Rabbit Eyelid Response Research Articles

A Variable Oscillator Underlies the Measurement of Time Intervals in the Rostral Medial Prefrontal Cortex during Classical Eyeblink Conditioning in Rabbits.

The way in which brains generate working memories (those used for the transient processing and storage of newly acquired information) is still an intriguing question. Here, we report that the firing activities of neurons located in the rostromedial prefrontal cortex recorded in alert behaving rabbits are controlled by a dynamic oscillator. This oscillator generated firing frequencies in a variable band of 3-12 Hz depending on the conditioned stimulus-unconditioned stimulus intervals (1 s, 500 ms, 250 ms) selected for classical eyeblink conditioning of behaving rabbits. Shorter (50 ms) and longer (2 s) intervals failed to activate the oscillator and prevented the acquisition of conditioned eyelid responses. This is an unexpected mechanism to generate sustained firing activities in neural circuits generating working memories.

Red Nucleus Neurons Actively Contribute to the Acquisition of Classically Conditioned Eyelid Responses in Rabbits

The red nucleus (RN) is a midbrain premotor center that has been suggested as being involved in the acquisition and/or performance of classically conditioned nictitating membrane/eyelid responses. We recorded in rabbits the activity of RN and pararubral neurons during classical eyeblink conditioning using a delay paradigm. Neurons were identified by their antidromic activation from contralateral facial and accessory abducens nuclei and by their synaptic activation from the ipsilateral motor cortex (MC) and the contralateral cerebellar interpositus (IP) nucleus. For conditioning, we used a tone as a conditioned stimulus (CS) followed 250 ms later by a 100 ms air puff as an unconditioned stimulus (US) coterminating with it. Conditioned responses (CRs) were determined from the evoked changes in the electromyographic activity of the orbicularis oculi (OO) muscle. Recorded neurons were classified by their antidromic activation and by their changes in firing rate during the CS-US interval. Identified neurons increased their firing rates in relation to the successive conditioning sessions, but their discharge rates were related more to the EMG activity of the OO muscle than to the learning curves. Reversible inactivation of the IP nucleus with lidocaine during conditioning evoked a complete disappearance of both conditioned and unconditioned eyelid responses, and a progressive decrease in CR-related activity of RN neurons. In contrast, MC inactivation evoked a decrease in the acquisition process and an initial disfacilitation of neuronal firing (which was later recovered), together with the late appearance of CRs. Thus, RN neurons presented learning-dependent changes in activity following MC inactivation.

Involvement of cerebral cortical structures in the classical conditioning of eyelid responses in rabbits

The classical conditioning of the eyelid motor system in alert behaving rabbits has been used to study the expression of Fos in the hippocampus, and in the occipital, parietal, piriform and temporal cortices. Animals were classically conditioned with both delay and trace conditioning paradigms. As conditioned stimulus, both short and long (20 and 100 ms) tones (600 Hz, 90 dB) or short, weak (20 ms, 1 kg/cm 2) air puffs were used. The unconditioned stimulus was always a long, strong (100 ms, 3 kg/cm 2) air puff that started 250–270 ms after the onset of the conditioned stimulus. The expression of Fos was significantly increased after both delayed and trace conditioning in the hippocampus, and in the parietal and piriform cortices contralateral to the unconditioned stimulus presentation side, compared with equivalent ipsilateral structures in conditioned animals, or with Fos production in the same contralateral structures in pseudo-conditioned and control animals. Fos expression in some cortical sites was specific to tone versus air puff stimuli when used as conditioned stimulus. Thus, Fos expression was significantly increased in the contralateral temporal lobe when tones were used as conditioned stimulus, for both delayed and trace conditioning paradigms, but not when animals were conditioned to short, weak air puffs. The present results indicate a specific Fos activation in several cerebral cortical structures during associative eyelid conditioning.

A nitric oxide synthase inhibitor delays the formation of learning-related neural activity in the cerebellar interpositus nucleus during rabbit eyelid conditioning

Inhibiting nitric oxide synthesis with l-nitro arginine methyl ester ( l-NAME) has been found to retard the acquisition of the classically conditioned rabbit eyelid response. In the present study, rabbits received an SC injection of either l-NAME (an inhibitor of nitric oxide synthase) or its inactive stereoisomer, d-NAME, an hour before each daily training session. l-NAME retarded both the acquisition of conditioned responses and associated learning-related neural activity in the interposilus nucleus. There was no effect of l-NAME on spontaneous interpositus activity recorded during a period of time before trial onset. These findings indicate that nitric oxide may play a role in neuronal processes associated with the acquisition of the classically conditioned rabbit eyelid responses.

Involvement of the ventrolateral thalamic nucleus in rabbit classical eyeblink conditioning

The involvement of the ventrolateral nucleus of the thalamus in relaying learning-related activity to higher brain structures during classical conditioning of the rabbit eyelid response was examined in two experiments. In the first study, multiple-unit ventrolateral thalamic nucleus activity was monitored before and after lesions of either the cerebellar interpositus nucleus or red nucleus were given. Before the lesions were given, conditioned response-related activity was observed in the ventrolateral thalamic nucleus. Lesions of the interpositus nucleus, but not the red nucleus, disrupted the conditioning-related activity in the ventrolateral thalamic nucleus, thus suggesting that an efferent copy to conditioned response-related activity is projected directly from the interpositus nucleus to higher brain areas by way of the ventrolateral thalamic nucleus. In the second study, multiple unit activity in the hippocampus was monitored before and after lesions were placed in the ventrolateral thalamic nucleus or red nucleus. Conditioning-related activity in the hippocampus was not affected by either lesion, thus suggesting that maintenance of training-related activity in the hippocampus is not critically dependent on cerebellar information relayed through the ventrolateral thalamic nucleus or red nucleus.

Extinction of conditioned eyelid responses requires the anterior lobe of cerebellar cortex

We test the hypothesis that the cerebellar cortex is required for the extinction of conditioned eyelid responses in rabbits trained using standard Pavlovian delay procedures. Following 10 daily training sessions during which rabbits achieved asymptotic performance, lesions of the ipsilateral hemisphere of the cerebellar cortex were made by aspiration. The target of these lesions was the anterior lobe, as suggested by previous observations that this region is necessary for the learning-dependent timing of conditioned eyelid responses (Perrett et al., 1993). We report that anterior lobe damage, as indicated by disrupted response timing and confirmed by tissue analysis, produces severe deficits in conditioned response extinction. Postlesion responses show no significant decline over ten training sessions, whereas response timing and extinction are unaffected by lesions that do not include the anterior lobe. These conditioned responses that do not extinguish display stimulus specificity, excluding the possibility that they are unlearned responses unmasked by cerebellar cortex lesions. These observations suggest that Pavlovian eyelid conditioning is mediated by synaptic plasticity in at least two sites and the anterior lobe of the cerebellar cortex influences one of these sites during extinction. Based on these and previous data, we propose the hypothesis that eyelid conditioning can involve plasticity in both the cerebellar cortex and interpositus nucleus and that plasticity in the nucleus is controlled by input from Purkinje cell activity in the cortex. This hypothesis is consistent with observations that the cerebellar cortex may not always be required for the expression of conditioned responses, but it is necessary for response timing and for extinction.

Classical discrimination conditioning of the rabbit's eyelid response using pontine stimulation as a conditioned stimulus

Classical discrimination conditioning of the nictitating membrane/eyelid response was performed on seven rabbits using stimulation of the pontine nuclei or middle cerebellar peduncle as the conditioned stimulus (CS) and an air puff as the unconditioned stimulus (US). The rabbits learned to discriminate between a CS paired with a US and delivered to one pontine nucleus (the CS+) and a CS presented alone and delivered to the contralateral pontine nucleus (the CS-). Subsequent reversal of the discrimination was also achieved when the CS+ and CS- stimulation sites were interchanged. The results are interpreted as support for the idea that essential plasticity for classical eyelid conditioning occurs efferent to the pontine nuclei, possibly in regions of the cerebellum.

Classical conditioning of the rabbit eyelid response with a mossy-fiber stimulation CS: II. Lateral reticular nucleus stimulation.

Stimulation of mossy fibers arising from the pontine nuclei can be used as a conditioned stimulus (CS) during classical conditioning of the eyelid/nictitating membrane response (NM). In the present experiment we stimulated another source of mossy fibers, the lateral reticular nucleus (LRN), as a CS for NM conditioning. LRN stimulation was an effective CS, resulting in learning, and the conditioned response to LRN stimulation showed normal extinction. Unpaired presentation of CS and UCS did not result in pseudo-conditioning. Lesions of the cerebellar dentate-interpositus region abolished the conditioned response but left the unconditioned reflex response intact. We suggest that mossy fibers may normally carry CS information to the cerebellum.

Classical conditioning of the rabbit eyelid response with a mossy-fiber stimulation CS: II. Lateral reticular nucleus stimulation.

Classical conditioning of the rabbit eyelid response with a mossy-fiber stimulation CS: II. Lateral reticular nucleus stimulation.

Classical conditioning of the rabbit eyelid response with a mossy-fiber stimulation CS: I. Pontine nuclei and middle cerebellar peduncle stimulation.

The nictitating membrane/eyelid responses of 18 rabbits were classically conditioned using cerebellar mossy-fiber stimulation as a conditioned stimulus (CS) and air puff as an unconditioned stimulus (US). The dorsolateral, lateral, and medial pontine nuclei and the middle cerebellar peduncle were effective stimulation-CS sites for training. In one group of rabbits, robust conditioned eyelid responses were produced with paired trials and subsequently extinguished with CS-alone and explicitly unpaired presentation of the CS and US. In a second group of rabbits, no conditioned responses were evident for 4 days of unpaired CS and US presentations. Conditioned responses did develop, however, after paired training was begun. Lesions of the interpositus nucleus of the cerebellum completely abolished the conditioned responses of a third group of rabbits overtrained with the mossy-fiber CS and air-puff US. These results support previous studies which have demonstrated that the cerebellum is critically involved in acquisition and retention of simple learned responses. In addition, the present results support previous theories of cerebellar function which have proposed that mossy fibers supply critical "learning" input to the cerebellum for acquisition and retention of motor skills.

Classical conditioning of the rabbit eyelid response with a mossy-fiber stimulation CS: I. Pontine nuclei and middle cerebellar peduncle stimulation.

Classical conditioning of the rabbit eyelid response with a mossy-fiber stimulation CS: I. Pontine nuclei and middle cerebellar peduncle stimulation.

Classical conditioning of the rabbit eyelid response with mossy fiber stimulation as the conditioned stimulus

The nictitating membrane responses of 12 rabbits were classically conditioned using mossy fiber stimulation as a conditioned stimulus (CS) and airpuff as an unconditioned stimulus. The dorsolateral pontine nucleus and the lateral reticular nucleus were effective stimulation-CS sites for learning. Robust conditioned responses were obtained with relatively few paired trials and extinguished with subsequent CS-alone presentations. In addition, explicitly unpaired presentations of stimulation and airpuff failed to produce response increments. These results support previous theories of cerebellar function that have proposed that mossy fibers supply “learning” input to the cerebellum for motor learning.

Classical conditioning of the rabbit eyelid response increases glutamate receptor binding in hippocampal synaptic membranes.

Hippocampal pyramidal neurons exhibit a rapid within-trial increase in firing frequency during classical conditioning of the rabbit eyelid response. It has been proposed that the cellular mechanisms responsible for hippocampal long-term potentiation (LTP) may also mediate this learning-dependent increase in neuronal activity. The induction of LTP in rat hippocampal slices results in an increase in the number of [3H]glutamate-binding sites in the potentiated region. The present study investigates the kinetics of [3H]glutamate binding to hippocampal synaptic membranes after eyelid conditioning in the rabbit. We report that the regional distribution of [3H]glutamate binding across the layers of rabbit hippocampus is compatible with a dendritic localization. The pharmacological and ionic properties of the binding suggest that it is associated with an excitatory amino acid receptor. After eyelid conditioning, the maximal number of hippocampal [3H]glutamate-binding sites is increased in animals receiving paired presentations of the tone conditioned stimulus and corneal air-puff unconditioned stimulus relative to that found in naive or unpaired control animals. These results strengthen the hypothesis that an LTP-like mechanism underlies the increase in hippocampal firing frequency during rabbit eyelid conditioning.

Backward conditioning of the rabbit eyelid response: A test using second-order conditioning

Investigations studying fear in the rat have reported that a CS paired in a backward relationship with the US can acquire excitatory properties. This procedure does not appear to be effective in rabbit eyelid conditioning. The present studies examined Rescorla’s proposal that an excitatory effect might be observed with US-CS pairings in eyelid conditioning if the CS was tested in a second-order conditioning paradigm. The results provided no support for this proposal because second-order eyelid conditioning was just as difficult to demonstrate as was backward eyelid conditioning.

Spontaneous recovery in rabbit eyelid conditioning.

Spontaneous recovery of the classically conditioned rabbit eyelid response increased in a negatively accelerated function with an increase of postextinction rest from .17 to 24 hr. The data were obtained from six groups of male New Zealand rabbits which received an extinction session separated by rest intervals of varying duration from a spontaneous recovery test.

Second-order conditioning: The importance of stimulus overlap on second-order trials

Second-order conditioning was examined using the rabbit eyeblink paradigm and the gerbil CER paradigm. Pavlov’s hypothesis that stimulus overlap on second-order trials produces conditioned inhibition and that nonoverlap leads to second-order conditioning was not confirmed. Our results also revealed that the manner in which first-order and second-order trials are intermixed has an important influence on the properties of the second-order CS. A within-session mixture of first- and second-order trials tended to produce second-order conditioning, and a between-session mixture tended to produce conditioned inhibition. Second-order conditioning was more prominent with the gerbil fear response than with the rabbit eyelid response.

Classical conditioning of the rabbit eyelid response as a function of interstimulus interval.

Classical conditioning of the rabbit eyelid response as a function of interstimulus interval.

Rabbit eyelid conditioning as a function of the intertrial interval

Single session conditioning of the rabbit eyelid response was dependent on the intertrial interval (ITI); in general, the shorter the ITI, the greater the mean and variance of the trial at which conditioning began. Conditioning was facilitated by massing the initial CS-US pairings and then continuing training with a spaced trial procedure. No ITI effect was observed with multiple session conditioning.

Differential conditioning of the rabbit's eyelid response with an examination of Pavlov's induction hypothesis.

Differential conditioning of the rabbit's eyelid response with an examination of Pavlov's induction hypothesis.