Blocking glutamate-mediated inferior olivary signals abolishes expression of conditioned eyeblinks but does not prevent their acquisition

Abstract

The inferior olive (IO) is considered a crucial component of the eyeblink conditioning network. The cerebellar learning hypothesis proposes that the IO provides the cerebellum with a teaching signal that is required for the acquisition and maintenance of conditioned eyeblinks. Supporting this concept, previous experiments showed that lesions or inactivation of the IO blocked CR acquisition. However, these studies were not conclusive. The drawback of the methods used by those studies is that they not only blocked task-related signals, but also completely shut down the spontaneous activity within the IO, which affects the rest of the eyeblink circuits in a nonspecific manner. We hypothesized that more selective blocking of task-related IO signals could be achieved by using injections of glutamate antagonists, which reduce, but do not eliminate, the spontaneous activity in the IO. We expected that if glutamate-mediated IO signals are required for learning, then blocking these signals during training sessions should prevent conditioned response (CR) acquisition. To test this prediction, rabbits were trained to acquire conditioned eyeblinks to a mild vibrissal airpuff as the conditioned stimulus while injections of the glutamate antagonist γ-d-glutamylglycine were administered to the IO. Remarkably, even though this treatment suppressed CRs during training sessions, the postacquisition retention test revealed that CR acquisition had not been abolished. The ability to acquire CRs with IO unconditioned stimulus signals that were blocked or severely suppressed suggests that mechanisms responsible for CR acquisition are extremely resilient and probably less dependent on IO-task-related signals than previously thought.

Figure 1.

Locations of injection sites in the IO for the experimental group (stars) and the control animals (circles). The injection sites for individual animals were plotted on a set of standardized coronal sections of the rabbit medulla arranged in rostral-to-caudal order with the upper left section being the most rostral. The numbers on the lower right side of each section represent the anterior–posterior distance in millimeters from the rostral part of the dorsal accessory IO. All injection sites were located in or adjacent to the rostral portion of the IO complex. IOD indicates dorsal accessory IO; IOM, medial IO; IOPr, principal IO; Sp5N, spinal trigeminal nucleus; sp5, spinal trigeminal tract; Pr, prepositus hypoglossal nucleus; 7N, facial nucleus; icp, inferior cerebellar peduncle; MVe, medial vestibular nucleus; SolN, solitary nucleus; Amb, ambiguous nucleus; DCN, dorsal cochlear nucleus.

Figure 2.

Individual examples from the same animal showing the effects of injecting DGG or vehicle in the IO on expression of tone CS-evoked CRs in the IO mapping part of the study. Each experiment starts at the top and each blink represents one trial of the 200-trial experiment. Upward deflections of the trace between the vertical CS and US markers denote conditioned eyeblinks. The timing of injections is shown by an arrow in each stack plot. A, Effect of DGG injected in the IO after 40 preinjection trials. DGG immediately abolished CRs and this effect lasted for the remainder of the experiment. B, Control experiment in which aCSF was injected in the IO. The vehicle had no effect on CRs.

Figure 3.

Group data for tone CS-evoked CR incidence (± SEM) after DGG or vehicle injections in the IO for the experimental (n = 5) and control (n = 4) groups. The dashed vertical line indicates the time of injection. The abscissa represents blocks of 10 trials, which are numbered separately for the preinjection and postinjection periods. DGG injections in both the experimental group (black triangles) and control group (black squares) rapidly abolished CRs and CR expression did not recover until the end of the experiment. Injections of vehicle had no effect on CR incidence.

Figure 4.

Individual examples of vibrissal and tone CS trials from day 3 of acquisition with DGG or vehicle injections in the IO. A, Example showing a 100-trial vCS acquisition experiment after a DGG injection. The rabbit was exhibiting no CRs on this last day of acquisition. B, The probe tone CS trials delivered to the rabbit during the same acquisition session as in A. The DGG injection prevented expression of previously learned CRs. C, Stack plot of eyeblinks of a control animal that was injected with vehicle. At this stage of acquisition, the control rabbit expressed well developed CRs. D, Eyeblinks in the tone CS trials presented in the same session as in C. In these trials, the control animal showed well timed CRs to the previously learned tone CS.

Figure 5.

A complete printout of all eyeblinks generated by one DGG-injected experimental rabbit during 3 days of vCS training and in the retention test. A, With the exception of several spontaneous responses, DGG in this rabbit suppressed the expression of CRs throughout the acquisition phase of the study. B, Despite showing no CRs during acquisition, the same animal exhibited well formed vCS-evoked CRs in the vCS-alone retention test. Dashed vertical line shows where the onset of the US would normally occur in a paired stimuli experiment.

Figure 6.

Group effects on vCS-evoked CR incidence (± SEM) during acquisition, retention test (RET), and postacquisition training for the experimental group (diamonds, n = 5) and control group (squares, n = 4). A, DGG injections suppressed expression of any vCS-evoked CRs during acquisition, but did not block learning, as shown by the presence of relatively high CR incidence in the retention test. The control group acquired CRs to the vCS quickly and CR expression reached asymptote by day 2. Both groups showed asymptotic levels of CR incidence from the first day of the no-injection, postacquisition training. B, Tone CR incidence during the 3 d of acquisition. Both groups showed high levels of CRs during the preinjection (PI) trials. DGG injections in the experimental group blocked expression of tone-evoked CRs in both the preacquisition (PA) and acquisition (AQ) trials. Vehicle injections in the control group had no effect on expression of tone-evoked CRs.