Hippocampal lesions in rats differentially affect long- and short-trace eyeblink conditioning

Extensive previous research has implicated the hippocampus as an important structure for the acquisition of trace eyeblink conditioning. Evidence from multiple species and various lesioning methods shows that the disruption of conditioned responding (CR) may be partially dependent on the relative lengths of the conditioned stimulus (CS) period and the trace interval. The present study systematically manipulated the length of the CS and the trace interval while matching the interstimulus intervals (ISI) in rats with or without ibotenic acid hippocampal lesions. The long-trace interval condition had a CS duration of 50 ms and a trace interval of 500 ms. The short-trace interval condition had a 500 ms CS and a 50 ms trace interval. We found that control animals in the long-trace interval condition learned at a slower rate than the control animals in the short-trace interval condition. Lesioned animals in both the trace conditions showed deficits in acquisition. Lesioned animals in the short-trace interval condition acquired conditioned responses at a rate almost identical to that of the control animals in the long-trace interval condition. CR onset latencies were impaired for lesioned animals. Peak latencies were not different, indicating no difference in the adaptiveness of the CRs. These results suggest that while the hippocampus is important for acquisition of trace eyeblink conditioning, performance also depends on the parameters used for the task. In particular, the relative lengths of the CS period and the trace interval appear to be important.     

Startle reflex facilitation as a function of classical eyeblink conditioning in humans

I investigated whether classical eyeblink conditioning increases the activating effect of the conditional stimulus (CS) on the relevant alpha motoneuron pool in the facial nucleus. The activating effect was measured by the reflex modification method, where startle reflexes, sharing the facial nerve motor pathway with conditional and unconditional eyeblink reflexes, were elicited in the presence of reinforced and nonreinforced CSs (CS+ and CS?). To reduce influences of conditional arousal on startle, a weak airpuff to the eye was used as unconditional stimulus and relatively short stimulus onset asynchronies (SOAs) of 300 and 600 ms were used in three startle tests presented while conditioning was in progress. Two groups (N= 72) received 100% or 0% reinforcement (Groups 100 and 0) of a tone CS+. The CS? was not reinforced. Three startle tests, consisting of acoustic startle probes presented alone and after CS+ and CS? onset, were conducted early, middle, and late in conditioning. Results showed increased startle reflex facilitation after CS+ onset in the late startle test in Group 100, indicating that startle increased as a function of classical conditioning. Although reflex facilitation at the 300-ms SOA in Group 100 is consistent with the hypothesis that CS presentations activated motoneurons in the facial nucleus, the present study cannot itself definitively support this hypothesis. Reflex facilitation at the 600-ms SOA in Group 100 could be influenced by conditional arousal.     

Awareness is necessary for differential trace and delay eyeblink conditioning in humans

Squire et al. have proposed that trace and delay eyeblink conditioning procedures engage separate learning systems: a declarative hippocampal/cortical system associated with conscious contingency awareness, and a reflexive sub-cortical system independent of awareness, respectively ( [Clark and Squire, 1998] and [Smith et al., 2005]). The only difference between these two procedures is that the conditioned stimulus (CS) and the unconditioned stimulus (US) overlap in delay conditioning, whereas there is a brief interval (e.g., 1 s) between them in trace conditioning. In two experiments using the same procedure as Clark and Squire's group, we observed differential conditioning only in participants who showed contingency awareness in a post-experimental questionnaire, with both trace and delay procedures. We interpret these results to suggest that, although there may be multiple brain regions involved in learning, these regions are organized as a coordinated system rather than as separate, independent systems.     

Trace eyeblink conditioning in patients with cerebellar degeneration: comparison of short and long trace intervals

To elucidate whether the cerebellar cortex may contribute to trace eyeblink conditioning in humans, eight patients with degenerative cerebellar disorders (four with sporadic adult onset ataxia, three with autosomal dominant cerebellar ataxia type III and one with spinocerebellar ataxia type 6) and eight age- and sex-matched healthy control subjects were investigated. Individual high resolution three-dimensional MRI data sets were acquired. As revealed by volumetric measurements of the cerebellum using ECCET software, patients showed cerebellar atrophy to various degrees. No abnormalities were observed in the control subjects. Eyeblink conditioning was performed twice using a tone of 40 ms as conditioned stimulus, followed by a short (400 ms) and a long (1,000 ms) trace interval and an air-puff of 100 ms as unconditioned stimulus. Using the short trace interval, eyeblink conditioning was significantly impaired in cerebellar patients compared to controls, even in those who fulfilled criteria of awareness. Using the long trace interval no significant group differences could be observed. The present findings of impaired trace eyeblink acquisition in patients with cortical cerebellar degeneration suggest that the cerebellar cortex in humans, in addition to the interposed nucleus, is involved in trace eyeblink conditioning, if the trace interval is relatively short. Using a long trace interval, the cerebellum appears to be less important.     

Trace eyeblink conditioning in human subjects with cerebellar lesions

Trace eyeblink conditioning was investigated in 31 patients with focal cerebellar lesions and 19 age-matched controls. Twelve patients presented with lesions including the territory of the superior cerebellar artery (SCA). In 19 patients lesions were restricted to the territory of the posterior inferior cerebellar artery (PICA). A 3D magnetic resonance imaging was used to determine the extent of the cortical lesion and possible involvement of cerebellar nuclei. Eyeblink conditioning was performed using a 40 ms tone as conditioned stimulus (CS) followed by a stimulus free trace-interval of 400 ms and a 100 ms air-puff as unconditioned stimulus (US). In SCA patients with lesions including parts of the cerebellar interposed nucleus trace eyeblink conditioning was significantly impaired. Pure cortical lesions of the superior cerebellum were not sufficient to reduce acquisition of trace conditioned eyeblink responses. PICA patients were not impaired in trace eyeblink conditioning. Consistent with animal studies the findings of the present human lesion study suggest that, in addition to forebrain areas, the interposed nucleus is of importance in trace eyeblink conditioning. Although cortical cerebellar areas appear less important in trace compared with delay eyeblink conditioning, the present data strengthen the view that cerebellar structures contribute to different forms of eyeblink conditioning paradigms.     

Impaired long-trace eyeblink conditioning in a Tg2576 mouse model of Alzheimer's disease

Eyeblink conditioning has been used for assessing cognitive performance in cases of human neurodegenerative diseases including Alzheimer's disease (AD). Here, we tested and compared the delay and long-trace interval (TI = 500 ms) eyeblink conditionings in a Tg2576 mouse model of AD, at the age of 3, 6, and 12 months. Tg2576 mice exhibited significant impairment in trace conditioning at 6 months of age. In contrast, delay conditioning was not impaired in Tg2576 mice even at 12 months. These findings indicate that the long-TI eyeblink conditioning is more susceptible to age-related cognitive deterioration than delay conditioning in Tg2576 mice. The long-trace eyeblink conditioning could be a potential tool for detecting early cognitive deficits in AD mouse model.     

Nucleus basalis lesions attenuate aquisition,but not retention,of Pavlovian heart rate conditioning and have no effect on eyeblink conditioning

Summary Rabbits with lesions of the anterior nucleus basalis of Meynert (nBM) were compared with animals with sham lesions or unoperated control animals on a classical conditioning task in which heart rate (HR) and eyeblink (EB) conditioned responses (CRs) were as sessed. The nBM lesions impaired the magnitude of the decelerative HR CR, but had no effect on the EB CR. A second experiment, in which animals were lesioned af ter acquisition was complete, showed that anterior nBM lesions had no effect on retention of either the HR or EB CR. These data suggest that the anterior nBM may participate in the early stages of information processing in which stimuli are evaluated for their significance based on their association with a reinforcer. However, the ante rior nBM is apparently not involved in the selection of a somatomotor response to deal effectively with such changing stimulus contingencies.     

Assessment of forebrain-dependent trace eyeblink conditioning in chronic cannabis users

While CB1 knockout mice exhibit striking impairments on a cerebellar-dependent task called delay eyeblink conditioning (dEBC), these animals demonstrate intact forebrain-dependent trace EBC (tEBC). Although heavy human cannabis users also show impaired delay EBC, their performance on tEBC is currently unknown. Therefore, 13 heavy cannabis users and 13 cannabis naive controls completed a tEBC procedure. The cannabis group exhibited similar rates of conditioned responding compared to controls in the acquisition and extinction phase. Consistent with reports of overt attentional abnormalities, the cannabis group exhibited decreased N100 ERP amplitudes to the tone CS that were unrelated to mean levels of conditioning across blocks during the acquisition phase. The lack of a significant effect of heavy cannabis use on tEBC reported here, combined with the previous report of impaired dEBC in such users, mirrors the findings observed in CB1 knockout mice, and suggests that the cannabinoid system differentially mediates forebrain- and cerebellar-dependent learning processes in both humans and animals.     

Normal delay eyeblink conditioning in mice devoid of astrocytic S100B

S100B is a small calcium binding protein synthesized and secreted mostly by astrocytes. Mice devoid of S100B (S100B-KO) develop without detectable anatomic abnormalities of the brain, but exhibit enhanced hippocampal long-term potentiation and enhanced performance in hippocampus-dependent learning and memory tasks, indicating that S100B has a crucial role in hippocampal neuronal plasticity. In the present study, we examined whether S100B has a similar role in the cerebellar regions, because Bergmann glia, a specialized subset of astrocytes in the cerebellar cortex, express a particularly large amount of S100B under physiologic conditions. Unlike in the hippocampus-dependent tasks, S100B-KO mice were indistinguishable from wild-type mice in both cerebellum-dependent motor coordination and delay eyeblink conditioning, a well-established paradigm for cerebellum-dependent learning and memory. These results suggest that S100B has differential roles in the hippocampus and cerebellum.     

Differential and reversal Pavlovian conditioning in rabbits with mediodorsal thalamic lesions: assessment of heart rate and eyeblink responses

Rabbits received ibotenic acid lesions of the mediodorsal nucleus of the thalamus (MD), or sham lesions. These animals were compared on acquisition and reversal of a Pavlovian conditioned discrimination task in which tones were the conditioned stimuli and paraorbital electric shock was the unconditioned stimulus. Eyeblink and heart rate conditioned responses were assessed. Lesions of MD impaired the reversal, but not original acquisition, of the eyeblink discrimination. Heart rate discrimination was somewhat impaired during both acquisition and reversal. There were no differences between lesion and sham animals on control measures assessing general activity, somatomotor shock thresholds, or heart rate unconditioned responses.     

Neonatal maternal separation-induced changes in glucocorticoid receptor expression in posterior interpositus interneurons but not projection neurons predict deficits in adult eyeblink conditioning

Neonatal maternal separation impairs adult eyeblink conditioning. This impairment is correlated with increases in adult glucocorticoid receptor (GR) expression in the posterior interpositus nucleus [A.A. Wilber, C. Southwood, G. Sokoloff, J.E. Steinmetz, C.L. Wellman, Neonatal maternal separation alters adult eyeblink conditioning and glucocorticoid receptor expression in the interpositus nucleus of the cerebellum, Developmental Neurobiology 67 (2007) 1751–1764], a key structure in the neural circuitry controlling eyeblink conditioning. To further localize this effect, we assessed adult eyeblink conditioning and GR expression in projection versus interneurons in the interpositus of rats that had undergone standard rearing or maternal separation (1 h/day) on postnatal days 2–14. At 3 months of age, interpositus neurons were labeled with the retrograde tracer biotinylated dextran amine (BDA). After delay eyeblink conditioning, brains were processed immunohistochemically for GR and BDA labeling of interpositus neurons. GR expression was quantified in BDA-labeled and unlabeled neurons. Neonatal maternal separation impaired adult eyeblink conditioning. Control rats had significantly less GR expression in posterior interpositus BDA-unlabeled versus BDA-labeled neurons, but this difference was absent in maternally separated rats. While neonatal separation significantly increased GR expression in BDA-labeled and unlabeled posterior interpositus neurons, only GR expression in non-BDA-labeled neurons was associated with eyeblink conditioning. Thus, neonatal maternal separation may alter interneuronal modulation of interpositus output neurons, producing deficits in adult eyeblink conditioning.     

Effect of delay interval on classical eyeblink conditioning in 5-month-old human infants

Associative learning was evaluated in human infants with simple delay classical eyeblink conditioning. A tone conditioned stimulus (CS) was paired with an airpuff unconditioned stimulus (US) at three different delay intervals (250, 650, and 1,250 ms). Independent groups of healthy, full-term 5-month-old human infants were assigned to these three paired conditions and received two identical training sessions 1 week apart. The two longer delays resulted in associative conditioning, as confirmed by comparison with unpaired control groups. However, only at the 650-ms delay were associative eyeblinks adaptively timed to avoid the airpuff. The delay function at 5 months of age appears much sharper than is observed in adults. Together with the findings of A. H. Little, L. P. Lipsitt, and C. Rovee-Collier (1984), the present study suggests a downward shift in the optimal delay interval for associative eyeblink conditioning between 1 and 6 months of age. However, this delay remains longer than what is typically reported in adults.     

Fear-potentiated startle conditioning in humans: Explicit and contextual cue conditioning following paired versus unpaired training

Conditioned fear in response to explicit and contextual cues was examined using the startle reflex in three groups of participants over two sessions separated by 4–5 days. The conditioned stimulus (CS) was paired with an aversive unconditioned stimulus (US) (shock) during conditioning in the paired but not in the unpaired group. In the reaction time (RT) group, the US was a nonaversive visual signal for an RT task. In the paired group, the CS potentiated startle in the postconditioning phase. This conditioned response was fully retained over the retention interval. There was no substantial change in baseline startle (startle delivered in the absence of CS). By contrast, startle was not potentiated by the CS in the unpaired group, but baseline startle was increased from Session 1 to Session 2. In the RT group, startle was not affected by the CS, and baseline startle was reduced from Session 1 to Session 2. These results suggest that paired presentations of a CS and an aversive US result in conditioned fear in response to the CS but little contextual fear, whereas unpaired presentations of a CS and US leads to poor explicit cue conditioning but substantial contextual fear.     

Eyeblink conditioning in patients with hereditary ataxia: a one-year follow-up study

Delay eyeblink conditioning was examined in patients with genetically-defined heredoataxias and age-matched control subjects. 24 patients with spinocerebellar ataxia type 6 (SCA6), type 3 (SCA3), and Friedreichs ataxia (FRDA) participated. SCA6 affects primarily the cerebellum, whereas extracerebellar involvement is common in SCA3 and FRDA. Testing was performed in three sessions six months apart. Severity of ataxia was defined based on the International Ataxia Cooperative Rating Scale (ICARS). As expected, cerebellar patients were significantly impaired in eyeblink conditioning compared to controls. Signs of retention and further learning across sessions were present in controls, but not in the cerebellar patients. In addition, findings of disturbed timing of conditioned responses were observed. Both onsets and peaks of the conditioned responses (CRs) occurred significantly earlier in cerebellar patients. Shortened CR responses were most prominent in patients with primarily cerebellar cortical disease (SCA6). In the group of all cerebellar patients, the SCA3 and the FRDA group correlations between learning deficits and clinical findings were weak. Moderate-to-strong correlations were found in the SCA6 patients. There was no significant change, however, in clinical ataxia scores and CR incidence across the three sessions. In summary, impaired learning of conditioned eyeblink responses is a stable finding across multiple sessions in patients with degenerative cerebellar disorders. Eyeblink conditioning may be a useful measure of cerebellar impairment in patients with hereditary ataxias that primarily affect the cerebellum (such as SCA6). In other heredoataxias (such as SCA3 and FRDA), extracerebellar involvement not assessed by ICARS likely contributes to eyeblink conditioning abnormalities.     

Effects of ipsilateral cerebellum ablation on acquisition and retention of classically conditioned eyeblink responses in rats

The ipsilateral cerebellum to the trained eye has been reported to be essential for acquisition and retention of the conditioned response (CR) in rabbit eyeblink conditioning. Although pharmacological studies have suggested its important roles in other species too, to what degree does eyeblink conditioning in rats depend on the ipsilateral cerebellum is not clear. In this work, we ablated the ipsilateral cerebellum in rats before or after conditioning to examine its roles in acquisition and retention of the CR. In the first experiment, rats received ablation of the ipsilateral cerebellum and recovered for more than 3 weeks. They then underwent eyeblink conditioning for 7 days with a tone and a periorbital electrical shock. Consistent with other previous reports, hemicerebellectomized rats showed significant impairment compared to sham-lesioned rats. However, the hemicerebellectomized rats acquired CRs to some degree, and the acquired CR showed adaptive timing. In the second experiment, rats received the hemicerebellectomy after acquiring CR by 7 days of conditioning in a delay paradigm. After more than 3 weeks of recovery, they were again conditioned in a delay paradigm. Rats with ipsilateral cerebellar lesions showed severe impairment in retention of the pre-acquired CR; however, they reacquired CR to some degree during the subsequent reconditioning sessions. These results suggest that the ipsilateral cerebellum plays an important role in rat eyeblink conditioning as well but that other brain regions can partially compensate for its removal.     

Affective learning: Awareness and aversion

In two studies, we investigated the influence of aversive and nonaversive reinforcers on startle reactivity, visceral responses, and self-report during Pavlovian conditioning. Furthermore, we assessed how awareness of the stimulus contingencies affect conditioned discrimination in the different response systems. Conditioned potentiation of the startle response was only observed in the context of aversive learning. Moreover, blink potentiation occurred without awareness of the relationship between the conditioned and unconditioned stimulus. In contrast, skin conductance conditioning was independent of the aversiveness of the reinforcer and was only obtained for those individuals who could correctly verbalize the stimulus contingency in a postconditioning recognition test. Cardiac responses varied with the task demands of the situation and covaried with individual response stereotypes.     

Behavioral conditioning with interferon beta-1a in humans

Behavioral conditioning is one of the most impressive demonstrations of brain-immune system interaction. Numerous animal studies have demonstrated behavioral conditioned effects on immune functions, however, human studies are rare. We investigated whether it is possible to behaviorally condition the acute response to interferon (IFN)beta-1a. In a double-blind placebo-controlled study, 30 healthy subjects received a single injection of IFN(beta)-1a (6MIU of REBIF, Serono International) (unconditioned stimulus, UCS) together with a novel drink (conditioned stimulus, CS). Blood was drawn at baseline, 4, 8, and 24 h after drug administration. Within the first 8 h peripheral granulocytes significantly increased, while monocytes, lymphocytes, T-, B- and natural killer (NK) cell numbers were significantly reduced. In parallel, body temperature, heart rate, norepinephrine and interleukin (IL)-6 plasma levels were heightened within 8 h after injection. 8 days later, all previously IFN(beta)-treated subjects received a subcutaneous placebo (NaCl) injection, but only 15 subjects were re-exposed to the CS (experimental group), while a control group (N=15) drank water and an additional group of subjects (n=8) remained untreated (untreated group). Blood sampling was performed at baseline and at 4, 8, and 24 h. Re-exposition to the CS did not elicit conditioned responses in the experimental group. Moreover, no differences were observed between groups. These data provide negative findings regarding behavioral conditioning of cytokine effects in humans employing a one-trial learning paradigm.     

Pavlovian aversive and appetitive odor conditioning in humans: subjective, peripheral, and electrocortical changes

The effects of presynaptic guanosine-5'-O-(3-thio)triphosphate (GTPγS) on GABAergic inhibitory postsynaptic currents (IPSCs) were studied in cultured hippocampal neurons using whole-cell recordings. Inclusion of GTPγS (0.5–1 mM) in the presynaptic electrode reduced both the amplitude and paired-pulse depression of IPSCs, indicating that the probability of GABA-release had been reduced. Presynaptic GTPγS increased the depression of IPSCs by the GABA_B-receptor-agonist baclofen (10 μM), and the effect of baclofen was poorly reversible after washing. Stimulation of the GABAergic neuron at 80 Hz for 1 s was accompanied by tetanic depression of the IPSCs by 52±6% and was followed by post-tetanic potentiation (PTP), reaching a peak value of 71±21% and lasting about 100 s. IPSCs evoked after tetanic stimulation were depressed and PTP was absent when tetanic stimulation was applied within 3 min after starting injection of GTPγS into the presynaptic neuron. At longer times, basal release underlying a single IPSC was depressed. This affected the ratios recorded in response to tetanic stimulations such that tetanic depression was abolished, while PTP increased to 117±34%. In conclusion, GTPγS reduces the probability of GABA-release in both a use- and time-dependent manner, most likely through an inhibitory action on presynaptic Ca²⁺-influx through voltage-gated Ca²⁺ channels or an interaction with small GTP-binding proteins in the nerve terminals. Electronic Publication     

The cerebellum and eye-blink conditioning: learning versus network performance hypotheses

Classical conditioning of the eye-blink reflex in the rabbit is a form of motor learning that is uniquely dependent on the cerebellum. The cerebellar learning hypothesis proposes that plasticity subserving eye-blink conditioning occurs in the cerebellum. The major evidence for this hypothesis originated from studies based on a telecommunications network metaphor of eye-blink circuits. These experiments inactivated parts of cerebellum-related networks during the acquisition and expression of classically conditioned eye blinks in order to determine sites at which the plasticity occurred. However, recent evidence revealed that these manipulations could be explained by a network performance hypothesis which attributes learning deficits to a non-specific tonic dysfunction of eye-blink networks. Since eye-blink conditioning is mediated by a spontaneously active, recurrent neuronal network with strong tonic interactions, differentiating between the cerebellar learning hypothesis and the network performance hypothesis represents a major experimental challenge. A possible solution to this problem is offered by several promising new approaches that minimize the effects of experimental interventions on spontaneous neuronal activity. Results from these studies indicate that plastic changes underlying eye-blink conditioning are distributed across several cerebellar and extra-cerebellar regions. Specific input interactions that induce these plastic changes as well as their cellular mechanisms remain unresolved.