Conditioning Of Nictitating Membrane Response Research Articles

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.

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.

Recruitment in Retractor Bulbi Muscle During Eyeblink Conditioning: EMG Analysis and Common-Drive Model

To analyze properly the role of the cerebellum in classical conditioning of the eyeblink and nictitating membrane (NM) response, the control of conditioned response dynamics must be better understood. Previous studies have suggested that the control signal is linearly related to the CR as a result of recruitment within the accessory abducens motoneuron pool, which acts to linearize retractor bulbi muscle and NM response mechanics. Here we investigate possible recruitment mechanisms. Data came from simultaneous recordings of NM position and multiunit electromyographic (EMG) activity from the retractor bulbi muscle of rabbits during eyeblink conditioning, in which tone and periocular shock act as conditional and unconditional stimuli, respectively. Action potentials (spikes) were extracted and classified by amplitude. Firing rates of spikes with different amplitudes were analyzed with respect to NM response temporal profiles and total EMG spike firing rate. Four main regularities were revealed and quantified: 1) spike amplitude increased with response amplitude; 2) smaller spikes always appeared before larger spikes; 3) subsequent firing rates covaried for spikes of different amplitude, with smaller spikes always firing at higher rates than larger ones; and 4) firing-rate profiles were approximately Gaussian for all amplitudes. These regularities suggest that recruitment does take place in the retractor bulbi muscle during conditioned NM responses and that all motoneurons receive the same command signal (common-drive hypothesis). To test this hypothesis, a model of the motoneuron pool was constructed in which motoneurons had a range of intrinsic thresholds distributed exponentially, with threshold linearly related to EMG spike amplitude. Each neuron received the same input signal as required by the common-drive assumption. This simple model reproduced the main features of the data, suggesting that conditioned NM responses are controlled by a common-drive mechanism that enables simple commands to determine response topography in a linear fashion.

Evidence From Retractor Bulbi EMG for Linearized Motor Control of Conditioned Nictitating Membrane Responses

Classical conditioning of nictitating membrane (NM) responses in rabbits is a robust model learning system, and experimental evidence indicates that conditioned responses (CRs) are controlled by the cerebellum. It is unknown whether cerebellar control signals deal directly with the complex nonlinearities of the plant (blink-related muscles and peripheral tissues) or whether the plant is linearized to ensure a simple relation between cerebellar neuronal firing and CR profile. To study this question, the retractor bulbi muscle EMG was recorded with implanted electrodes during NM conditioning. Pooled activity in accessory abducens motoneurons was estimated from spike trains extracted from the EMG traces, and its temporal profile was found to have an approximately Gaussian shape with peak amplitude linearly related to CR amplitude. The relation between motoneuron activity and CR profiles was accurately fitted by a first-order linear filter, with each spike input producing an exponentially decaying impulse response with time constant of order 0.1 s. Application of this first-order plant model to CR data from other laboratories suggested that, in these cases also, motoneuron activity had a Gaussian profile, with time-of-peak close to unconditioned stimulus (US) onset and SD proportional to the interval between conditioned stimulus and US onsets. These results suggest that for conditioned NM responses the cerebellum is presented with a simplified "virtual" plant that is a linearized version of the underlying nonlinear biological system. Analysis of a detailed plant model suggests that one method for linearising the plant would be appropriate recruitment of motor units.

Time-specific extinction and recovery of the rabbit's (Oryctolagus cuniculus) conditioned nictitating membrane response using mixed interstimulus intervals.

Extinguishing a conditioned response (CR) has entailed separating the conditioned stimulus (CS) from the unconditioned stimulus (US). This research reveals that elimination of the rabbit nictitating membrane response occurred during continuous CS-US pairings. Initial training contained a mixture of 2 CS-US interstimulus intervals (ISIs), 150 ms and 500 ms. The CRs showed double peaks, one for each ISI. When the 150-ms ISI was removed, its CR peak showed 2 hallmarks of extinction: a decline across sessions and spontaneous recovery between sessions. When a further stage of training was introduced with a distinctive CS using the 150-ms ISI, occasional tests of the original, extinguished CS revealed another hallmark of extinction, specifically, strong recovery of the 150-ms peak. These results support both abstract and cerebellar models of conditioning that encode the CS into a cascade of microstimuli, while challenging theories of extinction that rely on changes in CS processing, US representations, and contextual control.

Recovery of the rabbit's conditioned nictitating membrane response without direct reinforcement after extinction.

Three experiments demonstrated that, following the extinction of an established conditioned stimulus (CSA--e.g., tone), the pairing of a novel, cross-modal stimulus (CSB--e.g., light) with the unconditioned stimulus (US) results in strong recovery of responding to the extinguished CSA. Experiment 1 demonstrated that the recovery of responding to CSA is not the result of US reinstatement but is attributable to pairings of CSB with the US. Experiment 2 demonstrated that the recovery of responding is specific to CSA and is not the result of cross-modal generalization. Experiment 3 revealed that a large number of CSB-US pairings in Stage 1 significantly reduced the amount of recovery to CSA during subsequent CSB-US trials. Experiment 3 also provided unexpected evidence of cross-modal secondary extinction. The extinction and subsequent recovery of responding seen in the present experiments is discussed with respect to possible contributions from contextual associations, CS processing, US processing, conditioned response expression, and layered excitatory associations.

Effects of a Corneal Anesthetic on Extinction of the Classically Conditioned Nictitating Membrane Response in the Rabbit (Oryctolagus cuniculus).

Rabbits (Oryctolagus cuniculus) were presented with 7 daily sessions of tone-alone training after conditioning. Before the beginning of each of the first 4 extinction sessions, an artificial tear solution or tetracaine hydrochloride was administered to the cornea of rabbits in the control group (n = 6) and experimental group (n = 7), respectively. There were no between-group differences in the percentage of conditioned responses between both groups. However, the amplitude of the conditioned response was notably reduced in the tetracaine group (M = 0.40, SEM +/- 0.216) relative to the control group (M = 1.32, SEM +/- 0.639) early in extinction. Results seem to suggest that although motor output has been found to play an important role in extinction, corneal sensory feedback is not necessary.

The effect of brachium conjunctivum transection on a conditioned limb response in the cat

Seven cats were trained to perform a forelimb conditioned response to a paired tone conditioned stimulus (CS)/shock unconditioned stimulus (UCS). Brachium conjunctivum section ipsilateral to the trained limb was carried out following criterion conditioned response (CR) performance. Lesion sites were identified histologically and further confirmed by observation of cellular changes in the dentate and interpositus nuclei ipsilateral to the section. The brachium conjunctivum was found to have been sectioned in four of the seven subjects. Each of these animals demonstrated a total or near-total loss of the CR. Extended postoperative training resulted in no increase in CR performance levels. The unconditioned response (UCR) remained unaffected, as did limb placing, accuracy of striking at moving objects, grooming, running and walking. The results are discussed in the context of an earlier report by McCormick et al. [Bull Psychonom Soc 1981;18:103–5], in which section of the superior cerebellar peduncle was found to abolish a conditioned nictitating membrane response in the rabbit. Taken together, they support the contention of Lavond [Annu Rev Psychol 1993;44:317–42], Thompson [In: Sprague JM, Epstein AN, editors. Progress in Psychobiology and Physiological Psychology. New York: Academic Press 1983, pp. 167–96], Yeo et al. [Behav Brain Res 1984;13:261–66; Exp Brain Res 1985;60:87–98; Exp Brain Res 1985;60:99–113; Exp Brain Res 1992;88:623–38.] and others that the cerebellum represents a critical site for acquisition and retention of a conditioned memory trace.

The effect of feature-target intervals in conditional discriminations on acquisition and expression of conditioned nictitating membrane and heart rate responses in the rabbit

Nictitating membrane (NM) and heart rate (HR) responses were investigated in a conditional discrimination (A→X+ vs. B→X−), using feature-target intervals of 0, 5, 15, and 45 sec. Conditional control of NM responses, but not of HR responses, was acquired to the 400-msec X stimulus in all the groups tested. However, differential conditioning of both the NM and the HR responses to A versus B feature cues appeared for the three shorter intervals. Following acquisition, all the rabbits were tested with the four different feature-target intervals. All the groups showed a gradient of NM responding to X, in which the highest level of responding occurred at or near the interval used in training. The results are discussed with respect to the relationship of simple conditioning of the feature cues to their control over responding during presentation of the target stimulus, the putative role of HR as an index of preparatory processes during presentation of feature cues, and mechanisms of temporal specificity in conditional discriminations.

Cerebellar Cortical AMPA–Kainate Receptor Blockade Prevents Performance of Classically Conditioned Nictitating Membrane Responses

Classical conditioning of the nictitating membrane-eye blink response of rabbits is a simple form of associative motor learning. Lesion studies have shown that performance of learned responses is dependent on the cerebellum, but they have not shown whether there is storage of memories within the cerebellum or distinguished the roles of the cerebellar cortex and nuclei. Reversible inactivations of the cerebellar nuclei have directly implicated the cerebellum in the acquisition of nictitating membrane conditioning, but previously the cerebellar cortex has not been reversibly inactivated to assess its contribution to the performance or acquisition of conditioned responses. Here we use the water-soluble disodium salt of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reversibly to block cerebellar cortical AMPA-kainate receptors in lobule HVI and quantitative autoradiography to map its distribution. Conditioned responses are completely, but reversibly, abolished for 10-60 min depending on the concentration of the CNQX infusion and its location within HVI. Zebrin immunohistochemistry was used to define the optimal cortical infusion site that, we suggest, corresponds to the location of the eye blink control regions. We confirm that areas in HVI are essential for the expression of classically conditioned nictitating membrane responses, and we establish a method to analyze the role of cerebellar cortex in the acquisition of this form of motor learning.

Conditioning and reflex modification of the rabbit nictitating membrane response using electrical stimulation in auditory nuclei

Electrical brain stimulation (EBS) was applied to four nuclei in the auditory system, namely, the cochlear nucleus (CN), superior olive (SO), inferior colliculus, and medial geniculate. EBS was also applied to the pontine nuclei, which are the main relays for transmitting auditory conditioned stimuli (CSs) into the cerebellar pathways for conditioning of the nictitating membrane response (NMR). EBS of the CN, but no other site, yielded reflex modification, which was an increase in the unconditioned NMR to an airpuff unconditioned stimulus (US) when preceded by EBS. Throughout the experiment, EBS of the SO produced a distinctive distribution of NMRs, in which a high proportion had latencies less than 50 ms. When EBS was repeatedly paired with the airpuff US, conditioned responses (CRs) were acquired to comparable levels across all sites. At each site, response likelihood was an increasing function of the EBS parameters of pulse amplitude, pulse frequency, and pulse width. Combined with anatomical findings, these results indicate that multiple encodings of an auditory CS are sent to the pathways for the NMR.

Acute inactivation of the inferior olive blocks associative learning.

Can acute inactivation of the inferior olive block associative learning? We anaesthetized the inferior olive with lidocaine while rabbits simultaneously: (i) performed conditioned nictitating membrane responses to a flashing light to which they had already been trained; and (ii) underwent their first experience with classical conditioning of the same response to a tone. Inactivation of the inferior olive immediately and reversibly abolished the performance of conditioned responses and prevented learning during rabbits' initial conditioning with a tone-conditioned stimulus. When olivary function was restored, rabbits showed no signs of having learned under olivary anaesthesia. The experiment demonstrates that an acute disruption in olivary function can block learning, in addition to severely degrading motor control. The results are interpreted to indicate the importance of the inferior olive in optimizing learning, perhaps through a general role in regulating temporal processing.

Blocking of the Rabbit's Classically Conditioned Nictitating Membrane Response: Effects of Modifications of Contextual Associative Strength

The effects of contextual manipulations on the magnitude of discrete cue blocking of the rabbit's nictitating membrane response were investigated in three experiments. Experiment 1 showed that changing or extinguishing the single stimulus training context prior to compound training attenuated the blocking effect. In contrast, extinction of the context following compound conditioning did not attenuate blocking. In Experiment 2, attenuation of blocking was found to be greater following extinction of both the discrete CS and the context, in comparison to extinction of the discrete cue alone. Interpolation of US-alone presentations (context conditioning) between single stimulus training and compound conditioning produced no effects. In Experiment 3, enhanced responding to the blocked stimulus was observed following a retention interval interposed between compound training and testing. However, the blocking effect was preserved if subjects were repeatedly exposed to training context cues during the interval (i.e., context extinction) and tested in the presence of those cues. The results of the present investigation support the conclusion that the CS complex producing the blocking effect is composed of both discrete and contextual components. The observed effects were discussed in terms of associative and comparator models of conditioning.

Acquisition of a new-latency conditioned nictitating membrane response--major, but not complete, dependence on the ipsilateral cerebellum.

Classical conditioning of the nictitating membrane response (NMR) of rabbits is simple associative learning of a motor response. In several two-stage experiments, reversible inactivations of the deep cerebellar nuclei in stage 1 appeared to prevent acquisition of NMR conditioning in naive rabbits--no conditioned responses (CRs) were evident after inactivations were lifted in stage 2. Results of a three-stage experiment were different. When subjects were first trained with a light conditional stimulus (CS) in stage 1, reversible cerebellar inactivations during conditioning to a different, tone CS during stage 2 did not appear to prevent new learning because CRs to the tone CS were evident when the inactivation was lifted. Results from the two-stage experiments support the suggestion that the cerebellum is essential for the acquisition of NMR conditioning, but results from the three-stage experiment do not. Here, we use a three-stage design with different interstimulus intervals (ISIs) in stages 1 and 2. Because CRs develop with latencies-to-peak dependent on the ISI, learning during stage 1 can be dissociated from that accruing in stage 2. Complete inactivation of the ipsilateral cerebellar nuclei with muscimol substantially but not completely prevented learning with the second ISI during stage 2 because small CR peaks around the stage 2 ISI could be detected in some subjects after the inactivation had been lifted in stage 3. We suggest that the weak levels of conditioning possible during unilateral inactivation depend on the contralateral cerebellum or on extracerebellar circuitry and that these may be capable of supporting transfer of conditioning in a previous three-stage experiment. But, we confirm that normal NMR conditioning is critically dependent on the ipsilateral cerebellum.

Reversible inactivations of the cerebellum prevent the extinction of conditioned nictitating membrane responses in rabbits.

1. Studies show that reversible inactivation of the anterior interpositus nucleus (AIP) of the cerebellum with muscimol (a GABAA agonist) prevents acquisition of the classically conditioned nictitating membrane response (NMR) in the rabbit. Here, we have used reversible inactivations of the AIP with muscimol to investigate the role of the cerebellum in the extinction of this response. 2. Experimental subjects were implanted with cannulae targeted to the AIP, through which muscimol could be infused via an injector cannula. This experiment was divided into three phases lasting 4 days, separated by 3 day intervals. Experimental and unoperated control subjects received acquisition training in phase 1; in phases 2 and 3 they received extinction training. 3. Presentation of the conditioned stimulus (CS) alone in phase 2 produced normal extinction in control subjects. Muscimol inactivation of the AIP in experimental subjects during phase 2 prevented extinction of conditioned responses (CRs), shown by initial high CR frequency in the first post-drug session of phase 3, which then extinguished in a manner indistinguishable from controls in phase 2. 4. Our findings support the suggestion that similar cerebellar circuitry is engaged in acquisition and extinction of NMR conditioning.

Reversible inactivations of the cerebellum with muscimol prevent the acquisition and extinction of conditioned nictitating membrane responses in the rabbit.

Lesions of the cerebellum severely impair the classically conditioned nictitating membrane response (NMR) in rabbits. Thus, the cerebellum is essential for the production of conditioned responses (CRs), either because it is actively involved in NMR conditioning or because damage to it causes motor or other general deficits. To distinguish between these alternatives, the cerebellum may be inactivated during training. Inactivation of the cerebellum during acquisition training might result in the absence of CRs on initial trials of subsequent training without the neuronal blockade. The blockade may have prevented learning but it may have produced other deficits that require time or further training to overcome. This problem can be addressed by inactivating the cerebellum during extinction training. If inactivation during extinction training results in the immediate production of CRs when training is resumed without the blockade, then it may be concluded that extinction learning was prevented by the blockade-the presence of CRs argues against any deficits not associated with learning. We used muscimol to inactivate the cerebellum and test its involvement in acquisition and extinction of NMR conditioning in the same subjects. We injected muscimol close to the interpositus nucleus of the cerebellum 1 h before each of four daily training sessions of delay conditioning. Almost no CRs were produced in these training sessions-there was little or no acquisition of NMR conditioning during cerebellar inactivation. The subjects were then trained for four daily sessions without injections of muscimol. There were no CRs on initial trials of the first session of retraining, but all subjects produced CRs by the end of this session. The subjects then received four daily sessions of extinction training with muscimol inactivation of the nuclei-no CRs were produced. Extinction training then continued for four daily sessions without muscimol inactivation. On the first of these sessions, all subjects immediately produced high levels of CRs. These responses then extinguished within and between sessions with characteristic beginning-of-session spontaneous recovery. There was little or no extinction of NMR conditioning during cerebellar inactivation. After inactivation, the muscimol- inactivated subjects went on to acquire and extinguish NM responses at rates similar to those of appropriate controls. We conclude that cerebellar circuitry is essential for, and actively engaged in, both acquisition and extinction of this simple form of motor learning.

Metabotropic Glutamate Receptor Activation in Cerebellar Purkinje Cells as Substrate for Adaptive Timing of the Classically Conditioned Eye-Blink Response

To understand how the cerebellum adaptively times the classically conditioned nictitating membrane response (NMR), a model of the metabotropic glutamate receptor (mGluR) second messenger system in cerebellar Purkinje cells is constructed. In the model, slow responses, generated postsynaptically by mGluR-mediated phosphoinositide hydrolysis and calcium release from intracellular stores, bridge the interstimulus interval (ISI) between the onset of parallel fiber activity associated with the conditioned stimulus (CS) and climbing fiber activity associated with unconditioned stimulus (US) onset. Temporal correlation of metabotropic responses and climbing fiber signals produces persistent phosphorylation of both AMPA receptors and Ca(2+)-dependent K+ channels. This is responsible for long-term depression (LTD) of AMPA receptors. The phosphorylation of Ca(2+)-dependent K+ channels leads to a reduction in baseline membrane potential and a reduction of Purkinje cell population firing during the CS-US interval. The Purkinje cell firing decrease disinhibits cerebellar nuclear cells, which then produce an excitatory response corresponding to the learned movement. Purkinje cell learning times the response, whereas nuclear cell learning can calibrate it. The model reproduces key features of the conditioned rabbit NMR: Purkinje cell population response is timed properly; delay conditioning occurs for ISIs of up to 4 sec, whereas trace conditioning occurs only at shorter ISIs; mixed training at two different ISIs produces a double-peaked response; and ISIs of 200-400 msec produce maximal responding. Biochemical similarities between timed cerebellar learning and photoreceptor transduction, and circuit similarities between the timed cerebellar circuit and a timed dentate-CA3 hippocampal circuit, are noted.

A neural model of timed response learning in the cerebellum

A spectral timing model is developed to explain how the cerebellum learns adaptively timed responses during the rabbit's conditioned nictitating membrane response (NMR). The model posits two learning sites that respectively enable conditioned excitation and timed disinhibition of the response. Long-term potentiation of mossy fiber pathways projecting to interpositus nucleus cells allows conditioned excitation of the response's adaptive gain. Long-term depression of parallel fiber-Purkinje cell synapses in the cerebellar cortex allows learning of an adaptively timed reduction in Purkinje cell inhibition of the same nuclear cells. A spectrum of partially timed responses summate to generate an accurately timed population response. In agreement with physiological data, the model Purkinje cell activity decreases in the interval following the onset of the conditioned stimulus, and nuclear cell responses match conditioned response (CR) topography. The model reproduces key behavioral features of the NMR. including the properties that CR peak amplitude occurs at the unconditioned stimulus (US) onset: a discrete CR peak shift occurs with a change in interstimulus interval (ISI) between conditioned stimulus (CS) and US; mixed training at two different ISIs produces a double-peaked CR; CR acquisition and rate of responding depend unimodally on the ISI; CR onset latency decreases during training; and maladaptively timed, small-amplitude CRs result from ablation of cerebellar cortex.

MK801 retards acquisition of a classically conditioned response without affecting conditioning-related alterations in perforant path-granule cell synaptic transmission

The contributions of N-methyl-d-aspartate (NMDA) receptor activity to classical conditioning of the rabbit nictitating membrane (NM) response and conditioning-related alterations of the hippocampal perforant path-granule cell response were examined. Subcutaneous injections of the non-competitive NMDA antagonist MK801 (0.00, 0.05, or 0.10 mg/kg) were administered 75 min prior to each daily training session in a standard delay paradigm. MK801 administration resulted in a significant dose-dependent retardation in acquisition of the classically conditioned NM response. There were no consistent dose-dependent effects of MK801 on NM topography or on conditioned or unconditioned stimulus sensitivity. MK801 administration did not disrupt conditioning-related potentiation of perforant path-granule cell responses. The within-trial decrease in spike amplitude, relative to the between-trial response, was also not affected by MK801 administration. These results suggest that NMDA-mediated activity contributes to classical conditioning of the rabbit NM response and raise the possibility that conditioning-related alterations of perforant path-granule cell synaptic transmission are not NMDA dependent.

Long-Term Memory and Extinction of the Classically Conditioned Rabbit Nictitating Membrane Response

The purpose of the present experiment was to examine the duration of long-term memory for classical conditioning of the rabbit nictitating membrane response (NMR). Anecdotal accounts of memory for the rabbit NMR suggest that a conditioned nictitating membrane response (CR) may occur as long as a year after acquisition. The data from the present between-subjects experiment indicate that CRs (1) are robust if tested after 1 month, (2) still occur if tested after 3 months, and (3) are initially absent if tested after 6 or 9 months but then reemerge over the course of extinction. The data also indicate that reacquisition of CRs 6 or even 9 months after training is significantly more rapid than original acquisition and thus represents a substantial level of savings. These results provide the first systematic, empirical assessment of the long-term memory capacity of the rabbit for classical conditioning of the NMR and suggest that the rabbit NMR preparation is a model suitable for studying the behavioral and physiological processes involved in long-term memory, retention, and forgetting.