Conditioning-specific Reflex Modification Research Articles

Inactivation of the interpositus nucleus during unpaired extinction does not prevent extinction of conditioned eyeblink responses or conditioning-specific reflex modification.

For almost 75 years, classical eyeblink conditioning has been an invaluable tool for assessing associative learning processes across many species, thanks to its high translatability and well-defined neural circuitry. Our laboratory has adapted the paradigm to extensively detail associative changes in the rabbit reflexive eyeblink response (unconditioned response, UR), characterized by postconditioning increases in the frequency, size, and latency of the UR when the periorbital shock unconditioned stimulus (US) is presented alone, termed conditioning-specific reflex modification (CRM). Because the shape and timing of CRM closely resembles the conditioned eyeblink response (CR) to the tone conditioned stimulus (CS), we previously tested whether CRs and CRM share a common neural substrate, the interpositus nucleus of the cerebellum (IP), and found that IP inactivation during conditioning blocked the development of both CRs and the timing aspect of CRM. The goal of the current study was to examine whether extinction of CRs and CRM timing, accomplished simultaneously with unpaired CS/US extinction, also involves the IP. Results showed that muscimol inactivation of the IP during extinction blocked CR expression but not extinction of CRs or CRM timing, contrasting with the literature showing IP inactivation prevents CR extinction during CS-alone presentations. The continued presence of the US throughout the unpaired extinction procedure may have been sufficient to overcome IP blockade, promoting plasticity in the cerebellar cortex and/or extracerebellar components of the eyeblink conditioning pathway that can modulate extinction of CRs and CRM timing. Results therefore add support to the distributed plasticity view of cerebellar learning. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Inactivation of the interpositus nucleus blocks the acquisition of conditioned responses and timing changes in conditioning-specific reflex modification of the rabbit eyeblink response.

Conditioning-specific reflex modification (CRM) of the rabbit eyeblink response is an associative phenomenon characterized by increases in the frequency, size, and peak latency of the reflexive unconditioned eyeblink response (UR) when the periorbital shock unconditioned stimulus (US) is presented alone following conditioning, particularly to lower intensity USs that produced minimal responding prior to conditioning. Previous work has shown that CRM shares many commonalities with the conditioned eyeblink response (CR) including a similar response topography, suggesting the two may share similar neural substrates. The following study examined the hypothesis that the interpositus nucleus (IP) of the cerebellum, an essential part of the neural circuitry of eyeblink conditioning, is also required for the acquisition of CRM. Tests for CRM occurred following delay conditioning under muscimol inactivation of the IP and also after additional conditioning without IP inactivation. Results showed that IP inactivation blocked acquisition of CRs and the timing aspect of CRM but did not prevent increases in UR amplitude and area. Following the cessation of inactivation, CRs and CRM latency changes developed similarly to controls with intact IP functioning, but with some indication that CRs may have been facilitated in muscimol rabbits. In conclusion, CRM timing and CRs both likely require the development of plasticity in the IP, but other associative UR changes may involve non-cerebellar structures interacting with the eyeblink conditioning circuitry, a strong candidate being the amygdala, which is also likely involved in the facilitation of conditioning. Other candidates worth consideration include the cerebellar cortex, prefrontal and motor cortices.

Propranolol produces short-term facilitation of extinction in a rabbit model of post-traumatic stress disorder

Post-traumatic stress disorder (PTSD) is a learning-based anxiety disorder with significant public health challenges due to difficulties in treating the complex, multiple symptomology. We have developed an animal model of PTSD, based on Pavlovian eyeblink conditioning in rabbits, that addresses two key features: conditioned responses (CRs) to cues associated with an aversive event and a form of conditioned hyperarousal referred to as conditioning-specific reflex modification (CRM). We have found previously that unpaired extinction is ideal for reducing both CRs and CRM simultaneously and shows sensitivity to systemic serotonergic and glutamatergic manipulations. The following study aimed to extend our work to examine the role of the noradrenergic system, dysregulation of which is strongly implicated as part of the neurobiology of PTSD and which may also play a role in the balance shift from fear reconsolidation to extinction during treatment. The goal of the following two studies was to examine whether the β-adrenergic receptor antagonist propranolol combined with either a full or brief course of unpaired extinction treatment could enhance extinction of CRs and/or CRM. Results showed a within-session facilitation of propranolol on extinction of CRs, particularly during the first extinction session, and a short-term enhancement of extinction of CRM when extinction treatment was brief. However, neither benefit translated to long-term extinction retention for the majority of subjects. Findings suggest that propranolol may provide the most therapeutic benefit in situations of high arousal early in treatment, which may be more important for future patient compliance rather than long-term treatment outcomes.

Grouping subjects based on conditioning criteria reveals differences in acquisition rates and in strength of conditioning-specific reflex modification

Averaging behavioral data such as the nictitating membrane response (NMR) across subjects can conceal important individual and group differences. Analyses were conducted of NMR data from rabbits that were grouped based on the point during NMR conditioning when subjects produced 8 conditioned responses (CR) in a set of 10 trials. This resulted in five groups (Early Day 1, Late Day 1, Early Day 2, Late Day 2, Early Day 3) in which group differences in CR acquisition rates were found. Percent (%) CRs were not found to increase monotonically and between-session differences in % CR were found. Conditioning-specific reflex modification (CRM) of the NMR is a type of enhanced reflexive responding of the NMR that is detected when the unconditioned stimulus (US) is presented in the absence of the conditioned stimulus (CS) following paired classical conditioning. CRM occurred in some subjects in all five groups. Subjects from both the group that was fastest and the group that was slowest to reach the learning criterion had unconditioned response (UR) topographies following NMR conditioning that strongly resembled the CR-UR response sequence elicited during NMR conditioning. This finding was most pronounced when the US duration used to assess CRM was equivalent to that used during NMR conditioning, further evidence to support the hypothesis that CRM is a CR that has generalized from the CS to the US. While grouping data based on conditioning criteria did not facilitate identifying individuals more predisposed to exhibiting CRM, strong CRM only occurred in the groups that reached the conditioning criterion the fastest.

Effects of systemic glutamatergic manipulations on conditioned eyeblink responses and hyperarousal in a rabbit model of post-traumatic stress disorder.

Glutamatergic dysfunction is implicated in many neuropsychiatric conditions, including post-traumatic stress disorder (PTSD). Glutamate antagonists have shown some utility in treating PTSD symptoms, whereas glutamate agonists may facilitate cognitive behavioral therapy outcomes. We have developed an animal model of PTSD, based on conditioning of the rabbit's eyeblink response, that addresses two key features: conditioned responses (CRs) to cues associated with an aversive event and a form of conditioned hyperarousal referred to as conditioning-specific reflex modification (CRM). The optimal treatment to reduce both CRs and CRM is unpaired extinction. The goals of the study were to examine whether treatment with the N-methyl-D-aspartate glutamate receptor antagonist ketamine could reduce CRs and CRM, and whether the N-methyl-D-aspartate agonist D-cycloserine combined with unpaired extinction treatment could enhance the extinction of both. Administration of a single dose of subanesthetic ketamine had no significant immediate or delayed effect on CRs or CRM. Combining D-cycloserine with a single day of unpaired extinction facilitated extinction of CRs in the short term while having no impact on CRM. These results caution that treatments may improve one aspect of the PTSD symptomology while having no significant effects on other symptoms, stressing the importance of a multiple-treatment approach to PTSD and of animal models that address multiple symptoms.

Effects of extinction treatments on the reduction of conditioned responding and conditioned hyperarousal in a rabbit model of posttraumatic stress disorder (PTSD).

We have previously characterized a model of posttraumatic stress disorder (PTSD), based on classical conditioning of the rabbit nictitating membrane response (NMR), that focuses on 2 key PTSD-like features: conditioned responses to trauma-associated cues and hyperarousal. In addition to the development of conditioned NMRs (CRs) to a tone conditioned stimulus (CS) associated with a periorbital shock unconditioned stimulus (US), we have observed that rabbits also exhibit a conditioning-specific reflex modification (CRM) of the NMR that manifests as an exaggerated and more complex reflexive NMR to presentations of the US by itself, particularly to intensities that elicited little response prior to conditioning. Previous work has demonstrated that unpaired presentations of the CS and US are successful at extinguishing CRs and CRM simultaneously, even when a significantly weakened version of the US is utilized. In the current study, additional extinction treatments were tested, including continued pairings of the CS with a weakened US and exposure to the training context alone, and these treatments were contrasted with the effects of unpaired extinction with a weakened US and remaining in home cages with no further treatment. Results showed that continued pairings only slightly decreased CRs and CRM, while context exposure had no effect on CRs and marginal effects on reducing CRM. Unpaired extinction was still the most effective treatment for reducing both. Findings are discussed in terms of applications to cognitive-behavioral therapies for treatment of PTSD, such as incorporating mild, innately stressful stimuli into virtual reality therapy.

Subacute fluoxetine enhances conditioned responding and conditioning-specific reflex modification of the rabbit nictitating membrane response

Extensive research on the rabbit nictitating membrane response (NMR) has shown that the NMR reflex can become exaggerated following classical fear conditioning. This learning-related change is referred to as conditioning-specific reflex modification (CRM) and is observed in the absence of the conditioned stimulus. The aim of the current study was to examine the sensitivity of the CRM paradigm to serotonergic manipulation with fluoxetine, a commonly prescribed selective serotonin reuptake inhibitor for anxiety disorders. To assess the effect of fluoxetine on exaggerated reflexive responding indicative of CRM and on conditioned cued fear, rabbits underwent delay NMR conditioning (pairings of tone and periorbital shock) and were tested for CRM, followed by 5 days of daily fluoxetine (0.03, 0.3, or 3.0 mg/kg) or saline injections. CRM was reassessed 1 day and 1 week later, followed by a retention test of conditioned responses (CRs) to the tone. Fluoxetine (3.0 mg/kg) enhanced CRM and retention of conditioned responses, a week after treatment ceased, and this is in agreement with the reports on increased anxiety-like behaviors in other animal models and humans. The CRM paradigm, therefore, may provide important insight into the mechanisms underlying the paradoxical selective serotonin reuptake inhibitor effects.

Inactivation of the central nucleus of the amygdala blocks classical conditioning but not conditioning-specific reflex modification of rabbit heart rate

Heart rate (HR) conditioning in rabbits is a widely used model of classical conditioning of autonomic responding that is noted for being similar to the development of conditioned heart rate slowing (bradycardia) in humans. We have shown previously that in addition to HR changes to a tone conditioned stimulus (CS), the HR reflex itself can undergo associative change called conditioning-specific reflex modification (CRM) that manifests when tested in the absence of the CS. Because CRM resembles the conditioned bradycardic response to the CS, we sought to determine if HR conditioning and CRM share a common neural substrate. The central nucleus of the amygdala (CeA) is a critical part of the pathway through which conditioned bradycardia is established. To test whether the CeA is also involved in the acquisition and/or expression of CRM, we inactivated the CeA with muscimol during HR conditioning or CRM testing. CeA inactivation blocked HR conditioning without completely preventing CRM acquisition or expression. These results suggest that the CeA may therefore only play a modulatory role in CRM. Theories on the biological significance of conditioned bradycardia suggest that it may represent a state of hypervigilance that facilitates the detection of new and changing contingencies in the environment. We relate these ideas to our results and discuss how they may be relevant to the hypersensitivity observed in fear conditioning disorders like post-traumatic stress.

Predictors of susceptibility and resilience in an animal model of posttraumatic stress disorder.

Animal models of posttraumatic stress disorder (PTSD) are based on fear conditioning where innocuous cues elicit reactions that originally occur to traumatic events--a core feature of PTSD. Another core feature is hyperarousal--exaggerated reactions to stressful events. One limitation of animal models of PTSD is that group effects do not model the sporadic incidence of PTSD. We developed an animal model of PTSD in which rabbit nictitating membrane responses become exaggerated as a function of classical conditioning to a tone conditioned stimulus (CS) paired with a shock unconditioned stimulus (US). Exaggerated responses to the US are a form of hyperarousal termed conditioning-specific reflex modification (CRM) and occur in the absence of the CS. Inspecting data across several experiments, we determined 25% of our rabbits exhibit strong CRM despite all subjects having high levels of conditioning. To determine how prone rabbits were to CRM (susceptibility) or how resistant (resilience), we examined data from 135 rabbits analyzing for factors during CS-US pairings and during US prescreening that would predict CRM. We found the magnitude of CRM was correlated with the onset latency and area of conditioned responding during CS-US pairings and with the peak latency of a response during US pretesting. In an animal model of PTSD that more accurately reflects clinical prevalence, we can begin to predict susceptibility not only during responding to a stressful conditioning situation but also during a screening process before the stressful situation takes place. The results suggest relatively innocuous testing may help detect PTSD after trauma and screen for it before trauma occurs.

Incubation of conditioning-specific reflex modification: Implications for post traumatic stress disorder

Incubation of fear has been used to account for the delayed manifestation of symptoms of fear and anxiety including the delayed onset of Post Traumatic Stress Disorder (PTSD). We have shown the utility of classical conditioning-specific modification of the rabbit nictitating membrane response (NMR) as a model of PTSD. This modification includes an exaggeration in the size and a change in the timing of the unconditioned NMR after several days of classical conditioning. To assess the effects of incubation on conditioning-specific modification, we measured changes in responding as a function of the time between classical conditioning and NMR testing. After just one day of classical conditioning resulting in modest levels of learning, increases in response size were an inverted-U shaped function of days of incubation with little if any change occurring one and ten days after training but significant change occurring after six days. The incubation effect persisted for a week. An unpaired control group showed no change in the size of the response confirming the incubation effect was associative. The results bear a striking resemblance to symptoms of PTSD that do not always occur immediately after trauma and become exacerbated over time and then persist. They point to a window when incubation can exacerbate symptoms and speak to the vulnerability of re-experiencing trauma too soon. This could be a serious problem for military or emergency personnel recalled to combat or a disaster site without sufficient time to deal with the effects of their initial experiences.

Classical conditioning and conditioning-specific reflex modification of rabbit heart rate as a function of unconditioned stimulus location.

Heart rate conditioning is used as an index of conditioned fear and is important for understanding disorders of anxiety and stress, including post traumatic stress disorder (PTSD). One important feature of PTSD is that patients generalize conditioned fear from danger signals to safety signals especially when the two signals have overlapping features. What has not been determined is whether generalization occurs between unconditioned stimuli with overlapping features. In the current experiment, heart rate conditioning and conditioning-specific reflex modification of rabbit heart rate were examined as a function of two different unconditioned stimulus locations. Heart rate conditioning occurred at identical terminal levels whether electrical stimulation was presented near the eye or on the back. Despite different heart rate response topographies to electrical stimulation at the two locations, conditioning-specific reflex modification was detected near the eye and on the back and appeared to generalize between the locations. Interestingly, only conditioning-specific reflex modification detected on the back persisted for a week after heart rate conditioning. This persistence may be a model for some features of post traumatic stress disorder. Overgeneralization of unconditioned responses to unconditioned stimuli similar to the trauma may also be an important aspect of PTSD modeled here.

Unpaired extinction: Implications for treating post-traumatic stress disorder

Extinction of fear is important for treating stress-related conditions particularly post-traumatic stress disorder (PTSD). Although traditional extinction presents the feared stimulus by itself, there is evidence from both clinical and basic research that repeatedly presenting the feared stimulus by itself does not prevent fear from returning. This renewal or relapse can be “thwarted” by unpaired extinction–presentations of the feared stimulus and the event producing the fear. However, no matter how effective standard unpaired extinction may be in the laboratory, repeated presentation of a traumatic event is untenable. To make an unpaired extinction procedure more clinically relevant, we classically conditioned the rabbit nictitating membrane response using electrical stimulation or air puff as the unconditioned stimulus and then during unpaired extinction reduced both the intensity of the unconditioned stimulus and the days of unpaired stimulus presentations. We found unpaired extinction reduced conditioned and exaggerated unconditioned responding (an animal analog of PTSD called conditioning-specific reflex modification) and could be accomplished with a weak unconditioned stimulus as long as extended presentations were used. Surprisingly, brief presentations of a weak unconditioned stimulus or extended presentations of a strong one made the exaggerated responses stronger. One implication is that brief treatment may not just be ineffectual; it may heighten the symptoms of PTSD. Another implication is that using strong stimuli may also heighten those symptoms.

Effects of extinction on classical conditioning and conditioning-specific reflex modification of rabbit heart rate

Understanding the mechanisms of fear extinction has become increasingly important for treating a number of disorders, particularly post-traumatic stress disorder. Conditioning of rabbit heart rate (HR) is an established model for studying fear, yet little is known about procedures for extinguishing it other than repeated presentations of cue(s) associated with the fear-inducing event. The following study examined the effects of conditioned stimulus (CS) alone, unconditioned stimulus (US) alone, unpaired CS/US presentations, continued CS–US pairings, or no further stimulation on rabbit HR following HR conditioning. We have previously shown the rabbit HR response to the US can change as a function of learning when measured in the absence of the CS, a phenomenon referred to as conditioning-specific reflex modification (CRM). More specifically, the HR exhibits a deceleration in response to the US reminiscent of the conditioned bradycardia that develops to the CS. Consequently, the following study also examined the effects of extinction treatments on HR CRM. For HR conditioned responses (CRs), CS-alone and unpaired CS/US presentations were the most successful extinction treatments. For HR CRM, all conditions led to a reduction in CRM except for a subset of rabbits that maintained high levels following unpaired extinction, indicating a dissociation between extinction of HR CRs and CRM. The findings highlight the parameters of HR extinction, the transient nature of HR CRM, vagal involvement in both acquisition and extinction of HR CRM, and suggest that HR CRM cannot be fully explained as a CR that has generalized from the CS to the US.

Inactivation of the central nucleus of the amygdala abolishes conditioning-specific reflex modification of the rabbit (Oryctolagus cuniculus) nictitating membrane response and delays classical conditioning.

The central nucleus (CE) of the amygdala has been gaining attention for its importance in the plasticity underlying conditioned emotional responding. Already known for its role in nictitating membrane response (NMR) reflex facilitation, the CE may also be involved in conditioning-specific reflex modification (CRM)--changes in the NMR to the unconditioned stimulus (US) when tested in the absence of the conditioned stimulus following classical conditioning. To examine the CE's role in acquisition and/or expression of CRM, the authors temporarily inactivated the CE of rabbits (Oryctolagus cuniculus) with muscimol during NMR conditioning and/or during US testing. Results show that CRM was abolished by inactivation during US testing but intact following inactivation during NMR conditioning, suggesting that the CE is involved in CRM expression. Also, inactivation during conditioning delayed the development of conditioned NMRs. These findings show that the CE may act as an output center for expression of emotional responding in one situation (CRM) but is involved in facilitating plasticity in another (NMR conditioning). The authors propose that analysis of CRM may be an important corollary to current models for the treatment of posttraumatic stress disorder.

Conditioning-specific reflex modification of the rabbit's nictitating membrane response and heart rate: Behavioral rules, neural substrates, and potential applications to posttraumatic stress disorder.

Interest in classical conditioning is usually focused on anticipatory responses to a stimulus associated with a significant event, and it is assumed that responses to the event itself are reflexive, involuntary, and relatively invariant. However, there is compelling evidence that both the rabbit nictitating membrane response (NMR) and heart rate response (HR), well-known reflexive reactions to aversive events, can change quite dramatically as a function of learning when measured in the absence of the conditioned stimulus. In the case of NMR conditioning, a simple blink is transformed into a larger and more complex response. For HR conditioning, reflexive heart rate acceleration can actually change to heart rate deceleration. In both cases, the reflex comes to resemble the conditioned response and follows some of the same behavioral laws. This change in response to the aversive event itself or weaker forms of that event is called conditioning-specific reflex modification (CRM). CRM may force us to reevaluate the behavioral and neural consequences of classical conditioning and may have important consequences for the treatment of conditions such as posttraumatic stress disorder.

Classical conditioning of the rabbit's nictitating membrane response is a function of the duration of dietary cholesterol

Modifying dietary cholesterol may improve learning and memory but very high cholesterol can cause pathophysiology and death. Rabbits fed 2% cholesterol for 8, 10 or 12 weeks with 0.12 ppm copper added to distilled water and rabbits fed a normal diet without copper added to distilled water (0 weeks) were given a difficult trace classical conditioning task and an easy delay conditioning task pairing tone with corneal air puff. The majority of cholesterol-fed rabbits survived the deleterious effects of the diet but survival was an inverse function of the diet duration. Compared to controls, the level of classical conditioning and conditioning-specific reflex modification were an inverted "U"-shaped function of diet duration. Highest levels of responding occurred in rabbits on cholesterol for 10 weeks and trace conditioning was negatively correlated with the number of hippocampal β-amyloid-positive neurons. Rabbits on the diet for 12 weeks responded at levels comparable to controls. The data provide support for the idea that dietary cholesterol may facilitate learning and memory but there is an eventual trade off with pathophysiological consequences of the diet.

High dietary cholesterol facilitates classical conditioning of the rabbit's nictitating membrane response

Studies have shown that modifying dietary cholesterol may improve learning and that serum cholesterol levels can be positively correlated with cognitive performance. Rabbits fed a 0, 0.5, 1 or 2% cholesterol diet for eight weeks and 0.12 ppm copper added to their drinking water received trace and then delay classical conditioning pairing tone with corneal air puff during which movement of the nictitating membrane (NM) across the eye was monitored. We found that the level of classical conditioning and conditioning-specific reflex modification (CRM) as well as the number of beta amyloid-labeled neurons in the cortex and hippocampus were a function of the concentration of cholesterol in the diet. The data provide support for the idea that dietary cholesterol may facilitate learning and memory.

Conditioning-specific reflex modification of the rabbit (Oryctolagus cuniculus) nictitating membrane response is sensitive to context

Conditioning-specific reflex modification occurs when an unconditioned response is modified in the absence of the conditioned stimulus as a result of pairings of the conditioned stimulus and an unconditioned stimulus. In two experiments, we assessed conditioning-specific reflex modification in either a novel context (Experiment 1) or a context different from, but equally familiar in relation to, the training context (Experiment 2). Conditioning-specific reflex modification did not demonstrate sensitivity to a novel context but did demonstrate sensitivity to a change in familiar context. The data cannot be explained by unconditioned stimulus preexposure, overtraining, or context insensitivity. The results suggest that conditioning-specific reflex modification models normal stress and may be used to evaluate theories of and treatments for posttraumatic stress disorder.

Effects of 4-aminopyridine on classical conditioning of the rabbit (Oryctolagus cuniculus) nictitating membrane response

A large body of data suggests that potassium channels may play an important role in learning and memory. Previous in-vitro research in a number of species including Hermissenda and the rabbit suggests that a 4-aminopyridine-sensitive transient potassium channel may be involved in classical conditioning. We investigated the effects of in-vivo 4-aminopyridine administration (0.5 mg/kg) on classical conditioning of the rabbit nictitating membrane response using a battery of tests designed to assess the associative, sensory, and motor contributors of 4-aminopyridine to responding. 4-Aminopyridine enhanced both classical conditioning and conditioning-specific reflex modification compared with a saline vehicle control, and these effects had several nonassociative components including an increase in the frequency of responding to both the conditioned and the unconditioned stimuli, suggesting a sensitizing effect of the drug. Although 4-aminopyridine can have peripheral effects, it may also modify cerebellar excitability or hippocampal neurotransmitter balance resulting in heightened responsiveness to stimulation.

Heart rate changes during conditioning-specific reflex modification of the rabbit’s ( Oryctolagus cuniculus) nictitating membrane response

Conditioning-specific reflex modification (CRM) of the rabbit’s nictitating membrane response (NMR) involves changes in responding to an unconditioned stimulus (US) when the US is tested in the absence of the conditioned stimulus. Previous experiments have shown that CRM is a function of the type and intensity of the aversive US used during classical conditioning. As a result, it has been suggested that CRM may be mediated, at least in part, by the aversiveness of the US. Here, we show that by using a moderately intense electrical pulse to the skin as a US, CRM of the rabbit NMR is accompanied by an increase in heart rate. The largest changes in heart rate occur at US intensities that produce the strongest levels of CRM. The heart rate data show that there may be an increased emotional/arousal component to the US that is correlated with CRM and support the use of CRM as a potential model for posttraumatic stress disorder.