrieval of serial feature-negative discrimination; however, in its absence feature-negative discrimination can still be acquired.

It is well known that a velocity perturbation can travel through a mass spring chain with strongly nonlinear interactions as a solitary and antisolitary wave pair. In recent years, nonlinear wave propagation in 2D structures have also been explored. Here we first consider the propagation of such a velocity perturbation for cases where the system has a 2D "Y"-shaped structure. Here each of the three pieces that make up the "Y" are made of a small mass spring chain. In addition, we consider a case where multiple "Y"-shaped structures are used to generate a "tree." We explore the early time dynamical behavior associated with the propagation of a velocity perturbation initiated at the trunk and at the extremities for both cases. We are looking for the energy transmission properties from one branch to another of these "Y"-shaped structures. Our dynamical simulations suggest the following broad observations: (i) for strongly nonlinear interactions, mechanical energy propagation resembles pulse propagation with the energy propagation being dispersive in the linear case; (ii) for strong nonlinear interactions, the tree-like structure acts as an energy gate showing preference for large perturbations in the system while the behavior of the linear case shows no such preference, thereby suggesting that such structures can possibly act as switches that activate at sufficiently high energies. The study aspires to develop insights into the nature of nonlinear wave propagation through a network of linear chains.

The fear-potentiated startle paradigm has been used with great success to examine conditioned fear in both rats and humans. The purpose of the present experiment was to extend the authors' previous findings and further validate the fear-potentiated startle paradigm in mice. In Experiments 1 and 2, C57BL/6J mice were given Pavlovian fear conditioning with either an auditory or a visual conditioned stimulus. Similar to data collected with rats, fear-potentiated startle was observed for both stimulus modalities. In Experiment 3, posttraining lesions of the amygdala disrupted fear-potentiated startle in both conditioned stimulus modalities. These data are consistent with amygdala lesion studies in rats and suggest that fear-potentiated startle in mice requires an intact amygdala. Together, these results extend the authors' previous results and provide the basis for using this well-understood behavioral paradigm for examining the molecular mechanisms of conditioned fear in transgenic and knockout mice.

The auditory thalamus is part of a neural circuit that mediates the expression of fear to auditory stimuli. Bilateral lesions of the auditory thalamus prevent the expression of fear to an auditory stimulus paired with shock. The present study assessed whether bilateral lesions of the auditory thalamus would also disrupt the inhibition of fear to an auditory stimulus paired with the absence of shock. Rats were given bilateral lesions of the auditory thalamus followed by Pavlovian conditioned inhibition training in which a light was paired with shock and a noise and light compound was presented in the absence of shock. Fear and the inhibition of fear were measured with the fear-potentiated startle effect. Lesions of the auditory thalamus did not disrupt the ability of the noise to inhibit the expression of fear to the light. However, these lesions did disrupt the ability of the noise to produce fear-potentiated startle after it had been subsequently paired with shock. These results suggest that although the auditory thalamus is an essential part of a neural circuit that mediates the expression of fear to auditory stimuli, it is not an essential part of the circuit that mediates the inhibition of fear to auditory stimuli.

The elicitation and reduction of fear were indexed with fear-potentiated startle and corticosterone release and induction of the immediate-early gene c-fos as a marker of neural activity in male Sprague-Dawley rats. Conditioning consisted of pairing one stimulus with footshock, which was withheld when the conditioned stimulus was preceded by a different modality stimulus, the conditioned inhibitor. On the test day, approximately 60% of the rats were used for c-fos in situ hybridization, and were presented with either the conditioned stimulus alone, the conditioned inhibitor alone, a compound of the two stimuli, or no stimuli, and killed 30 min following the presentation of 10 such stimuli. The remaining rats were tested with the fear-potentiated startle paradigm. Rats displayed reliable fear-potentiated startle and corticosterone release to the conditioned stimulus, and both measures were reduced when the conditioned stimulus was preceded by the conditioned inhibitor. The ventral bed nucleus of the stria terminalis, septohypothalamic nucleus, some tegmental nuclei, and the locus coeruleus had particularly high c-fos induction in rats that received the conditioned inhibitor, providing one of the first functional indication that these nuclei might be important in behavioural or endocrine inhibition. Conditioning specific c-fos induction in the three groups that received a stimulus on the test day was observed in many hypothalamic areas, the medial geniculate body and the central gray, structures previously involved in fear and anxiety. The cingulate, infralimbic and perirhinal cortex, nucleus accumbens, lateral septum, dorsal endopiriform nucleus, and ventral tegmental area had higher c-fos induction in rats presented with the fearful conditioned stimulus, confirming previous studies. The amygdala and hippocampus of conditioned rats did not show higher c-fos induction than in rats repeatedly exposed to the context. Many regions displayed c-fos messenger RNA induction in the control condition, suggesting that processes other than fear and anxiety participate in c-fos induction.

The fear-potentiated startle paradigm has been used with great success to examine conditioned fear in both rats and humans. The purpose of this study was to examine fear-potentiated startle in inbred mice. One-month-old C57BL/6J (C57) and DBA/2J (DBA) mice were given tone + foot shock training trials. The amplitude of the acoustic startle reflex was measured in the presence and absence of the tone both before and after training. Both strains showed fear-potentiated startle after training as evidenced by larger startle amplitudes in the presence of the tone than in its absence. However, the magnitude of fear-potentiated startle was greater in DBA mice than in C57 mice. These results not only demonstrate fear-potentiated startle in mice for the first time but also suggest that fear-potentiated startle can be influenced by characteristics of the mouse strain.

The capacities of three different conditioned stimulus modalities (light, noise, and airflow produced by a fan) to produce fear-potentiated startle were evaluated. Previous experiments have shown that following either light-shock or noise-shock pairings, both the light and noise conditioned stimuli acquire the ability to potentiate the acoustically elicited startle response in rats (the so-calledfear-potentiated startle effect). In Experiment 1, the ability of airflow produced by a fan to act as a conditioned stimulus was investigated. Rats were given either paired or impaired fan-shock training followed by a test for fear-potentiated startle. The fan conditioned stimulus potentiated startle only in the group given explicit fan-shock pairings. In Experiment 2, we evaluated the discriminability of the three conditioned stimulus modalities. Rats were given light, noise, or fan-shock pairings and were subsequently tested for fear-potentiated startle with the trained conditioned stimulus as well as the two remaining novel conditioned stimuli. Only the trained conditioned stimulus potentiated startle. These results show that fear-potentiated startle can be produced with three discriminable conditioned stimulus modalities, allowing the future use of fear-potentiated startle in the investigation of higher order conditioning phenomena.

Following observations in the literature that sensory cortex ablations prevent extinction of conditioned fear, the present experiments tested the generality of this finding by examining whether visual cortex ablations would prevent extinction of conditioned fear as assessed by fear-potentiated startle using a visual conditioned stimulus. Consistent with previous reports, visual cortex ablations did not prevent the acquisition or expression of fear-potentiated startle to a visual conditioned stimulus. More importantly, visual cortex ablations did not prevent extinction of fear-potentiated startle to a visual conditioned stimulus, nor did they reverse preoperatively established extinction, indicating that sensory cortex is not required for extinction of conditioned fear in all situations.

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.

In the fear-potentiated startle procedure, conditioned fear is defined as elevated startle amplitude in the presence versus the absence of a stimulus that was previously paired with shock. Owing to the fact that it involves the modulation of the acoustic startle response – a ubiquitous cross-species reflex that is easily and objectively quantified in rats, mice, and the human – fear-potentiated startle has been used quite successfully to examine the behavioral and neurobiological correlates of fear across these species. Anxiety also potentiates startle in rodents and the human, and recent work utilizing anxiety-potentiated startle procedures suggests that the neurobiological substrates of anxiety are separate and distinct from those of conditioned fear.

The present study is part of an ongoing series of experiments aimed at delineation of the neural pathways that mediate fear-potentiated startle, a model of conditioned fear in which the acoustic startle reflex is enhanced when elicited in the presence of a light previously paired with shock. A number of cortical areas that might be involved in relaying information about the visual conditioned stimulus (the light) in fear-potentiated startle were investigated. One hundred thirty-five rats were given 10 light-shock pairings on each of 2 consecutive days, and 1–2 d later electrolytic or aspiration lesions in various cortical areas were performed. One week later, the magnitude of fear-potentiated startle was measured. Complete removal of the visual cortex, medial prefrontal cortex, insular cortex, or posterior perirhinal cortex had no significant effect on the magnitude of fear-potentiated startle. Lesions of the frontal cortex attenuated fear-potentiated startle by approximately 50%. However, lesions of the anterior perirhinal cortex completely eliminated fear-potentiated startle. The effective lesions included parts of the cortex both dorsal and ventral to the rhinal sulcus and extended from approximately 1.8 to 3.8 mm posterior to bregma. Lesions slightly more posterior (2.3–4.8 mm posterior to bregma) or lesions that included only the perirhinal cortex dorsal to the rhinal sulcus had no effect. The region of the perirhinal cortex in which lesions blocked fear-potentiated startle projects to the amygdala, and thus may be part of the pathway that relays the visual conditioned stimulus information to the amygdala, a structure that is also critical for fear-potentiated startle. In addition, the present findings are in agreement with numerous studies in primates suggesting that the perirhinal cortex may play a more general role in memory.

The present study evaluated the effects of intraventricular or intracerebral administration of pertussis toxin on fear-potentiated startle (a measure of conditioned fear) and shock sensitization (a measure of unconditioned fear). In Experiment 1 all animals were unilaterally implanted with cannulae into the lateral ventricle 1 week prior to 2 days of fear conditioning (ten light-shock pairings on each of 2 days). Five days later, animals were infused with either 1 microgram pertussis toxin or saline and tested for fear-potentiated startle 24 h after infusion and tested for shock sensitization 26 or 50 h after infusion. Pertussis toxin blocked the ability of a light conditioned stimulus to facilitate startle but did not alter the ability of acute footshock to increase startle amplitude in the same animals. In Experiment 2 bilateral infusion of 1 microgram pertussis toxin into the basolateral nuclei of the amygdala, but not the interpositus nuclei of the cerebellum, also blocked fear-potentiated startle when animals were tested 6 h after infusion. These findings suggest a role for pertussis toxin sensitive G-proteins, perhaps within the amygdala, in the expression of conditioned but not unconditioned fear.

Intracerebroventricular infusion of corticotropin-releasing factor (CRF) (0.1–1.0 micrograms) produced a pronounced, dose-dependent enhancement of the acoustic startle reflex in rats. This excitatory effect began about 20–30 min after infusion, grew steadily over the 2 hr test period, and lasted at least 6 hr. Higher doses of CRF (10 micrograms) often produced marked facilitation and then inhibition of startle that oscillated repeatedly with a period of 10–20 min. CRF- enhanced startle did not result from an increase in sensitization produced by repetition of the startle stimulus or from a blockade of habituation. Peripheral injections of the autonomic ganglionic blockers hexamethonium (10 mg/kg) or chlorisondamine (3 mg/kg) slightly attenuated the magnitude of CRF-enhanced startle, suggesting a partial role of peripheral sympathetic activation. Intracerebroventricular infusion of the CRF antagonist alpha-helical CRF9–41 (alpha hCRF; 25 or 50 micrograms) blocked CRF-enhanced startle when infused 5 min prior to CRF, indicating a central site of action. CRF-enhanced startle also was reversed when alpha hCRF was given 90 min after infusion of CRF. This suggests that exogenously applied CRF remains in the brain for a very long time after administration or that CRF given exogenously initiates a process that results in a long-lasting activation of endogenous CRF. Because the startle reflex is elevated by both conditioned and unconditioned fear, these data lend further support to the idea that CRF infusion produces a behavioral state that resembles fear or anxiety. Because startle is mediated by a well-defined neural pathway, CRF-enhanced startle may provide a useful behavioral assay to analyze the neural systems upon which exogenous CRF acts to produce its behavioral effects.

Intracerebroventricular (icv) infusion of corticotropin-releasing factor (CRF) was previously found to produce a long-lasting, dose- dependent (0.1–1.0 microgram) increase in the amplitude of the acoustic startle reflex. The present study sought to determine where in the CNS CRF acts to increase startle. Intracisternal infusion of CRF (0.1–1.0 microgram) increased startle with a time course and magnitude similar to that produced by icv CRF, unlike intrathecal infusion, which produced a small, more rapid enhancement of startle. While lesions of the paraventricular nucleus of the hypothalamus had no effect on icv CRF-enhanced startle, bilateral lesions of the central nucleus of the amygdala significantly attenuated the excitatory effect of icv CRF but had no effect on intrathecal CRF-enhanced startle. Even though lesions of the amygdala blocked icv CRF-enhanced startle, local infusion of CRF into the amygdala did not significantly elevate startle. The present data indicate that the amygdala is part of the neural circuitry required for icv CRF to elevate startle, but does not appear to be the primary receptor area where CRF acts. The involvement of the amygdala in icv CRF-enhanced startle is consistent with the hypothesis that both the amygdala and CRF are critically involved in fear and stress.

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 anterograde transport of horseradish peroxidase and tritiated amino acids was used to examine the distribution and morphology of spinal afferent fibers terminating in the rat spinal trigeminal complex. The results confirm the existence of a direct, ipsilateral projection from the spinal cord which is distributed exclusively to the deepest layers of the medullary dorsal horn narrow regions subjacent to the spinal trigeminal tract in trigeminal nucleus interpolaris, trigeminal nucleus oralis and the trigeminal main sensory nucleus. Spinal inputs also terminated in the insular trigeminal-cuneatus lateralis nucleus which is a distinct component of the interstitial system of the spinal trigeminal tract. The spinal afferent fibers which terminated in the dorsolateral parts of the spinal trigeminal complex arose from the dorsal column funiculi, while those that terminated in ventral parts of the complex arose from both the dorsal column and lateral funiculi. The tritiated amino acid experiments indicate that at least part of the spinotrigeminal pathway originates from cells located in the cervical spinal dorsal horn. The present findings also document a complex spatial relationship between the spinotrigeminal and trigeminospinal pathways which includes an extensive overlap between spinotrigeminal fibers and spinal projecting neurons in each of the lateralmost regions of the complex. This spatial overlap supports the existence of anatomical substrates which may underlie functional reciprocal loops between the spinal trigeminal complex and cervical spinal cord. Since these regions are primarily concerned with the processing of sensory information from lateral and posterior parts of the face, it follows that the spinotrigeminal pathway may be primarily concerned with the integration of head and neck functions. In addition, the spatial convergence of spinal inputs and the distribution of other trigeminal efferent neurons suggests that part of the spinotrigeminal pathway may be involved in spino-trigemino-thalamic and spino-trigemino-cerebellar pathways in parallel with other spinobulbar pathways in the medulla.

The retrograde transport of horseradish peroxidase was used to examine and compare the distribution and morphology of thalamic, cerebellar and spinal projecting neurons in rat trigeminal nucleus interpolaris following large injections into their respective targets. The regional distribution of these three populations was evaluated in relation to the six cytoarchitecturally distinct regions which characterize the nucleus. Cerebellar projecting neurons were distributed throughout the rostrocaudal extent of trigeminal nucleus interpolaris, but were infrequently present in its dorsolateral region and in the rostral pole of the nucleus. Thalamic projecting neurons exhibited a distribution pattern that extensively overlapped with that of the trigeminocerebellar neurons; however, they were particularly concentrated in caudal, dorsomedial and rostral, ventrolateral regions of the nucleus. Trigeminospinal projecting neurons exhibited a more restricted distribution within ventral and lateral regions of trigeminal nucleus interpolaris. Although the three populations of projection neurons could not be distinguished solely on the basis of somatic size or shape, distinct regional variations in the distribution and somatodendritic and axonal morphology of these neurons indicated that they arise largely from independent cell populations. However, several regions were identified in which specific cell types were likely to contribute to axonal collateralization among these pathways. In the ventrolateral magnocellular region of the nucleus, for example, more than half of the large multipolar-shaped neurons were retrogradely labeled after injections into each of the three target sites. The results of the present study indicate that the thalamic, cerebellar and spinal projections of trigeminal nucleus interpolaris arise from a morphologically heterogeneous group of neurons. In addition, regional variations in the distribution and morphology of these neurons provide evidence for the existence of functionally distinct regions that parallel the cytoarchitecturally defined regions of the nucleus. This study also provides indirect evidence for and against collateralization among these three projections within specific regions of the nucleus.

The present experiments assessed the necessity of central CRF in reinstatement of extinguished fear. Using the fear-potentiated startle procedure, rats were given light-shock pairings (fear conditioning) followed by light-alone extinction training. Rats were then given unsignaled shocks to reinstate fear to the light conditioned stimulus (CS). Intracerebroventricular administration of the CRF antagonist α-Helical CRF9-41 prior to reinstatement training dose-dependently prevented reinstatement. Further, α-Helical CRF9-41 administration prior to reinstatement training or the test for reinstatement of fear to the extinguished CS prevented reinstatement at both treatment times, suggesting that CRF activity is critical for this type of return of fear to an extinguished CS. The abolition of reinstatement by drug administration was not due to state-dependent learning, as rats treated with the drug prior to both reinstatement training or testing also failed to show reinstatement of fear. Local administration of α-Helical CRF9-41 in the bed nucleus of the stria terminalis suggested that this area is a site at which central CRF is involved in this form of relapse.

Voluntary exercise has been associated with reduced anxiety across several animal models. Manipulation of central 5-HT can alter anxiety-like behaviors and administration of the 5-HT agonist metachlorophenylpiperazine (mCPP) increases anxiety in rodents and humans. To examine whether the anxiolytic effect of exercise is associated with an alteration in 5-HT systems, we examined the anxiogenic effect of mCPP in exercising and nonexercising mice. C57BL/6J mice were given 2 weeks of free access to either a functioning or nonfunctioning running wheel. Mice were then tested for acoustic startle following systemic injection of either 0, 0.1, 0.3, or 1 mg/kg of mCPP. Consistent with its anxiogenic properties, mCPP produced a dose-dependent increase in acoustic startle in nonexercising mice. However, this anxiogenic effect was blunted in exercising mice. These findings suggest that exercise may help to reduce anxiety by altering 5-HT systems, perhaps by down-regulating postsynaptic 5HT 2B/2C receptors.

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.

Pavlovian conditioning is a useful tool for elucidating the neural mechanisms involved with learning and memory, especially in regard to the stimuli associated with aversive events. The amygdala has been repeatedly implicated as playing a significant role in the acquisition and expression of fear. If the amygdala is critical for the acquisition of fear, then it should contribute to this processes regardless of the parameters used to induce or evaluate conditioned fear. A series of experiments using reversible inactivation techniques evaluated the role of the amygdala in the acquisition of conditioned fear when training was conducted over several days in rats. Fear-potentiated startle was used to evaluate the acquisition of conditioned fear. Pretraining infusions of N-methyl-d-aspartic acid (NMDA) or non-NMDA receptor antagonists alone into the amygdala interfered with the acquisition of fear early in training, but not later. Pretraining infusions of a cocktail consisting of both an NMDA and non-NMDA antagonist interfered with the acquisition of conditioned fear across all days of training. Taken together these results suggest the amygdala may potentially be critical for the acquisition of conditioned fear regardless of the parameters utilized.

There is considerable interest in examining the genes that may contribute to anxiety. We examined the function of ERK/MAPK in the acquisition of conditioned fear, as measured by fear-potentiated startle (FPS) in mice as a model for anticipatory anxiety in humans. We characterized the following for the first time in the mouse: (1) the expression of the ERK/MAPK signaling pathway components at the protein level in the lateral amygdala (LA); (2) the time course of activation of phospho-activated MAPK in the LA after fear conditioning; (3) if pharmacological inhibition of pMAPK could modulate the acquisition of FPS; (4) the cell-type specificity of pMAPK in the LA after fear conditioning. Using western blot and immunohistochemistry techniques and injecting the MEK inhibitor U0126 in the LA, we showed the following: (1) both MEK1/MEK2 and ERK1/ERK2 were co-expressed in the LA of the adult mouse brain; (2) there is a peak of pMAPK at 60 min after fear conditioning; (3) the ERK/MAPK signaling pathway activation is essential for the acquisition of an FPS response; (4) at 60 min, the pMAPK are exclusively neuronal and not glial. These results emphasize the importance of this signaling pathway in the acquisition of conditioned fear in the mouse. Given the widely held view that conditioned fear models the essential aspects of anxiety disorders, the results confirm the ERK/MAPK signaling pathway as a molecular target for the treatment of anxiety disorders in the clinic.

Pavlovian fear conditioning is frequently used to assess the behavioral, physiological, genetic and molecular correlates of learning and memory. In the typical Pavlovian conditioned fear procedure a neutral stimulus, such as a tone, is paired with a mildly aversive stimulus such as a foot shock. The tone conditioned stimulus (CS) comes to elicit a variety of behaviors that are indicative of learned fear. One of the more prominent of these behaviors is a potentiated acoustic startle response. While fear-potentiated startle in mice is qualitatively similar to that in rats, the stimulus parameters and procedures for producing optimum fear-potentiated startle in mice differ considerably from those used in rats. Procedures outlined in this unit include initial assessment of startle, fear conditioning and fear-potentiated startle testing. Special attention is paid to the parameters that affect the magnitude of fear-potentiated startle and procedures designed to systematically examine these parameters are included.