A brain stem region essential for the classically conditioned but not unconditioned nictitating membrane response

Radio-frequency lesions of the right dorsolateral pons severely impaired both acquisition and retention of the ipsilateral conditioned nictitating membrane response to both visual and auditory conditioned stimuli. Unconditioned responses were unaffected, and conditioning of the contralateral (left) eye was not impaired. These data suggest a critical role of the dorsolateral pons in the generation of conditioned responding in this preparation.     

Efferent neuronal control of the nictitating membrane response in rabbit (Oryctolagus cuniculus): a reexamination

Four Albino rabbits acquired a classically conditioned nictitating membrane response (NMR) to light, tone, and back shock conditioned stimuli using a paraorbital shock applied to the right eye as the unconditioned stimulus. Electrocoagulation lesions were made in the ipsilateral abducens nucleus of the pons in two animals, and the pontine reticular formation in two animals. Reinstitution of conditioning following surgery (retention test) indicated that the lesions failed to eliminate conditioned or unconditioned responses. This result is surprising in light of recent evidence that efferent control of the rabbit NMR derives solely from motoneurons of the ipsilateral abducens nucleus. Alternative hypotheses are considered.     

Classical conditioning of the nictitating membrane response of the rabbit

Summary The nictitating membrane response (NMR) of 15 rabbits was conditioned to light and white noise conditional stimuli (CSs) using a periorbital shock unconditional stimulus (US). Unilateral lesions of the inferior olive were then made. Lesions restricted to the medial parts of rostral dorsal accessory olive (DAO) and principal olive (PO) abolished conditioning and prevented subsequent acquisition on either side. Unconditional responses to the US were intact. Lesions in all other parts of the olive did not impair conditioning. The effective lesions were located in that part of the olive which supplies somatosensory information from the face to cerebellar lobule HVI. Lobule HVI is also essential for NMR conditioning. We suggest that this region of the inferior olive is part of a circuit which provides US information to the cerebellar cortex during NMR conditioning.     

The temporary inactivation of the red nucleus affects performance of both conditioned and unconditioned nictitating membrane responses in the rabbit

These experiments are part of a series of studies examining the role of the red nucleus in the performance of the conditioned and unconditioned nictitating membrane reflexes in the rabbit. Specifically, the experiments test the hypothesis that the temporary inactivation of the red nucleus selectively affects the performance of the conditioned reflex. The experiments were designed to assess the effects of lidocaine and control saline microinjections on conditioned as well as unconditioned responses in both paired and unpaired trials. Rabbits were chronically implanted with cannulae through which small injecting tubes were passed stereotaxically to the red nucleus. The animals were conditioned using a delay paradigm in which a 1 kHz tone and an air puff applied to the cornea were used as the unconditioned and conditioned stimulus, respectively. Once conditioned, the effects of either lidocaine or saline injection were evaluated while alternating paired trials with unpaired trials in which only the air puff was applied. The principal finding of this study was that the amplitudes of both the conditioned and unconditioned responses were reduced following lidocaine injection into the red nucleus. The effect on the unconditioned response amplitude could not be ascribed to any interaction between the conditioned and unconditioned responses, since it also was present in the unpaired trials. The reduction in amplitude of the conditioned and unconditioned responses was shown to be correlated with changes in other characteristics of the same responses. The data suggest that the red nucleus contributes to the performance of both the conditioned and unconditioned nictitating membrane reflexes and consequently is not likely to be involved only in pathways responsible for mediating and/or storing the engram for the conditioned reflex.     

Role of extraocular muscles in the rabbit (Oryctolagus cuniculus) nictitating membrane response

The contributions of the extraocular and retractor bulbi muscles to the nictitating membrane response (NMR) were assessed using transections of the abducens nerve or the extraocular muscles. Transection of the six extraocular muscles or the muscle that attaches to the nictitating membrane had no effect on the NMR in otherwise intact animals. Transections of the abducens nerve reduced the NMR by 50%, and transections of the abducens nerve and the extraocular muscles abolished the NMR. These results indicate that the NMR can be effected by the six extraocular muscles, but that these six muscles may play a role that is redundant in the intact animal. The anatomical location of the motoneurons innervating the retractor bulbi is discussed.     

Cerebellar Purkinje cell activity related to the classically conditioned nictitating membrane response

Summary Because of the purported critical role of cerebellar lobule HVI in classical conditioning of the nictitating membrane response of the rabbit, we recorded extracellularly from HVI Purkinje cells (PCs) during differential conditioning. Rabbits were trained using tonal conditioned stimuli (CSs) and stimulation of the periocular region as the unconditioned stimulus (US). Many PCs responded to the US, the most frequently observed response being a burst of simple spikes. PCs in HVI showed a variety of responses to CSs that were related to conditioned responses (CRs). The most frequently observed response was an increase in simple spikes correlated with CRs. The activity of many of these cells antedated CRs by 20–200 ms. A smaller proportion of cells exhibited inhibition of simple spike activity that antedated CRs. The existence of PCs that alter their firing before CRs suggests that they may be causally involved in this behavior, and in this respect they reinforce reports that lesions of HVI or its connections disrupt nictitating membrane CRs. Although complex spike activity was not generally related to the US or to CRs, a few PCs responded in relation to CRs with only complex spikes. In demonstrating CR-related activity in cerebellar PCs, this study supports theories of cerebellar learning such as those of Marr and Albus.     

Role of the levator palpebrae superioris (LPS) muscle in effecting nictitating membrane movement in the rabbit

Examination of the origin and course of the muscle fibers attached to the nictitating membrane (NM) of the rabbit demonstrated that these muscle fibers constitute part of the nasal branch of the levator palpebrae superioris (LPS) muscle. Other fibers of the nasal branch of the LPS muscle were observed to terminate in the nasal half of the upper eyelid. No further anatomical division of the nasal LPS into distinct eyelid and NM branches was observable by dissection. Contraction of the fibers inserting in the NM, produced by direct electrical stimulation, resulted in movement of the extended membrane towards its resting position in the corner of the eye. The observed anatomical relationships demonstrate in part that functional control of NM movement is structurally linked to control of upper eyelid elevation in this species. The origin and distribution of fibers inserting in the NM, and the effects of contraction of the muscle fibers on movement of the NM, indicate that contraction of these fibers could both oppose extension of the NM and/or initiate or contribute to retraction of the membrane following extension. Because of their position and the direction of NM movement caused by their contraction, these muscle fibers cannot actively cause or contribute to extension of the NM. Therefore, it is suggested that the LPS has a role in effecting movement of the rabbit NM by actively contributing to its retraction or reopening.     

Single-unit activity in red nucleus during the classically conditioned rabbit nictitating membrane response

Previous investigations have suggested that the cerebellum and associated brainstem structures, including the red nucleus, are essential for the expression of the classically conditioned nictitating membrane (NM) response. The present study examined the firing patterns of extracellularly-recorded single units in the red nucleus of the awake rabbit during differential conditioning. Tones were used as conditioned stimulus (CS+ and CS−) and periocular electrostimulation was used as the unconditioned stimulus (US). Most units exhibited one or more changes in firing rate during the presentation of the CS, and increases in firing were much more common than decreases. The onset of some of these changes appeared to be time-locked to the onset of the CS (‘CS-locked’ responses), while other changes were time-locked to the onset of the CR (‘CR-locked’ responses). About one-third of all CS-locked changes were CR-dependent, meaning that the neuronal response was reduced when the CR did not occur. About two-thirds of all CR-locked responses preceded the onset of the CR, and lead times varied considerably across units. Many CR-locked units were located in what has been described as a dorsal face region of the red nucleus. Most units responded to the US, and some of the US responses were CR-dependent: i.e., a smaller US response was evoked when a CR preceded the US than when the CR was absent. Our results support the notion that cerebellum-brainstem circuits are involved in generating NM CRs.     

Retardation of rabbit nictitating membrane conditioning by subseizure electrical stimulation of hippocampus.

The experiment involved classical conditioning of the nictitating membrane response of rabbits with subseizure, posttrial electrical stimulation of hippocampus. Hippocampal electrical activity was recorded before, during and after each trial to assure that no seizure activity was present. Stimulated animals, in comparison with unstimulated controls, showed a marked retardation of CR onset which paralleled that of previous research employing seizure producing stimulation. This implies that brain seizures are not the critical element in the disruption effect, but rather the anatomical locus of stimulation. Results are discussed in the context of recent lesion, recording and stimulation research employing the conditioned nictitating membrane response of rabbits as a model system.     

Latency of the nictitating membrane response to periocular electro-stimulation in unanesthetized rabbits

The latency of the nictitating membrane response (NMR) in rabbit (Oryctolagus cuniculus) to periocular electro-stimulation is a negative exponential function of stimulus current with an asymptote of approximately 17 msec. The NMR was recorded by means of a precision low-torque potentiometer like that employed in previous studies of NMR latency, and the criterion of response initiation employed here was similar to that employed in studies of classical conditioning in this preparation. Using estimates from physiological studies on surgically acute preparations, the minimum latency of the NMR of 17 msec can be decomposed as follows: 4 msec to fire motoneurons of the accessory abducens nucleus; 9 msec for conduction, synaptic transmission, and recruitment of retractor bulbi muscle fibers; 4 msec for the nictitating membrane to initiate its sweep after eyeball retraction. The implications of these estimates for chronic unit-recording studies of the conditioned NMR are discussed.     

Dorsolateral pontine tegmentum and the classically conditioned nictitating membrane response: analysis of CR-related single-unit activity

Summary Previous investigations have suggested that the dorsolateral pontine tegmentum (DLP) may be part of a system essential for classical conditioning of the nictitating membrane response. The present study examined CR-related firing patterns of extracellularly-recorded single units in the DLP. Differential conditioning, using tonal CSs and periocular electrostimulation as the US, was employed so that firing patterns on CR and non-CR trials could be compared. Cells that exhibited CR-related increases in firing (excitatory cells) were found in reticular formation surrounding the motor trigeminal nucleus (zone h), including the supratrigeminal region, and in a region dorsal and dorsomedial to the brachium conjunctivum. Cells that exhibited CR-related decreases in firing (inhibitory cells) were observed in subcoeruleus/medial parabrachial regions and dorsal nucleus reticularis pontis oralis and caudalis. A third class of cells (temporal cells) exhibited CR-related shifts in the temporal distribution of spikes; these cells were located in various brain stem regions. The firing of many of the excitatory, inhibitory, and temporal cells preceded the behavioral CR by an amount of time sufficient for causal involvement. In light of evidence indicating that the cerebellum is critically involved in conditioning, the present study suggests that two systems, the cerebellum and the DLP, may be in control of CRs. The relationship of the two systems and their possible roles in conditioning are discussed.     

Habituation of the nictitating membrane reflex response in the intact frog.

Habituation of the nictitating membrane reflex response was measured in the intact bullfrog. A stimulus map was created, and stimulating electrode pairs directly opposed across the eye proved to be the optimal loci for cutaneous electrical stimulation. The response was found to exhibit all of the parametric characteristics of habituation. To date, this represents what is perhaps the simplest intact vertebrate preparation demonstrating habituation. The response was also found to exhibit a surprising periodicity in response amplitude when unchanging stimuli were infrequently presented over long periods of time. The convenience, reliability and successful behavioral assessment strongly recommend this preparation as an ideal simple vertebrate model system for further examination of the neural basis of behavioral plasticity.     

Brachium conjuntivum and rubrobulbar tract: brain stem projections of red nucleus essential for the conditioned nictitating membrane response

The trajectory of the rubrobulbar tract in rabbit to the level of the accessory abducens nucleus is described: orthograde labeling of fibers of the rubrobulbar tract following horseradish peroxidase implants into red nucleus of 8 animals permitted ad hoc analysis of the effects of brain stem lesions on the rabbit's conditioned nictitating membrane response. Twenty-four rabbits, trained to give conditioned NM responses from both eyes, received unilateral lesions of the right pontine brain stem. Six of the 7 cases of post-lesion disruption of ipsilateral conditioned responding involved either ipsilateral brachium conjunctivum or the rubrobulbar tract. This finding, together with a reexamination of data from two related studies from this laboratory strongly support the conclusion that an essential premotor component of the conditioned NM response is a doubly decussating circuit from the interpositus nucleus of the cerebellum to magnocellular red nucleus, via brachium conjunctivum and its decussation, and from red nucleus caudally, via the ventral tegmental bundle and the rubrobulbar tract, to the accessory abducens nucleus, where motoneurons involved in the NM response are located. These findings are consistent with published reports on the essential role of interpositus nucleus of the cerebellum in NM conditioning. The possible role of the supratrigeminal reticular formation in this learned behavior is also discussed.     

Electrical brain stimulation as the reinforced CS in Pavlov's conditioned inhibition paradigm

Rabbits received Pavlovian conditioned inhibition training (nictitating membrane response) using ESB as the reinforced CS and a tone as the conditioned inhibitor. Summation, retardation, and generalization tests indicated that the use of ESB in the paradigm did not interfere with the development of inhibitory control by the tone.     

Attenuation of latent inhibition by electrical stimulation of hippocampus

The effects of hippocampal stimulation on the development and expression of latent inhibition were investigated employing classical conditioning of the nictitating membrane response of rabbits. Hippocampal stimulation overlapping conditioned stimulus preexposure produced attenuation of latent inhibition, as did stimulation presented during conditioning after preexposure. However, hippocampal stimulation during conditioning in the absence of preexposure had little effect. The results support the hypotheses that in classical conditioning a major function of the hippocampus is the modulation of sensory input, especially input involved in the learned irrelevance of stimuli.     

Disruption of rabbit (Oryctolagus cuniculus) nictitating membrane conditioning by posttrial electrical stimulation of hippocampus

Two experiments were conducted involving classical conditioning of the nictitating membrane response of rabbits with posttrial electrical brain stimulation. Hippocampal posttrial stimulation (PTS) presented immediately after each CS-US paired trial was found to cause massive disruption of conditioning by comparison with performance of either a cortical PTS group, an operated but unstimulated group, or an unoperated control group. The major effect of PTS was to postpone CR emergence. Once CRs began to occur, conditioning seemed to proceed normally regardless of the presence or absence of stimulation. Hippocampal PTS initiated after conditioning had no effect on the elicitation of conditioned responses. The results indicated that hippocampal PTS disrupted consolidation but did not affect either registration of stimuli or execution of unconditioned or conditioned responses. It was concluded that classical conditioning of the rabbit nictitating membrane response is an excellent model neural system for the investigation of neurophysiological aspects of memory.     

Disrupted retention of the classically conditioned nictitating membrane response in the aluminum-intoxicated rabbit using electrical stimulation of the brain as a conditioned stimulus

Rabbits were classically conditioned to emit an eyeblink conditioned response (CR) to electrical stimulation (ESB) of the medial geniculate nucleus (MGN) paired with a corneal air puff until they attained a criterion of two consecutive days of greater than 90% CRs. They then received intraventricular injections of 1% AlCl3, HCL, or normal saline. Ten days postinjection, each animal underwent a retention test consisting of 50 ESB alone presentations. Whereas all saline and HCL animals gave at least 90% CRs during retention, no aluminum rabbit emitted more than 30% CRs. Considered with the results of previous work, these data suggest that aluminum-induced neurofibrillary degeneration disrupts retention of the CR by affecting central associative processes.     

Activity of deep cerebellar nuclear cells during classical conditioning of nictitating membrane extension in rabbits

Summary The activity of neurons in the interposed and dentate nuclei of the cerebellum was investigated during differential classical conditioning of the rabbit eye blink/ nictitating membrane response. Forty-seven percent of the 165 cells in the study responded to the orbital stimulation used as the unconditioned stimulus (US). The latency distribution of US-elicited responses was bimodal with peaks at 7 and 19 ms. Twenty-one percent of the cells responded with short latencies to the tones used as conditioned stimuli (CSs). These cells typically responded to both the reinforced and nonreinforced CSs. Forty-one percent of the cells responded on conditioned response (CR) trials but not on trials without CRs. The average lead of the neural response to the CR was 71.4 ms. Cells that responded on CR trials were more likely to respond to the CSs, or to the CSs and the US, than cells that did not respond on CR trials. For about half of the cells that responded on CR trials the latency of response followed trial-by-trial variations of CR latency. For the remainder, the response was time-locked to CS-onset. Cells whose responses paralleled the CR may be involved in the initiation or modulation of the CR, while those whose responses were time-locked to the CS may be involved in sensory processing underlying the initiation of the movement. The pathways that may underlie the US- and CS-elicited responses are also discussed.     

Cerebellar cortex and eyeblink conditioning: bilateral regulation of conditioned responses

We examined the role of the cerebellum in classical conditioning of the nictitating membrane response (NMR) of rabbits by comparing the effects of unilateral and bilateral cerebellar cortical lesions. Using extended preoperative conditioning to ensure high levels of learning, we confirmed that unilateral lesions of lobules HVI and ansiform lobe impaired conditioned responses (CRs) previously established to an auditory conditioned stimulus, but did not prevent some relearning with post-operative retraining. Bilateral lesions of HVI and ansiform lobe produced similar impairments of CRs, but also prevented subsequent relearning. Unilateral cortical lesions produced significant enhancement of unconditioned response (UR) amplitudes to periorbital electrical stimulation. Bilateral cortical lesions enhanced UR amplitudes to a lesser extent. Because there was no correlation between the degree of CR impairment and UR enhancement across the unilateral and bilateral lesion groups, the suggestion that the lesions impaired CRs due to general effects upon performance, rather than due to losses of learning, is not supported. Both sides of the cerebellar cortex contribute towards learning a unilaterally trained CR. This finding is important for the re-interpretation of unilateral, reversible inactivation studies that have found no involvement of the cerebellar deep nuclei in the acquisition of NMR conditioning. In addition, we found conditioning-dependent modifications of unconditioned responses that were particularly apparent at low intensities of periorbital electrical stimulation. This finding is important for the re-interpretation of studies that have found apparent changes in the UR of conditioned subjects after cerebellar lesions.