Pontine stimulation overcomes developmental limitations in the neural mechanisms of eyeblink conditioning

Abstract

Pontine neuronal activation during auditory stimuli increases ontogenetically between postnatal days (P) P17 and P24 in rats. Pontine neurons are an essential component of the conditioned stimulus (CS) pathway for eyeblink conditioning, providing mossy fiber input to the cerebellum. Here we examined whether the developmental limitation in pontine responsiveness to a CS in P17 rats could be overcome by direct stimulation of the CS pathway. Eyeblink conditioning was established in infant rats on P17-P18 and P24-P25 using pontine stimulation as a CS. There were no significant age-related differences in the rate or level of conditioning. Eyeblink conditioned responses established with the stimulation CS were abolished by inactivation of the ipsilateral cerebellar nuclei and overlying cortex in both age groups. The findings suggest that developmental changes in the CS pathway play an important role in the ontogeny of eyeblink conditioning.

Figure 1.

Mean (± standard error of the mean, s.e.m.) conditioned response (CR) percentage for rat pups trained with pontine stimulation (left) or a 2-kHz tone (Nicholson and Freeman Jr. 2004) (right) as the conditioned stimulus (CS) on postnatal days (P) P17-P18 (white symbols) or P24-P25 (black symbols). The pups were given either paired (circles) or unpaired (triangles) presentations of the CS and a periorbital shock unconditioned stimulus (US). The amount of associative learning in each paired group was determined by the increase in responding across training sessions and by the difference in CR percentage between the paired and unpaired conditions in both age groups.

Figure 2.

Mean (± s.e.m.) conditioned response (CR) percentage for rat pups trained with pontine stimulation as the CS on postnatal days (P) P17-P18 (white symbols) or P24-P25 (black symbols). The pups were given paired presentations of pontine stimulation and a shock US. Muscimol was infused into the cerebellar nuclei prior to session 6 to inactivate the cerebellar nuclei and overlying cortex ipsilateral to the conditioned eye. CRs established by paired training of pontine stimulation and the US were abolished by muscimol inactivation. Response recovery was evident in both groups on session 7.

Figure 3.

Traces of eyelid EMG activity recorded from a rat pup trained on P17 and P18 during test trials of pontine stimulation without the US on the first (S1), third (S3), fifth (S5), muscimol inactivation (session 6, MUS), and recovery (session 7, REC) sessions. The arrows indicate the onset time of pontine stimulation. Scale bar, 100 msec.

Figure 4.

Coronal sections of the rat basilar pontine nuclei (PN) depicting the effective stimulating electrode placements. The electrode placements for groups trained on P17-P18 (left) or P24-P25 (right) in the paired (black dots) or unpaired conditions (gray dots) are shown. The numbers indicate the stereotaxic coordinates in the anterior-posterior dimension relative to lambda.

Figure 5.

Coronal sections of the rat basilar pontine nuclei (PN) depicting the ineffective stimulating electrode placements. The electrode placements for groups trained on P17-P18 (gray dots) or P24-P25 (black dots) are shown. The numbers indicate the stereotaxic coordinates in the anterior-posterior dimension relative to lambda for rats trained on P17-P18 and P24-P25.

Figure 6.

Coronal sections of the rat cerebellum depicting cannula placements. The cannula placements for the groups trained on P17-P18 (gray dots) or P24-P25 (black dots) were in or just dorsal to the anterior interpositus nucleus (IP). D, dentate nucleus; F, fastigial nucleus.