Expression Of Conditioned Freezing Research Articles

The activity of discrete sets of neurons in the posterior insula correlates with the behavioral expression and extinction of conditioned fear.

The interoceptive insular cortex is known to be involved in the perception of bodily states and emotions. Increasing evidence points to an additional role for the insula in the storage of fear memories. However, the activity of the insula during fear expression has not been studied. We addressed this issue by recording single units from the posterior insular cortex (pIC) of awake behaving rats expressing conditioned fear during its extinction. We found a set of pIC units showing either significant increase or decrease in activity during high fear expression to the auditory cue ("freezing units"). Firing rate of freezing units showed high correlation with freezing and outlasted the duration of the auditory cue. In turn, a different set of units showed either significant increase or decrease in activity during low fear state ("extinction units"). These findings show that expression of conditioned freezing is accompanied with changes in pIC neural activity and suggest that the pIC is important to regulate the behavioral expression of fear memory. NEW & NOTEWORTHY Here, we show novel single-unit data from the interoceptive insula underlying the behavioral expression of fear. We show that different populations of neurons in the insula codify expression and extinction of conditioned fear. Our data add further support for the insula as an important player in the regulation of emotions.

Dopamine D2 receptors modulate the expression of contextual conditioned fear

Although dopaminergic systems are more commonly associated with the reinforcing effects of various stimuli, numerous reports have demonstrated a relationship between changes in dopaminergic transmission and aversive situations. In the present study, we examined the involvement of D1-like and D2-like receptors in the expression of conditioned freezing using the context as the conditioned stimulus. Intraperitoneal injections of the D1 agonist SKF38393 or the D1 antagonist SCH23390 did not change the conditioned freezing in rats subjected to the contextual fear paradigm. In contrast, intraperitoneal injections of the D2 agonist quinpirole and the D2 antagonist sulpiride caused a significant dose-dependent reduction in the expression of contextual conditioned freezing. As these data may reflect that the systemic manipulations acted on dopaminergic receptors in different brain areas, the effects of administration of quinpirole and sulpiride into the ventral tegmental area (VTA) and the basolateral amygdala complex (BLA) on the expression of contextual conditioned freezing were also evaluated. Intra-VTA quinpirole and intra-BLA sulpiride injections reduced the conditioned freezing response; intra-VTA sulpiride and intra-BLA quinpirole injections had no significant effects. These data suggest that D2-like receptors, but not D1-like receptors, play an important role in the expression of contextual conditioned freezing. Quinpirole may act at D2 presynaptic receptors located in the VTA, decreasing dopamine levels in the terminal fields of the mesolimbic pathway. The effects of sulpiride, in contrast, appear to be triggered by an action on postsynaptic dopaminergic receptors located in the BLA. However, it cannot be totally excluded that the injected solutions did not also affect neighboring amygdalar regions. Together with previous findings, the present data suggest the need to consider dopaminergic mechanisms in the mesolimbic circuit as novel targets for the pharmacological treatment of fear-related disorders, especially post-traumatic stress disorder.

Effects of acute citalopram on the expression of conditioned freezing in naive versus chronic citalopram-treated rats

An acute challenge with selective serotonin (5-HT) reuptake inhibitors (SSRIs) reduces the conditioned freezing in rats, a model of anxiety. The increase in the 5-HT levels in the nerve terminal induced by SSRIs is closely related to its pharmacological effects. Clinically, SSRIs exert an anxiolytic effect after chronic treatment. The effects of repeated treatment with citalopram on conditioned freezing in rats were examined in the present study. Acute citalopram (10 mg/kg) reduced freezing at a short post-training interval (1 day) significantly. While the effect of citalopram (10 mg/kg) on freezing was diminished by prolonging the interval between conditioning and the exposure to conditioned fear stress, repeated citalopram (10 mg/kg) injection twice daily for 7 days restored the inhibitory effect of acute challenge of citalopram (10 mg/kg) on freezing. By prolonging the period between conditioning and exposure to conditioned fear stress, this model may have a more precise predictive validity of anxiety disorder as an animal model.

Facilitation of contextual fear memory extinction and anti-anxiogenic effects of AM404 and cannabidiol in conditioned rats

The present study investigated the central effects of the eCB uptake/metabolism inhibitor AM404 and the phytocannabinoid cannabidiol (CBD) on the extinction of contextual fear memories in rats. Rats were conditioned and 24 h later subjected to three consecutive 9-min non-reinforced exposures to the conditioning context (extinction sessions, 24 h intervals). AM404 or CBD was injected i.c.v. 5 min before each extinction session and a 3-min drug-free test of contextual memory was performed 24 h after the last extinction session. AM404 (1.0 µg/µl, i.c.v.) and CBD (2.0 µg/µl, i.c.v.) facilitated extinction of contextual fear memory, with persistent effects. These responses were antagonized by the CB1-selective antagonist SR141716A (0.2 mg/kg, i.p.), but not by the TRPV1-selective antagonist capsazepine (5.0 µg/µl, i.c.v.). The effect of the anxiolytic drug Diazepam (DZP) on the extinction of contextual fear memory was also investigated. In contrast with the CBD and AM404 results, DZP induced a general reduction in the expression of conditioned freezing. Both AM404 and CBD induced anti-anxiogenic effect in the fear-potentiated plus-maze test, whereas DZP was anxiolytic in conditioned and unconditioned rats. In conclusion, CBD, a non-psychoactive phytocannabinoid could be an interesting pharmacological approach to reduce the anxiogenic effects of stress and promote the extinction of fear memories.

Effect of co-administration of the selective 5-HT 1A receptor antagonist WAY 100,635 and selective 5-HT 1B/1D receptor antagonist GR 127,935 on anxiolytic effect of citalopram in conditioned fear stress in the rat

This study investigated the effect of co-administration of the selective 5-HT 1A receptor antagonist WAY 100,635 and selective 5-HT 1B/1D receptor antagonist GR 127,935 with a subactive dose of citalopram [selective serotonin (5-HT) reuptake inhibitor (SSRI)] on the expression of conditioned freezing, an index of fear. In the present study, acute administration of citalopram (s.c.) reduced freezing significantly at high doses (10, 30 and 100 mg/kg), while showing no significant effect at low doses (1 and 3 mg/kg). Co-administration of WAY 100,635 (0.15 mg/kg) with citalopram (3 mg/kg) reduced freezing markedly and significantly, as compared with either drug alone. However, the addition of GR 127,935 (4 mg/kg) did not potentiate the effects of citalopram (3 mg/kg) on freezing and did not enhance the effect of WAY 100,635 (0.15 mg/kg) with citalopram (3 mg/kg). Co-administration of WAY 100,635 (0.15 mg/kg) or GR 127,935 (4 mg/kg) gave no effect on high-dose citalopram (30 mg/kg)-induced inhibition of freezing behavior. These results suggest that co-administration of WAY 100,635 (0.15 mg/kg) strengthens the anxiolytic effect of citalopram (3 mg/kg) by facilitating central 5-HT neurotransmission. Since GR 127,935 (4 mg/kg) failed to accelerate the inhibition of freezing induced by citalopram (3 mg/kg) with WAY 100,635 (0.15 mg/kg) or citalopram (3 mg/kg) alone, it is suggested that blocking 5-HT 1A receptors is more effective in facilitating the anxiolytic effect of citalopram than blocking 5-HT 1B/1D receptors.

The central nucleus of the amygdala is essential for acquiring and expressing conditional fear after overtraining

The basolateral complex of the amygdala (BLA) is critical for the acquisition and expression of Pavlovian fear conditioning in rats. Nonetheless, rats with neurotoxic BLA lesions can acquire conditional fear after overtraining (75 trials). The capacity of rats with BLA lesions to acquire fear memory may be mediated by the central nucleus of the amygdala (CEA). To examine this issue, we examined the influence of neurotoxic CEA lesions or reversible inactivation of the CEA on the acquisition and expression of conditional freezing after overtraining in rats. Rats with pretraining CEA lesions (whether alone or in combination with BLA lesions) did not acquire conditional freezing to either the conditioning context or an auditory conditional stimulus after extensive overtraining. Similarly, post-training lesions of the CEA or BLA prevented the expression of overtrained fear. Lastly, muscimol infusions into the CEA prevented both the acquisition and the expression of overtrained fear, demonstrating that the effects of CEA lesions are not likely due to the destruction of en passant axons. These results suggest that the CEA is essential for conditional freezing after Pavlovian fear conditioning. Moreover, overtraining may engage a compensatory fear conditioning circuit involving the CEA in animals with damage to the BLA.

Hitting Ras where it counts: Ras antagonism in the basolateral amygdala inhibits long‐term fear memory

Several studies have implicated the Ras/mitogen-activated protein kinase (MAPK) pathway in Pavlovian fear conditioning. RasGRF1 knockout mice show significant deficits in acquisition of long-term fear memories and long-term potentaition (LTP) in the basolateral amygdala (BLA). MAPK kinase inhibition also impairs fear conditioning and amygdaloid LTP. However, there is no direct evidence to date for the involvement of Ras itself in fear conditioning. To address this issue, we examined the effects of intra-amygdala infusions of the selective Ras antagonist farnesylthiosalicylic acid (FTS) on the acquisition and expression of conditional freezing in rats. Micro-infusions of FTS into the BLA prior to contextual fear conditioning significantly impaired acquisition of long-term contextual fear memory in a dose-dependent manner. Post-training FTS infusions had no effect on acquisition of long-term fear memory. The effects of FTS on fear conditioning were specific for the BLA. Finally, intra-amygdala infusions of FTS inhibited MAPK activation in BLA. Collectively, these results provide further evidence for the involvement of amygdaloid Ras in the acquisition of long-term conditional fear memory.

Auditory-Evoked Spike Firing in the Lateral Amygdala and Pavlovian Fear Conditioning: Mnemonic Code or Fear Bias?

Amygdala neuroplasticity has emerged as a candidate substrate for Pavlovian fear memory. By this view, conditional stimulus (CS)-evoked activity represents a mnemonic code that initiates the expression of fear behaviors. However, a fear state may nonassociatively enhance sensory processing, biasing CS-evoked activity in amygdala neurons. Here we describe experiments that dissociate auditory CS-evoked spike firing in the lateral amygdala (LA) and both conditional fear behavior and LA excitability in rats. We found that the expression of conditional freezing and increased LA excitability was neither necessary nor sufficient for the expression of conditional increases in CS-evoked spike firing. Rather, conditioning-related changes in CS-evoked spike firing were solely determined by the associative history of the CS. Thus, our data support a model in which associative activity in the LA encodes fear memory and contributes to the expression of learned fear behaviors.

Effect of chronic treatment with the protein kinase C inhibitor staurosporine on the acquisition and expression of contextual fear conditioning

The present study investigated the effects of acute and chronic administration of the protein kinase C inhibitor, staurosporine, on the acquisition and expression of conditioned freezing behavior, an index of anxiety induced by conditioned fear stress. Results revealed that acute staurosporine (0.01 and 0.1 mg/kg, i.p.) did not affect either acquisition or expression of conditioned freezing. Chronic staurosporine administration (0.01 or 0.1 mg/kg, i.p., for 14 days) significantly reduced the acquisition of conditioned freezing at a dose of 0.1 mg/kg, but failed to affect the expression of conditioned freezing at any dose. These results suggest the involvement of protein kinase C in synaptic and cellular plasticity underlying emotional learning and memory.

Is There Savings for Pavlovian Fear Conditioning after Neurotoxic Basolateral Amygdala Lesions in Rats?

Considerable evidence indicates an important role for amygdaloid nuclei in both the acquisition and expression of Pavlovian fear conditioning. Recent reports from my laboratory have focused on the impact of neurotoxic lesions of the basolateral complex of the amygdala (BLA) on conditional freezing behavior in rats. In these studies, I have observed severe effects of posttraining BLA lesions on the expression of conditional freezing even after extensive presurgical overtraining (25–75 trials). Moreover, I have found no evidence for sparing of fear memory (i.e., savings) in these rats when I assess their rate of reacquisition relative to BLA rats receiving minimal training (1 trial). In these experiments, freezing behavior was assessed using a conventional time-sampling procedure and expressed as a response probability. Although this measure is well established in the literature, it is conceivable that it is not sensitive to spared memory in rats with BLA lesions. To address this issue, I present a more detailed analysis of freezing behavior that quantifies latency to freeze, the number of freezing bouts, the duration of freezing bouts, and the probability distribution of bout lengths. I also include control data from untrained (no-shock) rats. Consistent with my earlier reports, I find no evidence of savings of fear memory in rats with neurotoxic BLA lesions using several measures of freezing behavior. These results reiterate the conclusion that fear memory, as it is expressed in freezing behavior, requires neurons in the BLA.

Effect of chronic administration of flesinoxan and fluvoxamine on freezing behavior induced by conditioned fear

The present study investigated the acute effects of flesinoxan (a selective 5-HT 1A receptor agonist), fluvoxamine (a selective serotonin reuptake inhibitor) and their co-administration on the expression of conditioned freezing, and index of anxiety in rats. This study also examined the acute effects of fluvoxamine and flesinoxan following chronic flesinoxan or chronic fluvoxamine on the expression of conditioned freezing. Acute administration of flesinoxan (s.c.; 0.1–3 mg/kg) reduced freezing dose dependently, and fluvoxamine (i.p.) at a high dose (60 mg/kg) reduced freezing significantly. Acute co-administration of fluvoxamine (30 mg/kg) and flesinoxan (0.3 mg/kg) showed an additive inhibitory effect on freezing. Chronic flesinoxan treatment (0.3 mg/kg, for 13 days) did not affect the inhibitory effect of acute flesinoxan treatment, but enhanced that of acute fluvoxamine (30 mg/kg) on conditioned freezing. Chronic fluvoxamine treatment (30 mg/kg, for 13 days) enhanced the inhibitory effect of acute fluvoxamine (30 mg/kg) and the inhibitory effect of acute flesinoxan (0.3 mg/kg) on conditioned freezing. These results suggest that co-administration of a selective serotonin reuptake inhibitor and a 5-HT 1A receptor agonist is useful for the treatment of anxiety disorders.

Amygdalar NMDA Receptors Are Critical for the Expression of Multiple Conditioned Fear Responses

There is conflicting evidence regarding the issue of whether NMDA receptors in the basolateral amygdalar complex (BLA) are critically involved in the expression of conditioned fear. This matter was addressed by infusing the rat BLA with d,l-2-amino-5-phosphonovaleric acid (APV), a competitive NMDA receptor antagonist. APV infusion into the BLA was reported to block the expression of conditioned fear when measured by freezing but not when measured by fear-potentiated startle response to a loud noise. To examine this issue further, here we used multiple indices of conditioned fear, including analgesia, 22 kHz ultrasonic vocalization (USV), defecation, and freezing. Rats with bilateral BLA cannula implants underwent fear conditioning consisting of 10 tone-footshock pairings. Before context and tone fear-retention tests, animals received intra-BLA infusions with APV (2.5 microg/side) or artificial CSF. Both tone and context tests demonstrated that the expression of conditioned freezing, USV, defecation, and analgesia were significantly impaired by intra-amygdalar infusions of APV. In a second set of experiments, intra-BLA infusions of APV markedly impaired the normal expression of postshock fear responses during training, as measured by freezing, USV, and defecation. Immediate postshock fear expression was predictive of subsequent fear retention to the tone and context when the animals were not infused. These results are consistent with the hypothesis that amygdalar NMDA receptors participate in normal synaptic transmission and therefore the overall functioning of the amygdala.

Effect of subchronic lithium carbonate treatment on anxiolytic-like effect of citalopram and MKC-242 in conditioned fear stress in the rat

We investigated the effect of citalopram [selective serotonin (5-HT) reuptake inhibitor] and MKC-242 (5-[3-{(2 S)-(1,4-Benzodioxan-2-ylmethyl) amino}propoxy]-1, 3-benzo-dioxol hydrochloride; a selective 5-HT 1A receptor agonist) on the expression of conditioned freezing, an index of anxiety, following treatment with subchronic lithium carbonate (LiCO 3). Male Sprague–Dawley rats were used in these experiments. Acute administration of citalopram (subcutaneously, s.c.) reduced freezing significantly at a high dose (30 mg/kg), while showing no effect at lower doses (3 and 10 mg/kg). Acute administration of MKC-242 (s.c.; 0.1–10 mg/kg) dose dependently reduced freezing. Subchronic LiCO 3 treatment (1 week; 0.05% or 0.2% LiCO 3 in diet; p.o.) showed no effect on freezing behavior. Acute treatment with both citalopram (3 and 30 mg/kg) and MKC-242 (1 mg/kg) after subchronic treatment with the higher, but not the lower concentration of LiCO 3 (1 week), reduced freezing markedly and significantly, as compared with either drug alone. These results suggest that subchronic LiCO 3 treatment enhanced the anxiolytic-like effects of these serotonergic drugs by facilitating central 5-HT neurotransmission at clinically therapeutic plasma lithium levels.

Effect of citalopram, a selective serotonin reuptake inhibitor, on the acquisition of conditioned freezing

The present study examined the effects of the selective serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitor citalopram on the acquisition of conditioned freezing, an index of anxiety. Acute treatment with citalopram (1–10 mg/kg) dose dependently prevented the acquisition of conditioned freezing, while acute treatment with noradrenaline or dopamine reuptake inhibitors failed. The acute effect of citalopram was not antagonized by the 5-HT 1A receptor antagonist NAN190, 1-(2-methoxyphenyl)-4-[4-(2-phthalimmido)butyl]piperazine or the 5- HT 2A 2C receptor antagonist ICI169,369, 2-(2-dimethylaminoethylthio)-3-phenylquinoline hydrochloride. These results indicate that selective 5-HT reuptake inhibitors reduce not only the expression of conditioned freezing as reported previously, but also the acquisition of conditioned freezing. Both these effects of selective 5-HT reuptake inhibitors may be related to their clinical efficacy in the treatment of anxiety disorders.