Neurotransmitter release during delay eyeblink classical conditioning: role of norepinephrine in consolidation and effect of age

Abstract

Delay classical eyeblink conditioning (EBC) is an important model of associative, cerebellar-dependent learning. Norepinephrine (NE) plays a significant modulatory role in the acquisition of learning; however, other neurotransmitters are also involved. The goal was to determine whether NE, gamma-aminobutyric acid (GABA) and glutamate (GLU) release are observed in cerebellar cortex during EBC, and whether such release was selectively associated with training. Further studies examined the role of the beta-noradrenergic receptor in consolidation of the learned response by local infusion of propranolol at 5-120 min following training into the cerebellar cortex. In vivo microdialysis coupled to EBC was performed to examine neurotransmitter release. An increase in the extracellular level of NE was observed during EBC and was maximal on day 1 and diminished in amplitude with subsequent days of training. No changes in baseline NE release were observed in pseudoconditioning indicating that NE release is directly related to the associative learning process. The extracellular levels of GABA were also increased selectively during paired training however, the magnitude of GABA release increased over days of training. GLU release was observed to increase during both paired and unpaired training, suggesting that learning does not occur prior to the information arriving in the cerebellum. When propranolol was administered at either 5-, 60-, or 120-min post-training, there was an inhibition of conditioned responses, these data support the hypothesis that NE is important for consolidation of learning. In another set of experiments we demonstrate that the timing of release of NE, GABA and glutamate are significantly delayed in onset and lengthened in duration in the 22-month-old F344 rats. Over days of training the timing of release becomes closer to the timing of training and this is associated with increased learning of conditioned responses in the aged rats.

Figure 1. Conditioning vs Pseudo-conditioning: Eyelid Conditioning

Eyelid conditioning was performed over 5 days. The Y-axis shows the percentage of conditioned response (% CR’s), the x-axis represents daily training sessions of 50 trials. Rats in the conditioning group learned this task over days (represented by higher % CR) whereas rats who under went pseudoconditioning did not learn the task. Open boxes represent conditioning rats with microdialysis probes implanted (N=12), black boxes represent pseudoconditioning rats with microdialysis probes implanted (N=12). Closed triangles represent conditioniong rats with no surgery for microdialysis probe implantation (N=6). The conditioning group learned significantly more than pseudoconditioning rats (F 175.41 [1, 40] p<0.001) and there was no significant difference between rats with or without canula implantation.

Figure 2. Temporal release of norepinephrine during eyelid conditioning

Microdialysis was performed in the cerebellar cortex on rats during training in the delay eyelid conditioning task. The time window of NE release once eyelid conditioning starts can be observed on day 1 (A; N=5 Condition, 4 Pseudocondition), day 2 (B; N=5 condition, 4 pseudocondition), day 3 (C; N= 4 condition, 3 pseudocondition), day 4 (D; N=4 condition, 3 pseudocondition) and day 5 (E; N= 4 condition; 4 pseudocondition). On day one of training (A) there was an increase in NE that peaked at the end of the behavioral training and remains significantly above baseline for 70 minutes after the training session. On all days (A–E) a clear increase in NE levels was observed with the onset of training in the conditioning group. Data are expressed as NE [nM] (y-axis) over time in 10 minute dialysate samples (x-axis). Black squares represent conditioning, open squares represent pseudoconditioning. F) Area under the curve (AUC units) representation of NE release for each day of training during Eyelid conditioning. Note that the pseudoconditioning group (Ps-Cd) did not show this pattern of transient NE release (indicates that the NE release observed (conditioning group) is directly related to the learning process in the eyeblink task.) The solid bar underneath the curves indicates the time when the rats were receiving training trials.

Figure 3. Temporal release of GABA during eyelid conditioning

Microdialysis was performed through the cerebellar cortex and interpositus nuclei on rats during training in the delay eyelid conditioning task. The time window of GABA release once eyelid conditioning starts can be observed on day 1 (A; N=5 condition, 4 pseudocondition), day 2 (B; N=5 condition, 4 pseudocondition), day 3 (C; N= 4 condition, 3 pseudocondition), day 4 (D; N=4 condition, 3 pseudocondition) and day 5 (E; N= 4 condition; 4 pseudocondition).. GABA release was significantly greater in the conditioning group on day 1 compared to days 2, 3 and 5 (p<0.05). Peak magnitude of release increases over time and the time frame of the response sharpens so that GABA release is sharply timed with the performance of CR’s by day 3 and sharpens further up to day 5. Data are expressed as percent of baseline (% baseline) (y-axis). Black squares represent conditioning, open squares represent pseudoconditioning. (F) Area under the curve representation of GABA release for each day of training during eyeblink conditioning. As was observed with NE, the conditioning group resulted in significantly higher AUC for GABA on all days except day 5, when compared to the pseudoconditioning group (Ps-Cd) (p<0.05), suggesting that the GABA release is associated with learning of the conditioned response. The solid bar underneath the curves indicates the time when the rats were receiving training trials.

Figure 4. Temporal release of glutamate during eyelid conditioning

Microdialysis was performed in the cerebellar cortex on rats during training in the delay eyelid conditioning task. The release of Glu over time during eyelid conditioning is shown on day 1 (A; N=4 condition, 4 pseudocondition), day 2 (B; N=4 condition, 4 pseudocondition), day 3 (C; N= 4 condition, 3 pseudocondition), day 4 (D; N=4 condition, 3 pseudocondition) and day 5 (E; N= 4 condition; 4 pseudocondition). Comparable levels of glutamate were released during training on the eyeblink task for both the conditioning and the pseudoconditioning groups. Data are expressed as Glu [µM] (y-axis). Black squares represent conditioning, open squares represent pseudoconditioning. (F) Area under the curve representation of Glu release for each day of training during eyelid conditioning and pseudoconditioning (Ps-Cd, average of days). There were no differences in AUC of Glu. The solid bar underneath the curves indicates the time when the rats were receiving training trials.

Figure 5

Propranolol administered 5, 60 or 120 minutes following training significantly disrupts learning of CR’s. The X-axis shows training sessions over time, with session 1 being Day 1 AM, 2 Day 1 PM, 3 Day 2 AM, 4 Day 2 PM, 5 Day 3 AM, and 6 Day 3 PM. Learning was attenuated (shown as % CR) when propanolol was administered 5 (N= 6), 60 (N=6), or 120 (N=6) minutes post training, compared to aCSF (N=14) on sessions 3, 4, 5 and 6 (indicated by *). Pseudoconditioning did not differ over sessions or as a result of drug administration (N= 6 psuedocondition control, 6 pseudocondtion propranolol at 5 mintues post training). These data show robust learning in aCSF treated rats and partial learning in propranolol treated rats when administered 5, 60 or 120 minutes post training.

Figure 6

Amplitude of the conditioned response (A,B, C,) or unconditioned response (D, E, F) were analyzed for all drug groups compared with aCSF. There were not differences between control and drug administration groups in either the 5 minute post-training administration (A, D) the 60 minute (B, E) or the 120 minute post-training groups (C, F).

Figure 7

Timing of the conditioned responses was measured for all groups. The time to onset of the CR (A, B, C) and the time of peak amplitude of the CR (D,E,F) are illustrated for both the aCSF and proranolol treatement groups. No significant differences were observed between groups for either measure.

Figure 8

Eyelid conditioning was performed over 5 days. The Y-axis shows the percentage of conditioned response (% CR’s), the x-axis represents 5 daily training sessions of 50 trials. Black triangles represent young conditioning, open triangles are young pseudoconditioning, black squares represent aging conditioning, open squares are aging pseudoconditioning. Both aging and young rats performed significantly better than their age-matched pseudoconditioning groups. For the conditioning groups, young rats learned the task faster and to a higher performance level than aged rats (p<0.01). * indicates difference between young and aged, # indicates difference between aged conditioning and aged pseudoconditioning.

Figure 9

Microdialysis was performed in the cerebellar cortex of aged rats during training in the delay eyelid conditioning task. The time window of NE release once eyelid conditioning starts can be observed on day 1 (A; N= 6 condition, 5 pseudocondition), day 3 (B; N= 6 condition, 5 pseudocondition) and day 5 (C; N= 6 condition, 5 pseudocondition). Data are expressed as NE [nM] (y-axis) over time in 10 minute dialysate samples (x-axis). Black squares represent conditioning, open squares represent pseudoconditioning. D) Area under the curve (AUC units) representation of NE release for each day of training during eyelid conditioning.

Figure 10

Microdialysis was performed in the cerebellar cortex on aged rats during training in the delay eyelid conditioning task. The time window of GABA release once eyelid conditioning starts can be observed on day 1 (A; N= 5 condition, 4 pseudocondition), day 3 (B; N= 5 condition, 4 pseudocondition) and day 5 (C; N= 5 condition, 5 pseudocondition). GABA release was significantly greater in the conditioning group on day 1 compared to day 5 (p<0.05). The pattern of release in aged rats changes across days so that by day 5 GABA remains elevated 2 hours after training. Data are expressed as percent of baseline (% baseline) (y-axis). Black squares represent conditioning, open squares represent pseudoconditioning. (D) Area under the curve representation of GABA release for each day of training during eyeblink conditioning

Figure 11

Microdialysis was performed in the cerebellar cortex on aged rats during training in the delay eyelid conditioning task. The release of Glu over time during eyelid conditioning is shown on day 1 (A; N= 4 condition, 4 pseudocondition), day 3 (B; N= 4 condition, 4 pseudocondition) and day 5 (C; N= 4 condition, 4 pseudocondition). Comparable levels of glutamate are released during training on the eyeblink task for both the conditioning and the pseudoconditioning groups. Data are expressed as Glu [µ M] (y-axis). Black squares represent conditioning, open squares represent pseudoconditioning. (D) Area under the curve representation of Glu release for each day of training during eyelid conditioning and pseudoconditioning (Ps-Cd, average of days). There were no differences in AUC of Glu.