John T. Green

Eyeblink classical conditioning: hippocampal formation is for neutral stimulus associations as cerebellum is for association-response.

Extensive evidence has been amassed that the cerebellum, hippocampus, and associated circuitry are activated during classical conditioning of the nictitating membrane/eyeblink response. In this article, the authors argue that the cerebellum is essential to all eyeblink classical conditioning paradigms. In addition, the septohippocampal system plays a critical role when the classical conditioning paradigm requires the formation of associations in addition to the simple association between the conditioned and unconditioned stimuli. When only a simple conditioned stimulus--unconditioned stimulus association is needed, the septohippocampal system has a more limited, modulatory role. The neutral stimulus association versus simple association-response distinction is one of the ways in which declarative or relational memory can be separated from nondeclarative or nonrelational memory in classical conditioning paradigms.

Neonatal ethanol produces cerebellar deep nuclear cell loss and correlated disruption of eyeblink conditioning in adult rats

Binge-like neonatal exposure to ethanol (EtOH) in rats, during the period of brain development comparable to that of the human third trimester, produces significant, dose-dependent Purkinje cell loss in the cerebellum and deficits in eyeblink classical conditioning. There are currently no published reports of whether neuronal loss in the cerebellar deep nuclei also results from binge-like neonatal exposure to EtOH and what the functional consequences of any cell loss might be. Since eyeblink conditioning requires cerebellar deep nuclear cells for normal learning to occur, we examined the effects of binge-like neonatal EtOH exposure on the total number of deep nuclear cells and eyeblink conditioning in adult rats. Group Ethanol (n=11) received EtOH doses of 5.25 g/kg/day on postnatal days 4-9, producing average peak blood alcohol concentrations of 363 mg/dl. Group Sham Intubated (n=11) underwent acute intragastric intubation on postnatal days 4-9 but did not receive any EtOH infusions. Group Unintubated Control (n=10) did not receive any intubations. When rats were at least 3 months old, they received either paired eyeblink conditioning or unpaired training. Following training, estimates of the total number of cerebellar deep nuclear cells were obtained using the optical fractionator, an unbiased stereological counting procedure. Rats in Group Ethanol had approximately 50% fewer deep nuclear cells compared to rats in Groups Sham Intubated and Unintubated Control, which did not differ. For 21 rats that received paired eyeblink conditioning, a highly significant correlation (+0.80) was found between the number of deep nuclear cells and learning rate in eyeblink conditioning.

The effects of ethanol on the developing cerebellum and eyeblink classical conditioning.

In rats, developmental ethanol exposure has been used to model the central nervous system deficits associated with human fetal alcohol syndrome. Binge-like ethanol exposure of neonatal rats depletes cells in the cerebellum, including Purkinje cells, granule cells, and deep nuclear cells, and produces deficits in simple tests of motor coordination. However, the extent to which anatomical damage is related to behavioral deficits has been difficult to estimate. Eyeblink classical conditioning is known to engage a discrete brain stem-cerebellar circuit, making it an ideal test of cerebellar functional integrity after developmental ethanol exposure. Eyeblink conditioning is a simple form of motor learning in which a neutral stimulus (such as a tone) comes to elicit an eyeblink when repeatedly paired with a stimulus that evokes an eyeblink prior to training (such as mild periorbital stimulation). In eyeblink conditioning, one of the deep cerebellar nuclei, the interpositus nucleus, as well as specific Purkinje cell populations, are sites of convergence for tone conditioned stimulus and somatosensory unconditioned stimulus information, and, together with brain stem nuclei, provide the necessary and sufficient substrate for the learned response. A series of studies have shown that eyeblink conditioning is impaired in both weanling and adult rats given binge-like exposure to ethanol as neonates. In addition, interpositus nucleus neurons from ethanol-exposed rats showed impaired activation during eyeblink conditioning. These deficits are accompanied by a permanent reduction in the deep cerebellar nuclear cell population. Because particular cerebellar cell populations are utilized in well-defined ways during eyeblink conditioning, conclusions regarding the underlying neural substrates of behavioral change after developmental ethanol exposure are greatly strengthened.

Classical Eyeblink Conditioning: Clinical Models and Applications

In this paper, we argue that the main reason that classical eyeblink conditioning has proven so useful when applied to clinical situations, is that a great deal of information is known about the behavioral and neural correlates of this form of associative learning. Presented here is a summary of three lines of research that have used classical eyeblink conditioning to study three different clinical conditions; autism, fetal alcohol syndrome, and obsessive-compulsive disorder. While seemingly very different clinical conditions, classical eyeblink conditioning has proven very useful for advancing our understanding of these clinical pathologies and the neural conditions that may underlie them.

A nicotinic cholinergic agonist (GTS-21) and eyeblink classical conditioning: acquisition, retention, and relearning in older rabbits.

Previously we demonstrated that GTS-21, a nicotinic cholinergic agonist, ameliorated eyeblink classical conditioning deficits in older rabbits. The present experiment was undertaken to replicate and extend these results by examining the effects of GTS-21 on retention and relearning. Retired breeder rabbits received 15 daily injections of 0.5 mg/kg GTS 21 (n = 8) or sterile saline vehicle (n = 8) during acquisition training, and no further injections occurred. Acquisition of conditioned responses (CRs) was significantly better in GTS-21-treated rabbits. During the first tone-alone retention session in week 6 of the experiment, rabbits initially treated with GTS-21 produced significantly more CRs than vehicle-treated rabbits. There were no group differences in retention at the 13-week retest. Differences in relearning were in the predicted direction but did not attain statistical significance. Results indicate that treatment with GTS-21 ameliorates learning beyond the period when the drug is actually administered.Previously we demonstrated that GTS-21, a nicotinic cholinergic agonist, ameliorated eyeblink classical conditioning deficits in older rabbits. The present experiment was undertaken to replicate and extend these results by examining the effects of GTS-21 on retention and relearning. Retired breeder rabbits received 15 daily injections of 0.5 mg/kg GTS 21 (n = 8) or sterile saline vehicle (n = 8) during acquisition training, and no further injections occurred. Acquisition of conditioned responses (CRs) was significantly better in GTS-21-treated rabbits. During the first tone-alone retention session in week 6 of the experiment, rabbits initially treated with GTS-21 produced significantly more CRs than vehicle-treated rabbits. There were no group differences in retention at the 13-week retest. Differences in relearning were in the predicted direction but did not attain statistical significance. Results indicate that treatment with GTS-21 ameliorates learning beyond the period when the drug is actually administered.

Timing in Eyeblink Classical Conditioning and Timed-Interval Tapping

The cerebellum is implicated in interval timing for diverse tasks including eyeblink classical conditioning (EBCC) and repetitive tapping. We examined performance on both tasks across identical intervals ranging from 325 to 550 ms. In five weekly sessions, 23 participants used a different interval each week, both as the target for tapping and as the delay interval in EBCC. Changes in variability as a function of the tapping or delay interval were assessed using regression analyses. The slope for repetitive tapping was comparable to two measures of temporal acuity in EBCC, onset and peak latency of the conditioned response. Each of 80 additional participants was assessed in one session at one of four tapping and delay intervals. Results were similar to those observed in the repeated measures group. These findings provide further evidence that EBCC and repetitive tapping utilize common mechanisms for representing temporal information.

Discrimination Reversal Conditioning of an Eyeblink Response is Impaired by NMDA Receptor Blockade

In the present study we examined the effects of the specific NMDA receptor antagonist CPP on discrimination reversal learning in rabbits. We report two primary findings. First, the institution of NMDA receptor blockade had no effect on a learned discrimination. Second, after stimulus reversal, CPP treatment impaired acquisition of the discrimination reversal. This impairment manifested itself early in training as a retardation in acquisition of a CR to the new CS+ and late in training as an inability to suppress responsiveness to the new CS-. Given the comparability of the present results with previously published results for phenytoin-treated rabbits, we suggest that the effects of phenytoin on learning in this paradigm is at least in part mediated by its effects on NMDA receptors. We further suggest that these findings emphasize the need to better define the role of NMDA receptor activation and hippocampally-mediated circuits in a variety of associative learning paradigms.

Abnormal Topography and Altered Acquisition of Conditioned Eyeblink Responses in a Rodent Model of Attention-Deficit/Hyperactivity Disorder

The spontaneously hypertensive rat (SHR) has been suggested as a possible animal model of attention-deficit/hyperactivity disorder (ADHD). Reductions in the volume of the cerebellum and impairments in cerebellar-dependent eyeblink conditioning have been observed in ADHD, prompting investigation into whether SHRs also exhibit eyeblink conditioning impairments. In Experiment 1, SHRs and a control strain, Wistar, were trained on a long-delay eyeblink conditioning task in which a tone conditioned stimulus was paired with a periorbital stimulation unconditioned stimulus (750-ms delay paradigm). SHRs exhibited faster acquisition of eyeblink conditioned responses (CRs) and displayed mistimed (early onset and peak latency) and larger CRs in comparison with Wistar rats. In subsequent extinction training, SHRs were slower to extinguish CRs. The authors conducted Experiment 2 using separate rats to rule out the possibility that the results of Experiment 1 were due to nonassociative responding. SHRs and Wistar rats were presented with explicitly unpaired tone and periorbital stimulation stimuli. There was no evidence of conditioning in either group, nor were there differences between the groups in terms of the number of eyeblink responses elicited by the tone. The current results support the hypothesis of cerebellar abnormalities in this rodent model of ADHD.

Evaluation of behavioral disinhibition in P/NP and HAD1/LAD1 rats.

1. Two lines of rats specifically bred for alcohol preference were exposed to two different behavioral tasks that required behavioral inhibition to successfully solve. 2. Learning and performance of a step-down passive avoidance task and a differential reinforcement of low-rate responding task were studied in P/NP and HAD1/LAD1 rats. 3. While the P rats had difficulty in learning both tasks, HAD1, LAD1 and NP rats performed at control levels. 4. These data suggest that P rats, but not HAD1 rats, may have problems learning tasks that require inhibition of ongoing or previously learned behaviors.

The long-term effects of nefiracetam on learning in older rabbits

Classical conditioning of the nictitating membrane (NM)/eyeblink response has proven utility in the study of age-related memory disorders. The 750 ms delay eyeblink conditioning procedure was used to investigate the magnitude and duration of the nootropic drug nefiracetams effect on retention and relearning. After administering daily injections of 0 (vehicle), 5, 10, or 15 mg/kg nefiracetam to 34 retired breeder rabbits during 15 days of acquisition, we tested retention and relearning 1, 5, and 12 weeks post-training. Rabbits received no drug after the initial 15 daily injections. Significant relearning was observed in the 10 mg/kg nefiracetam group 1 and 5 weeks after initial acquisition. Differences in tone-alone retention did not achieve statistical significance, although responses were numerically greater in the 10 mg/kg nefiracetam group. The effect of nefiracetam upon the ability of older rabbits to relearn a previously learned task is apparent up to 5 weeks after drug administration. Under normal conditions, a drug is administered continuously. In this experiment, nefiracetam had a significant effect long after drug administration had ceased. Prolonged administration of nefiracetam may have ameliorating effects greater than those observed in only 15 days of drug administration.