Delayed unpaired extinction as a treatment for hyperarousal of the rabbit nictitating membrane response and its implications for treating PTSD.

Treatment for PTSD (Post-traumatic stress disorder) is rarely available immediately after trauma and often delayed for weeks or months after an event. In a rabbit eyeblink conditioning model of PTSD, we have previously shown that presentations of a tone conditioned stimulus (CS) and shock unconditioned stimulus (US) in an explicitly unpaired manner known as unpaired extinction is effective in reducing CS responding and US hyperarousal even if shock intensity is reduced eight-fold and elicits only minimal responding. Here we determined if delayed delivery of unpaired extinction would still be effective in extinguishing hyperarousal. Rabbits were tested for sensitivity to shock before CS-US pairings and after six days of unpaired extinction presented a day, a week or a month after CS-US pairings. Hyperarousal was extinguished a day and a week after conditioning but not after a month suggesting a significant delay in "treatment" can make hyperarousal persist. We next assessed if this persistence of hyperarousal was associative by comparing rabbits given CS-US pairings to those given explicitly unpaired CS and US presentations, measuring hyperarousal a day and a month later, followed by unpaired extinction and hyperarousal assessment. After four weeks, there was an increase in responding for all rabbits but only rabbits receiving CS-US pairings showed a significant increase in associatively-mediated hyperarousal. Importantly, both paired and unpaired groups showed increased levels of responding after unpaired extinction suggesting treatment delayed for too long may no longer be effective and could cause generalized hyperarousal.

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 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.

Time course of the rabbit's conditioned nictitating membrane movements during acquisition, extinction, and reacquisition.

The present experiment tested whether or not the time course of a conditioned eyeblink response, particularly its duration, would expand and contract, as the magnitude of the conditioned response (CR) changed massively during acquisition, extinction, and reacquisition. The CR duration remained largely constant throughout the experiment, while CR onset and peak time occurred slightly later during extinction. The results suggest that computational models can account for these results by using two layers of plasticity conforming to the sequence of synapses in the cerebellar pathways that mediate eyeblink conditioning.

Acute exposure of rabbit eyes to artificial light in vivo: effect on corneal and third eyelid conjunctival histology and the gene expression of PAFR.

PURPOSE: To study the effect of acute exposure of rabbit eyes to artificial sunlight in vivo, on the integrity of corneal and conjunctival tissue as well as on the gene expression of the receptor for platelet activating factor (PAFR). METHODS: New Zealand albino rabbits were immobilized opposite a 300 W Osram Ultra-Vitalux® light bulb with an emission radiation spectrum similar to that of normal sunlight at noon, and exposed to ultraviolet B radiation in the range of the reported threshold for corneal damage. Corneal and third eyelid tissue samples were removed from exposed eyes at 2, 6 and 24 h following the end of the exposure to the bulb light and were subsequently processed for histochemical staining and RNA extraction. The gene expression of PAFR was detected with real time reverse transcription polymerase chain reaction. RESULTS: Some epithelial shedding was detected in the corneal tissue as a result of acute exposure to artificial sunlight. In the eyelid conjunctiva, a marked accumulation of eosinophils was noticed, as early as 2 h post-exposure, apparently directed toward the upper part of the epithelial layer. This effect appears to subside by hour 24. No statistically significant changes in gene expression were detected in the corneal tissue, whereas in the third eyelid, PAFR gene expression was significantly induced, most prominently at t = 2 and 6 h post-exposure. CONCLUSION: Acute exposure of rabbit eyes to artificial sunlight induced a marked infiltration of eosinophils into the epithelial layer of the conjunctiva but no gross alterations in the cornea or the third eyelid. The gene expression of PAFR was upregulated, as an effect of light exposure, in the third eyelid but not in the cornea.

Dietary cholesterol degrades rabbit long term memory for discrimination learning but facilitates acquisition of discrimination reversal.

We have shown previously that feeding dietary cholesterol before learning can improve acquisition whereas feeding cholesterol after learning can degrade long term memory. To examine these different findings within a single paradigm, we fed groups of rabbits 2% cholesterol or normal chow with or without 0.12 ppm copper added to the drinking water following two-tone discrimination learning of the nictitating membrane response in which a 8-kHz tone (conditioned stimulus, CS+) was followed by air puff and a 1-kHz tone (CS-) was not. After eight weeks on the diet, we assessed the rabbits' conditioned responding during testing and retraining. We then reversed the two-tone discrimination and assessed responding to the 1-kHz tone CS+ and the 8-kHz CS-. During testing, rabbits given cholesterol without copper had lower levels of responding to CS+ than rabbits in the other groups suggesting they did not retain the discrimination as well. However, during a brief discrimination retraining session, their response levels to the CS+ returned to the level of the other groups, demonstrating a return of the memory of the original discrimination. At the end of discrimination reversal, these same rabbits exhibited superior discrimination indexed by lower response levels to CS- but similar levels to CS+, suggesting they were better able to acquire the new relationship between the two tones by inhibiting CS- responses. These results add to our previous data by showing cholesterol diet-induced degradation of an old memory and facilitation of a new memory can both be demonstrated within a discrimination reversal paradigm. Given discrimination reversal is a hippocampally-dependent form of learning, the data support the role of cholesterol in modifying hippocampal function as we have shown previously with in vitro brain slice recordings.

Timing and cue competition in conditioning of the nictitating membrane response of the rabbit (Oryctolagus cuniculus).

Rabbits were classically conditioned using compounds of tone and light conditioned stimuli (CSs) presented with either simultaneous onsets (Experiment 1) or serial onsets (Experiment 2) in a delay conditioning paradigm. Training with the simultaneous compound reduced the likelihood of a conditioned response (CR) to the individual CSs ("mutual overshadowing") but left CR timing unaltered. CR peaks were consistently clustered around the time of unconditioned stimulus (US) delivery. Training with the serial compound (CSA→CSB→US) reduced responding to CSB ("temporal primacy/information effect") but this effect was prevented by prior CSB→US pairings. In both cases, serial compound training altered CR timing. On CSA→CSB test trials, the CRs were accelerated; the CR peaks occurred after CSB onset but well before the time of US delivery. Conversely, CRs on CSB- trials were decelerated; the distribution of CR peaks was variable but centered well after the US. Timing on CSB- trials was at most only slightly accelerated. The results are discussed with respect to processes of generalization and spectral timing applicable to the cerebellar and forebrain pathways in eyeblink preparations.

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.

Pavlovian conditioning: pigeon nictitating membrane.

A new Pavlovian conditioning preparation was developed using the nictitating membrane of the restrained pigeon. Either visual or auditory stimuli served as conditioned stimuli (CSs) with an unconditioned stimulus (US) of a puff of air to the cornea. Movement of the nictitating membrane constituted the conditioned and unconditioned responses (CR and UR). Conditioning was studied with the Kamin blocking procedure. In agreement with findings from other conditioning preparations, responding to the redundant stimulus was attenuated relative to a stimulus that received the same number of CS-US pairings in a compound-conditioning procedure. Although response attenuation occurred, substantial individual variation was observed within the blocking procedure, a finding with some precedent in the experimental literature. Theoretical analysis and neural-network simulations indicate that inter-subject variation in response attenuation may result from differences in the extent to which contextual stimuli contribute to the functional CS.

Two streams make a river: the rabbit in Richard F. Thompson's laboratory.

The rabbit nictitating membrane (NM) preparation was developed in 1962 by Gormezano as a model system for classical conditioning. It had many features that made it ideal for use in exploring the laws of this conditioning paradigm, including easily obtainable subjects, good control of stimulus delivery and precise response specifications. Most importantly, the preparation evidenced very low spontaneous response rates and showed no evidence of nonassociative effects of sensitization and pseudoconditioning and had highly predictable learning functions. In contrast, previous human and animal models that had been utilized to explore the features of classical conditioning, such as the dog salivary model were far less easy to use, showed high spontaneous response rates, and had high nonassociative components. Over the ensuing years, Gormezano and his students characterized most of the parametric characteristics of classical conditioning with the use of the preparation. In 1970, Richard Thompson began exploring the use of the rabbit NM preparation in his laboratory as a model system with which to explore the brain substrates of classical conditioning. At the time, his work was centered around exploring the neural substrates of sensitization and habituation in spinal reflexes. Soon, however, he turned his attention to the brain substrates of classical conditioning almost exclusively, and produced an impressive amount of data detailing the neural underpinnings of classical conditioning. His work has generated perhaps the most detailed and complete picture of the neural mechanisms of learning currently available, and has led to countless other research efforts in the area of brain and behavior. Current understandings of neural mechanisms of associative learning owe much to Thompson and his various colleagues.

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.

Timing in trace conditioning of the nictitating membrane response of the rabbit (Oryctolagus cuniculus): scalar, nonscalar, and adaptive features.

Using interstimulus intervals (ISIs) of 125, 250, and 500 msec in trace conditioning of the rabbit nictitating membrane response, the offset times and durations of conditioned responses (CRs) were collected along with onset and peak latencies. All measures were proportional to the ISI, but only onset and peak latencies conformed to the criterion for scalar timing. Regarding the CR's possible protective overlap of the unconditioned stimulus (US), CR duration increased with ISI, while the peak's alignment with the US declined. Implications for models of timing and CR adaptiveness are discussed.

Magnitude and timing of conditioned responses in delay and trace classical conditioning of the nictitating membrane response of the rabbit (Oryctolagus cuniculus).

The present experiment characterized conditioned nictitating membrane (NM) movements as a function of CS duration, using the full range of discernible movements (>.06 mm) rather than movements exceeding a conventional criterion (>.50 mm). The CS-US interval was fixed at 500 ms, while across groups, the duration of the CS was 50 ms (trace), 550 ms (delay), or 1050 ms (extended delay). The delay group showed the highest level of acquisition. When tested with the different CS durations, the delay and extended delay groups showed large reductions in their responses when their CS was shortened to 50 ms, but the trace group maintained its response at all durations. Timing of the conditioned movements appeared similar across all manipulations. The results suggest that the CS has both a fine timing function tied to CS onset and a general predictive function tied to CS duration, both of which may be mediated by cerebellar pathways.

The role of the cerebellar interpositus nucleus in short and long term memory for trace eyeblink conditioning.

In previous studies the cerebellar interpositus (IP) nucleus, but not the hippocampus, was shown to be necessary both for initial learning and retention and for long-term retention of the standard delay eyeblink conditioned response (CR). However, in the trace eyeblink CR procedure, the hippocampus is also necessary for initial learning and retention, but not for long-term retention. Here the authors evaluate the role of the IP nucleus in both initial learning and retention, and in long-term retention of the trace eyeblink CR, using muscimol infusion to reversibly inactivate the IP nucleus. For the short-term study, there were two subgroups, the first sequentially passed through acquisition, inactivation, and reacquisition phases, whereas the second subgroup went through inactivation, acquisition, and inactivation phases. For the long-term study, the rabbits acquired the CR and then rested for a month. Next, they were distributed into two subgroups: with or without retention training, and finally went through inactivation and reacquisition phases. The results showed that the prelearning IP nucleus inactivation prevented the acquisition of the trace CR, whereas the postlearning inactivation reversibly abolished the expression of both the short- and long-term CR.

Scalar timing varies with response magnitude in classical conditioning of the nictitating membrane response of the rabbit (Oryctolagus cuniculus).

The present experiment was aimed at characterizing the timing of conditioned nictitating membrane (NM) movements as function of the interstimulus interval (ISI) in delay conditioning for rabbits (Oryctolagus cuniculus). Onset latency and peak latency were approximately, but not strictly, scalar for all but the smallest movements (<.10 mm). That is, both the mean and standard deviation of the timing measures increased in proportion to the ISI, but their coefficients of variation (standard deviation/mean) tended to be larger for shorter ISIs. For all ISIs, the absolute timing of the NM movements covaried with magnitude. The smaller movements (approximately, .11-.50 mm) were highly variable, and their peaks tended to occur well after the time of US delivery. The larger movements (>.50 mm) were less variable, and their peaks were better aligned with the time of US delivery. These results are discussed with respect to their implications for current models of timing in eyeblink conditioning.

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.

Magnitude and timing of nictitating membrane movements during classical conditioning of the rabbit (Oryctolagus cuniculus).

A trial-by-trial, subject-by-subject analysis was conducted to determine whether generation of the conditioned response (CR) occurs on a continuous or all-or-none basis. Three groups of rabbits were trained on different partial reinforcement schedules with the conditioned stimulus presented alone on 10%, 30%, or 50%, respectively, of all trials. Plots of each rabbit's nictitating membrane movements revealed that their magnitude rose in a continuous fashion. Response growth during acquisition followed a sigmoidal curve, and the timing of CR-sized movements was largely stable throughout the experiment. The results are discussed with respect to alternative models of CR generation.

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.

The role of sensory pathways in Pavlovian conditioning in rabbit.

In an earlier experiment we showed that selective attention plays a critical role in rabbit eye blink conditioning (Steele-Russell et al. in Exp Brain Res 173:587-602, 2006). The present experiments are concerned to examine the extent to which visual recognition processes are a separate component from the motor learning that is also involved in conditioning. This was achieved by midline section of the optic chiasma which disconnected the direct retinal projections via the brainstem to the cerebellar oculomotor control system. By comparing both normal and chiasma-sectioned rabbits it was possible to determine the dependence or independence of conditioning on the motor expression of the eye blink response during training. Both normal and chiasma-sectioned animals were tested using a multiple test battery to determine the effect of this redirection of the visual input pathways on conditioning. All animals were first tested for any impairment in visual capability following section of the optic chiasma. Despite the loss of 90% of retinal ganglion cell fibres, no visual impairment for either intensity or pattern vision was seen in the chiasma animals. Also no difference was seen in nictitating membrane (NM) conditioning to an auditory signal between normal and chiasma animals. Testing for motor learning to a visual signal, the chiasma rabbits showed a complete lack of any NM conditioning. However the sensory tests of visual conditioning showed that chiasma-sectioned animals had completely normal sensory recognition learning. These results show that NM Pavlovian conditioning involves anatomically separate and independent sensory recognition and motor output components of the learning.

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 beta-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.