Monitoring kinetic and frequency-domain properties of eyelid responses in mice with magnetic distance measurement technique

Classical eye-blink conditioning in mutant mice can be used to study the molecular mechanisms underlying associative learning. To measure the kinetic and frequency domain properties of conditioned (tone - periorbital shock procedure) and unconditioned eyelid responses in freely moving mice, we developed a method that allows adequate, absolute, and continuous determination of their eyelid movements in time and space while using an electrical shock as the unconditioned stimulus. The basic principle is to generate a local magnetic field that moves with the animal and that is picked up by either a field-sensitive chip or coil. With the use of this magnetic distance measurement technique (MDMT), but not with the use of electromyographic recordings, we were able to measure mean latency, peak amplitude, velocity, and acceleration of unconditioned eyelid responses, which equaled 7.9 +/- 0.2 ms, 1.2 +/- 0.02 mm, 28.5 +/- 1 mm/s, and 637 +/- 22 mm/s(2), respectively (means +/- SD). During conditioning, the mice reached an average of 78% of conditioned responses over four training sessions, while animals that were subjected to randomly paired conditioned and unconditioned stimuli showed no significant increases. The mean latency of the conditioned responses decreased from 222 +/- 40 ms in session 2 to 127 +/- 6 ms in session 4, while their mean peak latency increased from 321 +/- 45 to 416 +/- 67 ms. The mean peak amplitudes, peak velocities, and peak acceleration of these responses increased from 0.62 +/- 0.02 to 0.77 +/- 0.02 mm, from 3.9 +/- 0.3 to 7.7 +/- 0.5 mm/s, and from 81 +/- 7 to 139 +/- 10 mm/s(2), respectively. Power spectra of acceleration records illustrated that both the unconditioned and conditioned responses of mice had oscillatory properties with a dominant peak frequency close to 25 Hz that was not dependent on training session, interstimulus interval, or response size. These data show that MDMT can be used to measure the kinetics and frequency domain properties of conditioned eyelid responses in mice and that these properties follow the dynamic characteristics of other mammals.     

Temporal discrimination in the cerebellar cortex during conditioned eyelid responses

Little is known about mechanisms used by the nervous system to encode time. In light of recent evidence, cerebellar cortex involvement in the learned timing of conditioned eyelid responses shows promise as an area of investigation into neural timing mechanisms. Lesion studies indicate that the cerebellar cortex is necessary for response timing, but do not rule out the possibility that response timing is encoded afferent to the cerebellum. To differentiate between precerebellar and cerebellar cortical timing mechanisms, rabbits were trained by pairing direct stimulation of mossy fibers in the cerebellum as the conditioned stimulus (CS) with an eyeshock unconditioned stimulus (US). We find that individual animals can produce diffently timed conditioned responses when trained with a mossy fiber CS that has been paired with the US at various interstimulus intervals. The fact that differently timed responses can be conditioned using constant-frequency stimulation of an invariant subset of mossy fibers as the CS suggests that timing information in the afferent input to the cerebellum is not essential. Two rabbits trained with single-pulse stimulation in the cerebellum as the CS also learned differently timed conditioned responses; suggesting that fiber recruitment during a stimulus train does not convey the necessary temporal coding to the cerebellar cortex. Together with the lesion data, these findings suggest that the learned timing of conditioned eyelid responses occurs in the cerebellar cortex. Received: 25 August 1997 / Accepted: 12 January 1998     

Bilateral cerebellar lesions disrupt conditioned eyelid responses in unrestrained rats

Electromyographic eyelid responses in unrestrained rats were classically conditioned in a Pavlovian delay paradigm by using a tone conditioned stimulus (CS) and periorbital shock unconditioned stimulus (US). After eyelid conditioning was complete, bilateral electrolytic lesions were made in the dentate-interpositus region of the cerebellar nuclei. Initial eyelid conditioning was reliable and very similar to that previously observed in the rabbit, although the asymptotic eyelid responses contained a short-latency startle response in addition to the usual conditioned and unconditioned responses (CR and UR). Substantial decrements in CRs were observed in 13 of the 14 rats with accurately placed lesions. In contrast, startle responses and URs were unaffected. The results replicate the effects of cerebellar lesions on eyelid CRs in the rabbit and suggest that the anatomical basis of eyelid conditioning in both species is similar.     

Classically conditioned withdrawal reflex in cerebellar patients. 1. Impaired conditioned responses

The role of the cerebellum in the classically conditioned, human lower-limb-withdrawal reflex was studied in ten patients with pure cerebellar diseases (CBL), ten patients showing additional extracerebellar symptoms (CBL+), and in 11 sex- and age-matched normal controls (CTRL). Where conditioning was successful, the electrically evoked, unconditioned response was preceded by a tone-conditioned response (CR). CR incidence was variable, with best results in the CTRL, significantly less in CBL, and lowest in CBL+. Although CRs could be established in subjects in all groups, a continuous increase in the CR incidence in the course of the recording session was observed primarily in CTRL. In CBL and CBL+, such a characteristic reflex acquisition was rather the exception. CR onsets in CBL were within the range of those in CTRL, but CR amplitude was significantly lower in CBL. Cerebellar patients with circumscribed lesions behaved differently in our motor-learning paradigm, depending on the lesion site. Patients suffering from pathology of the posterior inferior cerebellum showed a mean CR incidence within the lower range of CTRL. In contrast, if the anterior and superior cerebellum was affected, few or even no CRs were observed. Our findings thus provide evidence that the human cerebellum is required for the acquisition and the retention of this specific conditioned limb-withdrawal reflex. In particular, anterior and superior parts of the cerebellum appear to be involved. Thus, an expansion of the current concept of clinically based, functional compartmentalization is suggested, such that anterior and superior cerebellar regions must be intact to establish plastic changes required for the acquisition of the conditioned withdrawal response.     

Chaotic components in the high-frequency EEG of rabbit cortex during formation of a conditioned defensive reflex

The chaotic component of the high-frequency EEG of the rabbit cortex was studied during the development of a conditioned defensive reflex to nonrhythmic light stimulation with electric reinforcement applied to the animal's paw. Regular changes in the dynamic parameters of the chaotic component of the high-frequency EEG were demonstrated. Characteristic changes in the asymptotic evaluations of the correlational dimensionality of the attractor of the chaotic component (DCC) of the high-frequency EEG were detected in the cortical regions studied both during the action of the conditioned stimulus and in background conditions before presentation of combinations. These changes affected both the afferent and efferent cortical regions of the conditioned reflex arc, as well as regions not directly addressed by the conditioned or unconditioned stimuli. These results allow the dynamics of changes in the morphofunctional structures of the conditioned reflex to be seen at the cortical level during learning. Significant differences in the early and late stages of conditioned reflex formation were detected. Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Moscow. Translated from Zhurnal Vysshei Nervnoi Deyatel'nosti imeni I. P. Pavlova, Vol. 47, No. 5, pp. 858–869, September–October, 1997.     

Cerebellar posterior interpositus nucleus as an enhancer of classically conditioned eyelid responses in alert cats

Cerebellar posterior interpositus neurons were recorded in cats during delayed and trace conditioning of eyeblinks. Type A neurons increased their firing in the time interval between conditioned and unconditioned stimulus presentations for both paradigms, while type B neurons decreased it. The discharge of different type A neurons recorded across successive conditioning sessions increased, with slopes of 0.061-0.078 spikes/s/trial. Both types of neurons modified their firing several trials in advance of the appearance of eyelid conditioned responses, but for each conditioned stimulus presentation their response started after conditioned response onset. Interpositus microstimulation evoked eyelid responses similar in amplitude and profiles to conditioned responses, and microinjection of muscimol decreased conditioned response amplitude. It is proposed that the interpositus nucleus is an enhancer, but not the initiator, of eyelid conditioned responses.     

Classically conditioned withdrawal reflex in cerebellar patients. 2. Impaired unconditioned responses

The study addresses the issue of the role of the cerebellum in human withdrawal-reflex conditioning by comparing data from patients with pure cerebellar diseases (CBL, n = 10) and from cerebellar patients showing additional extracerebellar symptoms (CBL+, n = 10) with those from 11 control subjects (CTRL). During recording sessions, the standard delay-conditioning paradigm with paired-trials was used with tone as the conditioned stimulus (CS). Parameters of the conditioned muscle responses are analyzed in an accompanying paper. Here, we focus on the unconditioned muscle response. A train of current pulses (unconditioned stimulus, US) evoked a lower-limb withdrawal reflex (unconditioned response, UR), which was recorded electromyographically from leg muscles. During the recording sessions with CTRL subjects, UR amplitudes decayed from initially 100% to approximately 50% at the end of the session. This type of decay was clearly less pronounced in the CBL group and minimal in the CBL+ group. Furthermore, the CBL group exhibited UR onsets that were delayed by 20 ms compared with those from CTRL subjects. Although the ranges of measurements characterizing the URs of a given cerebellar patient tested in the paired-trial paradigm overlapped with those of control subjects, the statistically significant differences observed at the group level suggest deficits in the performance of the reflex responses. The delayed URs in patients and the different type of decay of UR amplitudes in repetitively evoked withdrawal reflexes constitute evidence that the cerebellum is critically involved in the control of these UR parameters.     

Trunk-ocular reflex in the rabbit

The trunk-ocular reflex of the rabbit was investigated by recording EMGs of right lateral rectus and splenius muscles. The skull and the chest of the rabbit were restrained and the lower part of the trunk was flexed laterally around the axis at the TH10--11 intervertebral joint. Two kinds of eye movements, small with short latencies and large with longer latencies, were induced counter to the direction of the lateral flexion. The latency of the former was 158 msec (range: 64--200 msec) and that of the latter was 255 msec (range: 180--380 msec).     

Local changes in the redox potential in the rabbit cerebral cortex accompanying the acquisition of a conditioned defensive reflex

The oxidative-reductive (redox) potential (E) of brain tissue depends on the ratio of the speeds of processes occurring in the glycolysis (the evolutionarily ancient energy compartment operating without oxygen) and oxidative metabolism (evolutionarily younger and energetically more efficient) compartments. E in the cortex was recorded using implanted platinum electrodes. A conditioned defensive reflex (CDR) was developed by combination of a light and electrocutaneous stimulation (ECS) of the ear. The results showed that after a series of combinations of the light and the ECS, the light started to elicit a change in E. By 200 combinations, the brain developed both increases and decreases in E during combinations. As the number of combinations increased, increases in E were gradually replaced by decreases. We believe that this dynamic of the balance of the major sources of brain energy supply suggests that formation of the CDR may involve a significant role for subcellular structures receiving energy from oxidative metabolism formed at the relatively young evolutionary level, while the major source of energy for brain function during performance of the acquired CDR is the older evolutionary compartment — glycolysis. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 92, No. 3, pp. 273–283, March, 2006.     

Role of proprioception in the control of lid position during reflex and conditioned blink responses in the alert behaving cat

The contribution of the orbicularis oculi muscle to the determination of lid position, and the putative role of eyelid proprioception in the control of reflex and conditioned eye blinks, were studied in alert behaving cats. Upper lid movements and the electromyographic activity of the orbicularis oculi muscle were recorded during reflexively evoked blinks and during the classical conditioning of the eyelid response. Blinks were evoked by air puffs, flashes and electrical stimulation of the supraorbitary branch of the trigeminal nerve. Eyelid responses were conditioned with a trace classical conditioning paradigm consisting of a short, weak air puff, followed 250 ms later by a long, strong air puff. Orbicularis oculi muscle activation during reflex blinks was independent of lid position and was not modified by the presence of weights acting in the upward or downward directions. Local anesthesia of the supraorbital nerve reduced blinks evoked by air puffs applied to the lower jaw, but did not affect flash-evoked blinks. No relationship was established between initial lid position and the first downward component of conditioned eyelid responses. In contrast, initial lid position was related to the first upward component of the same conditioned response.

It is concluded that orbicularis oculi motor units receive no feedback proprioceptive signals from the eyelid, other than those coming from cutaneous receptors, and that lid position is determined by the activity of the levator palpebrae superioris muscle. The lack of sensory information about lid position in facial motoneurons probably has some functional implications on the central control of cognitive and emotional facial expressions.     

The conditioned reflex regulation of the blood pressure

Summary The conditioned reflexes formed on the basis of unconditioned changes of the blood pressure in stimulation of the proprioceptors with the aid of static load were studied in experiments on 3 dogs. It was demonstrated that the reflexes referred to above are rapidly, acquired, reestablished and changed, but in comparison with this their extinction is slower. It is thus assumed that these conditioned reflexes provide the adjustment of the function of the cardiovascular system to the constantly changing conditions of the body existence.Presented by Academician K. M. Bykov     

Sex difference in rabbit eyelid conditioning

The rabbit eyelid conditioned reflex has been used to compare associative learning in males and ovariectomized females. A new method for monitoring eyelid movements is described. Rabbits were trained on simple delay classical conditioning. Conditioned responses were recorded during 8 acquisition days and 6 days of extinction training. Analysis of variance (ANOVA) and the least significant difference (LSD) post hoc test was used to analyze the data. The results showed that males achieved significantly better learning than females during the first day of acquisition but later they slowly attained the best result, contrary to females. Moreover, extinction of the conditioned reflex was significantly faster in females than in males. It is postulated that females learn and extinguish faster than males because of a higher level of brain plasticity.     

Classically conditioned postural reflex in cerebellar patients

The aim of the current study was to compare postural responses to repetitive platform-evoked perturbations in cerebellar patients with those of healthy subjects using a classical conditioning paradigm. The perturbations consisted of tilting of the platform (unconditioned stimulus: US) at random time intervals, preceded by an auditory signal that represented the conditioning stimulus (CS). Physiological reactions were recorded biomechanically by measuring the vertical ground forces, yielding the center of vertical pressure (CVP), and electrophysiologically by EMG measurements of the main muscle groups of both legs. The recording session consisted of a control section with US-alone trials, a testing section with paired stimuli and a brief final section with US-alone trials. Healthy control subjects were divided into those establishing conditioned responses (CR) in all muscles tested (strategy I) and those with CR in the gastrocnemius muscles only (strategy II), suggesting an associative motor-related process is involved. Patients with a diffuse, non-localized disease were almost unable to establish CR. This was also true for a patient with a focal surgical lesion with no CR on the affected side but who, simultaneously, showed an essentially normal CR incidence on the intact side. During US-alone trials healthy controls exhibited a remarkable decay of the UR amplitude due to a non-associative motor-related process such as habituation. The decay was most prominent in the paired trials section. In contrast, patients showed no significant differences in the UR amplitude throughout the entire recording session. Analysis of the CVP supported the electrophysiological findings, showing CR in the controls only. The differences between the responses of control subjects and those of the cerebellar patients imply strongly that the cerebellum is involved critically in controlling associative and non-associative motor-related processes.This study was supported by the Wilhelm Sander-Stiftung (AZ: 94.090.1, 2, 3), by the Friedrich-Baur-Stiftung (AZ: 46/93), and by the Deutsche Forschungsgemeinschaft (AZ: TI 239/7–1)     

Temporally specific extinction of conditioned responses in the rabbit (Oryctolagus cuniculus) nictitating membrane preparation

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 (Oryctolagus cuniculus) nictitating membrane response occurred during continuous CS-US pairings. Initial training contained a mixture of 2 CS-US interstimulus intervals (ISIs): 200 ms and 1,200 ms. The CRs showed double peaks, one for each ISI. When 1 ISI was removed, its CR peak showed the hallmarks of extinction: a decline across sessions, spontaneous recovery between sessions, and rapid reacquisition when the absent ISI was reintroduced. These results support real-time models of conditioning that segment the CS into microstimuli while challenging theories that rely on contextual control, US representations, CS processing, and response inhibition.