Expression Of Fear-potentiated Startle Research Articles

Behavioral Expression of Contextual Fear in Male and Female Rats.

The study of fear conditioning has led to a better understanding of fear and anxiety-based disorders such as post-traumatic stress disorder (PTSD). Despite the fact many of these disorders are more common in women than in men, the vast majority of work investigating fear conditioning in rodents has been conducted in males. The goal of the work presented here was to better understand how biological sex affects contextual fear conditioning and expression. To this end, rats of both sexes were trained to fear a specific context and fear responses were measured upon re-exposure to the conditioning context. In the first experiment, male and female rats were given context fear conditioning and tested the next day during which freezing behavior was measured. In the second experiment, rats were trained and tested in a similar fashion while fear-potentiated startle and defecation were measured. We found that males showed more freezing behavior than females during a fear expression test. The expression of fear-potentiated startle did not differ between sexes, while males exhibited more defecation during a test in a novel context. These data suggest that the expression of defensive behavior differs between sexes and highlight the importance of using multiple measures of fear when comparing between sexes.

Endocannabinoids and Fear-Related Behavior in Mice Selectively Bred for High or Low Alcohol Preference.

Alcohol use disorders (AUDs) have a high incidence of co-morbidity with stress-related psychopathologies, such as post-traumatic stress disorder (PTSD). Genetic and pharmacological studies support a prominent role for the endocannabinoid system (ECS) in modulating stress-related behaviors relevant to AUDs and PTSD. Mouse lines selectively bred for high (HAP) and low (LAP) alcohol preference show reproducible differences in fear-potentiated startle (FPS), a model for PTSD-related behavior. The first experiment in this study assessed levels of the endocannabinoids, anandamide (AEA) and sn-2 arachidonylglycerol (2-AG), in the prefrontal cortex (PFC), amygdala (AMG), and hippocampus (HIP) of male and female HAP1 and LAP1 mice following the expression of FPS to determine whether ECS responses to conditioned-fear stress (FPS) were correlated with genetic propensity toward high or low alcohol preference. The second experiment examined effects of a cannabinoid receptor type 1 agonist (CP55940) and antagonist (rimonabant) on the expression of FPS in HAP1 and LAP1 male and female mice. The estrous cycle of females was monitored throughout the experiments to determine if the expression of FPS differed by stage of the cycle. FPS was greater in male and female HAP1 than LAP1 mice, as previously reported. In both experiments, LAP1 females in diestrus displayed greater FPS than LAP1 females in metestrus and estrus. In the AMG and HIP, AEA levels were greater in male fear-conditioned HAP1 mice than LAP1 mice. There were no line or sex differences in effects of CP55940 or rimonabant on the expression of FPS. However, surprisingly, evidence for anxiogenic effects of prior treatment with CP55940 were seen in all mice during the third drug-free FPS test. These findings suggest that genetic differences in ECS function in response to fear-conditioning stress may underlie differences in FPS expression in HAP1 and LAP1 selected lines.

No effect of sex and estrous cycle on the fear potentiated startle response in rats

The prevalence of anxiety disorders is higher in women than in men. Yet preclinical studies on anxiety are mostly performed in male subjects. This may have limited our understanding of mechanisms contributing to anxiety disorders. Since fear conditioning is considered an important factor in the etiology of anxiety disorders, the present study aimed to investigate the effect of sex and estrous cycle on conditioned fear and the anxiolytic effect of benzodiazepines in rats.We measured the fear-potentiated startle response in male and female rats during different estrous cycle stages and performed a replication study in a separate cohort. In addition, we assessed the response to diazepam (0–3.0 mg/kg IP) and chlordiazepoxide (0–10 mg/kg IP) in male and female rats in proestrous/estrous and diestrous stage.Our results showed that there were no sex differences in the expression of fear-potentiated startle. The estrous cycle also did not affect the fear-potentiated startle response. In addition, male and female rats did not differ in their fear-potentiated startle response following treatment with either diazepam or chlordiazepoxide.In conclusion, the current study shows that male and female rats do not differ in their conditioned fear response and the responsiveness to benzodiazepines. The results further indicate that conditioned fear-related processes are not affected by gonadal hormone fluctuations in this paradigm. These findings may suggest that the higher prevalence of anxiety disorders in women more likely results from differences in responding to previous experiences or differences in other predisposing factors, rather than differences in conditioned fear per se.

Genetic correlation between alcohol preference and conditioned fear: Exploring a functional relationship.

Post-traumatic stress disorder (PTSD) and alcohol-use disorders have a high rate of co-occurrence, possibly because they are regulated by common genes. In support of this idea, mice selectively bred for high (HAP) alcohol preference show greater fear potentiated startle (FPS), a model for fear-related disorders such as PTSD, compared to mice selectively bred for low (LAP) alcohol preference. This positive genetic correlation between alcohol preference and FPS behavior suggests that the two traits may be functionally related. This study examined the effects of fear conditioning on alcohol consumption and the effects of alcohol consumption on the expression of FPS in male and female HAP2 and LAP2 mice. In experiment 1, alcohol consumption (g/kg) under continuous-access conditions was monitored daily for 4 weeks following a single fear-conditioning or control treatment (foot shock and no shock). FPS was assessed three times (once at the end of the 4-week alcohol access period, once at 24h after removal of alcohol, and once at 6-8 days after removal of alcohol), followed by two more weeks of alcohol access. Results showed no change in alcohol consumption, but alcohol-consuming, fear-conditioned, HAP2 males showed increased FPS at 24h during the alcohol abstinence period compared to control groups. In experiment 2, alcohol consumption under limited-access conditions was monitored daily for 4 weeks. Fear-conditioning or control treatments occurred four times during the first 12 days and FPS testing occurred four times during the second 12 days of the 4-week alcohol consumption period. Results showed that fear conditioning increased alcohol intake in both HAP2 and LAP2 mice immediately following the first conditioning session. Fear-conditioned HAP2 but not LAP2 mice showed greater alcohol intake compared to control groups on drinking days that occurred between fear conditioning and FPS test sessions. FPS did not change as a function of alcohol consumption in either line. These results in mice help shed light on how a genetic propensity toward high alcohol consumption may be related to the risk for developing PTSD and co-morbid alcohol-use disorders in humans.

Lifelong disturbance of serotonin transporter functioning results in fear learning deficits: Reversal by blockade of CRF1 receptors

The inability to associate aversive events with relevant cues (i.e. fear learning) may lead to maladaptive anxiety. To further study the role of the serotonin transporter (SERT) in fear learning, classical fear conditioning was studied in SERT knockout rats (SERT−/−) using fear potentiation of the startle reflex. Next, fear acquisition and concomitant development of contextual conditioned fear were monitored during training. To differentiate between developmental and direct effects of reduced SERT functioning, effects of acute and chronic SSRI treatment were studied in adult rats. Considering the known interactions between serotonin and corticotropin-releasing factor (CRF), we studied the effect of the CRFR1 antagonist CP154,526 on behavioral changes observed and determined CRF1 receptor levels in SERT−/− rats. SERT−/− showed blunted fear potentiation and enhanced contextual fear, which resulted from a deficit in fear acquisition. Paroxetine treatment did not affect acquisition or expression of fear-potentiated startle, suggesting that disturbed fear learning in SERT−/− results from developmental changes and not from reduced SERT functioning. Although CRF1 receptor levels did not differ significantly between genotypes, CP154,526 treatment normalized both cue- and contextual fear in SERT−/− during acquisition, but not expression of fear-potentiated startle. The disrupted fear acquisition and concomitant increase in contextual conditioned fear-potentiated startle fear in SERT−/− resembles the associative learning deficit seen in patients with panic disorder and suggests that normal SERT functioning is crucial for the development of an adequate fear neuro-circuitry. Moreover, the normalization of fear acquisition by CP154,526 suggests a role for central CRF signaling in the generalization of fear.

Medial prefrontal cortex serotonergic and GABAergic mechanisms modulate the expression of contextual fear: Intratelencephalic pathways and differential involvement of cortical subregions

Several lines of evidence indicate that the dorsal hippocampus (dH) and medial prefrontal cortex (mPFC) regulate contextual fear conditioning. The prelimbic (PrL), infralimbic (IL) and the anterior cingulate cortex (ACC) subregions of the mPFC likely play distinct roles in the expression of fear. Moreover, studies have highlighted the role of serotonin (5-hydroxytryptamine, 5-HT)- and γ-aminobutyric acid (GABA)-mediated mechanisms in the modulation of innate fear in the mPFC. The present study characterized dH-mPFC pathways and investigated the role of serotonergic and GABAergic mechanisms of the PrL, IL and ACC-area 1 (Cg1) in the elaboration of contextual fear conditioning using fear-potentiated startle (FPS) and freezing behavior in Rattus norvegicus. The results of neurotracing with microinjections of biotinylated dextran amine into the dH revealed a neural link of the dH with the PrL and ACC. Intra-PrL injections of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and the GABAA receptor-selective agonist muscimol reduced contextual FPS and freezing responses. Intra-Cg1 injections of muscimol but not 8-OH-DPAT decreased FPS and freezing responses. However, neither intra-IL injections of a 5-HT1A agonist nor of a GABAA agonist affected these defensive responses. Labeled neuronal fibers from the dH reached the superficial layers of the PrL cortex and spread to the inner layers of PrL and Cg1 cortices, supporting the pharmacological findings. The present results confirmed the involvement of PrL and Cg1 in the expression of FPS and freezing responses to aversive conditions. In addition, PrL serotoninergic mechanisms play a key role in contextual fear conditioning. This study suggests that PrL, IL and Cg1 distinctively contribute to the modulation of contextual fear conditioning.

Effects of alcohol on the acquisition and expression of fear-potentiated startle in mouse lines selectively bred for high and low alcohol preference

Anxiety disorders and alcohol use disorders frequently co-occur in humans perhaps because alcohol relieves anxiety. Studies in humans and rats indicate that alcohol may have greater anxiolytic effects in organisms with increased genetic propensity for high alcohol consumption. The purpose of this study was to investigate the effects of moderate doses of alcohol (0.5, 1.0, 1.5 g/kg) on the acquisition and expression of anxiety-related behavior using a fear-potentiated startle (FPS) procedure. Experiments were conducted in two replicate pairs of mouse lines selectively bred for high- (HAP1 and HAP2) and low- (LAP1 and LAP2) alcohol preference; these lines have previously shown a genetic correlation between alcohol preference and FPS (HAP > LAP; Barrenha and Chester, Alcohol Clin Exp Res 31:1081-1088, 2007). In a control experiment, the effect of diazepam (4.0 mg/kg) on the expression of FPS was tested in HAP2 and LAP2 mice. The 1.5 g/kg alcohol dose moderately decreased the expression of FPS in both HAP lines but not LAP lines. Alcohol had no effect on the acquisition of FPS in any line. Diazepam reduced FPS to a similar extent in both HAP2 and LAP2 mice. HAP mice may be more sensitive to the anxiolytic effects of alcohol than LAP mice when alcohol is given prior to the expression of FPS. These data collected in two pairs of HAP/LAP mouse lines suggest that the anxiolytic response to alcohol in HAP mice may be genetically correlated with their propensity toward high alcohol preference and robust FPS.

Gone but not forgotten

GENERAL COMMENTARY article Front. Behav. Neurosci., 18 August 2011Sec. Learning and Memory https://doi.org/10.3389/fnbeh.2011.00051

Nucleus accumbens carbachol disrupts olfactory and contextual fear-potentiated startle and attenuates baseline startle reactivity

Although the nucleus accumbens (NAc) typically is not considered a primary component of the circuitry underlying either the acquisition or retrieval of conditioned fear, evidence suggests that this region may play some role in modulating fear-related behaviors. The goal of the present study was to explore a potential role for NAc cholinergic receptors in the expression of fear-potentiated startle (FPS) and baseline startle reactivity. Intra-NAc infusion of the broad-acting cholinergic receptor agonist, carbachol, suppressed FPS elicited by re-exposure to both a discrete odor previously paired with footshock and the conditioning context. Although carbachol elevated spontaneous motor activity, activity bouts did not account for startle suppression in carbachol-treated Ss. In addition, intra-NAc carbachol suppressed baseline startle over a range of acoustic pulse intensities in the absence of explicit fear conditioning. Collectively, these findings suggest that NAc cholinergic receptors play a role in the modulation of baseline startle reactivity, rather than in the retrieval of learned fear, and that this role is independent of overt motor activity.

Effects of the novel endocannabinoid uptake inhibitor, LY2183240, on fear-potentiated startle and alcohol-seeking behaviors in mice selectively bred for high alcohol preference

Alcohol-use disorders often occur together with anxiety disorders in humans which may be partly due to common inherited genetic factors. Evidence suggests that the endocannabinoid system (ECS) is a promising therapeutic target for the treatment of individuals with anxiety and/or alcohol-use disorders. The present study assessed the effects of a novel endocannabinoid uptake inhibitor, LY2183240, on anxiety- and alcohol-seeking behaviors in a unique animal model that may represent increased genetic risk to develop co-morbid anxiety and alcohol-use disorders in humans. Mice selectively bred for high alcohol preference (HAP) show greater fear-potentiated startle (FPS) than mice selectively bred for low alcohol preference (LAP). We examined the effects of LY2183240 on the expression of FPS in HAP and LAP mice and on alcohol-induced conditioned place preference (CPP) and limited-access alcohol drinking behavior in HAP mice. Repeated administration of LY2183240 (30 mg/kg) reduced the expression of FPS in HAP but not LAP mice when given prior to a second FPS test 48 h after fear conditioning. Both the 10 and 30 mg/kg doses of LY2183240 enhanced the expression of alcohol-induced CPP and this effect persisted in the absence of the drug. LY2183240 did not alter limited-access alcohol drinking behavior, unconditioned startle responding, or locomotor activity. These findings suggest that ECS modulation influences both conditioned fear and conditioned alcohol reward behavior. LY2183240 may be an effective pharmacotherapy for individuals with anxiety disorders, such as post-traumatic stress disorder, but may not be appropriate for individuals with co-morbid anxiety and alcohol-use disorders.

The Role of Neuropeptide Y in the Expression and Extinction of Fear-Potentiated Startle

Neuropeptides are a promising target for novel treatments for anxiety and other psychiatric disorders and neuropeptide Y (NPY) has emerged as a key component of anxiolytic circuits in the brain. For this reason, we have evaluated the role of NPY in the expression and extinction of conditioned fear. We found that intracerebroventricular administration of NPY inhibits both baseline acoustic startle and the expression of fear-potentiated startle. Infusion of NPY (10 pmol/side) into the basolateral, but not the medial, nucleus of the amygdala reproduced the intracerebroventricular effect. Central administration of NPY (10 microg) also enhanced within-session extinction of fear-potentiated startle. This finding, coupled with the growing body of literature correlating NPY with resilience in humans, led us to the hypothesis that NPY may enhance the extinction of conditioned fear. When NPY (10 microg) is administered intracerebroventricularly before extinction training, extinction retention for both the contextual and cued components of conditioned fear is enhanced when tested 48 h later off drug. Additionally, we found that intra-basolateral amygdala administration of the NPY Y(1) receptor antagonist BIBO 3304 (200 pmol/side) before extinction training led to a profound deficit in extinction retention. This is the first evidence that NPY facilitates and an NPY antagonist blocks the extinction of conditioned fear. We believe that the role of NPY in the extinction of conditioned fear may, at least in part, explain the mechanism underlying the association between NPY and psychobiological resilience in humans.

Role of the Primate Amygdala in Fear-Potentiated Startle: Effects of Chronic Lesions in the Rhesus Monkey

In experiment 1, we assessed the role of the primate amygdala and hippocampus in the acquisition of learned fear measured with fear-potentiated startle. Three groups of six rhesus monkeys were prepared with bilateral ibotenic acid lesions of the amygdaloid complex and the hippocampus or were sham operated. Selective ibotenic acid lesions of the amygdala, but not the hippocampus, blocked the acquisition of fear-potentiated startle. In experiment 2, we assessed the role of the primate amygdala in the expression of fear-potentiated startle. Surprisingly, animals that sustained amygdala damage after they successfully learned fear-potentiated startle expressed normal fear-potentiated startle, despite a complete amygdala lesion based on magnetic resonance imaging assessments. These results suggest that although the amygdala is necessary for the initial acquisition of fear-potentiated startle, it is not necessary for the retention and expression of fear-potentiated startle. These findings are discussed in relation to the role of the amygdala in emotional learning and in cross-species comparisons of emotional behavior.

Carbachol injections into the nucleus accumbens disrupt acquisition and expression of fear-potentiated startle and freezing in rats

The nucleus accumbens is involved in different types of emotional learning, ranging from appetitive instrumental learning to Pavlovian fear conditioning. In previous studies, we found that temporary inactivation of the nucleus accumbens blocked both the acquisition and expression of conditioned fear. This was not due to altered dopaminergic activity as we have also found that intra-nucleus accumbens infusions of the dopamine agonist amphetamine do not affect either the acquisition or the expression of conditioned fear. Therefore, in the present study we examined whether cholinergic activity in the nucleus accumbens is involved in the acquisition and expression of conditioned fear. Specifically, the effect of intra-nucleus accumbens infusions of the unselective cholinergic agonist carbachol on the acquisition and expression of conditioned fear was assessed. Across several experiments, we measured fear to visual and acoustic conditioned stimuli and to the experimental context. Further, two different measures of conditioned fear were recorded: fear potentiation of startle and freezing. Intra-nucleus accumbens carbachol infusions disrupted acquisition as well as expression of conditioned fear, regardless of the modality of the fear-eliciting stimulus or of the specific measure of conditioned fear. This disruption of conditioned fear was not simply a by-product of enhanced motor activity which also occurred after intra-nucleus accumbens carbachol infusions. Interestingly, despite the substantial effect of intra-nucleus accumbens carbachol on expression of conditioned fear, the results of the final experiment suggest that these rats extinguish similarly to control rats. Taken together, the present results indicate that acetylcholine within the nucleus accumbens is important for the learning and retrieval of conditioned fear.

The expression of fear-potentiated startle during development: Integration of learning and response systems.

Relative to freezing, fear-potentiated startle (FPS) is developmentally delayed. Rats trained on Postnatal Day (PD) 18 expressed conditioned stimulus learning on PD 19 in freezing but not in FPS, whereas rats trained on PD 24 and tested on PD 25 expressed both freezing and FPS (Experiment 1). According to a neural maturation hypothesis, this delay results from functional immaturity of pathways mediating FPS. When rats were trained on PD 18, neither delaying the FPS test, allowing FPS pathways to develop, nor administrating the "reminder" treatment, the expression of FPS was promoted (Experiments 1, 2, and 2A). PD 18 learning was expressed in FPS on PD 25 when nontarget conditioned stimulus-unconditioned stimulus training occurred prior to the test, and this effect was modality dependent (Experiments 3 and 4). The authors conclude that engaging mechanisms of associative encoding when FPS pathways are functional is a critical condition for integrating learning and FPS response systems in development.

Effects of clonidine injections into the bed nucleus of the stria terminalis on fear and anxiety behavior in rats

Emotions such as fear and anxiety are mediated by a neural network containing nuclei like the amygdala, the bed nucleus of the stria terminalis and the periaqueductal gray. Noradrenaline is a neurotransmitter closely connected with the processing of stimuli eliciting these emotions. The bed nucleus of the stria terminalis contains the highest density of noradrenaline within the brain. In the present study, we investigated effects of injections of the noradrenergic α 2-adrenoceptor agonist clonidine into the bed nucleus of the stria terminalis on learned and unlearned fear (anxiety) in rats on different animal models of fear and anxiety: acquisition and expression of fear-potentiated startle, sensitization of the acoustic startle response by foot shocks and light-enhanced startle. Clonidine injections disrupted acquisition and expression of fear-potentiated startle, as well as light-enhanced startle, whereas sensitization was not affected. These results indicate that noradrenaline within the bed nucleus of the stria terminalis mediates both fear and anxiety. We suggest that there is rather a neurochemical than a neuroanatomical dissociation between learned fear and anxiety as hypothesized by Walker and Davis (Walker, D.L. and M. Davis, 1997b, Double dissociation between the involvement of the bed nucleus of the stria terminalis and the central nucleus of the amygdala in startle increases produced by conditioned versus unconditioned fear, J. Neurosci. 17, 9375–9383.).

Temporary inactivation of the nucleus accumbens disrupts acquisition and expression of fear-potentiated startle in rats

Recent research suggests that in addition to its prominent role in appetitive learning, the nucleus accumbens (NAC) may also be involved in fear conditioning. In the present study, we investigated whether temporary inactivation of the NAC, by injection of tetrodotoxin (TTX), affects acquisition and expression of conditioned fear, as measured by fear-potentiated startle (FPS). TTX injection into the NAC totally blocked acquisition and markedly decreased expression of conditioned fear to a discrete visual conditioned stimulus (CS). Interestingly, temporary inactivation of the NAC did not affect shock sensitization of startle, indicating that both the perception of the shock and short-term contextual conditioning was not affected by intra-accumbal TTX injection. Taken together, these results show that the NAC is crucial for acquisition and expression of long-term conditioned fear, as measured by fear-potentiated startle, to discrete CSs, but not short-term conditioned fear to a context.

The nucleus accumbens is not critically involved in mediating the effects of a safety signal on behavior.

Although considerable progress has been made towards understanding the neural systems mediating conditioned fear, little is known about the neural mechanisms underlying conditioned inhibitors of fear (or safety signals). The present series of experiments examined the involvement of the nucleus accumbens (NAC) in mediating the effects of safety signals on behavior using a conditioned inhibition of fear-potentiated startle paradigm. Neither increasing dopaminergic nor decreasing glutamatergic function in the NAC altered the magnitude of conditioned fear or conditioned inhibition of fear in rats. Furthermore, large pre- or post-training electrolytic lesions of the NAC did not affect acquisition or expression of fear-potentiated startle or conditioned inhibition of fear-potentiated startle. Taken together, these data suggest that the NAC is not critically involved in the acquisition or expression of fear-potentiated startle or conditioned inhibition of fear-potentiated startle. Previous research has implicated the NAC in 'reward-attenuated startle' in which presentation of a stimulus paired with food decreased startle responding. The present results, therefore, indicate important neural dissociations between the processing of appetitive and safety signals, even though behavioral studies and learning theories have suggested that these two forms of learning share some commonalities.

Inhibition of fear potentiated startle in rats following peripheral administration of secretin.

Previous results indicate that peripheral administration of secretin leads to robust Fos protein expression in the central nucleus of the rat amygdala. The implications of this observation on rat brain function, if any, remain unclear. We examined the effect of systemic secretin administration on the expression of fear-potentiated startle in rats, a behavioral response known to require an intact, functional central nucleus of the amygdala. Rats were trained to associate a neutral light conditioned stimulus (CS) with footshock, a fear-inducing unconditioned stimulus (US). Twenty-four hours later, rats were administered secretin or vehicle and were tested immediately for their startle response to a loud noise in the presence or absence of the light. Within a dose range relevant to its clinical use in autistic children, secretin dose-dependently decreased the magnitude of fear-potentiated startle in rats. This investigation provides additional evidence that systemically administered secretin can influence a neural network implicated in the acquisition and expression of emotional behaviors, including fear and anxiety.

CREB and the enhancement of long-term memory

Studies performed on invertebrates and mammals indicate that temporally spaced behavioural training is more effective for eliciting long-term memory (LTM) than massed training. At the molecular level, LTM, but not short-term memory (STM), requires new protein synthesis for its formation. How is the formation of LTM tuned to specific temporal patterns of training?A study by Josselyn et al. 1xLong-term memory is facilitated by cAMP response element binding protein overexpression in the amygdala. Josselyn, S.A. et al. J. Neurosci. 2001; 21: 2404–2412PubMedSee all References now provides the first direct evidence that overexpression of CREB (cAMP response element-binding protein) in the mammalian brain improves the formation of LTM after massed training. CREB is a transcription factor that is known to regulate the synthesis of proteins important for the formation of LTM. In their study, Josselyn et al. hypothesized that overexpression of CREB in one region of the rat brain, the basolateral amygdala (BLA), should enhance LTM for fear conditioning following massed training trials, which normally do not elicit robust LTM. In fear conditioning, rats learn to associate a conditioned stimulus (e.g. a light) with a harmful stimulus, such as footshock. Successful retention of this association is evident from enhanced startle responses displayed by rats when the startle reflex is subsequently elicited in the presence of the conditioned stimulus. Expression of fear-potentiated startle, and acquisition of fear conditioning, are dependent upon the BLA. Josselyn et al. showed that temporally spaced training trials elicited robust LTM for fear-potentiated startle in control rats, whereas temporally massed training produced weaker LTM in the same animals. They then used herpes simplex virus (HSV) vector-mediated gene transfer to overexpress CREB (‘HSV-CREB’) in the BLA of rats before behavioural training. Josselyn et al. confirmed that CREB overexpression was specific to the lateral (but absent from the central) nucleus of the amygdala and that injection of HSV vectors per se had no harmful effects on their behavioural measurements. In rats that overexpressed CREB in the BLA during massed fear conditioning, LTM was significantly enhanced to levels seen following spaced training. Rats that received HSV-CREB injections in adjacent brain regions that are not crucial for fear conditioning did not show memory improvement. Injection of mutant CREB into the BLA also did not enhance LTM compared with saline-injected animals. More importantly, HSV-CREB injection into the BLA did not enhance STM and it did not improve LTM following spaced training using both weak and strong footshock intensities. The latter finding shows that enhancement of LTM is specific to massed training.These data support the notion that CREB exists as activator and repressor isoforms. Activator isoforms are hypothesized to inactivate more slowly than repressor isoforms. Spaced training might elicit more effective LTM because activator isoforms have time to accumulate during the rest intervals between training trials. Massed training elicits weaker LTM presumably because the lack of rest intervals does not permit activator isoforms to accumulate (i.e. repressor isoforms outweigh the activators). The finding by Josselyn et al. that CREB overexpression enhanced LTM after massed training (but not after spaced training using weak or strong shock intensities) supports this model. Spaced training might allow sufficient accumulation of activators (even at weak shock intensities) such that artificial overexpression of CREB might not produce further improvement of LTM. Some questions that are ripe for future exploration include: which downstream genes regulated by CREB are crucial for LTM? Can CREB over-expression in other specific brain regions enhance other distinct types of behavioural memory?

Muscimol in the deep layers of the superior colliculus/mesencephalic reticular formation blocks expression but not acquisition of fear-potentiated startle in rats.

Deposits of the retrograde tracer Fluoro-Gold into the ventrolateral nucleus reticularis pontis caudalis labeled neurons in the deep layers of the superior colliculus/mesencephalic reticular formation (deep SC/Me). To test the involvement of this area in the fear-potentiated startle effect, rats were implanted with cannulas into the deep SC/Me and trained for fear-potentiated startle after infusion of the GABA(A) agonist muscimol (0.1 microg/0.5 microl). Two days later, they were tested for fear-potentiated startle. Rats then received a 2nd training session without any infusions, and 2 days later they were reinfused with muscimol (0.1 microg/0.5 microl) and tested for fear-potentiated startle. Local infusion of muscimol into the deep SC/Me completely blocked the expression but not the acquisition of fear-potentiated startle. These results indicate that a synapse in the midbrain is critical for the expression of fear-potentiated startle.